CN116606818A

CN116606818A - Salmonella phage salmonella phage vB-SalP-JXAU-W3 and application thereof

Info

Publication number: CN116606818A
Application number: CN202310597605.2A
Authority: CN
Inventors: 舒梅; 吴国平; 潘红; 钟婵
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2023-05-25
Filing date: 2023-05-25
Publication date: 2023-08-18

Abstract

The invention discloses salmonella phage salmonella phage vB _SalP_JXAU_W3 and application thereof. salmonella phage vB SalP JXAU W3, accession number GDMCC No. 62838-B1, at month 26 of 2022, was deposited with the Guangdong province microorganism strain collection. Has the purposes of cracking salmonella and preparing bacteriostatic agent.

Description

Salmonella phage salmonella phage vB-SalP-JXAU-W3 and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to salmonella phage vB_SalP_JXAU_W3 and application thereof.

Background

Salmonella (Salmonella) is an important food-borne pathogenic bacterium, causing significant economic losses worldwide, affecting public health safety. In recent years, the salmonella resistance rate tends to rise year by year due to abuse and unreasonable use of antibiotics, and the spread of antibiotic resistant bacteria, and even resistant pathogenic bacteria (including salmonella) is more rapid, presenting a situation of hidden pandemic, leading to an increase in human mortality and economic loss vectors. To maintain food and public health safety, the world-wide country has issued the "forbidden reactance limit" regulations in succession since 1986. The Chinese agricultural rural bulletin shows that the feed 'forbidden' command is implemented from 7 months and 1 day in 2020, and the use of antibiotics is gradually 'restricted' in the field of public medical and health. Therefore, the search for safe, effective "tibody" bacteriostats that are specific to salmonella, especially resistant salmonella, is a focus of attention in the fields of livestock breeding and food safety.

The virulent phage is virus which specifically infects and kills bacteria, and has the advantages of high specificity and good safety. As a natural bacteriostat, phage has high genetic diversity and natural adaptability, and has been applied to prevention and treatment of pathogenic bacteria including multi-drug resistant bacteria infection in the fields of clinical medicine, livestock epidemic disease, aquaculture and the like. In the 21 st century, there were many commercial phage products licensed on the market for the prevention and control of food-borne pathogens such as E.coli O157H 7, salmonella, listeria monocytogenes, etc. in foods. In 2006, the U.S. Food and Drug Administration (FDA) approved the first food additive product consisting of listeria phage. Since then, other phage products have been approved for biocontrol in foods.

Jiang Gongxia et al (application publication No. CN 114921423A) disclose a multi-drug resistant Salmonella kentucky virulent phage and a preparation method and application thereof. The phage can lyse 92 strains of multidrug-resistant Kentucky salmonella.

Chen Deguo et al (application publication No. CN 15505577A) propose a novel phage for controlling diseases of E.coli and Salmonella in poultry animals and its application. The phage can lyse 12 serotype escherichia coli 54 strains and 3 serotype salmonella 14 strains, can more effectively solve the problem of bacterial disease infection in poultry cultivation, and can be used as a microecological preparation for preventing and treating bacterial diseases of poultry animal escherichia coli, salmonella and the like.

WuGuo Ping et al (application publication No. CN 112029732A) discloses a salmonella phage JNwz02 and use thereof. The phage can lyse 8 serotypes of salmonella such as stent Li Weier, hirspanus, typhoid and the like, can lyse escherichia coli O157:H27, and can also be used as a bacteriostatic agent for food storage.

Salmonella butralis Lu Bai, salmonella rison and Salmonella nchang are more common in foods such as pork, raw poultry, eggs, tuna, etc., but no phage capable of simultaneously lysing Salmonella butralis Lu Bai, salmonella rison and Salmonella nchang is available in the prior art.

Disclosure of Invention

The invention aims to provide phage for lysing the salmonella bufalin Lu Bai, salmonella enteritidis, salmonella debi, salmonella kentucky, salmonella typhimurium, salmonella rison and salmonella nchang.

To achieve the above object, salmonella phage (salmonella phage) vb_SalP_JXAU_W3 is characterized in that the salmonella phage (salmonella phage) vb_SalP_JXAU_W3 is deposited with the microorganism culture Collection in Guangdong province at month 09 of 2022 under accession number GDMCCNo 62838-B1.

The invention also protects the salmonella phage (salmonella phage) vb_SalP_JXAU_W3 for use in lysing salmonella.

Further, the salmonella is at least one of Salmonella buflomedin Lu Bai, salmonella enteritidis, salmonella debenzoica, salmonella kentucky, salmonella typhimurium, salmonella rison, and Salmonella nchang.

The invention also protects the application of the salmonella phage (Sallmonella phage) vB_SalP_JXAU_W3 in preparing a bacteriostatic agent.

Further, the use refers to the use in preparing a salmonella bacteriostat.

Further, the salmonella is at least one of salmonella enteritidis, bundler Lu Bai salmonella, delbrueckia salmonella, kentucky salmonella, salmonella typhimurium, salmonella rison and mchina.

Strain name: salmonella phage vB _SalP_JXAU_W3;

preservation date: 2022, 09, 26;

preservation unit: the Guangdong province microorganism strain collection center (GDMCC) of Guangdong province, a building of the China, which is the institute of experiments for microorganisms, building 5, national institute of advanced No. 100, university 59;

preservation number: GDMCC No. 62838-B1.

The phage (salmonella phase) vB_SalP_JXAU_W3 of the present invention belongs to the family of short-tailed phages, and has a polyhedral head with a diameter of about 56nm and a tail length of about 27nm. The one-step growth curve of the phage vB_SalP_JXAU_W3 shows that when the host bacterium is the Salmonella bufalin Lu Bai, the incubation period of the phage is 0-5min, the outbreak period is 100min, the cracking amount is 222PFU/cell, and the cracking capacity is high.

Further, phage vB_SalP_JXAU_W3 was placed at 30-50deg.C for 30min and 60min, and its titer was 10 ⁹ PFU/mL; placing at 60deg.C for 30min and 60min, respectively with titers of 10 ⁷ PFU/mL、10 ⁶ PFU/mL; the cells were left at 70℃for 30min and 60min, and the potency was undetected. Phage vB_SalP_JXAU_W3 has acid-base resistance, is relatively stable under pH 3.0-12.0, and has potency of 10 ⁹ PFU/mL。

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) Phage vB_SalP_JXAU_W3 lyses 7 serotypes of Salmonella, one of the most important food-borne pathogens worldwide. It causes food-borne diseases that often account for the first two of the bacterial food poisoning events.

(2) Phage vB_SalP_JXAU_W3 has better temperature and pH tolerance.

(3) In the present invention, the optimal multiplicity of infection of phage vB_SalP_JXAU_W3 with the host Salmonella Lu Bai is 0.0001.

(4) The phage vB_SalP_JXAU_W3 can be used as a biological bacteriostatic agent in food storage.

Drawings

FIG. 1 is a plaque plot of phage vB_SalP_JXAU_W3 on a double-layered agar plate;

FIG. 2 is a transmission electron microscope image of phage vB_SalP_JXAU_W3;

FIG. 3 is a graph showing the whole genome analysis result of phage vB_SalP_JXAU_W3;

FIG. 4 is a graph of one-step growth of phage vB_SalP_JXAU_W3;

FIG. 5 is a graph of the temperature stability of phage vB_SalP_JXAU_W3;

FIG. 6 is a graph of the pH stability of phage vB-SalP-JXAU-W3;

FIG. 7 is a graph showing the bacteriostatic effect of phage vB-SalP-JXAU-W3 on simulated contamination in milk.

FIG. 8 is a graph of the bacteriostatic effect of phage vB_SalP_JXAU_W3 on lettuce to simulate contamination.

FIG. 9 is a graph showing the therapeutic effect of phage vB-SalP-JXAU-W3 on larvae of Chilo suppressalis.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the examples to be given below,

TSB broth: 15g of tryptone, 5g of soybean peptone and 5g of NaCl are dissolved in 800mL of distilled water, the pH is regulated to 7.2+/-0.2, the volume is fixed to 1000mL by using distilled water, and the solution is sterilized by steam at the temperature of 121 ℃ for use.

LB solid medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl are dissolved in 800mL of distilled water, the pH is regulated to 7.2+/-0.2, distilled water is added to fix the volume to 1000mL, 15g of agar is sterilized by steam at 121 ℃ and then used.

Semi-solid agar medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl are dissolved in 800mL of distilled water, the pH is regulated to 7.2+/-0.2, distilled water is added to fix the volume to 1000mL, 7g of agar is sterilized by steam at 121 ℃ and then used.

SM buffer: naCl 8.5g, mg ₂ SO ₄ 2g, gelatin 0.25g,1mol/L Tris-HCl (pH 7.5) 50mL, dissolved in deionized water and fixed to 1000mL, and steam sterilized at 121 ℃.

0.01M PBS：NaCl 8g，KCl 0.2g，Na ₂ HPO ₄ ·12H ₂ O 3.63g，KH ₂ PO4, 0.24g, dissolved in 800mL deionized water, adjusted to pH7.4 with hydrochloric acid, then added with deionized water to volume to 1000mL, and sterilized by steam at 121 ℃.

Example 1: isolation and identification of phages

Isolation and purification of phages

Taking 500mL of pig farm sewage sample, adding 0.055g of calcium chloride, uniformly mixing, standing and precipitating for 2h, taking supernatant, centrifuging at 6000g and 4 ℃ for 10min, and filtering the supernatant with a 0.22 mu m filter membrane; 10mL of the filtrate was mixed with 10mL of TSB broth, and 600. Mu.L of the logarithmic phase (OD ₆₀₀ 2.5-0.6) in the above TSB broth, culturing at 37 ℃ for 12h at 220 rmp; collecting bacterial liquid, centrifuging at 4deg.C for 15min with 12000g, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain phage stock solution. Mixing 100 μL of phage stock solution with 10 times serial dilution and Lu Bai Salmonella solution obtained in logarithmic phase, incubating in a constant temperature incubator at 37deg.C for 15min, adding semisolid agar medium, rapidly mixing, pouring into a lower LB solid medium, culturing in an inverted manner in the constant temperature incubator at 37deg.C for 12-16 hr, and observing the result to form circular and transparent plaque on the medium if phage exists. The single large round transparent plaque is dug by a sterile gun head, added into 1mL SM buffer solution, kept stand for 12h at 4 ℃, centrifuged for 15min at 12000g and 4 ℃, and the supernatant is taken to obtain the single plaque by a double-layer agar plate method, and the steps are repeated for 5 times until the plaque with consistent size and shape is obtained.

Morphology of phage on double-layered agar plates, as shown in FIG. 1: the phage forms a circular, clear-centered phage plaque with a halo around it, all about 1.5mm in size, on the medium, which is a virulent phage designated vB_SalP_JXAU_W3.

Phage proliferation and titer assays

Picking single plaque on double-layer agar plate, adding into 1mL sterile SM buffer solution, standing at 4deg.C for 12 hr, centrifuging at 12000g at 4deg.C for 15min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain phage filtrate; adding 100 μl of phage filtrate into 100 μl of logarithmic phase buling Lu Bai Salmonella liquid, mixing, standing at 37deg.C for 15min, adding into 10mL TSB liquid culture medium, and shake culturing at 37deg.C at 220rpm for 5 hr; centrifuging 12000g at 4deg.C for 15min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain phage value-increasing liquid; continuous 10-fold gradient dilution of phage proliferation liquid with SM buffer solution, and measuring titer by double-layer agar plate method, measuring 10 ^-6 、10 ^-7 、10 ^-8 Three dilutions were made, 3 replicates each.

Plaques of the double-layer agar plates were counted, and the phage titer was calculated to be 2.8X10 ⁹ PFU/mL。

Morphological identification of phages

And (3) dripping 10 mu L of purified phage on the front surface of the copper net, drying at room temperature for 5min, dyeing with 2% phosphotungstic acid for 2min, and taking out and drying after dyeing. Phage morphology was observed with a FEI TalosF200X transmission electron microscope. The transmission electron microscope image shows that phage vB_SalP_JXAU_W3 belongs to the family of short-tailed phages, the head of which is polyhedral and has a diameter of about 56nm and the tail length of about 27nm. Phage morphology is shown in FIG. 2.

Gene sequence analysis of phage

The genome of phage vB_SalP_JXAU_W3 was extracted using a viral genome extraction kit, and the genomic samples were submitted to sequencing company for sequencing. Whole genome sequence analysis As shown in FIG. 3, the genome of phage vB-SalP-JXAU-W3 consisted of 39474bp double-stranded DNA (dsDNA) with 42 ORFs and a G+C content of 48.97%. According to the functional annotation, the proteins predicted by phage vB_SalP_JXAU_W3 can be divided into six classes: host lysis, DNA packaging, viral structural proteins, nucleotide metabolism, DNA replication and repair, and putative proteins. Genes encoding virulence, antibiotic resistance and phage lysogeny-related genes were not detected in the genome of phage vb_SalP_JXAU_W3. The NCBI Blast shows that the genome of phage vB_SalP_JXAU_W3 has the highest homology with Salmonella phage LPST (accession No. MN252582.1) of 98.45%.

Example 2: determination of phage vB_SalP_JXAU_W3 host Spectrum

The experimental selection involved 10 strains of Salmonella of different serotypes, and the host profile of phage vB-SalP-JXAU-W3 was determined. Wherein, 10 strains of salmonella are:

1) 1 salmonella enteritidis ATCC BAA-708;

2) 1 Buling Lu Bai Salmonella ATCC BAA-664;

3) 2 Salmonella delbrueck SeqrSC0049, seqrSC0045;

4) Salmonella 1 strain 161365;

5) Salmonella typhimurium ATCC 14028;

6) Salmonella rison SeqrSC0038;

7) 1 strain Enchang plus Salmonella SeqrSC0068;

8) 1 strain of agona salmonella SG17-135;

9) Salmonella anatipestifer NCTC8271 strain 1.

The above test strains were cultured separately to log phase (OD ₆₀₀ Respectively mixing 100 μl of each bacterial liquid with 7mL of LB semisolid culture medium, rapidly mixing, pouring into lower LB solid culture medium plate, cooling semisolid, and dripping 10 μl of phage vB_SalP_JXAU_W3 (10 ⁹ PFU/mL), plaque size was observed after culturing in an incubator at 37℃for 12-16 hours.

The results of the lysis pattern of phage vB-SalP-JXAU-W3 are shown in Table 1, which lyses 8 strains of 10 test strains, which 8 strains belong to the 7 serotypes of Salmonella, specifically Salmonella enteritidis ATCC BAA-664, salmonella enteritidis ATCC BAA-708, salmonella delbrueckii SeqrSC0049, seqrSC0045, salmonella kentucky 161365, salmonella typhimurium ATCC 14028, salmonella rison SeqrSC0038, salmonella enchang SeqrSC0068.

TABLE 1 results of the cleavage Spectrum of phage vB_SalP_JXAU_W3 Table

Strain name	Belonging to the genus	Cleavage Activity
			ATCC BAA-708	Salmonella Enteritidis	+
ATCC BAA-664	Salmonella Braenderup	+
			SeqrSC0049	Salmonella Derby	+
SeqrSC0045	Salmonella Derby	+
			161365	Salmonella Kentucky	+
ATCC 14028	Salmonella Typhimurium	+
			SeqrSC0038	Salmonella Rissen	+
SeqrSC0068	Salmonella Nchanga	+
			SG17-135	Salmonella Agona	—
NCTC8271	Salmonella Anatum	—

Note that: +: cracking; -: does not crack.

Example 3: determination of optimal multiplicity of infection (MOI) and one-step growth curve for phage vB_SalP_JXAU_W3

MOI determination:

100. Mu.L of phage (MOI of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, respectively) and 100. Mu.L of log-phase bundler were subjected to obtaining Lu Bai Salmonella ATCC BAA-664 (OD) ₆₀₀ =0.5-0.6), incubated at 37 ℃ for 15min, added to 10mL of TSB broth, and shake-cultured at 220rpm at 37 ℃ for 5h.12000g, centrifuging at 4deg.C for 15min, collecting supernatant, and filtering with 0.22 μm filter membrane for sterilization. The titer of phage under different infection complex numbers is measured by adopting a double-layer agar plate method, and the infection proportion with the highest titer is the optimal MOI.

The best MOI results are shown in Table 2, with a preferred infection of 0.0001 for phage vB_SalP_JXAU_W3 and Lu Bai Salmonella in the host.

TABLE 2 results Table of the multiplicity of infection (MOI) of phage vB_SalP_JXAU_W3

One-step growth curve determination:

100. Mu.L of phage vB-SalP-JXAU-W3 were mixed with 100. Mu.L of logarithmic phase of bundler to Lu Bai Salmonella ATCC BAA-664 at 37℃for 15min, incubated for 12000g, centrifuged at 4℃for 1min, the supernatant was discarded, and the pellet was resuspended in 5mL of TSB broth and shake-cultured at 220rpm at 37 ℃. Taking 300 μL of culture at intervals of 5min from 0 to 20min, and 300 μL of culture at intervals of 20min from 20 to 140 min; each culture was then serially diluted 10-fold in gradient and phage titers were determined on double-layer agar plates. The amount of lysis is calculated from the ratio of the final count of phage particles released during the incubation period to the initial count of infected bacterial cells.

The results of the one-step growth curve are shown in fig. 4: the host was Salmonella ATCC BAA-664, phage vB_SalP_JXAU_W3 with a latency of 0-5min, a outbreak of 100min, and a lysis of 222PFU/cell.

Example 4 analysis of phage vB_SalP_JXAU_W3 Heat stability and pH stability

Thermal stability determination: 1mL of phage solution (concentration 10) ⁸ PFU/mL) is added into a sterile 1.5mL centrifuge tube, and the mixture is placed in a constant temperature water bath kettle at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 30min and 60min respectively, and then is taken out for continuous 10-time gradient dilution, and the titer of phage is determined by adopting a double-layer agar plate method by taking the buling-obtained Lu Bai salmonella ATCC BAA-664 as host bacteria.

As shown in FIG. 5, the test results of the heat stability test show that the phage vB_SalP_JXAU_W3 was placed at 30-50deg.C for 30min and 60min, and its titer was 10 ⁹ PFU/mL; placing at 60deg.C for 30min and 60min, respectively with titers of 10 ⁷ PFU/mL、10 ⁶ PFU/mL; standing at 70deg.C for 30min and 60min, wherein the titers are 0. The phage vB_SalP_JXAU_W3 was shown to have better temperature tolerance.

Determination of pH stability: the pH of SM buffer was adjusted to 2-13 with HCl and NaOH, 100. Mu.L of phage vB_SalP_JXAU_W3 was added to 900. Mu.L of each pH buffer, water was used for 1h at 37℃and phage titer was determined by double-layer agar plate method using Lu Bai Salmonella ATCC BAA-664 as host bacteria.

As shown in FIG. 6, the results of the pH stability test are shown that the titers of phage vB_SalP_JXAU_W3 are relatively stable at pH 3.0-12.0, and are 10 ⁹ PFU/mL. The results showed a broader acid-base tolerance range for phage vB-SalP-JXAU-W3.

EXAMPLE 5 determination of bacteriostatic Effect of phage vB_SalP_JXAU_W3 in milk samples

Lu Bai Salmonella ATCC BAA-664 bacteria solution (final concentration 10) was prepared with 100. Mu.L of bufalin ⁴ CFU/mL、10 ² CFU/mL) was added to 50mL milk (commercial whole fat) to simulate contamination, and 100. Mu.L phage vB_SalP_JXAU_W3 (10) ⁸ PFU/mL), 100. Mu.L of SM buffer was added dropwise to the negative control, stored at 4℃and 1mL of the mixture was centrifuged at 12000g for 5min for 0 day, 1 day, 2 days, 3 days, and the pellet was resuspended in 1mL of sterile PBS (0.01M, pH 7.4), followed by continuous 10-fold gradient dilution, 100. Mu.L of the diluted solution was spread on LB solid medium plates, incubated at 37℃for 12-16h, and the colony numbers were counted.

The results of the bacteriostasis experiment are shown in fig. 7, wherein: a is phage vB_SalP_JXAU_W3 in milk against 10 ⁴ Bacteriostatic action of CFU/mL host bacteria; b is phage vB_SalP_JXAU_W3 in milk against 10 ² Bacteriostatic action of CFU/mL host bacteria. When the concentration of the simulated contaminant is 10 ⁴ At 3 days, the amount of Salmonella in the experimental group was continuously decreased by Lu Bai log on day 3 compared with the number of bacteria in the negative control group ₁₀ CFU/mL; when the concentration of the simulated contaminant is 10 ² The number of Salmonella in the experimental group was continuously decreased by Lu Bai as compared to the increase in the number of bacteria in the control group, wherein the decrease was 0.3log on day 3 ₁₀ CFU/mL; the result shows that the phage vB_SalP_JXAU_W3 has good antibacterial effect on the Salmonella bufalin Lu Bai in milk at 4 ℃.

EXAMPLE 6 determination of bacteriostatic Effect of phage vB_SalP_JXAU_W3 in raw vegetable samples

Salmonella ATCC BAA-664 strain solution obtained by 100 mu L of bunting Lu Bai(final concentration of 10) ⁴ CFU/mL、10 ² CFU/mL) bacterial liquid is respectively dripped on the surface of a lettuce (cut into 2X 2 cm) subjected to aseptic treatment for simulated pollution, and the lettuce is air-dried in a biosafety cabinet for 30min; 100. Mu.L of phage vB_SalP_JXAU_W3 (concentration 10) was added dropwise to the experimental group ⁹ PFU/mL), 100 mu L of SM buffer solution is dripped into the negative control group, and the mixture is air-dried for 30min again; then stored at 4℃and sampled at 0 day, 1 day, 2 days, 3 days, and thoroughly shaken 200 times with 0.85% NaCl+0.025% SDS, 1mL of the mixture was centrifuged at 12000g for 5min, the pellet was resuspended in 1mL of sterile PBS (0.01M, pH 7.4), followed by continuous 10-fold gradient dilution, 100. Mu.L of the diluted solution was spread on LB solid medium plates, and incubated at 37℃for 12-16 hours, and the colony count was counted.

The results of the bacteriostasis experiment are shown in fig. 8, wherein: a is phage vB_SalP_JXAU_W3 in lettuce to 10 ⁴ Bacteriostatic action of CFU/mL host bacteria; b is phage vB_SalP_JXAU_W3 in lettuce to 10 ² Bacteriostatic action of CFU/mL host bacteria. When the concentration of the simulated contaminant is 10 ⁴ At CFU/mL, the amount of Salmonella in the experimental group was continuously decreased by Lu Bai, 4.2log on day 3, compared to the bacterial count in the negative control group ₁₀ CFU/mL; when the concentration of the simulated contaminant is 10 ² At CFU/mL, the amount of Salmonella in the experimental group was continuously decreased by Lu Bai, 2.1log on day 3, compared to the increase in bacterial count in the negative control group ₁₀ CFU/mL; the results demonstrate that phage vB_SalP_JXAU_W3 has good bacteriostatic effect on Salmonella ATCC BAA-664 in lettuce.

EXAMPLE 7 treatment test of larvae of Paraphalocrocis medinalis of phage vB_SalP_JXAU_W3

The experimental larvae of Chilo suppressalis are randomly divided into 5 groups, wherein the 1 st and 2 nd groups are treatment groups, the 3 rd group is infection group, and the 4 th and 5 th groups are control groups. See in particular table 3. 10 larvae per group, 10. Mu.L of Buling was injected as per the protocol of Table 3 to obtain Lu Bai Salmonella ATCC BAA-664 (10 ⁶ CFU/10 μl) in the left hind leg of the larvae, phage vb_Salp_jxau_w3 was injected at MOI (100/10) two hours after infection in the right hind leg of the larvae for treatment. The survival of the larvae was observed at 24, 48, 72, 96 and 120 hours, respectively, and the survival rate was calculated.

The experimental results are shown in fig. 9, the survival rate of the larvae of the control group (group 4 and group 5) of the large wax moth is 100% after 120 hours of injection, the survival rate of the larvae of the infected group (group 3) of the large wax moth is 5% after 120 hours of injection, and the survival rates of the larvae of the treatment group 1 and the treatment group 2 are 93% and 90% respectively. The results demonstrate that phage vB-SalP-JXAU-W3 kills Boolean Lu Bai Salmonella ATCC BAA-664 in the larvae of Chilo suppressalis and is biosafety.

TABLE 3 in vivo injection of larvae of Chilo suppressalis salmonella and phage treatment tables

Group of	Treatment of	First injection	Second injection
				1	Salmonella + phage MOI 100	Salmonella: 10 ⁶ CFU/10μL	Phage: 10 ⁸ CFU/10μL
2	Salmonella + phage MOI 10	Salmonella: 10 ⁶ CFU/10μL	Phage: 10 ⁷ CFU/10μL
				3	Salmonella+physiological saline	Salmonella: 10 ⁶ CFU/10μL	Physiological saline 10. Mu.L
4	Physiological saline + phage	Physiological saline 10. Mu.L	Phage: 10 ⁸ CFU/10μL
				5	Physiological saline + physiological saline	Physiological saline 10. Mu.L	Physiological saline 10. Mu.L

Note that: salmonella is Buling Lu Bai Salmonella ATCC BAA-664;2: the phage was phage vB_SalP_JXAU_W3 3: MOI indicates the multiplicity of infection.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims

1. A salmonella phage (salmonella phage) vb_salp_jxau_w3, wherein the salmonella phage (salmonella phage) vb_salp_jxau_w3 is deposited under accession No. GDMCC No. 62838-B1, at 2022, 09, 26, with the cantonese collection of microorganism strains.

2. The salmonella phage (salmonella phase) vb_SalP_JXAU_W3 of claim 1, having use to lyse salmonella.

3. The use according to claim 2, wherein said salmonella is at least one of salmonella buterans Lu Bai, salmonella enteritidis, salmonella debi, salmonella kentuckii, salmonella typhimurium, salmonella rison and salmonella nchangensis.

4. Use of a salmonella bacteriophage (salmonella phase) vb_salp_jxau_w3 according to claim 1, for the preparation of a bacteriostatic agent.

5. The use according to claim 4, wherein the use is in the preparation of a salmonella bacteriostat.

6. The use according to claim 4, wherein the salmonella is at least one of salmonella digitata, salmonella buterae Lu Bai, salmonella debenzootic, salmonella kentucky, salmonella typhimurium, salmonella rison and salmonella nchangensis.