CN114196637A

CN114196637A - Salmonella phage (salmonella sp. phase) JNwz02 and application thereof

Info

Publication number: CN114196637A
Application number: CN202111583832.7A
Authority: CN
Inventors: 吴国平; 舒梅; 张慧珍; 钟婵
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-03-18
Anticipated expiration: 2041-12-22
Also published as: CN114196637B

Abstract

The invention discloses a salmonella phage (salmonella sp. phase) JNwz02 and application thereof. The strain is preserved in Guangdong province microorganism culture collection center at 12.05.2021, with the preservation number of GDMCC No. 61662-B1. It is a virulent phage capable of cleaving 8 serotypes of Salmonella such as Steiner, Hilden, typhoid, etc., including multi-strain resistant Salmonella, and also can cleave enterohemorrhagic Escherichia coli O157: H7. The invention also discloses application of the phage JNwz02 as a bacteriostatic agent in food storage.

Description

Salmonella phage (salmonella sp. phase) JNwz02 and application thereof

Technical Field

The invention relates to the field of microbial strains, in particular to a salmonella phage (salmonella sp. phase) JNwz02 and application thereof.

Background

Salmonella (Salmonella) and Escherichia coli (Enteromorrhagic Escherichia coli, EHEC) O157: H7 are two important food-borne pathogens that are co-morbid to humans and animals, causing significant economic loss worldwide and serious threats to public health and safety. Since the last century, a series of antibiotics after the discovery of penicillin by Fleming are discovered and synthesized and are applied to clinical practice and prevention and treatment of livestock and poultry diseases, and the infection of food-borne pathogenic bacteria is effectively controlled. However, in recent years, the emergence of a large number of resistant strains has been increased by the abuse of antibiotics, and there has been a serious trend toward high levels of resistance, multiple resistance and cross-resistance. Therefore, finding a biological bacteriostatic agent against food-borne pathogenic bacteria has become an elusive problem.

Bacteriophages are a class of viruses capable of specifically infecting and lysing bacteria, widely present in the environment and in living organisms, and the number of which is estimated to be as high as 10³⁰–10³². Since the discovery of phages in 1915, they were realized to have great potential for the treatment of bacterial infectious diseases and successfully controlled the spread of various types of bacteria. A large number of researches show that the phage is successfully applied to the prevention and control of bacterial infection in the fields of human medical treatment, livestock and poultry cultivation, aquaculture and the like, and has great potential in the prevention and control of food-borne pathogenic bacteria in food. Furthermore, in 2006, the U.S. Food and Drug Administration (FDA) approved the first product consisting of listeria phage as a food additive. In 2011, phage-based EcoSeeld was approved by FDA^TMThe product is used for preventing infection of EHEC O157: H7 in red-ground meat. In 2007, the United states department of agriculture (USDA/FSIS) approved phages for control of EHEC O157: H7 in livestock, and 2008 approved phages for control of Salmonella in birds. In recent years, many researchers in China also develop researches on applying the phage to control the pollution of food-borne pathogenic bacteria successively, and obtain good effects.

Mono Baolong et al (application publication No. CN107099511A) disclose a phage phi Sa-1 with Salmonella typhimurium control effect and application thereof. The bacteriophage has cracking effect on 10 salmonella in 18 bacteria (including salmonella, shigella and escherichia coli), and has no cracking effect on both shigella and escherichia coli; the bacteriophage is applied to the prevention and treatment experiment of mouse salmonella typhimurium infection, and the result shows that the prevention group and the treatment group have certain effect on mouse resistance to salmonella infection.

Biyan et al (grant No.: CN 109825479B) disclose a broad spectrum Salmonella phage LPSTLL and its application. The salmonella bacteriophage LPSTLL can crack 13 serotypes of salmonella such as typhimurium, enteritis, dublin and the like, and can also crack a plurality of strains of salmonella with drug resistance. The bacteriophage can inhibit contamination of milk and chicken caused by Salmonella proliferation.

Huangjie et al (application publication No.: CN 112029732A) disclose a high temperature resistant, broad lysis spectrum Salmonella bacteriophage SG8P3 which cleaves 153 strains of Salmonella of 34 serotypes of 160 strains of Salmonella, and compositions thereof. The chick breeding experiment shows that the salmonella phage SG8P3 can be used as a biological bactericide or a feed additive, and can effectively prevent and treat salmonella infection in the chick breeding process.

The technical scheme has the following defects: the research on the salmonella bacteriophage mainly focuses on the cracking and application aspects of different serotypes of salmonella, and the research on the aspects that the salmonella bacteriophage can also crack food-borne pathogenic bacteria of other genera or species and the application aspects is rarely reported. Therefore, there is a need to find a salmonella bacteriophage that can kill two or more food-borne pathogenic bacteria.

Disclosure of Invention

The invention aims to provide a salmonella bacteriophage capable of cracking salmonella and enterohemorrhagic Escherichia coli.

To achieve the above object, the present invention provides a salmonella phage (salmonella sp.phase) JNwz02, wherein the salmonella phage (salmonella sp.phase) JNwz02 has a deposit number of GDMCC No:61662-B1, and is deposited at the guangdong provincial collection of microorganisms at 12/05/2021.

The invention also protects the use of the salmonella bacteriophage (salmonella sp. phase) JNwz 02.

Further, the application refers to the application of salmonella phage (salmonella sp. phase) JNwz02 in cracking two food-borne pathogenic bacteria; preferably, the two food-borne pathogenic bacteria are salmonella and enterohemorrhagic escherichia coli.

Further, the salmonella is at least one of salmonella stainers, salmonella shiedensis, salmonella typhi, salmonella arizonensis, salmonella kovas, salmonella albani, salmonella kovas and salmonella albani.

Further, the enterohemorrhagic Escherichia coli refers to enterohemorrhagic Escherichia coli O157: H7.

Further, the application refers to the application in preparing bacteriostatic agents.

Furthermore, the application refers to the application in preparing the food-borne pathogenic bacteria bacteriostatic agent.

The phage has been deposited in the Guangdong province microorganism culture Collection (GDMCC) in 12.05.2021, the GDMCC is for short, the deposition number is GDMCC No:61662-B1, and the phage is classified and named as salmonella phage (salmonella sp. phase) JNwz02 with the address of: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

The phage (salmonella sp. phase) JNwz02 belongs to the family of long-tail bacteriophages, and has a polyhedral head with a diameter of about 92nm and a tail length of about 195 nm. By one-step growth curve representation, when the host is salmonella stainer RSE39, the incubation period of the phage Jnwz02 is 5-10min, the outbreak period is 100min, the cracking amount is 165PFU/cell, and the cracking capability is strong; when the host is enterohemorrhagic Escherichia coli O157: H7 AV4997, the incubation period of the phage JNwz02 is 0-5min, the outbreak period is 80min, and the cracking amount is 34 PFU/cell.

Further, phage JNwz02 was placed at 30-60 ℃ for 15min and 30min with titer of 10⁹–10¹⁰PFU/mL; standing at 70 deg.C for 15min to reduce the potency by 4 orders of magnitude, standing at 70 deg.C for 30min to reduce the potency by 6 orders of magnitude; the mixture was left at 80 ℃ for 15min, and the titer was not detected. The phage JNwz02 has acid-base tolerance and is at pH 2.0-11.0For stability, the potency is 10⁹PFU/mL。

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

(1) the phage JNwz02 can crack 8 serotypes of salmonella and enterohemorrhagic Escherichia coli O157: H7, wherein the salmonella and the enterohemorrhagic Escherichia coli O157: H7 are two very important food-borne pathogenic bacteria in the world. The food-borne diseases they cause usually account for the first two events of bacterial food poisoning.

(2) Phase JNwz02 has good temperature and pH tolerance.

(3) In the present invention, the optimal complex infection of the phage (salmonella sp. phase) JNwz02 and the host bacterium Salmonella stainers RSE39 is 0.01, and the optimal complex infection with the enterohemorrhagic Escherichia coli O157: H7 AV4997 is 10.

(4) The phage JNwz02 can be applied to food, and can safely and effectively prevent and control the pollution of salmonella and enterohemorrhagic Escherichia coli O157: H7.

The phage JNwz02 can be used as a biological bacteriostatic agent to be applied to food storage.

Drawings

Fig. 1 is a plaque map of phage (salmonella sp. phase) JNwz02 on a double layer agar plate;

fig. 2 is a transmission electron micrograph of phage (salmonella sp. phase) JNwz 02;

fig. 3 is a graph showing the results of whole genome analysis of phage (salmonella sp. phase) JNwz 02;

fig. 4 is a one-step growth plot of phage (salmonella sp. phase) JNwz 02;

fig. 5 is a temperature stability plot of phage (salmonella sp. phase) JNwz 02;

fig. 6 is a pH stability plot of phage (salmonella sp. phase) JNwz 02;

FIG. 7 is a diagram of the bacteriostatic effect of phage (salmonella sp. phase) JNwz02(25 ℃ condition);

fig. 8 is a diagram of the bacteriostatic effect of phage (salmonella sp. phase) JNwz02 on simulated pollution of the duck neck.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the following examples, the following examples are given,

TSB liquid medium: dissolving 15g of tryptone, 5g of soybean peptone and 5g of NaCl in 800mL of distilled water, adjusting the pH to 7.2 +/-0.2, then adding distilled water to reach a constant volume of 1000mL, and sterilizing by using pressure steam at 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 value is adjusted to 7.2 +/-0.2, then the distilled water is added to the solution to reach the constant volume of 1000mL, 15g of agar is added, and the solution is sterilized by steam at the temperature of 121 ℃ for use.

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 adjusted to 7.2 +/-0.2, then the distilled water is added to the solution to reach the constant volume of 1000mL, 7g of agar is added, and the solution is sterilized by steam at the temperature of 121 ℃ for use.

SM buffer solution: NaCl 8.5g, Mg₂SO₄2g, 0.25g of gelatin, 50mL of 1mol/L Tris-HCl (pH7.5), dissolving with deionized water, diluting to 1000mL, and sterilizing with 121 ℃ pressure steam.

0.01mol/L PBS：NaCl 8g，KCl 0.2g，Na₂HPO₄·12H₂O 3.63g，KH₂PO40.24g is dissolved in 800mL of deionized water, the pH value is adjusted to 7.4 by hydrochloric acid, then the deionized water is added to the solution to reach the volume of 1000mL, and the solution is sterilized by pressure steam at the temperature of 121 ℃ for use.

Example 1: isolation and characterization of phages

Isolation of a phage

(1) Taking 500mL of water sample from a lake near a farmer market of western and Jiang agriculture university, adding 0.055g of calcium chloride, uniformly mixing, standing and precipitating for 2 h; concentrating the supernatant, and centrifuging the concentrated solution at 6000g and 4 deg.C for 10 min; the supernatant was filtered through a 0.22 μm filter. Mixing 10mL of the above filtrate with 10mL of TSB liquid culture medium, and adding 600 μ L of host bacteria (Salmonella Stanvillensis RSE39) (OD)₆₀₀0.5-0.6) in the above TSB liquid medium, culturing at 37 deg.C and 220rmp for 12h, collecting the bacterial liquid, centrifuging at 4 deg.C for 15min at 12000g, and filtering the supernatant with 0.22 μm filter membrane. Repeating the steps for three times, and preserving the filtrate at 4 ℃ to obtain the phage stock solution.

(2) 100 μ L of log phase host bacteria (OD) were taken₆₀₀0.5-0.6) were spread on a plate of LB solid medium, after drying, the plate was divided into five regions of uniform size and marked, four of the regions were spotted with 10. mu.L of phage stock solution, and another region was spotted with 10. mu.L of 0.01M PBS solution as a control, and cultured at 37 ℃ for 12 hours, and the occurrence of plaques on the plate was observed. Simultaneously, the host bacterium solution (OD) is added₆₀₀0.5-0.6) and 100. mu.L of 10-fold serial diluted phage stock solution, incubating in a constant temperature incubator at 37 ℃ for 15 min; the mixture was added to semi-solid agar medium (about 45 ℃ C.), mixed rapidly and poured into the lower LB solid medium plate. After the semi-solid is cooled, the plate is placed upside down in a constant temperature incubator at 37 ℃ for 12-16h, and plaques are observed and counted.

The morphology of the phage on double-layer agar plates, as shown in FIG. 1: the bacteriophage forms a round and transparent bacteriophage plaque with a halo around, and the diameter of the bacteriophage plaque is about 0.5-1.5 mm on the solid culture medium; the phage is a virulent phage.

(3) Purification and potency determination: picking single large round transparent plaque on a double-layer agar plate, adding into 1mL sterile SM buffer solution, standing at 4 ℃ for 12h, centrifuging at 12000g and 4 ℃ for 15min, collecting supernatant, and filtering with a 0.22 mu m filter membrane to obtain phage filtrate a; 100 μ L of phage filtrate a was added to 100 μ L of host bacterial liquid (OD) in log phase₆₀₀0.5-0.6), standing and incubating for 15min at 37 ℃, adding into 10mL of TSB liquid culture medium, and shaking and culturing at 37 ℃ and 220rpm for 5 h;12000g, centrifuging for 15min at 4 ℃, collecting supernatant, and filtering by using a 0.22 mu m filter membrane to obtain phage filtrate b; using SM buffer solution to perform continuous 10-fold gradient dilution on the phage filtrate b, and taking the dilution degree as 10^–6The phage dilution and the host bacterial liquid (OD) in the logarithmic phase₆₀₀0.5-0.6), standing and incubating for 15min at 37 ℃, adding the mixed solution into a semisolid agar culture medium (about 45 ℃), quickly mixing and pouring into a lower layer LB solid culture medium plate, cooling the semisolid, and then inversely placing the plate in a constant temperature incubator at 37 ℃ for culturing for 12-16 h.

Repeating the steps for 5 times to obtain the purified phage named as the phage Jnwz 02. The plaque is counted on a double-layer agar plate, and the experimental result shows that the titer of the phage reaches 10¹⁰PFU/mL and above.

Identification of two phages

And (3) identifying the morphology of the transmission electron microscope: and (3) dripping 10 mu L of purified phage on the front surface of a copper net, drying at room temperature for 5min, dyeing with 2% phosphotungstic acid for 2min, taking out after dyeing, and drying. Phage morphology was observed with a FEI TalosF200X transmission electron microscope. Transmission electron microscopy shows that phage JNwz02 belongs to the family Long-tailed bacteriophages, with a polyhedral head with a diameter of about 92nm and a tail length of about 195 nm. The phage morphology is shown in FIG. 2.

Gene sequence analysis: the genome of the phage JNwz02 is extracted by adopting a virus genome extraction kit, and a genome sample is delivered to a sequencing company for sequencing. Whole genome sequence analysis As shown in FIG. 3, the genome of phage JNwz02 consisted of 114390 double-stranded DNA with 164 ORFs and a total G + C content of 40.22%. Of the 164 ORFs, 81 had significant homology to the reported functional genes, and the remaining 83 ORFs were annotated as hypothetical proteins. No genes encoding virulence, antibiotic resistance and phage lysogenicity related were detected in the genome of phage JNwz 02. NCBI Blast showed that phage JNwz02 has the highest homology of 98.43% with Salmonella phage Seabear (accession No. MK728824.1) and 96.57% with Enterobacter T5 phage Escherichia virus EPS7 (accession No. CP000917.1).

The strain name: phase) JNwz02,

the preservation date is as follows: the year 2021, the month 5 and the day 12,

the preservation unit: no. 59 building No. 5 building of the Fujiu No. 100 of the Pieli, Guangzhou city, Guangdong province microbial culture Collection center (GDMCC),

the preservation number is: GDMCC No. 61662-B1.

Example 2: determination of the lysis Profile of the phage JNwz02

The experiment selects 14 strains of bacteria including salmonella, escherichia coli and the like, and performs lysis spectrum determination on the phage Jnwz 02.

Among them, 14 strains of bacteria are:

1)1 strain of salmonella stainer RSE 39;

2)1 strain of salmonella schildiensis N1529-D3;

3)2 strains of Salmonella typhi ATCC BAA-664, NCTC 8271;

4)1 strain of salmonella arizonensis LHICA _ AZ 23;

5)1 strain of salmonella barrelii CFSAN 000189;

6)1 strain of salmonella hederabergii AR-0404;

7)1 strain of salmonella kovarenii AR-0406;

8)1 salmonella albonensis CVM N18S 2238;

9)1 strain of salmonella kentuckiensis 161365;

10)1 salmonella enteritidis strain BNCC 336875;

11)1 strain of salmonella typhimurium BNCC 185946;

12)1 strain escherichia coli ATCC 25922;

13)1 enterohemorrhagic Escherichia coli O157: H7 AV 4997.

The above test strains were cultured to logarithmic phase (OD) respectively₆₀₀0.5-0.6), mixing 100 mu L of phage JNwz02 with 100 mu L of log phase bacterial liquid respectively, incubating at 37 ℃ for 15min, mixing with 7mL of LB semisolid culture medium, adding semisolid agar culture medium (about 45 ℃) into the mixed liquid, quickly mixing, pouring into a lower layer LB solid culture medium plate, cooling the semisolid, inversely placing the plate in a 37 ℃ constant temperature incubator for culturing for 12-16h, and counting plaques. HoverThe main bacterium Salmonella stevesii RSE39 was tested in the same manner. Calculating the plaque forming rate (EOP) value according to the average plaque value of the test bacterium plate/the average plaque value of the host bacterium. When the EOP is more than or equal to 0.5, the test bacteria are classified as 'high efficiency'; EOP of 0.1 ≤<At 0.5, the test bacteria are classified as "intermediate effect", and EOP is 0.001 ≦ EOP<0.1, test bacteria were classified as "inefficient" efficiency; when EOP ≦ 0.001 was classified as "null", the test bacteria could not be lysed by the phage JNwz 02.

The results of the lysis profile of phage JNwz02 are shown in Table 1, and phage JNwz02 was able to lyse 11 of 14 test strains (9 Salmonella strains, 2 E.coli strains). The phage JNwz02 can efficiently crack 7 strains of salmonella (Salmonella stainers RSE39, Salmonella welldendridunnii N1529-D3, Salmonella typhi NCTC8271, Salmonella arizona LHICA _ AZ23 and Salmonella albanii CVM N18S2238), wherein 6 strains of salmonella are drug-resistant salmonella. Phage JNwz02 was able to "intermediate" lyse Salmonella Colavanii AR-0406, Escherichia coli ATCC 25922, EHEC O157: H79490. The phage JNwz02 was "inefficient" in cleaving Salmonella albanii CVM N18S 2238.

TABLE 1 lysis Profile results of phage JNwz02

Remarking: a: ampicillin; AC: amoxicillin; c: cefotaxime; t: a tetracycline; g: gentamicin; n: neomycin; f: florfenicol; CI: ciprofloxacin; na: nalidixic acid; s: compound sulfamethoxazole; -: no resistance.

Example 3: determination of the optimal multiplicity of infection (MOI) and one-step growth Curve for the phage JNwz02

MOI determination:

mu.L phage (

MOI

100, 10, 1, 0.1, 0.01, 0.001, 0.0001) and 100. mu.L log phase host bacterial liquid (OD)₆₀₀0.5-0.6), incubated at 37 ℃ for 15min, added to 10mL of TSB liquid medium at 37 ℃ and filteredShaking and culturing at 220rpm for 5 h. 12000g, 4 ℃ centrifugal 15min, the supernatant fluid using 0.22 m filter membrane filtration sterilization. Measuring the titer of the phage under different infection complex numbers by adopting a double-layer agar plate method, wherein the infection proportion with the highest titer is the optimal MOI;

the optimal MOI results are shown in Table 2, where the optimal multiplicity of infection for phage JNwz02 and host Salmonella steinvirens RSE39 is 0.01, and the optimal multiplicity of infection for EHEC O157: H7 AV4997 is 10.

One-step growth curve determination:

100. mu.L phage JNwz02 and 100. mu.L log phase Salmonella Stanvillensis RSE39 bacterial liquid or EHEC O157: H7(OD₆₀₀0.5-0.6), incubating at 37 ℃ for 15min, centrifuging at 12000g and 4 ℃ for 1min, discarding the supernatant, resuspending the pellet in 5mL of TSB liquid medium, and culturing at 37 ℃ and 220rpm with shaking. Respectively taking 300 μ L of culture every 5min at 0-20min, and taking 300 μ L of culture every 20min at 20-140 min; each culture was then serially diluted in 10-fold gradients and the titer of the phage was determined on double agar plates. The amount of lysis is calculated from the ratio of the final count of released phage particles in the incubation period to the initial count of infected bacterial cells.

The results of the one-step growth curve are shown in FIG. 4: when the host is Salmonella stainers RSE39, the incubation period of phage Jnwz02 is 5-10min, the outbreak period is 100min, and the cracking amount is 165 PFU/cell; when the host is EHEC O157: H7 AV4997, the incubation period of the phage JNwz02 is 0-5min, the outbreak period is 80min, and the lysis amount is 34 PFU/cell.

TABLE 2 Complex infection (MOI) results for phage JNwz02

Example 4 phage JNwz02 thermostability and pH stability assays

And (3) measuring the thermal stability: 1mL of phage solution (10 concentration)⁸PFU/mL) into a sterile 1.5mL centrifuge tube, and placing in a constant temperature water bath at 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 15min and 30minAfter min, taking out and carrying out continuous 10-fold gradient dilution, and determining the titer of the phage by using Salmonella steinernema RSE39 as a host bacterium and adopting a double-layer agar plate method.

The results of the thermal stability assay are shown in FIG. 5, in which the phage JNwz02 was placed at 30-40 deg.C for 15min and 30min, and its titer was 10¹⁰PFU/mL; standing at 50 deg.C for 15min and 30min to obtain titer of 10⁹PFU/mL; standing at 60 deg.C for 15min to obtain titer of 10⁹PFU/mL; standing for 30min to obtain a titer of 10⁸PFU/mL; standing at 70 deg.C for 15min to obtain a titer of 10⁶PFU/mL; standing for 30min to obtain a titer of 10³PFU/mL; standing at 80 deg.C for 15min and 30min, and the titer is 0. The phage JNwz02 is proved to have better temperature tolerance.

And (3) measuring the pH stability: the pH of SM buffer was adjusted to 2-13 with HCl and NaOH, 100. mu.L each of phage JNwz02 was added to 900. mu.L each of pH buffers, water bath was carried out at 37 ℃ for 1h, and then phage titer was determined by the double agar plate method using Salmonella Staunwell RSE39 as host bacteria.

The results of the pH stability assay are shown in FIG. 6, and the titer of the phage JNwz02 is relatively stable under the condition of pH 2.0-11.0, and is 10⁹PFU/mL; phage JNwz02 titer 10 at pH 12.0 buffer³PFU/mL. The results show that the phage JNwz02 has a wide range of acid-base tolerance.

Example 5 bacteriostatic assay of bacteriophage JNwz02 at 25 deg.C

Mixing 100 μ L phage JNwz02(

MOI

100, 10, 1, 0.1, 0.01, respectively) and 100 μ L Salmonella log-phase 2089b or EHEC O157: H7 bacterial solution (OD₆₀₀0.5-0.6), adding into 50mL of TSB liquid medium, performing static culture on salmonella 2089b at 25 ℃ for 24H, performing static culture on EHEC O157H 7 bacterial liquid at 25 ℃ for 16H, sampling at different time points, and measuring OD₆₀₀The values are respectively detected for the growth conditions of Salmonella stainer RSE39 and EHEC O157: H7 AV 4997. SM buffer was added to the negative samples instead of phage.

The results of the bacteriostatic experiments are shown in fig. 7, wherein: a is the bacteriostatic action of a bacteriophage Jnwz02 on Salmonella steinernema RSE 39; b is the bacteriostatic action of bacteriophage JNwz02 on EHEC O157: H7. When the MOI was 100, 10, 1, 0.1, 0.01 at 25 ℃, the number of bacteria in the experimental group was always kept at a lower level than that in the negative control group, and the number of bacteria was all 100CFU/mL or less. The result shows that the phage JNwz02 has good bacteriostatic effect on Salmonella steinernema RSE39, EHEC O157: H7 AV4997 at 25 ℃.

Example 6 evaluation of bacteriophage JNwz02 on inhibitory effect on the neck of marinated duck

Respectively dripping 100 μ L of Salmonella steinernema RSE39 and EHEC O157: H7 AV4997 bacterial solution on the sterilized surfaces of the necks of the marinated ducks for simulated pollution, wherein the final concentration of the bacterial solution is 10⁴CFU/g; air drying duck neck sample in biological safety cabinet for 30min, and adding 100 μ L bacteriophage JNwz02 (concentration of 10) dropwise into experimental group⁹PFU/mL), 100 μ L of SM buffer was added dropwise to the control group, and air-dried again for 30 min; then storing at 4 deg.C, sampling at 0 day, 1 day, 2 days, shaking with 0.85% NaCl + 0.025% SDS for 200 times, centrifuging 1mL of mixed solution at 12000g for 5min, continuously diluting the supernatant with 10-fold gradient, smearing 100 μ L of diluted solution on LB solid medium plate, culturing at 37 deg.C for 12-16h, and counting the colony number.

The experimental result is shown in fig. 8, wherein a is the bacteriostatic action of phage JNwz02 on salmonella steviolensis RSE39 in the duck neck marinated with water; b is the bacteriostasis of bacteriophage JNwz02 to EHEC O157: H7 AV4997 in the brine duck neck. Compared with the control group with 1.0log10 CFU/mL of bacteria number reduction, the experimental group has 3.0log10 CFU/mL of Salmonella stanlveri RSE39, EHEC O157: H7 AV4997 reduction. The result shows that the phage JNwz02 has good bacteriostatic effect on Salmonella steinernema RSE39 and EHEC O157: H7 AV4997 in the brine duck neck.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A salmonella phage (salmonella sp.phase) JNwz02, wherein the salmonella phage (salmonella sp.phase) JNwz02 has a deposit number GDMCC No 61662-B1, and is deposited at the guangdong provincial collection of microorganisms at 12/05/2021.

2. Use of the salmonella phage (salmonella sp. phase) JNwz02 according to claim 1.

3. The use according to claim 2, wherein the use is of salmonella phage (salmonella sp.phase) JNwz02 for lysing two food-borne pathogenic bacteria; preferably, the two food-borne pathogenic bacteria are salmonella and enterohemorrhagic escherichia coli.

4. The use of claim 3, wherein the Salmonella is at least one of Salmonella stainers, Salmonella shiedensis, Salmonella typhi, Salmonella arizona, Salmonella kovassii, Salmonella albanii, Salmonella kovassii, and Salmonella albanii.

5. The use according to claim 3, wherein the enterohemorrhagic Escherichia coli is EHEC O157: H7.

6. The use according to claim 2, in the preparation of bacteriostatic agents.

7. The use according to claim 2, in the preparation of a bacteriostatic agent for food-borne pathogenic bacteria.