CN108103031B

CN108103031B - Wide-spectrum phage preparation for aquaculture and preparation method thereof

Info

Publication number: CN108103031B
Application number: CN201810024877.2A
Authority: CN
Inventors: 刘莉; 卢淑娟; 吕孙建; 曹铮; 林锋
Original assignee: Zhejiang Institute of Freshwater Fisheries
Current assignee: Zhejiang Institute of Freshwater Fisheries
Priority date: 2018-01-10
Filing date: 2018-01-10
Publication date: 2021-09-07
Anticipated expiration: 2038-01-10
Also published as: CN108103031A

Abstract

The invention relates to the field of aquaculture, and discloses a wide-spectrum phage preparation for aquaculture and a preparation method thereof, wherein the phage preparation is prepared by fermenting a phage strain NTHP01 with the preservation number of CGMCC No.9623 and genetic engineering Escherichia coli DH5 alpha as host bacteria. The preparation method comprises the following steps: 1) adding escherichia coli DH5 alpha into a culture medium for fermentation culture to obtain host bacterium fermentation liquor; 2) phage and MgCl₂Adding the mother liquor into the host bacterium fermentation liquor, uniformly mixing, standing, and performing proliferation culture to obtain a fermentation mixed liquor; 3) and centrifuging the fermentation mixed solution to obtain the phage preparation. The wide-spectrum phage preparation has a cracking effect on various aquaculture pathogenic bacteria, and can be used independently or in combination with other microecological preparations. And the host bacteria adopt genetically engineered bacteria, the preparation method is simple and convenient, the use is safe, no side effect is caused, and the method can be popularized and applied to bacterial disease control of aquaculture.

Description

Wide-spectrum phage preparation for aquaculture and preparation method thereof

Technical Field

The invention relates to the field of aquaculture, in particular to a wide-spectrum phage preparation for aquaculture and a preparation method thereof.

Background

In recent years, the aquaculture of China develops rapidly, but the diseases are serious year by year. According to incomplete statistics, the current aquaculture diseases are more than 300, the aquaculture area with diseases generated per year accounts for 1/10 of the total aquaculture area, the annual loss accounts for 15% -30% of the total aquaculture output, the annual economic loss reaches hundreds of billions of yuan, and the sustainable development of the aquaculture industry is seriously threatened.

Since the discovery of penicillin by fleming in 1928, antibiotics were widely used and developed, after which time humans entered the high-rate development phase of antibiotics. Antibiotics in the broad sense are chemotherapeutic agents that inhibit or eliminate the growth of microorganisms. Because aquaculture has the characteristic of high risk of bacterial infection, the use of antibiotics in the aquaculture process has become a common practice in the aquaculture industry for both prevention and treatment purposes. The study of Amir Sapkota et al found that in 1990-2007 13 aquaculture countries including China, the national proportions of oxytetracycline, chloramphenicol and oxolinic acid used accounted for 92%, 69% and 69%, respectively. Antibiotics have been widely used over the past 60 years, resulting in the accelerated spread of drug resistant genes in bacteria, and the number of drug resistant bacteria has increased. In addition, the abuse of antibiotics also causes a plurality of serious consequences of aquatic product drug residue, water environment damage, beneficial microorganism harm and the like. With the knowledge of the harm of antibiotics and residues thereof to the safety and environmental safety of aquatic products and the global trend of trade, the species and use of antibiotics for aquatic products are strictly limited in China, and the phenomenon of abuse of antibiotics in aquaculture is well controlled. However, due to the fact that antibiotics are used as an important means for disease control in aquaculture for a long time, drug-resistant strains are ubiquitous. According to the research of the national flounder industry and technology system, more than 90 bacterial pathogens found in flounder cultivation in China have antibiotic resistance with different degrees. Therefore, the limited use of antibiotics is in conflict with the emergence of a large number of resistant strains, which often results in the production of no drug available. The antibiotic-free aquaculture is a trend of industrial development in the future.

Bacteriophages (phages) are a class of viruses that parasitize bacteria, mycoplasmas, spirochetes, actinomycetes, and cyanobacteria, also known as bacterial viruses. Wherein the virulent phage can proliferate and lyse in sensitive bacteria cells, which is the "natural killer" of the bacteria. Thus, bacteriophages are also one of the control approaches for bacterial diseases. Compared with antibiotics, the bacteriophage has the characteristics of strong specificity, small side effect, strong multiplication capacity, difficult generation of resistance, pathogenic bacteria dependence, convenient use and the like, and is a green biological control measure. The existing literature reports that the bacteriophage has better inhibition effect on Pseudomonas aeruginosa (Pseudomonas aeruginosa), Salmonella (Salmonella), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and other bacteria.

Because the bacteriophage has specificity to the sterilization of host bacteria, most bacteriophage has certain limitation to the prevention and treatment of pathogenic bacteria, namely, one bacteriophage only has sterilization to 1 or more pathogenic bacteria. In addition, the specificity of the phage preparation also causes the phage preparation to be prepared by taking pathogenic bacteria as host bacteria, has higher requirement on later purification and brings potential risks to production and application.

Disclosure of Invention

In order to solve the technical problems, the invention provides a wide-spectrum phage preparation for aquaculture and a preparation method thereof. The broad-spectrum phage preparation has a cracking effect on various aquaculture pathogenic bacteria, and can be used independently or in combination with other microecological preparations. And the host bacteria adopt genetically engineered bacteria, the preparation method is simple and convenient, the use is safe, no side effect is caused, and the method can be popularized and applied to bacterial disease control of aquaculture.

The specific technical scheme of the invention is as follows: a wide-spectrum bacteriophage preparation for aquaculture is prepared by fermenting bacteriophage strain NTHP01 with preservation number of CGMCC No.9623 and genetic engineering Escherichia coli DH5 alpha as host bacteria.

The inventor finds a phage strain NTHP01 in a prior patent (201410506817.6, a Chinese softshell turtle aeromonas phage and application thereof), and then carries out subsequent research to find that the phage strain has a lysis effect on aeromonas and also has broad spectrum.

In addition, in the prior art, usually, the bacteriophage preparation is prepared by taking harmful pathogenic bacteria as host bacteria due to the specificity of the bacteriophage, so that the requirement on later purification is high, and potential risks are brought to production and application. The invention adopts the auxotrophic escherichia coli DH5 alpha for genetic engineering as the host bacterium which is harmless or low in toxicity, so that the requirements for later purification are low, and the safety is high. And the invention also has good phagocytic effect on host bacteria.

In a word, the invention provides host bacteria with higher safety for subsequent large-scale production, and simplifies the purification and separation process. The preparation method of the phage preparation is simple and convenient, the use is safe, no side effect is caused, the prepared broad-spectrum phage preparation has the cracking effect on various aquaculture pathogenic bacteria, can be used independently or compounded with other microecological preparations, and can be popularized and applied to bacterial disease control of aquaculture.

The preparation method of the wide-spectrum phage preparation for aquaculture comprises the following steps:

1) adding a fresh bacterial liquid of escherichia coli DH5 alpha into a culture medium for fermentation culture to obtain a host bacterium fermentation liquid.

2) Bacteriophage strains NTHP01 and MgCl with the preservation number of CGMCC No.9623 and the preservation date of 2014, 8, 28₂Adding the mother liquor into the host bacterium fermentation liquor, uniformly mixing, standing, and performing enrichment culture to obtain a fermentation mixed liquor; wherein, the proliferation culture conditions are as follows: activation of E.coli DH5 alpha to OD upon addition of phage strain NTHP01₆₀₀0.3-0.5, and a multiplicity of infection of 10^-1-10^-2(ii) a Culturing at 30-37 deg.C for more than 5 hr.

3) And centrifuging the fermentation mixed solution, and separating the phage from the host bacteria to obtain the broad-spectrum phage preparation.

The prior patent (201410506817.6) of the invention group, a Chinese turtle aeromonas phage and application thereof, discloses a phage strain NTHP01 with the collection number of CGMCC No.9623, and discloses that the phage strain can form plaque on Chinese turtle aeromonas hydrophila, has a cracking effect on pathogenic aeromonas, can be used for treating and preventing diseases caused by aeromonas by adopting a biological therapy, and particularly has a remarkable treatment effect on the neck rot disease, white spot disease and the like of the Chinese turtle. In the patent technical scheme, only the bacteriophage strain NTHP01 is disclosed to have a lysis effect on aeromonas, and the broad spectrum is not disclosed: it has lytic effect on other bacteria (common bacteria such as Edwardsiella tarda, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Kbac bacteria, Citrobacter freundii, enterobacter) besides Aeromonas. Thus, the skilled person is not aware of the broad spectrum of phage strain NTHP01 from the disclosure of patent 201410506817.6.

Moreover, in practice the phage is not much studied by the remaining technicians in the field other than the team of the invention, and therefore the physiological characteristics of the phage NTHP01 are not known to the ordinary technicians in this field. Therefore, the invention largely explores the physiological characteristics of the broad-spectrum bacteriophage NTHP01, such as tolerance temperature, stability to different pH conditions, proliferation rule, infection number, magnesium chloride (MgCl)₂) Proliferation conditions are determined by adding concentration and the like, preparation conditions are determined by amplification culture, and a product capable of being stored at 4 ℃ is prepared.

The bacteriophage has a cracking effect on various common pathogenic bacteria in aquaculture; from the morphological analysis of an electron microscope, the Myoviridae belongs to the Myoviridae family, is named as NTHP01, and can survive under the condition of pH 4-10. The activity is stable after being stored for 6 months at normal temperature; adding 30% glycerol protectant, and storing at 4 deg.C for 1 year to obtain stable activity.

The broad-spectrum phage preparation can be applied to prevention and treatment of bacterial diseases in aquaculture, and can be used for inhibiting the growth of common pathogenic bacteria (including aeromonas hydrophila, aeromonas sobria, aeromonas caviae, edwardsiella tarda, vibrio parahaemolyticus, vibrio alginolyticus, vibrio cholerae, gasseri, citrobacter freundii, enterobacter and other common bacteria) in aquaculture.

Preferably, the culture medium is a nutrient broth culture medium; adding fresh bacterial liquid of Escherichia coli DH5 alpha into the culture medium, and performing fermentation culture at 37 ℃ for 3-4 h.

Preferably, in step 2), MgCl is added to the culture medium₂Mother liquor to MgCl in culture medium₂The concentration is 4-30mM, and the mixture is evenly mixed and then is kept stand for 20-40 min.

Preferably, in step 2), the propagation culture conditions are: multiplicity of infection is 10^-2，MgCl₂The final concentration was 4mM, the temperature was 37 ℃ and the culture was carried out for 5h to obtain OD₆₀₀Is 1.9.

Preferably, in step 3), in small scale production, the centrifugation conditions are: 5000-.

Preferably, after the fermentation mixed liquor is obtained in the step 2), before the fermentation mixed liquor enters the step 3), the fermentation mixed liquor is subjected to two-stage series fermentation treatment:

mixing the fermentation mixture with MgCl₂Adding the mother liquid into the fermentation liquid of large-scale host bacteria to obtain MgCl₂The final concentration is 4mM, the mixture is evenly mixed and then is kept stand for 20-40min, and secondary fermentation culture is carried out to obtain secondary fermentation mixed liquor; then entering step 3); wherein, the secondary fermentation culture conditions are as follows: at 37 ℃ with a multiplicity of infection of 10^-2When phage is added, the host bacteria are activated to OD₆₀₀Culturing for 5 hr at a temperature of 0.3-0.5%.

The invention provides a large-scale production method of a phage preparation, which comprises the steps of optimizing propagation conditions such as culture time, temperature, infection complex number and the like under the condition of shaking a flask in a laboratory, measuring the influence of ions with different concentrations on propagation, and finally determining the optimal propagation conditions of phage. On the basis, a two-stage series fermentation scheme is designed, and a 2-ton fermentation tank is adopted to successfully obtain 10⁹pfu/ml of phage liquid.

Preferably, the preparation method of the large-scale host bacterium fermentation liquid comprises the following steps: adding fresh bacterial liquid of Escherichia coli DH5 alpha into nutrient broth culture medium, and performing fermentation culture at 37 deg.C for 20-24 h; then transferring to a culture medium with 45-55 times volume and continuing fermentation culture at 37 ℃ for 3-4 h.

Preferably, in the step 3), in large-scale production, the secondary fermentation mixed liquor is centrifuged by a butterfly centrifuge to separate host bacteria, and the obtained liquid is a broad-spectrum phage preparation.

The wide-spectrum phage preparation for aquaculture is applied to prevention and treatment of aquaculture bacterial diseases, and is used for inhibiting the growth of common pathogenic bacteria for aquaculture; the pathogenic bacteria comprise aeromonas hydrophila, aeromonas sobria, aeromonas caviae, edwardsiella tarda, vibrio parahaemolyticus, vibrio alginolyticus, vibrio cholerae, gasseri, citrobacter freundii, enterobacter and other common bacteria.

Compared with the prior art, the invention has the beneficial effects that:

the broad-spectrum phage preparation has a cracking effect on various aquaculture pathogenic bacteria, and can be used independently or in combination with other microecological preparations. And the host bacteria adopt genetically engineered bacteria, the preparation method is simple and convenient, the use is safe, no side effect is caused, and the method can be popularized and applied to bacterial disease control of aquaculture.

The invention researches physiological characteristics of a broad-spectrum bacteriophage NTHP01, such as tolerance temperature, stability to different pH conditions, proliferation rule, infection complex number, and magnesium chloride (MgCl)₂) Proliferation conditions are determined by adding concentration and the like, preparation conditions are determined by amplification culture, and the prepared product can be stored at 4 ℃.

Drawings

FIG. 1 is a flow chart of a fermentation process for preparing phage preparations on a large scale according to the present invention;

FIG. 2 is a morphology of phage NTHP01 observed by electron microscopy (phage indicated by arrow);

FIG. 3 is a graph of one step growth of bacteriophage NTHP 01;

FIG. 4 is a graph of the effect of different pH on bacteriophage NTHP 01;

FIG. 5 is a graph of the effect of different temperatures on bacteriophage NTHP 01;

FIG. 6 is a graph of the effect of different multiplicity of infection on the proliferation of bacteriophage NTHP 01;

FIG. 7 is a graph of the effect of different magnesium chloride concentrations on the promotion of infection by bacteriophage NTHP 01;

FIG. 8 is a graph showing the bacteriostatic action of bacteriophage NTHP01 on part of common aquaculture pathogenic bacteria (Aeromonas hydrophila lysed by a double-plate method A, Aeromonas hydrophila and Vibrio parahaemolyticus lysed by a drip method B and C, respectively, and plaque shown by arrows).

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

A wide-spectrum bacteriophage preparation for aquaculture is prepared by fermenting bacteriophage strain NTHP01 with preservation number of CGMCC No.9623 and genetic engineering Escherichia coli DH5 alpha as host bacteria.

1) adding fresh bacterial liquid of Escherichia coli DH5 alpha into nutrient broth culture medium, and fermenting and culturing at 37 deg.C for 3.5h to obtain host bacteria fermentation liquid.

2) Bacteriophage strain NTHP01 and MgCl with the preservation number of CGMCC No.9623₂Adding the mother liquid into the fermentation liquid of host bacteria to make MgCl₂The final concentration is 4mM, the mixture is evenly mixed and then stands for 30min, and then proliferation culture is carried out to obtain fermentation mixed liquor; wherein, the proliferation culture conditions are as follows: activation of E.coli DH5 alpha to OD upon addition of phage strain NTHP01₆₀₀0.3 and a multiplicity of infection of 10^-2(ii) a Culturing at 37 deg.C for 5 hr to obtain OD₆₀₀Is 1.9.

3) And centrifuging the fermentation mixed solution (5500rpm/min, 15min), separating the phage from the host bacteria, and taking the supernatant to obtain the broad-spectrum phage preparation.

Example 2

Preparing large-scale host bacterium fermentation liquor simultaneously in the steps 1) and 2): adding fresh bacterial liquid of escherichia coli DH5 alpha into a nutrient broth culture medium, and performing fermentation culture at 37 ℃ for 22 h; then transferred to a 50-fold volume of medium and further fermented at 37 ℃ for 3.5 h.

3) Secondary series fermentation treatment: mixing the fermentation mixture with MgCl₂Mother liquor is respectively added to a large scaleAdding MgCl into fermentation liquor of model host bacteria₂The final concentration is 4mM, the mixture is evenly mixed and then is kept stand for 30min, and secondary fermentation culture is carried out to obtain secondary fermentation mixed liquor; wherein, the secondary fermentation culture conditions are as follows: at 37 ℃ with a multiplicity of infection of 10^-2When phage is added, the host bacteria are activated to OD₆₀₀The culture time is 0.3 and 5 hours.

4) And (3) centrifuging the secondary fermentation mixed solution by using a butterfly centrifuge to separate host bacteria, wherein the obtained liquid is a wide-spectrum phage preparation.

Example 3

1) adding fresh bacterial liquid of escherichia coli DH5 alpha into nutrient broth culture medium, and performing fermentation culture at 37 ℃ for 3h to obtain host bacterium fermentation liquid.

2) Bacteriophage strain NTHP01 and MgCl with the preservation number of CGMCC No.9623₂Adding the mother liquid into the fermentation liquid of host bacteria to make MgCl₂The final concentration is 30mM, the mixture is evenly mixed and then stands for 30min, and then propagation culture is carried out to obtain fermentation mixed liquor; wherein, the proliferation culture conditions are as follows: activation of E.coli DH5 alpha to OD upon addition of phage strain NTHP01₆₀₀0.4, multiplicity of infection of 10^-1(ii) a Culturing at 37 deg.C for 5 hr to obtain OD₆₀₀Is 1.9.

3) Centrifuging the fermentation mixture (5000rpm/min, 20min), separating bacteriophage from host bacteria, and collecting supernatant to obtain broad-spectrum bacteriophage preparation.

Example 4

1) adding fresh bacterial liquid of Escherichia coli DH5 alpha into nutrient broth culture medium, and fermenting and culturing at 37 deg.C for 4h to obtain host bacteria fermentation liquid.

2) Bacteriophage strain NTHP01 and MgCl with the preservation number of CGMCC No.9623₂Adding the mother liquid into the fermentation liquid of host bacteria to make MgCl₂The final concentration is 15mM, the mixture is evenly mixed and then stands for 30min, and then proliferation culture is carried out to obtain fermentation mixed liquor; wherein, the proliferation culture conditions are as follows: activation of E.coli DH5 alpha to OD upon addition of phage strain NTHP01₆₀₀0.5 and a multiplicity of infection of 10^-2(ii) a Culturing at 37 deg.C for 5 hr to obtain OD₆₀₀Is 1.9.

Preparing large-scale host bacterium fermentation liquor simultaneously in the steps 1) and 2): adding fresh bacterial liquid of escherichia coli DH5 alpha into a nutrient broth culture medium, and performing fermentation culture at 37 ℃ for 24 hours; then transferred to a 50-fold volume of medium and further cultured at 37 ℃ for 4 hours.

3) Secondary series fermentation treatment: mixing the fermentation mixture with MgCl₂Adding the mother liquid into the fermentation liquid of large-scale host bacteria to obtain MgCl₂The final concentration is 15mM, the mixture is evenly mixed and then is kept stand for 30min, and secondary fermentation culture is carried out to obtain secondary fermentation mixed liquor; wherein, the secondary fermentation culture conditions are as follows: at 37 ℃ with a multiplicity of infection of 10^-2When phage is added, the host bacteria are activated to OD₆₀₀The culture time is 0.5, and the culture time is 5 hours.

Examples of the research procedure

First, isolation and culture of bacteriophage NTHP01

Separating and purifying

1) Separating bacteriophage NTHP01 with tryptone and soybean agar culture medium, Aeromonas hydrophila separated from Chinese soft-shelled turtle culture pond as host bacteria, and separating lytic bacteriophage with double-layer plate separation method. The double-layer plate was prepared as follows: first, 2% tryptic soy agar medium and 0.7% semi-solid tryptic soy agar medium were prepared.

2) Pouring the melted 2% trypsin soybean agar culture medium into the bottom layer of the culture dish, and solidifying to obtain the bottom layer culture medium.

3) Melting 0.7% semisolid culture medium, keeping temperature at 46 deg.C, mixing a certain amount of bacteriophage with host bacteria, adsorbing for 20-30min, mixing with semisolid culture medium, pouring onto substrate culture medium, culturing at 28 deg.C for 6-8 hr, and observing plaques. After the phage is spotted, the spots are picked and cultured by liquid trypticase soybean broth, and the process is repeated until the phage is purified.

2. Amplification culture and harvest of bacteriophage NTHP01

Culturing fresh host bacterium liquid to OD₆₀₀MgCl was added at 0.3₂The final concentration is 4mM, the mixture is kept still and adsorbed for 20-30min, and the mixture is harvested after shaking at 150rpm/min for 5 hours. Transferring the culture solution into a centrifuge tube, and centrifuging at 5,000 g for 10 minutes to precipitate host bacteria; transferring the supernatant to a new tube, and removing residual bacteria with a bacterial filter with a pore size of 0.22 μm to obtain a phage stock solution without host bacteria. The titer is determined by a double-layer plate method, and the product is stored at 4 ℃ for later use.

Second, electron microscopy of bacteriophage NTHP01

Concentrating the prepared phage stock solution by an ultrafiltration tube with 30KD, washing by a proper amount of PBS buffer solution, negatively dyeing by phosphotungstic acid, dripping a copper net, and observing the form of the phage by a transmission electron microscope. As shown in FIG. 2, the phage heads were icosahedral, approximately 60-145nm, with varying length tails (80-455 nm). In combination with the report of the viral Classification-International Commission on viral Classification, 9 th time, NTHP01 phage was phage of the order Lepidoptera, Myoviridae.

Three, one step growth curve determination

The one-step growth curve can be used for obtaining information such as the latent period, the outbreak period and the outbreak amount of the phage through calculation. Mixing phage with 3mL of host bacteria according to the optimal MOI, incubating at 28 ℃ until mass adsorption, centrifuging at 13000g for 1min, discarding supernatant, resuspending precipitate with 10mL of liquid culture medium preheated at 28 ℃, and performing shake culture at 28 ℃ and 120 rpm/min. Sampling every 5min for the first 25 min, then sampling every 15min, continuously sampling for 3.5h, taking 500 μ L each time, filtering with 0.22 μm filter membrane, and determining the titer of phage in the filtrate by double-layer agar culture method. And drawing a one-step growth curve by taking the culture time as an abscissa and the bacteriophage titer as an ordinate. As shown in FIG. 3, the results showed that the incubation period and the burst period of the proliferation of phage NTHP01 were 150min and 60min, respectively.

IV, pH stability of bacteriophage NTHP01

The culture solution is used as a medium, and the pH value is adjusted to 4, 5, 6, 7, 8, 9, 10 and 11. The titer is taken to be 1 × 10⁸mu.L of phage stock solution of PFU/mL is added into 900 mu.L of TSB culture solution with different pH values, the titer of each tube of phage is measured after water bath at 28 ℃ for 2h, and the pH tolerance range of phage is calculated. As shown in fig. 4, the results showed that the bacteriophage NTHP01 has a wider pH adaptability. Under the conditions of pH7 to pH10, the titer was 10⁷PFU/mL is essentially identical to the initial titer, i.e., the phage retains higher biological activity in this pH interval. Activity was highest at pH 7. At a pH of less than 7 or greater than 10, the activity of the phage gradually decreases with increasing acidity or basicity, but overall there is some activity.

Fifth, thermal stability of bacteriophage NTHP01

The phage stock prepared above was diluted to 1x10⁷PFU/mL is divided into 45 mL centrifuge tubes, each 2mL, the centrifuge tubes are placed in a constant temperature water bath at 30 ℃, 40 ℃, 50 ℃ and 60 ℃ respectively, and the titer of the phage in each tube is determined every 20min until the 100 th min. After temperature equilibration, the titer was determined. As shown in FIG. 5, the results showed that the phage titer was 10 in all of the 30-40 ℃ intervals⁶PFU/mL or higher, compared to the original titer of 10⁷PFU/mL did not decrease significantly and overall survival was high. When the temperature reaches 50 ℃ or above, the bacteriophage is quickly inactivated within 20min, and the titer is only 10 at 20min⁵PFU/mL, at 100min time, phage titer at 50 ℃ of only 10⁴PFU/mL, mostly dead, showed that the phage was highly sensitive to temperatures above 50 ℃ with low activity.

Sixthly, optimum multiplicity of infection (MOI) of bacteriophage NTHP01

Shaking host bacteria to 1X10⁸CFU/mL, prepared at 1X10⁸PFU/mL phage stock, both were diluted to 1X10 gradient by 10-fold each⁵PFU/mL, at 7 different MOIs (1000, 10) according to Table 10, 10, 1, 0.1, 0.01, 0.001) was mixed in a 1.5mL Eppendorf tube in 100. mu.L each, and 800. mu.L of a fresh liquid medium was added and cultured at 28 ℃ for 3.5 hours. After being taken out, the titer of the phage in each tube is respectively determined, and the MOI corresponding to the highest titer of 3.5h is the optimal complex number of infection. As shown in FIG. 6, the results showed that the phage infectivity was the least effective against the host bacteria at 1, 10, 100, 1000 and 0.001 multiplicity of infection, and that the phage infectivity was better at 0.1 and 0.01 multiplicity of infection, with the optimum multiplicity of infection being 0.01.

Effect of seven, different magnesium chloride concentrations on bacteriophage NTHP01 proliferation

100. mu.L of each of 1X10 host cells were added to 6 sterilized 50mL centrifuge tubes containing 10mL of medium⁸CFU/mL, shake culture at 37 ℃ for 3 h. Each added with 1 × 10⁸The phage titer in each tube was determined by shaking culture at 37 ℃ for 4h after 30min of action of 100. mu.L of phage stock solution PFU/mL and different final concentrations of magnesium chloride (0, 2mM, 4mM, 8mM, 16mM, 32 mM). As shown in FIG. 7, the results show that the titer of phage with magnesium chloride added is significantly higher than that without, and that at a final concentration of magnesium chloride of 4mM, the optimal effect on phage infection is promoted.

Eight-stage and multistage series fermentation large-scale preparation of bacteriophage

Based on the experimental results of the above phage multiplication conditions, a multi-stage tandem fermentation scheme was designed, as shown in FIG. 1. The specific implementation is as follows:

1)30L of phage and host bacteria mixed solution is prepared. Firstly, preparing 600ml fresh host bacterial liquid, adding into 30L culture medium (50L fermentation tank), culturing at 37 deg.C for 3-4h to make bacterial liquid OD₆₀₀Reaching about 0.3, adding 600ml of phage stock solution and MgCl₂Mixing mother liquor 300ml (final concentration is 4mM), standing for 30min, culturing at 37 deg.C for 5h, and terminating fermentation to obtain 30L bacteriophage and host bacteria mixed liquor for subsequent fermentation.

2)1.5 ton phage liquid preparation. Adding 600ml of fresh host bacterium liquid into 30L of culture medium (50L fermentation tank), and culturing at 37 ℃ for 20-24 h; then adding into 1.5 ton fermentation medium (2 ton fermenter), culturing at 37 deg.C for 3-4 hr to make bacterial liquid OD₆₀₀Adding the prepared 30L phage and host to the mixture to reach about 0.3Adding MgCl while mixing the bacteria₂Mixing 1.5L of mother liquor (final concentration of 4mM), standing for 30min, culturing at 37 deg.C for 5 hr, and terminating fermentation. Centrifuging the fermentation product with a disk centrifuge, collecting liquid, and packaging. Phage titer was determined by double-layer plate method, with titer 2x10⁹pfu/ml。

Ninth, the bacteriostatic effect of the bacteriophage on different pathogenic bacteria

The bacteriophagic effect of the bacteriophage on common pathogenic bacteria of aquaculture is observed by adopting a double-layer flat plate method and a drip method. The pathogens tested were: aeromonas hydrophila, aeromonas sobria, Aeromonas caviae and Edwardsiella tarda which are separated from the Chinese softshell turtle; aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, and Gewass bacteria isolated from Penaeus vannamei; enterobacteria isolated from macrobrachium rosenbergii; citrobacter freundii isolated from a red crayfish; aeromonas hydrophila isolated from grouper, aeromonas sobria of weever, and the like. The double-layer plate method is to prepare 2% LB agar culture medium and 0.7% semisolid LB culture medium. Pouring the melted 2% LB culture medium into the bottom layer of the culture dish, and solidifying to obtain the bottom layer culture medium. Melting 0.7% semisolid culture medium, keeping the temperature at 46 ℃, mixing a certain amount of bacteriophage with pathogenic bacteria to be detected respectively, adsorbing for 20-30min, mixing with the semisolid culture medium, pouring onto the bottom layer culture medium, culturing at 28 ℃ for 6-8h, and observing the plaques. The dripping method comprises coating the plate with host bacteria, air drying the surface of the plate for 3-5min, dripping 5 μ L of phage solution on the plate, culturing at 28 deg.C for 8-12 hr, and observing phage effect. As shown in FIG. 8, the results show that the obvious plaques can be seen in the double-layer plate, and the phagocytic effect can be clearly observed when the pathogenic bacteria are tested by a drip method, which shows that the bacteriophage has a good lytic effect on the common pathogenic bacteria of aquaculture.

Meanwhile, the gene engineering auxotrophic escherichia coli DH5 alpha is used as a host bacterium, and the phage display effect is good. Provides host bacteria with higher safety for subsequent large-scale production, and simplifies the purification and separation process.

TABLE 1 bacteriophagic effect of bacteriophages on part of common bacteria in aquaculture

(Note: + indicates that the bacterium is less sensitive to the phage, + + indicates that the bacterium is moderately sensitive to the phage, and +++ indicates that the bacterium is strongly sensitive to the phage)

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. The application of the bacteriophage strain NTHP01 in preparing a broad-spectrum bacteriophage preparation for preventing and treating aquaculture bacterial diseases caused by Edwardsiella tarda, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Kbac bacteria, Citrobacter freundii or enterobacter is characterized in that: the broad-spectrum phage preparation is prepared by fermenting a phage strain NTHP01 with the preservation number of CGMCC No.9623 and genetic engineering Escherichia coli DH5 alpha serving as host bacteria.

2. The use of claim 1, wherein the broad spectrum phage preparation is prepared by a process comprising the steps of:

1) adding a fresh bacterial liquid of escherichia coli DH5 alpha into a culture medium for fermentation culture to obtain a host bacterium fermentation liquid;

2) bacteriophage strain NTHP01 and MgCl with the preservation number of CGMCC No.9623₂Adding the mother liquid into the fermentation liquid of host bacteriaUniformly mixing, standing, and performing proliferation culture to obtain a fermentation mixed solution; wherein, the proliferation culture conditions are as follows: activation of E.coli DH5 alpha to OD upon addition of phage strain NTHP01₆₀₀0.3-0.5, and a multiplicity of infection of 10^-1-10^-2(ii) a Culturing at 30-37 deg.C for more than 5 hr;

3. The use of claim 2, wherein the culture medium is a nutrient broth; adding fresh bacterial liquid of Escherichia coli DH5 alpha into the culture medium, and performing fermentation culture at 37 ℃ for 3-4 h.

4. The use according to claim 2, wherein in step 2) MgCl is added to the culture medium₂Mother liquor to MgCl in culture medium₂The concentration is 4-30mM, and the mixture is evenly mixed and then is kept stand for 20-40 min.

5. The use according to claim 2 or 4, wherein in step 2) the proliferation culture conditions are: multiplicity of infection is 10^-2，MgCl₂The final concentration was 4mM, the temperature was 37 ℃ and the culture was carried out for 5h to obtain OD₆₀₀Is 1.9.

6. The use according to claim 2, wherein in step 3), the centrifugation conditions are: 5000-.

7. The use of claim 2, wherein after the fermentation mixture is obtained in step 2), before entering step 3), the fermentation mixture is further subjected to a two-stage series fermentation treatment:

mixing the fermentation mixture with MgCl₂Adding the mother liquid into the fermentation liquid of large-scale host bacteria to obtain MgCl₂The final concentration is 4mM, standing for 20-40min after uniform mixing, and performing secondary fermentation culture to obtain a secondary fermentation mixed solution; then entering step 3); wherein, the secondary fermentation culture conditions are as follows: at 37 ℃ with a multiplicity of infection of 10^-2When phage is added, the host bacteria are activated to OD₆₀₀Culturing for 5 hr at a temperature of 0.3-0.5%.

8. The use of claim 7, wherein the large scale host strain fermentation broth is prepared by: adding fresh bacterial liquid of Escherichia coli DH5 alpha into nutrient broth culture medium, and performing fermentation culture at 37 deg.C for 20-24 h; then transferring to a culture medium with 45-55 times volume and continuing fermentation culture at 37 ℃ for 3-4 h.

9. The use of claim 7 or 8, wherein in step 3), the secondary fermentation mixture is centrifuged by a butterfly centrifuge to separate the host bacteria, and the resulting liquid is a broad-spectrum phage preparation.