CN114196637B - Salmonella phage Jnwz02 and application thereof - Google Patents
Salmonella phage Jnwz02 and application thereof Download PDFInfo
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- CN114196637B CN114196637B CN202111583832.7A CN202111583832A CN114196637B CN 114196637 B CN114196637 B CN 114196637B CN 202111583832 A CN202111583832 A CN 202111583832A CN 114196637 B CN114196637 B CN 114196637B
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A—HUMAN NECESSITIES
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- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10311—Siphoviridae
- C12N2795/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12N2795/00—Bacteriophages
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- C12N2795/10311—Siphoviridae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses salmonella phage JNwz02 and application thereof. The strain is deposited at the microorganism strain collection of Guangdong province at 2021, 05 and 12 days, and the deposit number is GDMCC No. 61662-B1. It is a virulent phage capable of lysing 8 serotypes of salmonella, including multi-strain resistant salmonella, such as stoneley Li Weier, hilingon, and also capable of lysing enterohemorrhagic Escherichia coli O157: H7. The invention also discloses application of the phage JNwz02 as a bacteriostatic agent in food storage.
Description
Technical Field
The invention relates to the field of microbial strains, in particular to a salmonella bacteriophage JNwz02 and application thereof.
Background
Salmonella (Salmonella) and enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli, EHEC) O157: H7 are two important zoonotic food-borne pathogens that cause significant economic losses worldwide and severely threaten public health safety. A series of antibiotics after penicillin is discovered and synthesized from fleming in the last century are applied to clinical practice and prevention and treatment of livestock and poultry diseases, and food-borne pathogenic bacteria infection is effectively controlled. However, in recent years, the abuse of antibiotics has led to the continued emergence of a large number of resistant strains, and there has been a serious trend towards high levels of resistance, multi-resistance and cross-resistance. Therefore, finding biological bacteriostasis agents against food-borne pathogens has become an unprecedented problem.
Phages are a class of viruses capable of specifically infecting and lysing bacteria, widely occurring in the environment and organisms, and estimated to be up to 10 in number 30 –10 32 . Since 1915 phages were discovered, people realized that they have great potential for the treatment of bacterial infections and successfully controlled the spread of various bacteria. A great deal of researches show that the phage has been successfully applied to the prevention and control of bacterial infection in the fields of human medicine, livestock breeding, aquaculture and the like, and has great potential in the prevention and control of food-borne pathogenic bacteria in foods. Furthermore, the U.S. Food and Drug Administration (FDA) approved the first product consisting of listeria phage as a food additive in 2006. In 2011, the FDA approved phage-based EcoShield TM The product is used for preventing EHEC O157:H27 infection in red 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 poultry. In recent years, many researchers in China have also continuously conducted research on the application of phage to the control of food-borne pathogenic bacteria pollution, and good effects are obtained.
Shan Baolong et al (application publication No. CN 107099511A) disclose a phage PhiSa-1 with Salmonella typhimurium control effect and its application. The phage has a lysis effect on 10 strains of salmonella in 18 strains of bacteria (including salmonella, shigella and escherichia coli), and has no lysis effect on shigella and escherichia coli; the phage is applied to the experiment of preventing and treating the salmonella typhimurium infection of the mice, and the result is that both the prevention group and the treatment group have a certain effect on the salmonella infection resistance of the mice.
Zhou Yang et al (grant bulletin number: CN 109825479B) disclose a broad-spectrum salmonella phage LPSTLL and use thereof. The salmonella phage LPSTLL can lyse 13 serotypes of salmonella such as typhimurium, enteritis, dublin and the like, and can lyse a plurality of strains of salmonella with drug resistance. The phage can inhibit the contamination of milk and chicken caused by salmonella proliferation.
Huang Jie et al (application publication No. CN 112029732A) disclose a Salmonella phage SG8P3 resistant to a broad spectrum of high temperature and a combination thereof, which lyses 153 strains of Salmonella, which are 160 strains of Salmonella of 34 serotypes. The chick raising experiment shows that salmonella phage SG8P3 can be used as a biological bactericide or a feed additive, and can effectively prevent and treat salmonella infection in the chicken raising process.
The technical scheme has the following defects: research on salmonella phage mainly focuses on the aspect of cracking and application of salmonella of different serotypes, and research on the aspect of cracking food-borne pathogenic bacteria of other genera or species and application of salmonella phage is reported. Therefore, there is a need to find a salmonella phage that kills two or more food-borne pathogens.
Disclosure of Invention
The invention aims to provide a salmonella bacteriophage which can lyse salmonella and enterohemorrhagic escherichia coli.
To achieve the above object, the present invention provides a salmonella bacteriophage (salmonella sp.phage) JNwz02, wherein the salmonella bacteriophage (salmonella sp.phage) JNwz02 has a accession number of GDMCCNo 61662-B1, which was deposited at the microorganism strain collection in guangdong province at about 12 months of 2021.
The invention also protects the use of the salmonella bacteriophage (salmonella sp.phage) JNwz02.
Further, the application refers to the application of salmonella phage (salmonella sp.phage) Jnwz02 for lysing 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 steni Li Weier, salmonella shilingerie, salmonella enteritidis (Salmonella Enteritidis) NCTC8271, salmonella arizonae, salmonella kovar, salmonella albani, salmonella kovar and salmonella albani.
Further, the intestinal hemorrhagic Escherichia coli refers to intestinal hemorrhagic Escherichia coli O157: H7.
Further, the use refers to the use in preparing a bacteriostatic agent.
Further, the application refers to the application in preparing a food-borne pathogenic bacteria inhibitor.
The phage has been deposited on 12 months 2021 at "Guangdong province microorganism strain collection", GDMCC for short, accession number GDMCC No. 61662-B1, classified and named as salmonella phage (Salmonella sp.phage) JNwz02, with the address: guangzhou city first middle road No. 100 college No. 59 building 5.
The bacteriophage (salmonella sp.phage) Jnwz02 of the present invention belongs to the family of long-tailed phages, and has a polyhedral head with a diameter of about 92nm and a tail length of about 195nm. By one-step growth curve characterization, when the host is salmonella RSE39 of the mycostam Li Weier, the incubation period of the phage Jnwz02 is 5-10min, the outbreak period is 100min, the cracking amount is 165PFU/cell, and the cracking capacity is strong; when the host is enterohemorrhagic Escherichia coli O157: H7AV4997, the incubation period of the phage JNwz02 is 0-5min, the outbreak period is 80min, and the lysis amount is 34PFU/cell.
Further, phage JNwz02 was placed at 30-60℃for 15min and 30min, with a titer of 10 9 –10 10 PFU/mL; placing at 70deg.C for 15min, decreasing the potency by 4 orders of magnitude, placing at 70deg.C for 30min, decreasing the potency by 6 orders of magnitude; the sample was left at 80℃for 15min, and the potency was undetected. Phage JNwz02 has acid-base tolerance, is relatively stable under the condition of pH 2.0-11.0, and has potency of 10 9 PFU/mL。
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) Phage JNwz02 lyses the 8 serotypes of Salmonella and enterohemorrhagic Escherichia coli O157:H27, which are two food-borne pathogens of paramount importance worldwide. They cause food-borne diseases that often account for the first two of the bacterial food poisoning events.
(2) Phage (salmonella sp.phage) JNwz02 has better temperature and pH tolerance.
(3) In the present invention, the optimal infection number of phage (salmonella sp.phage) JNwz02 and the host bacterium Salmonella RSE39 of Stannum Li Weier was 0.01, and the optimal infection number of intestinal hemorrhagic Escherichia coli O157: H7AV4997 was 10.
(4) The phage JNwz02 can be applied to food, and can safely and effectively prevent and control the pollution of salmonella and intestinal hemorrhagic escherichia coli O157:H27.
The phage JNwz02 can be used as a biological bacteriostat to be applied to food storage.
Drawings
FIG. 1 is a plaque plot of phage (salmonella sp.phage) Jnwz02 on double-layered agar plates;
FIG. 2 is a transmission electron microscope image of phage (salmonella sp.phage) Jnwz 02;
FIG. 3 is a graph showing the results of whole genome analysis of phage (salmonella sp.phage) Jnwz 02;
FIG. 4 is a graph of one-step growth of phage (salmonella sp.phage) Jnwz 02;
FIG. 5 is a graph of temperature stability of phage (salmonella sp.phage) Jnwz 02;
FIG. 6 is a graph of pH stability of phage (salmonella sp.phage) Jnwz 02;
FIG. 7 is a graph showing the bacteriostatic effect of phage (salmonella sp.phage) JNwz02 (25 ℃ C.);
fig. 8 is a graph showing the bacteriostatic effect of phage (salmonella sp.phage) JNwz02 on simulated contamination of a marinated duck neck.
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 ℃.
0.01mol/L 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 pH 7.4 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 of a phage
(1) Taking 500mL of water sample from a lake nearby the farmer market of the agricultural university of Jiangxi, adding 0.055g of calcium chloride, uniformly mixing, standing and precipitating for 2h; concentrating the supernatant, and centrifuging the concentrated solution at 6000g and 4 ℃ for 10min; the supernatant was filtered through a 0.22 μm filter. Mixing 10mL of the filtrate with 10mL of TSB liquid culture medium, and adding600. Mu.L of the log phase of the host bacterium (Salmonella Stans Li Weier RSE 39) (OD 600 =0.5 to 0.6) was cultured in the above TSB liquid medium at 37 ℃,220rmp for 12 hours, and the collected bacterial liquid was centrifuged at 12000g and 4 ℃ for 15 minutes, and the supernatant was filtered with a 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. Mu.L of log phase host bacteria (OD 600 =0.5-0.6) was plated on LB solid medium, and after drying, the plate was divided into five areas of uniform size and labeled, wherein four areas were dropped with 10 μl of phage stock solution, and the other area was dropped with 10 μl of 0.01M PBS solution as a control group, and the plate was incubated at 37 ℃ for 12 hours, and plaque was observed on the plate. Simultaneously, the host bacterial liquid (OD) 600 =0.5-0.6) was mixed with 100 μl of a 10-fold serial dilution of phage stock solution and incubated in a constant temperature incubator at 37 ℃ for 15min; the mixture was added to a semisolid agar medium (about 45 ℃) and rapidly mixed and poured into a lower LB solid medium plate. After semisolid cooling, the plates were placed upside down in a 37℃incubator for 12-16 hours, and plaques were observed and counted.
Morphology of phage on double-layered agar plates, as shown in FIG. 1: the phage forms a circular, transparent phage plaque with halo around on the solid culture medium, and the diameter is about 0.5-1.5mm; the phage is a virulent phage.
(3) Purification and potency determination: picking single large round transparent plaque on a double-layer agar plate, adding the plaque into 1mL sterile SM buffer solution, standing for 12h at 4 ℃, centrifuging for 15min at 12000g and 4 ℃, collecting supernatant, and filtering with a 0.22 μm filter membrane to obtain phage filtrate a; 100. Mu.L of phage filtrate a was added to 100. Mu.L of log phase host bacterial liquid (OD 600 =0.5-0.6), adding into 10mL of TSB liquid culture medium, mixing, standing at 37 ℃ for incubation for 15min, and shaking at 220rpm for 5h at 37 ℃; centrifuging 12000g at 4deg.C for 15min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain phage filtrate b; phage filtrate b was serially diluted 10-fold with SM buffer to a dilution of 10 –6 Phage dilution of (2) and log phase host bacterial liquid (OD 600 =0.5-0.6), and standing at 37deg.C for 15min, adding the mixtureSemi-solid agar medium (about 45 ℃) is quickly and evenly mixed and poured into a lower LB solid medium plate, and after semi-solid cooling, the plate is poured into a constant temperature incubator at 37 ℃ for culturing for 12-16 hours.
Repeating the above steps for 5 times to obtain purified phage named phage Jnwz02. Plaques were counted on double agar plates, and the experimental result showed that the phage titer reached 10 10 PFU/mL and above.
Identification of the two phages
And (3) morphological identification of a transmission electron microscope: 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 Jnwz02 belongs to the family of long-tailed phages, and has a polyhedral head with a head diameter of about 92nm and a tail length of about 195nm. Phage morphology is shown in FIG. 2.
Gene sequence analysis: the genome of phage JNwz02 is extracted by using a virus genome extraction kit, and a genome sample is handed over 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 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. Genes encoding virulence, antibiotic resistance and phage lysogeny-related genes were not detected in the genome of phage JNwz02. The NCBI Blast shows that the phage JNwz02 has the highest homology with Salmonella phage Seabear (accession number: no. MK728824.1) and 98.43 percent, and has 96.57 percent homology with enterobacter T5 phage Escherichia virus EPS7 (accession number: no. CP000917.1).
Strain name: phage (salmonella sp.phage) JNwz02,
preservation date: 2021, 5 and 12 days,
preservation unit: building 5, guangzhou city, first, middle road 100, institute 59, guangdong province microbiological culture collection center (GDMCC),
preservation number: GDMCC No. 61662-B1.
Example 2: determination of phage Jnwz02 cleavage Spectrum
The experiment selects 14 bacteria including salmonella, escherichia coli and the like, and the analysis of the lysis spectrum of phage JNwz02 is carried out.
Wherein, 14 strains of bacteria are:
1) 1 strain of Stant Li Weier Salmonella RSE39;
2) 1 strain of salmonella Hilingdani N1529-D3;
3) 2 Salmonella typhi ATCC BAA-664, NCTC 8271;
4) 1 strain of Salmonella arizona LHICA_AZ23;
5) 1 strain of Salmonella barley CFSAN000189;
6) 1 strain of Salmonella Haidelbergiae AR-0404;
7) 1 Salmonella vaccina AR-0406;
8) 1 strain of Salmonella albani CVM N18S2238;
9) Salmonella 1 strain 161365;
10 1 strain of salmonella enteritidis BNCC336875;
11 1 Salmonella typhimurium BNCC185946;
121 strain of Escherichia coli ATCC 25922;
13 1 strain of enterohemorrhagic Escherichia coli O157: H7AV 4997.
The above test strains were cultured separately to log phase (OD 600 100. Mu.L of phage JNwz02 was mixed with 100. Mu.L of each of the above bacterial solutions in logarithmic phase, incubated at 37℃for 15min, then mixed with 7mL of LB semisolid medium, the mixed solution was added to semisolid agar medium (about 45 ℃) and rapidly mixed evenly, poured into the lower LB solid medium plate, after semisolid cooling, the plate was placed upside down in a 37℃incubator for 12-16h, and plaque counting was performed. The host bacteria Stan Li Weier Salmonella RSE39 was tested in the same manner. Plaque formation rate (efficiency of plating, EOP) values were calculated as plaque average of the test plaques/plaque average of the host bacteria. When 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 "medium effect", when 0.001. Ltoreq. EOP<0.1, the test bacteria are classified as "inefficient" in efficiency; when EOP.ltoreq.0.001 was classified as "null", the test bacteria could not be lysed by phage JNwz02.
The results of the cleavage spectrum of phage JNwz02 are shown in Table 1, and phage JNwz02 can cleave 11 strains (9 strains of Salmonella, 2 strains of E.coli) of 14 test strains. Wherein, the phage JNwz02 can crack 7 strains of salmonella (Stannum Li Weier salmonella RSE39, hilington salmonella N1529-D3, salmonella enteritidis NCTC8271, salmonella arizona LHICA_AZ23, salmonella albani CVM N18S 2238) in a high-efficiency manner, wherein 6 strains of salmonella are drug-resistant salmonella. Phage JNwz02 "intermediate" lyses Salmonella kohlii AR-0406, escherichia coli ATCC 25922, EHEC O157:H24990. Phage JNwz02 "inefficiently" lyses salmonella albani CVM N18S2238.
TABLE 1 results of cleavage Spectrum for phage Jnwz02
Remarks: a: ampicillin; AC: amoxicillin; c: cefotaxime; t: tetracyclines; g: gentamicin; n: neomycin; f: florfenicol; CI: ciprofloxacin; na: nalidixic acid; s: compound neonolamine; -: no resistance.
Example 3: determination of optimal multiplicity of infection (MOI) and one-step growth curve for phage Jnwz02
MOI determination:
100. Mu.L of phage (MOI of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, respectively) were combined with 100. Mu.L of log phase host bacterial liquid (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. Determining 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, and the optimal multiplicity of infection of phage JNwz02 with Salmonella host, stent Li Weier, RSE39 is 0.01, and EHEC O157:H27 AV4997 is 10.
One-step growth curve determination:
mu.L of phage JNwz02 was combined with 100. Mu.L of Salmonella enterica RSE39 bacterial solution at log phase Li Weier or EHEC O157:H7 (OD) 600 =0.5-0.6), incubation at 37 ℃ for 15min, centrifugation at 12000g at 4 ℃ for 1min, discarding the supernatant, resuspending the pellet in 5mL TSB broth, shake culture at 37 ℃,220 rpm. 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: when the host is the salmonella RSE39 of the Stent Li Weier, the incubation period of the phage Jnwz02 is 5-10min, the outbreak period is 100min, and the cleavage amount is 165PFU/cell; when the host is EHEC O157:H27 AV4997, the incubation period of phage Jnwz02 is 0-5min, the outbreak period is 80min, and the lysis amount is 34PFU/cell.
TABLE 2 results table of the multiplicity of infection (MOI) of phage Jnwz02
Example 4 analysis of phage Jnwz02 Heat stability and pH stability
Thermal stability determination: 1mL of phage solution (concentration 10) 8 PFU/mL) was added to a sterile 1.5mL centrifuge tube, placed in a thermostat water bath at 30℃and 40℃and 50℃and 60℃and 70℃and 80℃for 15min and 30min, and then taken out for continuous 10-fold gradient dilution, and the titer of phage was determined by a double-layer agar plate method using Salmonella RSE39 of Stent Li Weier as the host bacteria.
The experimental results of the thermal stability measurement are shown in FIG. 5, and the phage JNwz02 is placed at 30-40deg.C for 15min and 30min, and its titer is 10 10 PFU/mL; placing at 50deg.C 15min and 30min, the potency is 10 9 PFU/mL; standing at 60deg.C for 15min with a potency of 10 9 PFU/mL; standing for 30min with a potency of 10 8 PFU/mL; standing at 70deg.C for 15min with a potency of 10 6 PFU/mL; standing for 30min to give a potency of 10 3 PFU/mL; standing at 80deg.C for 15min and 30min, and the potency is 0. The phage JNwz02 was shown to have better temperature tolerance.
Determination of pH stability: the SM buffer pH was adjusted to 2-13 with HCl and NaOH, 100. Mu.L of phage JNwz02 was added to 900. Mu.L of each pH buffer, water-bath was performed at 37℃for 1h, and phage titers were determined by double-layer agar plate method using Salmonella RSE39 of Stant Li Weier as host bacteria.
As shown in FIG. 6, the pH stability test results show that the titers of the phage JNwz02 are relatively stable under the condition of pH 2.0-11.0 and are 10 9 PFU/mL; phage JNwz02 titer was 10 in pH 12.0 buffer 3 PFU/mL. The results show that the acid-base tolerance range of phage Jnwz02 is wider.
Example 5 bacteriostasis assay of phage JNwz02 at 25℃
100. Mu.L of phage Jnwz02 (MOI 100, 10, 1, 0.1, 0.01, respectively) was combined with 100. Mu.L of Salmonella log-phase 2089b or EHEC O157:H27 bacterial fluid (OD 600 =0.5-0.6), adding into 50mL TSB liquid culture medium, standing at 25deg.C for 24 hr, standing at 25deg.C for 16 hr with EHEC O157:H7 bacterial liquid, sampling at different time points, and measuring OD 600 The values were measured for the growth of the salmonella RSE39, EHEC O157:H7AV4997, respectively, of Stein Li Weier. SM buffer was added to the negative samples instead of phage.
The results of the bacteriostasis experiment are shown in fig. 7, wherein: a is the antibacterial effect of phage Jnwz02 on the salmonella Stein Li Weier RSE39; b is the bacteriostasis of phage JNwz02 on EHEC O157:H7. The bacterial count in the experimental group was maintained at a lower level than that in the negative control group at 25℃and at MOI of 100, 10, 1, 0.1, 0.01, and the bacterial count was 100CFU/mL or less. The result shows that the phage Jnwz02 has good antibacterial effect on both the salmonella RSE39 and the EHEC O157:H7AV4997 of the Stein Li Weier at 25 ℃.
Example 6 evaluation of bacteriostasis of phage JNwz02 on Duck neck
Respectively dripping 100 mu L of bacteria solution of Stant Li Weier salmonella RSE39 and EHEC O157:H27 AV4997 on the surface of the sterilized marinated duck neck for simulated pollution, wherein the final concentration of the bacteria solution is 10 4 CFU/g; the duck neck sample was air-dried in a biosafety cabinet for 30min, and 100 μl of phage JNwz02 (concentration 10 9 PFU/mL), dropwise adding 100 mu LSM buffer solution into the control group, and air-drying for 30min again; then, the mixture was stored at 4℃and sampled at 0 day, 1 day and 2 days, and the mixture was centrifuged at 12000g for 5 minutes by 1mL shaking for 200 times with 0.85% NaCl+0.025% SDS, the supernatant was subjected to continuous 10-fold gradient dilution, 100. Mu.L of the dilution was spread on LB solid medium plates, and the culture was performed at 37℃for 12-16 hours, thereby counting the number of colonies.
The experimental results are shown in fig. 8, wherein A is the antibacterial effect of phage JNwz02 on the salmonella RSE39 of Stent Li Weier in the stewed duck neck; b is the bacteriostasis of phage JNwz02 on EHEC O157:H2 7AV4997 in the stewed duck neck. The reduction of the number of bacteria in the experimental group by 3.0log10 CFU/mL was compared with the reduction of the number of bacteria in the control group by 1.0log10 CFU/mL by the RSE39 of the Salmonella Steven Li Weier and the EHEC O157: H7AV 4997. The phage JNwz02 has good antibacterial effect on the RSE39 and the EHEC O157:H7AV4997 of the salmonella RSE Li Weier in the stewed duck neck.
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 (2)
1. A salmonella bacteriophage (salmonella sp.phage) JNwz02, wherein the salmonella bacteriophage JNwz02 has a deposit number of GDMCC No. 61662-B1, deposited with the cantonese collection of microorganism strain at 12 months 2021.
2. Use of salmonella bacteriophage JNwz02 according to claim 1 for the preparation of a medicament for inhibiting salmonella, which is at least one of salmonella stenlength Li Weier, salmonella hilum, salmonella enteritidis (Salmonella Enteritidis) NCTC8271, salmonella arizonae, salmonella kovassimae and salmonella albani, and enterohemorrhagic escherichia coli, which is EHEC O157:h7.
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| KR20170031516A (en) * | 2015-09-11 | 2017-03-21 | 서울대학교산학협력단 | Bacteriophage against Salmonella, pathogen E. coli O157, Shigella and composition threrof |
| CN110241091A (en) * | 2019-05-29 | 2019-09-17 | 华中农业大学 | Salmonella dublin bacteriophage D1-2 and its application in liquid eggs sample |
| CN110317793A (en) * | 2019-06-27 | 2019-10-11 | 华中农业大学 | A kind of main effect component is mix preparation and the application of bacteriophage LPEE17 and LPEK22 |
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