CN106995803B - Bacteriophage for preventing and treating vibrio parahaemolyticus disease of prawn and expanding culture method thereof - Google Patents
Bacteriophage for preventing and treating vibrio parahaemolyticus disease of prawn and expanding culture method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00051—Methods of production or purification of viral material
Abstract
The invention discloses a bacteriophage for preventing and treating prawn vibrio parahaemolyticus and an expanding culture method thereof, wherein the bacteriophage successfully screens out a strong lytic bacteriophage in seawater by utilizing vibrio parahaemolyticus infected by penaeus vannamei, and the titer of the bacteriophage is as high as 1010‑1012pfu/mL, fermentation period of 8-12h, obvious treatment effect, and great significance for treating diseases caused by vibrio parahaemolyticus and developing phage microecological preparation. The phage has a preservation unit code of CCTCC-China center for type culture Collection with the address as follows: the preservation date of the China center for type culture Collection in Wuhan university at Wuchang Lohonoa mountain is 2016, 12 and 9 days, and the preservation number is CCTCC NO: m2016740, survival as the result of preservation test, is named as Vibrio parahaemolyticus phage VP11 and is classified and named as Vibrio parahaemolyticus bacteriophage VP 11.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bacteriophage for preventing and treating vibrio parahaemolyticus disease of prawns and an expanding culture method thereof.
Background
The current primary means of controlling and eliminating pathogenic bacteria are various physical or chemical methods, including the use of various antibiotics. Physical or chemical methods have significant disadvantages. Firstly, the elimination effect of a physical method on various pathogenic bacteria can only be applied to a certain link of the related field, and the application of all links of the whole field cannot be realized, and secondly, the acid-base treatment in the chemical method can achieve certain control effect, but the effect of the methods is extremely undesirable after harmful bacteria form a biological membrane; finally, although the effect of the antibiotics is obvious when the antibiotics are used for a long time, the drug resistance of pathogenic bacteria is enhanced, beneficial bacteria are inhibited, and the pathogenic bacteria cannot be effectively controlled and eliminated finally.
In recent years, research and development of microbial ecological agents have been receiving attention from various parties, and in particular, research of bacteriophages has been favored. Since its discovery in 1896, phages have been of great interest because they lyse their host efficiently and are harmless to humans and animals. A bacteriophage is a bacterial virus that produces specific lysis of the host bacteria and forms plaques on bacterial growth plates, thus designating it as a bacteriophage. The phage is divided into temperate phage and virulent phage, wherein the virulent phage has strong cracking efficiency on host bacteria, and the replication efficiency and the reproductive capacity of the virulent phage are far higher than those of the host bacteria; after the virulent phage can be stably propagated in the host bacteria, one phage particle can kill hundreds of millions of host bacteria. Phage titer (titer) is the number of phage present in a unit volume (mL). Due to the high-efficiency lysis capacity of bacteriophage on host bacteria, the research on bacteriophage preparations for treating bacterial infection, especially drug-resistant bacterial infection, has become one of the hot spots of domestic and foreign research, and is paid more and more attention by biotechnology companies and research institutions.
The patent No. CN200910038392.X, entitled isolated Vibrio parahaemolyticus phage and its application in sterilization and prevention, the national invention patent discloses an isolated Vibrio parahaemolyticus phage (Vibrio parahaemolyticus) and its application in sterilization and prevention. The separated phage monomer is named as BPH-VP-1 and has broad-spectrum bactericidal capacity on vibrio parahaemolyticus.
Patent application No. 201510872234.X is named as a Vibrio parahaemolyticus phage, and a preparation method and application thereof, and relates to a Vibrio parahaemolyticus phage and sterilization application thereof as a biological bactericide in food and aquatic product culture environments. The separated vibrio parahaemolyticus phage has a preservation number of CCTCC M2015577, has high-efficiency cracking activity on vibrio parahaemolyticus, and the separated culture and the compound preparation thereof can be used as a food additive or an environmental disinfectant to prevent and control the pollution of vibrio parahaemolyticus in food.
Disclosure of Invention
The invention aims to solve the technical problem of providing a phage for preventing and treating prawn vibrio parahaemolyticus and an expanding culture method thereof, wherein the phage has the characteristics of strong phage lytic property, has the characteristics of high sterilization speed and high yield, and simultaneously performs solid-liquid separation on fermentation liquor when the fermentation is finished, so that the harm of vibrio parahaemolyticus to the environment is avoided. The phage has a preservation unit code of CCTCC-China center for type culture Collection, and the China center for type culture Collection has the following addresses: the preservation date of the China center for type culture Collection in Wuhan university at Wuchang Lohonoa mountain is 2016, 12 and 9 days, and the preservation number is CCTCC NO: m2016740, survival as the result of preservation test, is named as Vibrio parahaemolyticus phage VP11 and is classified and named as Vibrio parahaemolyticus bacteriophage VP 11.
In order to solve the technical problems, the technical scheme of the invention is as follows: the phage for preventing and treating the vibrio parahaemolyticus disease of the prawns is characterized by comprising the following steps of:
step A: separating and purifying host bacteria, namely separating and obtaining the vibrio parahaemolyticus from a large amount of diseased shrimp intestinal tracts of the penaeus vannamei;
and B: carrying out enlarged culture on host bacteria, adding 1-5% NaCl into NB or LB liquid culture medium, carrying out autoclaving at 121 ℃ for 20min, cooling to room temperature, and adopting two inoculation methods, wherein the first inoculation method is single colony inoculation, selecting a single colony on a seed-preserving plate under aseptic condition for inoculation, the fermentation temperature is 28-35 ℃, the rotation speed is 50-200rpm, and the fermentation time is 14-18 h; the second inoculation method is liquid inoculation, and PFU added to 10-20% of the culture medium volume is 106-108Liquid culture of pfu/mL vibrio parahaemolyticus liquid to NBFermenting in the medium at 28-35 deg.C at 50-200rpm for 10-16 h;
and C: separating phage from a large amount of seawater;
step D: expanding and culturing the phage, and expanding and culturing the separated phage;
step E: centrifuging the obtained fermentation liquid at 1000-8000rpm at 2-10 deg.C for 5-20min to allow host bacteria to settle at the bottom, collecting the upper clear part to obtain bacteriophage microecological preparation with titer of 1010-1012pfu/mL；
Step F: the bacteriophage is used for measuring the cracking growth curve of preventing and treating the vibrio parahaemolyticus disease of the prawn;
step G: performing a poison attacking experiment on the penaeus vannamei boone, namely culturing 60 healthy penaeus vannamei boone with the size specification of 50/jin in 15 glass jars respectively, wherein 9 of the healthy penaeus vannamei boone are experimental jars and are divided into A, B, C groups; the rest 6 are control cylinders which are divided into a negative control D group and a blank control E group, each group is repeated for 3 times, and 120L of seawater with the salinity of 25 is added into each glass cylinder; soaking and infecting the prawn, namely using the vibrio parahaemolyticus liquid fermented in the step B at the temperature of 28 ℃, calculating the volume of each group of bacteria liquid required to be added according to the concentration of the bacteria liquid and the volume of a glass jar, respectively adding A, B, C and a negative control group D, and finally enabling the bacteria content of water in the glass jar to reach 1 multiplied by 108pfu/mL, control group E without any treatment, 3 groups of experimental boxes were added phage 1X 105pfu/mL、1×104pfu/mL、1×103pfu/mL, A group, B group and C group, and recording the death condition of the penaeus vannamei boone.
Preferably, the vibrio parahaemolyticus in the step B is a prawn strong pathogenic bacterium, and the single colony inoculation fermentation time is 14-18 h; the inoculated bacterial liquid PFU of the second inoculation method is 106-108The inoculation proportion is 10-20%, and the fermentation time is 10-16 h.
Preferably, the PFU used for phage inoculation in step D is 104-108pfu/mL, the inoculation volume is between 1 and 20 percent of the volume of the fermentation broth.
Preferably, the expanding culture condition in the step D is that the temperature is 28-37 ℃, and the rotating speed is 50-200 rpm;
preferably, the time for the expanded culture is controlled to be between 8 and 12 hours.
Preferably, in the step E, the host bacteria are settled at the bottom by centrifugation at 8000rpm and 1000-10～1012pfu/mL。
A method for culturing the bacteriophage for preventing and treating the parahaemolyticus disease of prawn by applying the bacteriophage as claimed in any one of claims 1 to 6, wherein 1-20% of PFU is 104-108pfu/mL phage is added into the fermented vibrio parahaemolyticus fermentation liquid, the fermentation temperature is 28-37 ℃, the rotation speed is 50-200rpm, and the fermentation time is 8-12 h.
The invention has the advantages that: the invention separates a strong lytic bacteriophage in seawater by utilizing the vibrio parahaemolyticus infected by the penaeus vannamei, and the bacteriophage has high yield (the titer is up to 10)10-1012pfu/mL) and short fermentation period (8-12h), and has great significance for treating diseases caused by vibrio parahaemolyticus. The phage is named as VP11, and is preserved in China Center for Type Culture Collection (CCTCC) with the address: the preservation date of the China center for type culture preservation in Wuhan university at Wuchang Lojia mountain is 2016, 12 and 9 days, and the preservation number is CCTCCNO: m2016740. Has the following advantages: 1. the separated phage can crack vibrio parahaemolyticus with high titer, and has the characteristics of targeted specific sterilization, long-term continuous action, safety to environment and the like; 2. the growth cycle is short, the culture medium is simple, the cost is low, and the yield is high; 3. the phage is adopted to treat the culture water, and compared with antibiotics, the phage has the advantages of no water quality pollution, no generation of drug-resistant strains and low cost; 4. can be used for treating diseases caused by vibrio parahaemolyticus in the process of prawn culture and promoting high yield and harvest of prawn culture; 5. when the fermentation is finished, the solid-liquid separation is carried out on the fermentation liquor, so that the harm of vibrio parahaemolyticus to the environment is avoided.
Drawings
The invention is described in further detail below with reference to the figures and the detailed description.
FIG. 1 is a graph showing the one-step growth of the phage used for preventing and treating the vibrio parahaemolyticus disease of the prawn according to the invention and the amplification culture method thereof.
Detailed Description
The phage for preventing and treating the vibrio parahaemolyticus disease of the prawn comprises the following steps: step A: the host bacteria are separated and purified, and the vibrio parahaemolyticus is separated from a large amount of intestinal tracts of the penaeus vannamei. And B: performing amplification culture of host bacteria, adding 1-5% NaCl into NB or LB liquid culture medium, autoclaving at 121 deg.C for 20min, and cooling to room temperature. There are two methods of inoculation: firstly, single colony inoculation, namely picking single colony on a seed protection plate under aseptic condition for inoculation, wherein the fermentation temperature is 28-35 ℃, the rotation speed is 50-200rpm, and the fermentation time is 14-18 h; secondly, liquid inoculation is carried out, and PFU which accounts for 10 to 20 percent of the volume of the culture medium is added to be 106-108pfu/mL vibrio parahaemolyticus liquid is fermented in NB liquid culture medium at the fermentation temperature of 28-35 ℃, the rotation speed of 50-200rpm, and the fermentation time of 10-16 h; and C: separating phage from sea water. Step D: and (4) performing phage amplification, namely performing amplification on the separated phage. Step E: centrifuging the obtained fermentation liquid at 1000-8000rpm at 2-10 deg.C for 5-20min to allow host bacteria to settle at the bottom, collecting the upper clear part to obtain bacteriophage microecological preparation with titer of 1010-1012pfu/mL. Step F: the lytic growth curve of the phage for preventing and treating the vibrio parahaemolyticus disease of the prawn is determined and is shown in table 1. Step G: performing a poison attacking experiment on the penaeus vannamei boone, namely culturing 60 healthy penaeus vannamei boone with the size specification of 50/jin in 15 glass jars respectively, wherein 9 of the healthy penaeus vannamei boone are experimental jars and are divided into A, B, C groups; the remaining 6 are control jars, divided into negative control D and blank control E groups, each group has 3 replicates, and each glass jar is filled with 120L of seawater with salinity of 25. Soaking and infecting the prawn, namely using the vibrio parahaemolyticus liquid fermented in the step B at the temperature of 28 ℃, calculating the volume of each group of the vibrio parahaemolyticus liquid to be added according to the concentration of the vibrio parahaemolyticus liquid and the volume of a glass cylinder, respectively adding A, B, C groups and a negative control group D, and finally adding A, B, C groups and a negative control group DThe bacteria content of the water in the glass jar reaches 1 multiplied by 108pfu/mL, control group E without any treatment, 3 groups of experimental boxes were added phage 1X 105pfu/mL (group A), 1X 104pfu/mL (group B), 1X 103pfu/mL (group C), the mortality of Penaeus vannamei was recorded. As a result, see table 1, it was found that: the negative control group D without the applied phage is completely dead in 72h, and the sick prawns in the experimental group have lighter symptoms; the higher the using concentration of the phage is, the lower the morbidity degree of the prawns is, the higher the survival rate is, and the survival rate of an experimental group reaches more than 85 percent.
TABLE 1 toxicity test results (72h) of bacteriophage for controlling Vibrio parahaemolyticus of Penaeus vannamei
The invention separates a strong lytic phage from seawater by using the vibrio parahaemolyticus infected by the penaeus vannamei, and the titer of the phage after fermentation reaches 1010-1012pfu/mL, fermentation period of 8-12 h. Has the following advantages: the separated phage can be high-titer lytic phage and has the characteristics of targeted specific sterilization, long-term continuous action, safety to environment and the like; the fermentation period is short, the culture medium is simple, the cost is low, and the yield is high; the phage is adopted to treat the culture water, and compared with antibiotics, the phage has the advantages of no water quality pollution, no generation of drug-resistant strains and low cost; can be used for treating diseases caused by vibrio parahaemolyticus in the process of prawn culture and promoting high yield and harvest of prawn culture; when the fermentation is finished, the solid-liquid separation is carried out on the fermentation liquor, so that the harm of vibrio parahaemolyticus to the environment is avoided.
The phage for preventing and treating the prawn vibrio parahaemolyticus is adopted, the overnight cultured vibrio parahaemolyticus is uniformly coated on an LB flat plate by adopting a dot-drop method, 20-50ul of culture expanding solution is dripped after the flat plate is dried completely, the environmental temperature is kept at 28-37 ℃, standing culture is carried out for 8-18 hours, and the existence of plaque is observed; heating solidified NB or LB semisolid culture medium to be completely melted by adopting a double-layer flat plate method, adding 0.2mL of vibrio parahaemolyticus and 0.1mL of enrichment solution which are cultured to logarithmic phase when the temperature is reduced to 45 ℃, pouring the mixture into the upper layer of an LB flat plate, culturing the mixture for 8-18 hours at 28-37 ℃ after solidification, and observing whether the occurrence of the plaque is present; if the detection results are positive by adopting the two methods of the dropping method and the double-layer flat plate method, the existence of the phage is proved.
The titer identification of the phage for preventing and treating the prawn vibrio parahaemolyticus is characterized in that based on the detection of the phage for preventing and treating the prawn vibrio parahaemolyticus, phage filtrate is continuously diluted ten times, a double-layer plate method is adopted for overnight culture, then the number of plaques is counted, the number of plaques is accurate by 30-300 per NA or LB plate, the experiment needs to be repeated three times, the average value of the three times is taken as the final titer of the phage, wherein the titer conversion formula is as follows: phage titer-number of plaques 3 dilution fold/volume of phage filtrate added. If the number of the plaques of each NA or LB plate is more than or less than 30-300 plaques of each NA or LB plate in a doubling manner in the titer identification process, the dilution multiple of the phage filtrate needs to be increased or reduced in a doubling manner.
A method for expanding and culturing the bacteriophage used to prevent and treat the parahaemolyticus vibrio disease of prawn features that 1-20% of PFU is 104-108pfu/mL phage is added into the fermented vibrio parahaemolyticus fermentation liquid, the fermentation temperature is 28-37 ℃, the rotation speed is 50-200rpm, and the fermentation time is 8-12 h.
As shown in the phage one-step growth curve shown in fig. 1, the phage is inoculated into the bacterial suspension of the host bacteria, the time is 0, 25, 50, 75, 100, 125, 150, 175, 200, 720, 1440min, and the phage quantity is detected by using a double-layer plate method to obtain the phage one-step growth curve. The result shows that the bacteriophage VP11 has strong cracking performance on the Vibrio parahaemolyticus VP11, has extremely strong Vibrio killing capability which can reach 1010pfu/mL or more.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the technical solutions, and those skilled in the art should understand that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all the modifications and equivalent substitutions should be covered by the claims of the present invention.
Claims (2)
1. A bacteriophage (Vibrio parahaemolyticus bacteriophage) VP11 for preventing and treating Vibrio parahaemolyticus disease of prawns is deposited in China center for type culture Collection, and the preservation number is CCTCC NO: m2016740.
2. The method for expanding and culturing the phage for preventing and treating the vibrio parahaemolyticus disease of the prawns is characterized in that the phage is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: m2016740, characterized in that: the phage is expanded by setting PFU at 1-20% to 104-108pfu/mL phage is added into fermented vibrio parahaemolyticus fermentation liquid, the fermentation temperature is 28-37 ℃, the rotation speed is 50-200rpm, and the fermentation time is 8-12 h.
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