CN112063593B

CN112063593B - Pathogenic vibrio phage VyZU 10474 and application thereof

Info

Publication number: CN112063593B
Application number: CN202010981378.XA
Authority: CN
Inventors: 高璐; 陈静; 杨振泉; 饶胜其; 李熠; 郑香峰; 李华祥
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2021-08-31
Anticipated expiration: 2040-09-17
Also published as: CN112063593A

Abstract

The invention discloses a pathogenic vibrio phage VyZU 10474 and application thereof, the phage has unique genome structure characteristics through identification, belongs to a novel phage, has been preserved in China center for type culture collection, has the preservation time of 2020, 8, 12 days, and has the preservation number of CCTCC NO: m2020417. The phage of the high-efficiency cracking vibrio can inhibit pathogenic vibrio in various matrixes, such as vibrio mimicus, vibrio parahaemolyticus, vibrio alginolyticus, vibrio fluvialis and the like, and can be applied to preparing bacteriostatic agents for controlling the pollution of the pathogenic vibrio in aquatic products and environments thereof. The bacteriostatic agent prepared by the invention can effectively control the growth of pathogenic vibrios in culture medium, fish juice and aquatic product samples, can effectively inhibit the formation of pathogenic vibrio biofilms on solid such as equipment and vessels, is simple to prepare and convenient to use, and can reduce the risk of spreading the pathogenic vibrios and causing food-borne diseases.

Description

Pathogenic vibrio phage VyZU 10474 and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a pathogenic vibrio phage VyZU 10474 and bacteriostatic application thereof as a bacteriostatic agent in aquatic products and temporary rearing and storage environments and equipment thereof.

Background

Aquatic products are deeply favored by consumers due to delicious taste and high nutritional value, the aquatic product culture industry is rapidly developed along with the increasing demand of people on the aquatic products, and half of the global marine product supply comes from the aquatic product culture industry in 2020 according to the estimation of the Food and Agricultural Organization (FAO) of the United nations. However, bacterial diseases caused by high-density aquaculture models have become one of the major problems facing the aquaculture industry.

The vibrio is one of the most common, widely distributed and seriously harmful bacterial pathogens in aquaculture, is a group of gram-negative bacteria with short and small bacteria, rod-shaped bacteria or arc-shaped bacteria, is widely distributed in nature, and has more and more parasitic lives in aquatic animals in ocean, estuary and fresh water systems, so that the outbreak of the vibrio is more and more frequent along with the development of the aquaculture industry, the propagation speed is accelerated, and huge economic loss is brought to the world aquaculture industry. The vibrio is composed of more than 100 of 14 branches, and with the introduction of new bacterial taxonomic technologies such as multi-site sequence analysis, fluorescence amplification fragment length polymorphism and the like, new vibrio species are continuously discovered. At present, 12 kinds of vibrios are known to be related to human intestinal and extraintestinal infections, and epidemiological investigation results in recent years show that pathogenic vibrios are main pathogenic bacteria of diarrhea diseases of people in coastal and river cities in China, wherein the most harmful vibrios parahaemolyticus, cholera vibrio, vibrio vulnificus, vibrio alginolyticus and vibrio mimicus are most common, wherein the vibrio cholerae of type O1 is determined as a detection bacterium by WHO, and the vibrio cholerae and the vibrio parahaemolyticus are detection strains of international aquatic products. The novel biological antibacterial agent which is cheap and environment-friendly is screened, and the pollution of vibrio mimicus in animal breeding environment, food processing and storage environment and food raw materials is reduced, so that the method has very important significance for controlling diseases caused by the bacteria.

At present, antibiotics and the like are mainly applied to the aquaculture industry to inhibit the growth of pathogenic vibrios, however, bacterial drug resistance and superbacteria caused by the abuse of antibiotics become one of the most urgent public health problems in the world. Although chemical fungicides can be used to control bacterial growth in food production environments, chemical fungicide residues have a significant impact on human and animal safety and the environment. In the process of adapting to the growth of natural environment, pathogenic vibrios are easy to form biofilm on the surfaces of food and processing appliances, the resistance of bacteria in the biofilm to conventional antistaling agents and bacteriostats can be improved by hundreds of times and is difficult to remove, thereby causing continuous pollution and bringing serious harm to the safety of aquatic foods. Therefore, the development of natural harmless biological bacteriostatic agents to replace antibiotics and conventional chemical bacteriostatic agents for application in aquaculture water purification and aquatic food safety control is urgently needed.

The bacteriophage exists widely in nature, has specificity to host, no harm to human and animal body, rich resource, low cost, high safety and environment friendship. In recent years, research and development of novel phage bacteriostats have been paid much attention, and a great deal of research has been conducted in various countries in the world on phage resources, biological characteristics and control of bacteria in various substrates, so that commercial biological bacteriostats of pathogenic bacteria such as salmonella and listeria have been formed in the united states and european countries, and have the advantages of high efficiency, low cost, safety and the like in control of water production and infection and transmission of pathogenic bacteria in production environment. Although the research on the pathogenic vibrio phage at home and abroad is reported, the high-efficiency broad-spectrum pathogenic vibrio phage is reported less.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the phage with strong lytic effect on various pathogenic vibrios, and the phage can be used for controlling the pollution of the pathogenic vibrios in aquatic products and production environments.

The invention also provides application of the phage in preparation of pathogenic vibrios and biofilm inhibitors thereof. The bacteriophage can be singly or compounded into a bacteriostatic agent, is used for controlling the growth of pathogenic vibrios and the formation of biofilm in aquatic product temporary culture and production environment, and provides a safe, efficient and cheap biological antibacterial product for clean production of aquatic animals and food.

The technical scheme is as follows: in order to achieve the above object, the pathogenic Vibrio phage VmYZU10474 of the present invention is a Vibrio mimicus phage (Vibrio mimicus phage VmYZU10474), the host of which is Vibrio mimicus (CICC 10474), the host is identified to have unique genome structure characteristics, and the phage belongs to a novel phage, the phage of the present invention is obtained by separating from a sewage sample of farm and trade markets of hong kong of Jiangsu province, the phage VmYZU10474 with the function of efficiently cracking pathogenic Vibrio is deposited in China Center for Type Culture Collection (CCTCC), and the storage address is: wuhan university; the preservation number is: CCTCC NO: m2020417; the preservation date is as follows: 8/12/2020.

The pathogenic vibrio phage VmYZU10474 of the present invention has the following biological characteristics:

(1) morphological characteristics: the VyZU 10474 is head symmetric, about 54.84nm in diameter, tailless and morphologically characterized by the Capicophagae family as observed by transmission electron microscopy.

(2) Type of nucleic acid: VmYZU10474 is a dsDNA phage.

(3) Genome characterization: the total length of the genome of VmYZU10474 is 42883bp, wherein the total length of the coding gene is 39801bp, the average length is 884bp, the coding gene accounts for 92.81% of the total length, the GC content is 49.38%, 45 Open Reading Frames (ORFs) are provided, 7 ORFs have no homologous genes found in a database, and the homologous sequences of 41 ORFs coding proteins comprise 20 conservative hypothetical phage protein sequences and 21 sequences with known protein functions. Without any known virulence genes, the genomic data shows that VmYZU10474 is a new phage.

(4) Has the function of strongly cracking pathogenic vibrio.

(5) Can inhibit the formation of pathogenic vibrio biofilm.

The application of the phage YZU.P.A-5 in inhibiting pathogenic vibrios is provided.

Preferably, the pathogenic vibrio includes vibrio mimicus, vibrio parahemolyticus, vibrio alginolyticus, and vibrio fluvialis.

Preferably, the bacteriophage VmYZU10474 is used for inhibiting pathogenic vibrio in aquatic products and temporary rearing, storage and production environments and facilities thereof.

The invention relates to application of a bacteriophage VyZU 10474 in preparation of a pathogenic vibrio bacteriostatic agent.

Wherein the bacteriostatic agent takes the isolate or culture of bacteriophage VmYZU10474 as bacteriostatic agent component.

Preferably, the bacteriostatic agent is used for eliminating the pollution of pathogenic vibrio and biofilm thereof in aquatic products, temporary culture, storage and production environments and facilities.

Wherein, the bacteriostatic agent is prepared by the following steps: mixing the phage VyZU 10474 with vibrio mimicus at logarithmic growth phase, standing at room temperature, adding into LB liquid culture medium, and oscillating at constant temperature for overnight culture; transferring the culture to a sterilized centrifuge tube, centrifuging, collecting supernatant, filtering, collecting phage proliferation solution, adding PEG 8000 and NaCl, shaking to dissolve, and standing overnight; centrifuging to remove supernatant; adding SM buffer solution, and reacting at room temperature; extracting with chloroform; and centrifuging to recover a hydrophilic phase containing the VvYZU 10474 particles, and mixing the obtained VMYZU10474 particles with an SM buffer solution to prepare phage bacteriostatic agent mother liquor.

The application of the bacteriophage VyZU 10474 bacteriostatic agent provided by the invention refers to that: the mother liquor of the bacteriophage bacteriostatic agent is diluted by water and prepared into spraying liquid or leacheate which is used alone or in combination with other bactericides for spraying, soaking or washing temporary culture or storage aquatic products (including fresh water or seawater products such as fish, shrimp, shellfish, seaweed and the like) and various environments or facilities thereof, so that the amount of pathogenic vibrios and the formation of mycoderm in the temporary culture or storage environment of the aquatic products are reduced.

The preparation prepared by the invention can be used as a main component of a bacteriostatic agent singly or in a compound way, temporarily fosters or stores aquatic products and the growth and metabolism of pathogenic vibrios in the environment of the aquatic products, and provides a safe, efficient and cheap biological antibacterial product for the production and processing of the aquatic products.

Has the advantages that: compared with the prior art, the phage VmYZU10474 capable of efficiently cracking a plurality of pathogenic vibrios is obtained by separation, has unique morphological and genomic characteristics, is a brand-new broad-spectrum phage capable of effectively inhibiting the pathogenic vibrios, and can be used for preparing a green and cheap pathogenic vibrio inhibitor.

The phage has high-efficiency cracking activity on various pathogenic vibrios, and the prepared bacteriostatic agent can effectively control the growth of the pathogenic vibrios in various matrixes and the formation of biofilm, thereby effectively reducing the pollution and the propagation risk of the pathogenic vibrios. Specifically, the method comprises the following steps: the biological bacteriostatic agent prepared from the phage can effectively control the growth of pathogenic vibrios in a culture medium, a fish juice matrix, a temporary aquatic product and the like, can inhibit the formation of a pathogenic vibrio mycoderm on the surface of a production facility, and is simple to prepare and convenient to use. The spraying liquid or the leacheate can be easily prepared by the traditional method, so that pathogenic vibrios in the temporary culture and storage environment of aquatic products are removed, and the pollution of the vibrios is reduced; the bacteriophage of the present invention belongs to natural biological material, and the genome does not contain any virulence gene, has no toxic side effect, can be used as bacteriostat for aquatic products, storage equipment, vessels, environment, raw material solid surface, etc., and is used for the sterilization of aquatic products and temporary culture, storage and processing environments thereof, and the risk of pathogenic vibrio propagation and food-borne disease initiation is reduced.

Drawings

FIG. 1 phage VyZU 10474 plaque morphology.

FIG. 2 Transmission Electron microscopy images of phage VyZU 10474.

FIG. 3 phage VyZU 10474 Whole genome sequence features.

FIG. 4 the bacteriostatic effect of the bacteriophage VyZU 10474 on the host bacteria in the culture medium. The black dot data line in the figure represents the negative control, i.e., the control group that was not treated with phage VmYZU 10474; the light grey square data line represents the experimental group of host bacteria treated with the phage VmYZU 10474.

FIG. 5 inhibition of Vibrio biofilm formation by phage VyZU 10474. FIG. 5A shows the inhibition of biofilm formation by bacteriophage VyZU 10474 at different concentrations on mixed culture biofilms of representative strains of 4 arc species (Vibrio mimicus CICC10474, Vibrio parahaemolyticus CICC21617, Vibrio alginolyticus CICC10484, Vibrio fluvialis CICC 21612). FIG. 5B shows the inhibitory effect of the phage VyZU 10474 on the biofilm formation of 4 strains of Vibrio (Vibrio mimicus CICC10474, Vibrio parahaemolyticus CICC21617, Vibrio alginolyticus CICC10484, Vibrio fluvialis CICC21612), respectively. The black bar (Control) in FIG. 5A represents a Control group that was not treated with phage VyZU 10474; white bar chart (Blank) is Blank control without added bacterial liquid; columns A to E are each 10⁶、10⁷、10⁸、10⁹、10¹⁰PFU/mL concentration phage treated panel.

FIG. 6 shows the inhibitory effect of the bacteriophage VyZU 10474 on the growth of 4 strains of Vibrio (Vibrio mimicus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis) in a fish juice substrate. The square data points in the figure (control) represent the change in colony during incubation at 25 ℃ for each vibrio to which phage VyZU 10474 was not added; the round data points (test group) are the change in the total number of colonies during incubation at 25 ℃ for each vibrio treated with the added phage VmYZU 10474.

FIG. 7 inhibition of Vibrio during storage of commercial aquatic samples by phage VyZU 10474. In the figure, black square data lines indicate the change of the total number of colonies on the TCBS plate in the storage process of the aquatic product samples, and gray dot data lines indicate the change of the total number of colonies on the TCBS plate in the storage process of the aquatic product samples after being treated by the phage VyZU 10474.

Detailed Description

The invention is further illustrated by the following figures and examples.

The starting materials and reagents used in the present invention are commercially available unless otherwise specified.

The bacteriophages host bacteria Vibrio mimicus (Vibrio mimicus, strain number: CICC10474) for the test, and Vibrio parahaemolyticus (Vibrio parahaemolyticus, strain number: CICC21617), Vibrio alginolyticus (CICC 10484) and Vibrio fluvialis (CICC 21612) for the bacteriostatic test are purchased from China Center for Industrial Culture Collection (CICC).

Example 1

Phage separation and purification preparation

Phage isolation

The sewage sample was collected from the farmer market in Jiangsu Liyun harbor City. Taking 30mL of sewage sample, putting the sewage sample into a 50mL centrifuge tube, centrifuging for 10min at 5000 Xg, taking 5mL of supernatant, adding the supernatant into 5mL of LBS liquid culture medium (10 g of tryptone, 5g of yeast extract, 35g of NaCl, 950mL of deionized water, pH7.0), simultaneously adding 100 mu L of vibrio mimicus strain CICC10474 in logarithmic growth phase, and carrying out shake culture at 37 ℃ and 100rpm overnight; transferring the culture in the test tube into a sterile centrifuge tube, centrifuging at 4 deg.C and 5000 Xg for 10min, and filtering the supernatant with 0.22 μm filter membrane to obtain phage stock solution.

Using SM buffer (1L: NaCl 5.8g, MgSO)₄.7H₂O2.0 g, 1M Tris-HCl (pH7.4)50mL) is used for carrying out gradient dilution on phage stock solution by 10 times according to the volume ratio, 100 mu L of diluted phage suspension is taken to be mixed with 100 mu L of vibrio mimicus at logarithmic growth phase for 10min at room temperature, 5mL of LBS semisolid culture medium (10 g of tryptone, 5g of yeast extract, 35g of NaCl, 5g of agar powder, 950mL of deionized water and pH7.0) is added to be mixed evenly, the mixture is quickly poured on the LBS solid culture medium (10 g of tryptone, 5g of yeast extract, 35g of NaCl, 15g of agar powder, 950mL of deionized water and pH7.0) prepared in advance to be prepared into a double-layer plate, and the double-layer plate is placed in a constant temperature incubator at 37 ℃ after solidification and inversion.

Picking a transparent single plaque on the double-layer plate with the plaque to 1mL of SM buffer solution, uniformly mixing, and standing for 24h at 4 ℃; the next day phage liquid was diluted 10-fold in sterile SM buffer in a gradient (10)^-1-10^-7) Respectively taking 100 mu L of phage liquid of each dilution and 100 mu L of vibrio mimicus at logarithmic growth phase, mixing for 10min at room temperature, adding 5mL of LBS semisolid culture medium, mixing uniformly, and quickly pouring into LBS solid prepared in advancePreparing a double-layer flat plate on the culture medium, solidifying, inverting, and culturing in a constant temperature incubator at 37 ℃. The operation of the double-layer plate method is repeated for 3 times, and after the obtained plaques are consistent in size, the isolated phage is considered to be pure. Detecting the lysis effect of the purified phage by using a double-layer plate, selecting 50 phage plaques into 1mL SM buffer solution, uniformly mixing, and standing for 24h at 4 ℃; collecting supernatant on the next day, filtering with 0.22 μm microporous membrane to obtain bacteriophage separating medium, and storing at 4 deg.C. Adding 100 mu L of vibrio mimicus culture (namely vibrio mimicus strain CICC10474 bacterial liquid in logarithmic growth phase) into 5mL of melted LBS semisolid culture medium, immediately pouring the mixture on a bottom LBS solid culture medium, standing the mixture at room temperature for 10min, after the culture medium is solidified, respectively dripping 10 mu L of each phage separating medium into divided areas, drying the mixture in a sterile operating platform, then putting the plate into an incubator at 37 ℃ for culture, and screening phage with bright and clear lysis rings on the strain CICC10474 plate, wherein the phage is named as VmYZU 10474.

Purification of bacteriophages

Mixing 100 μ L phage VyZU 10474 separating medium (the preparation method is the same as above) with 100 μ L vibrio mimicus CICC10474 bacterial solution in logarithmic phase growth phase, standing at room temperature for 10min, adding 10mL LBS liquid culture medium, and performing constant temperature shaking culture at 37 deg.C and 150rpm overnight; transferring the culture to a sterilized centrifuge tube, centrifuging at 5000 Xg for 10min, collecting supernatant, and filtering with 0.22 μm microporous membrane for sterilization; collecting phage proliferation liquid, adding 0.93g PEG 8000 and 0.58g NaCl, shaking to dissolve, standing at 4 deg.C overnight; centrifuging at 4 deg.C at 10000 Xg for 20min, removing supernatant, adding 0.5mL SM buffer solution, and reacting at room temperature for 1 h; adding equal volume of chloroform for extraction for 30 s; centrifuging at 3000 Xg for 15min, and recovering hydrophilic phase containing phage particles to obtain phage purified solution; the double-layer plate is used for detecting the titer of the purified phage, and the specific procedure is as follows: phage purification was diluted 10-fold in SM buffer in a gradient (10)^-1-10^-7) Mixing 100 mu L of bacteriophage diluent with 100 mu L of vibrio mimicus CICC10474 bacterial liquid in logarithmic growth phase at room temperature for 10min, adding 5mL of LBS semisolid culture medium, mixing, pouring on the LBS solid culture medium, culturing in a constant temperature incubator at 37 ℃ for 8h after solidification and inversion,the plaques formed were counted manually and the titer was calculated. The results show that the purified VmYZU10474 titer reaches 10 to the strain CICC10474¹¹PFU/mL or more, the plaques formed on the dilution plates were bright, clear and uniform in size (as shown in FIG. 1).

Example 2

Phage VyZU 10474 characterization

Morphological characteristics of bacteriophages

And observing the microscopic morphological characteristics of the phage by using a transmission electron microscope. Using a phosphotungstic acid negative staining method, 10. mu.L of the purified solution of phage VyZU 10474 (10) obtained in example 1 was taken with the copper mesh side facing upward¹¹PFU/mL) is dropped on a copper net, water is absorbed after 15min of absorption, the copper net is taken out, and natural drying is carried out for 2-3 min. Then dropping 2% phosphotungstic acid (PTA) aqueous solution on a copper net for dyeing, taking down after 2min, absorbing water by absorbent paper, drying in the air for 5min, observing by a transmission electron microscope, and selecting a clear phage image for photographing analysis. Microscopic morphology of VmYZU10474 as shown in fig. 2, the phage VmYZU10474 exhibits head symmetry, a diameter of about 54.84nm, no tail, morphological features belonging to the capsphage family.

Genomic characterization of bacteriophages

The purified solution (10) of phage VyZU 10474 prepared as described above¹¹PFU/mL) was added DNase I to a final concentration of 5. mu.g/mL, RNase A to 1. mu.g/mL, incubated at 37 ℃ for 1 h; EDTA (pH 8.0) was added to a final concentration of 20 mmol/L; adding proteinase K to a final concentration of 50 μ g/mL, adding SDS to a final concentration of 0.5% (mg/mL), mixing, and incubating at 56 deg.C for 1 h; adding isovolumetric balance phenol (pH 8.0), extracting under shaking, centrifuging at 5000 × g for 10min, and collecting upper water phase; extracting with equal volume of chloroform, centrifuging at 5000 × g for 10min, and collecting upper water phase; 1/10 volumes of 3mol/L NaAc (pH 5.2) were added, and twice the volume of absolute ethanol was added to precipitate the nucleic acid, overnight at-20 ℃; centrifuging at 12000 Xg for 10min at 4 deg.C; washing the precipitate with 70% ethanol and anhydrous ethanol respectively, and drying the precipitate in air for 10 min; suspending and precipitating with appropriate amount of TE (pH 8.0), quantifying phage DNA with GeneQuant nucleic acid quantifier, and storing at-20 deg.C; the extracted phage DNA is sent to Shanghai Linn biological gene sequencing company Limited to carry out Illumina Hiseq sequencing.

Phage DNA sequencing results (shown in FIG. 3) show that the full length of the VmYZU10474 genome is 42883bp, wherein the total length of the coding genes is 39801bp, the average length is 884bp, which accounts for 92.81% of the total length, the GC content is 49.38%, and the genome has 45 Open Reading Frames (ORFs), wherein 7 ORFs have no homologous genes found in a database, and 41 ORFs code protein homologous sequences comprise 20 conservative hypothetical phage protein sequences and 21 sequences with known protein functions, and the homology is between 62.5% and 100%. Alignment with phage genome data in the GenBank database showed that VmYZU10474 is a new phage and does not contain any known virulence genes. Combining the physiological and biochemical characteristics of the phage, the phage is preliminarily identified as the Vibrio mimicus phage VmYZU10474(Vibrio mimicus phase VmYZU 10474). The phage is preserved in China center for type culture Collection (CCTCC for short), and the preservation address is as follows: wuhan university; the preservation number is: CCTCC NO: m2020417; the preservation date is as follows: 8/12/2020.

Example 3

Bacteriostatic action of phage VyZU 10474 on host bacteria in culture medium

Inoculating the bacterial liquid of vibrio mimicus CICC10474 into 10mL LBS liquid culture medium, and placing the liquid in an incubator at 25 ℃ for constant-temperature culture until the temperature reaches about 10 DEG⁷CFU/mL, phage treatment group added 100. mu.L VyZU 10474 (10)⁹PFU/mL) purified solution, adding an equal volume of SM buffer (0PFU/mL) to a negative control group, placing in a constant temperature culture for continuous culture, sampling 1mL every 2h, measuring the absorbance at a wavelength of 600nm with a spectrophotometer, setting 3 replicates in each group, and taking the average value for analysis.

As shown in FIG. 4, the OD of the negative control group was observed with the increase of the culture time_600nmThe rapid growth reaches 0.85 at 8h, and the treatment group added with the phage VmYZU10474 is maintained at the original concentration for 8h, which shows that the phage VmYZU10474 in the invention has a remarkable inhibition effect on the growth of host bacteria in the culture medium (P)<0.001)。

Example 4

Inhibition of Vibrio biofilm formation by bacteriophage VyZU 10474

Respectively taking Vibrio mimicus CICC10474, Vibrio parahaemolyticus CICC21617, Vibrio alginolyticus CICC10484 and Vibrio fluvialis CICC21612 in logarithmic growth phase, diluting with LBS culture medium, and adjusting final concentration to 1 × 10⁸CFU/mL, and mixing in equal volume to prepare vibrio mixed solution. The test was divided into VmYZU10474 treated group, negative control group, blank group. VmYZU10474 treatment group: 150 μ L of 10-concentrated solution was added to each well of a 96-well plate⁸CFU/mL Vibrio mixture and 150. mu.L phage VyZU 10474 purified solution (used concentrations of phage in A, B, C, D, E treatments were 10, respectively)⁵PFU/mL、10⁶PFU/mL、10⁷PFU/mL、10⁸PFU/mL、10⁹PFU/mL) are mixed evenly; negative control group: vibrio mixture (10) was added to each well of a sterile 96-well plate⁸CFU/mL) and SM buffer 150. mu.L each; blank group: adding 300 μ L LBS culture medium to each well; culturing at 37 deg.C for 24h, taking out 96-well plate, sucking out suspension liquid, washing with sterile PBS for 3 times, and sufficiently removing floating thallus; adding 200 μ L of crystal violet staining solution with concentration of 0.2% into each well, and staining for 30 min; sucking out the staining solution, washing the 96-well plate by PBS until the eluate is colorless, and drying at room temperature; adding 200 μ L of 33% acetic acid decolorizing agent into each well, shaking for dissolving, measuring OD value at 600nm with microplate reader, measuring each group for 5 times, and taking average value for analysis.

The results are shown in FIG. 5A, adding 10⁵PFU/mL、10⁶PFU/mL、10⁷PFU/mL、10⁸PFU/mL、10⁹OD of test group of PFU/mL phage VyZU 10474 suspension (A, B, C, D, E treated group)_600nmAre all significantly lower than the negative control group (p)<0.001), indicating that VmYZU10474 significantly inhibited vibrio biofilm formation.

In addition, phage VyZU 10474 (10) was measured by the above-described method, respectively⁶PFU/mL) can independently inhibit Vibrio mimicus CICC10474, Vibrio parahaemolyticus CICC21617, Vibrio alginolyticus CICC10484 and Vibrio fluvialis CICC21612 (the concentration of bacterial liquid is 1 × 10)⁸CFU/mL), and the implementation result is shown in FIG. 5B, the phage VmYZU10474 can obviously inhibit the formation of each vibrio biofilm, wherein the inhibition on the vibrio parahaemolyticus CICC21617 is most obvious.

Example 5

Bacteriostatic action of bacteriophage VyZU 10474 in fish juice matrix

Killing fresh marine hairtail, removing fishbone, taking fish, cutting the fish into thin strips, weighing, and mixing the water and the fish according to the mass ratio of 2: 1, adding water, boiling for 5min, filtering with gauze, adding water again, recovering to the first addition amount, boiling for 5min, filtering with filter paper, adjusting the pH of the filtrate to 7 with NaOH, adding 40mg L-cysteine and 40mg L-methionine into each liter of fish juice, subpackaging into conical flasks, and sterilizing at 121 ℃ for 15 min. Respectively mixing 100 μ L (10)⁷CFU/mL) of Vibrio mimicus CICC10474, Vibrio parahaemolyticus CICC21617, Vibrio alginolyticus CICC10484 and Vibrio fluvialis CICC21612 were inoculated into 30mL of fish juice, and 100. mu.L of phage VyZU 10474 purified solution (10. mu.L) was added to the experimental group⁹PFU/mL), 100 μ L SM buffer was added to the control group, the inocula were each incubated in a 25 ℃ incubator at constant temperature, 100 μ L was sampled every 4h, TCBS plates were spread, the total number of colonies was counted, 3 replicates per group were set, and the average was taken for analysis.

The implementation results are shown in FIG. 6, the growth curve of the phage treatment group is significantly lower than that of the negative control group, and the total number of vibrios can be reduced by 4 log values at most, which indicates that the phage VmYZU10474 in the invention can play a significant role in inhibiting 4 different vibrios in the fish juice matrix.

Example 6

The inhibition effect of the phage VyZU 10474 on vibrio in aquatic products.

5 parts of fish, shrimp and shellfish sold in farmer markets of Yangzhou city are collected respectively. The whole fish, shrimp and shellfish are put into the sample bag. Taking fish gills, intestines, contents thereof, viscera and the like; taking the whole shrimps; washing the shell of the shellfish with shell with tap water, and drying to remove water on the surface, wherein the shellfish takes all shellfish meat and body fluid. All samples are mixed according to the mass ratio of 1:1:1, then a beating type homogenizer is used for beating for 2min, water is added according to the mass ratio to prepare a sample uniform solution with the mass ratio of 1:10, the sample uniform solution is divided into two parts with the mass ratio of 0.9mL, 100 mu L phage VyZU 10474 (the PFU is 10)⁹PFU/mL), and adding 100 μ L SM buffer solution into another part, standing at 25 deg.C for 0 h; 6 h; 12 h; sampling 100 μ L for 24h, coating TCBS plates,the total number of colonies was calculated, 3 replicates were set for each group, and the average was taken for analysis.

As shown in FIG. 7, the growth curve of the phage-treated group is significantly lower than that of the untreated group, and the inhibition of the total number of Vibrio is about 2 log at most, indicating that the phage VyZU 10474 of the present invention can significantly inhibit the growth of the pathogenic Vibrio carried by itself in the aquatic sample.

Claims

1. A pathogenic vibrio phage VyZU 10474 is preserved in China Center for Type Culture Collection (CCTCC) with preservation number of CCTCC NO: m2020417.

2. Use of the bacteriophage VmYZU10474 according to claim 1 in the inhibition of pathogenic vibrio.

3. Use according to claim 2, wherein said pathogenic vibrio comprises vibrio mimicus: (vibrio mimicus)Vibrio mimicus) Vibrio parahaemolyticus: (Vibrio parahemolyticus) Vibrio alginolyticus (Vibrioalginolyticus) And Vibrio fluvialis: (Vibriofluvialis）。

4. The use according to claim 2, wherein the bacteriophage VmYZU10474 is used in the inhibition of pathogenic vibrio bacteria in aquatic products or in their temporary rearing, storage, production environments or facilities.

5. Use of the bacteriophage VmYZU10474 according to claim 1 in the preparation of a bacteriostatic agent for pathogenic vibrio.

6. Use according to claim 5, wherein the bacteriostatic agent is an isolate or culture of bacteriophage VmYZU10474 as a bacteriostatic agent component.

7. The use of claim 5, wherein the bacteriostatic agent is used for the removal of pathogenic vibrio and its biofilm contamination from aquatic products or their temporary rearing, storage, production environment or facilities.

8. The use according to claim 5, wherein the bacteriostatic agent is prepared as: mixing the phage VyZU 10474 with vibrio mimicus at logarithmic growth phase, standing at room temperature, adding into LB liquid culture medium, and oscillating at constant temperature for overnight culture; transferring the culture to a sterilized centrifuge tube, centrifuging, collecting supernatant, filtering, collecting phage proliferation solution, adding PEG 8000 and NaCl, shaking to dissolve, and standing overnight; centrifuging to remove supernatant; adding SM buffer solution, and reacting at room temperature; extracting with chloroform; and centrifuging to recover a hydrophilic phase containing the VvYZU 10474 particles, and mixing the obtained VMYZU10474 particles with an SM buffer solution to prepare phage bacteriostatic agent mother liquor.