CN109517804B - Vibrio mimicus bacteriophage OY-1 with cross-species lysis characteristic, preparation method and application thereof - Google Patents
Vibrio mimicus bacteriophage OY-1 with cross-species lysis characteristic, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a vibrio mimicus phage OY-1 with cross-species cracking characteristic, a preparation method and application thereof, wherein the separated vibrio mimicus phage is named as vibrio mimicus phage OY-1 (the preservation number is CCTCC NO: M2017588), and has high-efficiency cross-species cracking activity on vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus, and a phage culture and a preparation thereof can be used as a biological bacteriostatic agent to control the propagation and biofilm formation of pathogenic vibrios, so that the risk of vibrio pathogenic pollution in aquatic food and culture processing environment thereof is reduced.
Description
Technical Field
The invention relates to a vibrio mimicus bacteriophage with cross-species cracking characteristic, and bacteriostatic application of the vibrio mimicus bacteriophage serving as a biological bacteriostatic agent, a water purifying agent and a feed additive in aquatic product culture, storage, transportation and processing environments, which can reduce pollution caused by pathogenic vibrios and ensure food safety and sanitation, and belongs to the field of biological engineering.
Background
Vibrio is a gram-negative bacillus widely distributed in the world, is one of the most common bacterial pathogens, the most prevalent and the most serious harmful pathogens in aquaculture, and with the development of aquaculture industry, the outbreak of vibriosis is more and more frequent, the propagation speed is faster and faster, and huge economic loss is brought to the aquaculture industry all over the world. Among the Vibrio species currently found, Vibrio parahaemolyticus: (Vibrio parahemolyticus) Vibrio alginolyticus (Vibrio alginolyicus) And Vibrio mimicus: (Vibrio mimicus) Is an important zoonotic pathogenic bacterium related to human intestinal and parenteral infection. The aquatic product is mainly polluted to cause harm to human health, and can cause food poisoning, intestinal infectious diseases, wound infection, other tissue inflammation, septicemia and the like. Recent epidemiological investigation results show that the three pathogenic vibrios are main pathogenic bacteria of diarrhea in coastal river regions.
In the process of growing vibrio adapted to natural environment, biofilm is easily formed 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 the produced food. 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. 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.
Bacteriophages are a class of bacteria-dependent viruses, also known as bacterial viruses. After the lytic phage infects bacteria, it can quickly proliferate in host bacteria and lyse it, so it is also called as virulent phage. The bacteriophage exists widely in nature, has specificity to host bacteria, and has low preparation cost and good safety. In the safety control of aquatic products, the phage is used as a biological bacteriostatic agent to control the formation, growth and metabolism of a biofilm of pathogenic vibrio, and the potential development value is realized. Usually, the bacteriophage can only inhibit bacteria of a specific species, and has limited effect on mixed contamination of various vibrios, so that screening of bacteriophage with broad-spectrum lysis property for construction of bacteriostatic agents is urgently needed.
Disclosure of Invention
Aiming at the problems, the technical problem to be solved by the invention is to provide the phage with strong cracking effect on 3 pathogenic vibrios, the preparation can be used independently or in a compound way, can effectively control the formation of a biofilm, inhibit the reproduction and metabolism of the pathogenic vibrios in the process of culture, storage, transportation and processing of aquatic products, and provide a safe, efficient and cheap phage product for biological control of aquatic products.
The invention relates to a vibrio mimicus phage OY-1 with cross-species lysis characteristic, which is characterized in that the vibrio mimicus phage OY-1 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address: wuhan, Wuhan university; the preservation number is: CCTCC NO: m2017588; the preservation date is as follows: in 2017, 10 and 17 months, the classification names areBacteriophage of Vibrio mimicusOY-1 (i.e., the minimal Vibrio phage OY-1,Vibrio mimicusphase OY-1), wherein Vibrio mimicus is also called Vibrio mimicus.
The invention further discloses a vibrio mimicus bacteriophageVibrio mimicusphase) OY-1 in preparing bacteriostatic agent and its use as effective component of bacteriostatic agent.
The invention also discloses a vibrio mimicus bacteriophageVibrio mimicusphase) OY-1 in preventing and treating vibriosis of aquatic products. Namely, the application of the vibrio mimicus bacteriophage OY-1 in preparing a water purifying agent or a feed additive, wherein the vibrio mimicus bacteriophage OY-1 inhibits the growth of vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus, and is used for preventing and treating vibriosis caused by the vibrio mimicus, the vibrio parahaemolyticus, the vibrio alginolyticus and the like in aquaculture. Also, there is disclosed a Vibrio mimicus bacteriophage: (Vibrio mimicusphase) OY-1 as an effective component of a water body purifying agent or a feed additive.
The invention also discloses a vibrio mimicus bacteriophageVibrio mimicusphase) OY-1 is used for bacteriostasis in aquatic product storage, transportation and processing environments. I.e., Vibrio mimicus bacteriophage: (Vibrio mimicusphase) OY-1 in preparing bacteriostatic or degerming reagent for aquatic product culture, storage and transportation and processing.
The present invention also provides an isolate or culture of the Vibrio mimicus bacteriophage OY-1 having cross-species lytic properties.
The invention also provides a pathogenic vibrio bacteriostatic agent which takes the isolate or culture of the vibrio mimicus phage OY-1 as a bacteriostatic component.
The invention separates a vibrio mimicus bacteriophage from a sewage sample collected from a farmer marketVibrio mimicusphase) OY-1, deposited in China center for type culture Collection (CCTCC for short), and the deposition address is as follows: wuhan, Wuhan university; the preservation number is: CCTCC NO: m2017588; the preservation date is as follows: year 2017, month 10 and day 17.
The Vibrio mimicus bacteriophage OY-1 of the present invention has the following biological characteristics:
(1) morphological characteristics: through transmission electron microscope observation, the head is symmetrical, the diameter is about 55 nm, the tail length is about 8 nm, and the tail sheath is non-elastic, and belongs to the brachytrophagidae.
(2) Types of phage nucleic acids: the phage is dsDNA.
(3) Phage genome characterization: the whole length of the genome of the Vibrio mimicus bacteriophage OY-1 is 43479 bp, wherein the total lengths of the coding genes are 38403 bp respectively, the average lengths are 936.66 bp respectively, the total length is 88.33%, the GC content is 49.26%, and 41 Open Reading Frames (ORFs) are provided. All 41 proteins encoded by ORFs can be searched in a database to obtain homologous gene sequences, wherein 24 proteins encoded by ORFs are hypothetical proteins, 17 protein sequences have clear functions, and genome data show that the Vibrio mimicus bacteriophage OY-1 is a novel bacteriophage.
(4) Has strong cross-species cracking performance of vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus.
(5) Can inhibit biofilm formation of Vibrio mimicus, Vibrio parahaemolyticus and Vibrio alginolyticus.
The invention prepares the vibrio mimicus phage OY-1 into a pathogenic vibrio phage bacteriostatic agent which is used as a bacteriostatic agent in the environments of aquatic product culture, storage and transportation, production and processing, and the like, and the preparation method comprises the following steps: respectively mixing 100 μ L of vibrio mimicus phage OY-1 phage body fluid with 100 μ L of corresponding logarithmic phase growth phase mimicry arc bacteria liquid, standing at room temperature for 10 min, adding into 10 mL of LBS liquid culture medium, and performing constant temperature shaking at 25 deg.C and 150 rpm overnight culture; transferring the culture in the test tube into a sterilized centrifuge tube, centrifuging at 8000 Xg for 10 min, collecting supernatant, and filtering and sterilizing through a 0.22 mu m microporous filter membrane to obtain a phage proliferation solution. Adding 0.93g PEG 8000 and 0.58 g NaCl, shaking up to dissolve, and standing overnight at 4 ℃; centrifuging at 10000 Xg 4 deg.C for 20 min, and removing supernatant; 0.5 mL of SM (1L: NaCl 5.8g, MgSO)4.7H2O2.0 g, 50mL of 1M Tris-HCl (pH7.4) solution, and reacting at room temperature for 1 h; extracting for 30s by adding equal volume of chloroform; the hydrophilic phase containing the phage particles was recovered by centrifugation at 3000 Xg for 15min at 4 ℃. The obtained purified vibrio mimicus phage OY-1 phage particles (which are preserved CCTCC NO: M2017588) and SM buffer solution are mixed according to the ratio of 1: mixing the components in a ratio of 10 to form phage bacteriostat mother liquor.
The application of the bacteriostatic agent prepared from the vibrio mimicus bacteriophage is as follows: diluting the purified vibrio mimicus phage OY-1 phage particles with water and preparing a spraying liquid or a leacheate for storing and processing aquatic products and spraying or washing production instruments, and reducing the pollution of pathogenic vibrio such as vibrio mimicus, vibrio parahemolyticus, vibrio alginolyticus and the like in the processing process of the aquatic products; or the purified vibrio mimicus bacteriophage is used as a purifying agent of the temporary culture water body of marine products after being expanded and cultured, and is used for preventing and treating vibriosis caused by pathogenic vibrios such as vibrio mimicus, vibrio parahaemolyticus, vibrio alginolyticus and the like in the temporary culture process of the marine products.
The aquatic product in the invention refers to: edible or usable products in water bodies such as oceans, rivers, lakes and the like comprise aquatic animals such as fish, shrimps, shellfish, seaweeds and the like.
Because the invention adopts the vibrio mimicus phage OY-1 to crack the vibrio mimicus, the vibrio parahemolyticus and the vibrio alginolyticus, the metabolism and the propagation of various pathogenic vibrios such as the vibrio mimicus can be effectively inhibited, the food pollution caused by the pathogenic vibrios such as the vibrio mimicus is reduced, the sanitation and the safety of aquatic products and the environment are kept, and the occurrence of food-borne diseases is reduced; because the phage related to the invention is easily prepared into spraying liquid or leacheate by the traditional method, the phage can sterilize the environment or appliances, can inhibit the formation of mycoderm and reduce the pollution of pathogenic vibrios to food; as the bacteriophage of the invention belongs to natural biological materials, has no toxic or side effect and can be used as a food biological bacteriostatic agent.
Drawings
FIG. 1 shows the bacteriostatic results of phage OY-1 double-layer plate;
FIG. 2 lytic Performance of the bacteriophage OY-1 against other Vibrio strains, wherein the numbers of strains 1 to 18 are respectively: 1. CICC 21617; 2. vp 92; 3. ATCC 33842; 4. vp 68; 5. vp 71; 6. vp 76; 7. vp 101; 8. vp 110; 9. va F2-2-1; 10. va E1-3; 11. va H2-3; 12. va J1-4; 13. va J8-3; 14. ATCC 33809; 15. CICC 10474; 16. CICC 23794; 17. CICC 21613; 18. ATCC 17749;
FIG. 3 is an electron micrograph of phage OY-1;
FIG. 4 phage OY-1 genome-wide map;
FIG. 5 effect of bacteriophage OY-1 on inhibition of bacteria in CICC21613, CICC21617, J1-4 in culture medium for 6 h;
FIG. 6 shows the inhibitory effect of phage OY-1 on biofilm formation, wherein Control represents a Control group to which phage was not added(ii) a Blank represents a pure medium Blank control group; A. b, C, D, E, F groups represent respectively 105 PFU/mL、106 PFU/mL、107 PFU/mL、108 PFU/mL、109OY-1 treatment groups at PFU/mL concentration, 5 replicates in each group;
FIG. 7 inhibitory Effect of bacteriophage OY-1 on the formation of CICC21613, CICC21617, J1-4 biofilms;
the vibrio mimicus phage OY-1 with cross-species lysis characteristic is preserved in China Center for Type Culture Collection (CCTCC) at the preservation address: wuhan, Wuhan university; the preservation number is: CCTCC NO: m2017588; the preservation date is as follows: 10 and 17 months in 2017, and are named in classificationBacteriophage of Vibrio mimicusOY-1 (i.e., Vibrio mimicus phage OY-1).
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way.
The phage host bacterium vibrio mimicus (for testing) of the inventionVibrio mimicusThe strain number: CICC 21613) purchased from china industrial collection of microorganisms.
The Vibrio mimicus phage OY-1 (used in the test of the present invention)Vibrio mimicusphase OY-1) 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: m2017588; the preservation date is as follows: 2017, 10 and 17 months.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The culture media and test conditions used in the examples of the present invention are those conventional in the art unless otherwise specified. The reagents used in the examples of the present invention were all commercially available unless otherwise specified.
Example 1 phage isolation and purification preparation
Phage isolation
The sample is collected from sewage of world temple farmer market in Yangzhou Jiangsu, 30 mL of the sewage sample is put into a 50mL centrifuge tube, 8000 Xg is centrifuged for 10 min, 4mL of supernatant is added into 5 mL of LBS liquid culture medium, 100 mu L of vibrio mimicus CICC21613 in logarithmic phase is added at the same time, the centrifuge tube is placed on a 37 ℃ shaking table for overnight culture, the culture in the test tube is transferred into a sterile centrifuge tube on the next day, 8000 Xg is centrifuged for 10 min at 4 ℃, and the supernatant is taken to pass through a 0.22 mu m filter membrane to obtain phage stock solution which is placed in a 4 ℃ refrigerator for storage.
Diluting phage stock solution with sterile SM buffer solution in multiple proportion, mixing phage stock solution 100 μ L at appropriate dilution with vibrio mimicus CICC21613 in logarithmic growth phase 100 μ L at room temperature for 10 min, adding 5 mL LBS semisolid culture medium, mixing, rapidly pouring onto prepared LBS solid culture medium, making into double-layer plate, solidifying, and culturing in 37 deg.C constant temperature incubator.
Picking a transparent single plaque on the double-layer plate with the plaque to 1 mL of SM buffer solution, uniformly mixing, and standing for 24h at 4 ℃; diluting phage liquid with sterile SM buffer solution in multiple proportion the next day, mixing phage liquid 100 μ L at appropriate dilution with vibrio mimicus CICC21613 in logarithmic growth phase 100 μ L at room temperature for 10 min, adding 5 mL LBS semisolid culture medium, mixing, rapidly pouring onto prepared LBS solid culture medium, making into double-layer plate, solidifying, and culturing in 37 deg.C constant temperature incubator. The operation of the double-layer plate method is repeated for 5 times, and after the obtained plaques are consistent in size, the isolated phage is considered to be pure. The obtained phage was isolated and the lysis effect of the purified phage was examined with a double-layer plate. Adding 100 mu L of mimicry CICC21613 into 5 mL of melted LBS semisolid culture medium, immediately pouring the culture medium on a bottom LBS solid culture medium, standing the culture medium for 10 min at room temperature, dropwise adding 10 mu L of propagated phage after the culture medium is solidified, placing the culture medium in an aseptic operating platform until the surface of the culture medium is dried, then placing a flat plate upside down in an incubator at 37 ℃ for culture, and screening the phage which can generate bright and clear lysis rings for the mimicry vibrio CICC 21613.
Respectively adding 10 mu L of suspension of Vibrio mimicus phage isolates OY-1, OY-5, OY-6, OY-8 and OY-10 into 100 mu L of suspension of Vibrio mimicus CICC21613 in logarithmic phase growth phase, taking SM buffer solution with the same volume as a reference (Blank), standing at room temperature for 10 min, adding into 20 mL of nutrient broth culture medium, placing the inoculum in an incubator at 37 ℃ for constant-temperature culture, sampling 1 mL at 0 h and 24h respectively, measuring the light absorption value at 600 nm by using a spectrophotometer, setting 3 parallels in each group, taking an average value for comparing the bacteriostatic ability of different phage isolates, and screening the isolate with the strongest inhibitory ability to Vibrio mimicus CICC 21613. Obtaining a phage, which is OY-1.
Purification of bacteriophages
Respectively mixing 100 μ L of separated Vibrio mimicus bacteriophage OY-1 with 100 μ L of Vibrio mimicus CICC21613 liquid in logarithmic phase growth phase, standing at room temperature for 10 min, adding into 10 mL of LBS liquid culture medium, and culturing at 37 deg.C and 150 rpm under constant temperature shaking overnight; transferring the culture in the test tube into a sterilized centrifuge tube, centrifuging at 8000 Xg for 10 min, collecting supernatant, and filtering and sterilizing through a 0.22 mu m microporous filter membrane to obtain a phage proliferation solution. Adding 0.93g PEG 8000 and 0.58 g NaCl, shaking to dissolve, and standing at 4 deg.C overnight; centrifuging at 10000 Xg 4 deg.C for 20 min, and removing supernatant; adding 0.5 mL of SM (1L: NaCl 5.8g, MgSO4.7H2O 2.0g, 1M Tris-HCl (pH7.4) 50mL) solution, and reacting at room temperature for 1 h; extracting for 30s by adding equal volume of chloroform; the hydrophilic phase containing the phage particles was recovered by centrifugation at 3000 Xg for 15min at 4 ℃. Obtaining purified phage, and detecting the lysis effect of the purified phage by using a double-layer plate. The results showed that OY-1 produced a strong lytic effect on Vibrio mimicus CICC21613 (shown in FIG. 1).
Cracking characteristics
The spot-spotting method is used to determine the lysis spectrum of bacteriophage OY-1, and 5 mL LBS semisolid culture medium is added with 50 μ L of 18 strains of vibrio (10) including host bacteria8 CFU/mL) (as shown in FIG. 2), mixing by inversion, pouring onto solid medium, and after coagulation, dropwise adding phage OY-1 suspension (10)8 PFU/mL) 10. mu.L, placing the mixture in an incubator for culturing for 8-12 h, and observing the formation of a lysis ring.
Morphological characteristics of bacteriophages
And observing the morphological characteristics of the phage by using a transmission electron microscope. Adopting a phosphotungstic acid negative dyeing method, enabling the surface of the copper net with the film to face upwards, dripping 10 mu L of OY-1 phage on the copper net, adsorbing for 15min, then sucking water with absorbent paper, taking out the copper net, and naturally drying in the air for 2-3 min. And then, dripping a drop of 2% phosphotungstic acid (PTA) aqueous solution on a copper net for dyeing, taking down after 2 min, absorbing water by absorbent paper, drying in the air for 5min, observing by using a transmission electron microscope, and selecting a clear phage image for photographing analysis.
As shown in FIG. 3, the phage OY-1 was symmetric in head, about 55 nm in diameter, about 8 nm in tail length, and had no stretchy tail sheath, belonging to the family Brevibacterium.
Characterization of phage genome
Adding DNase I into the purified vibrio mimicus phage OY-1 suspension to a final concentration of 5 mu g/mL and RNase A to 1 mu g/mL, and incubating for 1h at 37 ℃; EDTA (pH 8.0) was added to a final concentration of 20 mmol/L; adding proteinase K to a final concentration of 50 mu g/mL, adding SDS to a final concentration of 0.5%, uniformly mixing, and multiplying for 1h at 56 ℃; adding isovolumetric balance phenol (pH 8.0), extracting under shaking, 5000 Xg, centrifuging for 10 min, and collecting upper water phase; extracting with equal volume of chloroform, centrifuging at 5000 × g for 10 min, and collecting upper water phase; adding isovolumetric isopropanol, storing at-20 deg.C for 1h, at 4 deg.C, centrifuging at 12000 Xg for 10 min; washing the precipitate with 70% ethanol twice, removing ethanol, and drying the precipitate in air; suspending the precipitate with appropriate amount of TE (pH 8.0), roughly quantifying on a GeneQuant nucleic acid quantifier, temporarily storing at-20 ℃ according to ds DNA set parameters; the extracted phage DNA is sent to Shenzhen Hengchuang Gen biology Limited company and is subjected to whole genome sequencing and analysis by an Illumina Hiseq 2500 PE150 platform.
As shown in FIG. 4, the total length of the genome of Vibrio mimicus bacteriophage OY-1 was 43479 bp, wherein the total length of the coding genes was 38403 bp, the average length was 936.66 bp, accounting for 88.33% of the total length, the GC content was 49.26%, and there were 41 Open Reading Frames (ORFs). The 41 proteins encoded by the ORFs can be searched in a database for homologous gene sequences, wherein 24 proteins encoded by the ORFs are hypothetical proteins, 17 protein sequences have definite functions, genome data show that OY-1 is a novel phage, and analysis shows that no known virulence related gene is detected in the genome of the minimal vibrio phage OY-1.
Example 2 inhibitory Effect of bacteriophage OY-1 in culture Medium
Taking 100 mu L of vibrio mimicus CICC21613, vibrio parahaemolyticus CICC21617 and vibrio alginolyticus isolate J1-4 in logarithmic phase growth period, adding 8mL of LBS liquid culture medium, and respectively adding 100 mu L of bacteriophage OY-1 (protobacteria + OY-1) with MOI value of 100; taking LBS liquid culture medium with the same volume as the reference (original bacteria), culturing at constant temperature of 25 ℃, measuring the light absorption value at 600 nm after 6h, setting 3 parallels in each group, taking the average value as a bar chart, and as shown in figure 5, the bacteriophage OY-1 has certain inhibition power on the growth of vibrio mimicus CICC21613 (the OD value is reduced from 0.39 to 0.05), vibrio parahaemolyticus CICC21617 (the OD value is reduced from 0.53 to 0.09) and vibrio alginolyticus J1-4 (the OD value is reduced from 0.47 to 0.1) in the culture medium.
Example 3 inhibition of Vibrio mimicus phage OY-1 on Vibrio mimicus biofilm
Diluting Vibrio mimicus CICC21613 in logarithmic growth phase with LBS culture medium to final concentration of 1 × 107CFU/mL, experimental group (A, B, C, D, E, F group): diluted bacterial solution (10) was added to each well of sterile 96-well plates7CFU/mL) and phage OY-1 suspension 109 PFU/mL、108 PFU/mL、107 PFU/mL、106 PFU/mL、105 PFU/mL、104PFU/mL 150. mu.L each; control group: diluted bacterial solution (10) was added to each well of sterile 96-well plates7CFU/mL) and LBS media each 150 μ L; blank group: adding 300 μ L LBS culture medium to each well; culturing at 37 deg.C for 24 hr; taking out the 96-well plate, sucking out the suspension bacteria liquid, cleaning the 96-well plate for 3 times by using sterile PBS, drying and fully removing floating bacteria; 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, thoroughly washing the 96-well plate by using sterile PBS until the eluate is colorless, and drying; 200 μ L of 33% acetic acid decolorant is added into each well, the mixture is shaken and fully dissolved, and the OD value at 600 nm is measured by a microplate reader. Each group was assayed in duplicate 5 times.
As a result, as shown in FIG. 6, OD measured by adding phage OY-1 suspension to test group (A, B, C, D, E group)600nm0.072, 0.07, 0.075, 0.073, 0.08 are significantly lower than the control group 0.13 (p)<0.001), indicating that105 The phage OY-1 with the concentration of more than PFU/mL can obviously inhibit the formation of the biomembrane of the vibrio mimicus CICC 21613.
The same method is used for detecting the inhibition of bacteriophage OY-1 on the formation of biological envelopes of vibrio parahaemolyticus CICC21617, CICC21613 and vibrio alginolyticus isolate J1-4, and the concentration of the bacterial liquid is adjusted to 107CFU/mL, concentration of phage OY-1 to 106PFU/mL. The results are shown in FIGS. 7A and 7B, which indicate that the Vibrio mimicus bacteriophage OY-1 has obvious inhibition capability on the growth of Vibrio parahaemolyticus CICC21617 (OD value is reduced from 0.17 to 0.10), CICC21613 (OD value is reduced from 0.22 to 0.095) and Vibrio alginolyticus J1-4 (OD value is reduced from 0.165 to 0.115) biofilms.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A Vibrio mimicus Bacteriophage (bacterio mimicus) OY-1 having cross-species lytic characteristics, which is deposited at the China center for type culture Collection under the accession number: CCTCC NO: m2017588; the preservation date is as follows: year 2017, month 10 and day 17.
2. Use of the vibrio mimicus phage OY-1 having cross-species lytic property of claim 1 in preparing bacteriostatic agent for inhibiting vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus.
3. The use of the vibrio mimicus phage OY-1 having cross-species lysis property in preparing a water purifying agent or a feed additive according to claim 1, wherein the vibrio mimicus phage OY-1 is used for inhibiting the growth of vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus.
4. The use of the vibrio mimicus phage OY-1 with cross-species lysis property of claim 1 in preparing bacteriostatic or degerming reagents required in aquaculture, storage, transportation and processing of aquatic products, wherein the vibrio mimicus phage OY-1 is used for inhibiting vibrio mimicus, vibrio parahaemolyticus and vibrio alginolyticus.
5. The culture of the Vibrio mimicus bacteriophage OY-1 having cross-species lytic property as set forth in claim 1.
6. A bacteriostatic agent for pathogenic vibrios, which is characterized in that: the culture of the Vibrio mimicus bacteriophage OY-1 having cross-species lytic property as set forth in claim 1, wherein the concentration of the Vibrio mimicus bacteriophage OY-1 in the bacteriostatic agent is 105 PFU/mL or higher.
7. The method for preparing the bacteriostatic agent according to claim 6, wherein the bacteriostatic agent is obtained by mixing and culturing the vibrio mimicus phage OY-1 and vibrio mimicus which have cross-species lytic characteristics.
8. The method for preparing the bacteriostatic agent according to claim 6, wherein the bacteriostatic agent stock solution is prepared by mixing the vibrio mimicus bacteriophage OY-1 with cross-species lysis property and vibrio mimicus, culturing and purifying to obtain the vibrio mimicus bacteriophage OY-1 bacteriophage particles, and mixing with a buffer solution.
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