Fermentation culture process and application of broad-spectrum high-pathogenicity vibrio bacteriophage

Abstract

The invention relates to the technical field of aquaculture, in particular to a broad-spectrum high-pathogenicity vibrio bacteriophage fermentation culture process and application, and the broad-spectrum high-pathogenicity vibrio bacteriophage fermentation culture process comprises the following steps: preparing a ZWHV liquid culture medium; the method comprises the following steps: inoculating host bacteria in a logarithmic phase into a ZWHV liquid culture medium, and inoculating high-pathogenicity vibrio phage hBP04 with an inoculation complex number of 0.01 MOI; continuously culturing and harvesting phage fermentation liquor; removing host bacteria and impurities to obtain a purified bacteriophage suspension; determining the titer of the purified bacteriophage, and evaluating the concentration and activity of the bacteriophage; according to the preliminary fermentation result, finely adjusting the culture conditions, and evaluating the influence of different culture media on the phage fermentation effect; the optimized bacteriophage fermentation process is used for producing the high-pathogenicity vibrio bacteriophage, the high-pathogenicity vibrio bacteriophage is applied to vibrio prevention and control tests, the actual application effect of the bacteriophage is evaluated by counting the death rate of juvenile shrimps, the titer of the bacteriophage is improved by optimizing the culture process, and application of the high-pathogenicity vibrio bacteriophage hBP04 to vibriosis prevention and control is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a fermentation and cultivation process of a broad-spectrum and highly pathogenic Vibrio phage and its application. Background Art

[0002] The use of antibiotics has caused the emergence of drug-resistant Vibrio, and the development of new and efficient Vibrio inhibitory products is of great significance. Bacteriophages are efficient, safe and environmentally friendly, and can effectively infect and kill drug-resistant Vibrio. They are an important direction for the development of Vibrio inhibitory products in the future, and phage therapy has been proven to be effective in preventing and treating diseases caused by Vibrio in aquaculture. However, for highly pathogenic Vibrio, there are currently few phages isolated, and there is a lack of relevant culture processes.

[0003] In the patent "A strain of Vibrio parahaemolyticus phage and its application against highly pathogenic Vibrio and drug-resistant Vibrio (patent application number: 202311054043.3)", a strain of phage against highly pathogenic Vibrio parahaemolyticus was isolated, which can be used for the prevention and treatment of drug-resistant Vibrio or highly pathogenic Vibrio, but more highly pathogenic Vibrio phages still need to be isolated to obtain phage cocktails, so as to more effectively inhibit the occurrence of highly pathogenic vibriosis. Regarding the fermentation culture of Vibrio phages, in the patent "A culture medium and method suitable for the culture and screening of Vibrio phages" (application number: CN202210908481.0), a method for culturing Vibrio parahaemolyticus phages was invented, using sucrose instead of salt ions, but for several strains of highly pathogenic Vibrio phages currently isolated in the culture laboratory, the culture medium did not effectively promote the increase in phage titer. Summary of the invention

[0004] The purpose of the present invention is to provide a broad-spectrum highly pathogenic Vibrio phage fermentation culture process and application, which solves the problem that the titers of several highly pathogenic Vibrio phages currently isolated in the culture laboratory are low when using known Vibrio phage fermentation culture medium and the effect of preventing and treating diseases caused by Vibrio is not good.

[0005] To achieve the above object, the present invention provides a broad-spectrum and highly pathogenic Vibrio phage fermentation and cultivation process, comprising the following steps:

[0006] Prepare ZWHV liquid culture medium under sterile conditions for fermentation culture of highly pathogenic Vibrio phages;

[0007] The host bacteria in the logarithmic phase were inoculated into ZWHV liquid culture medium at a ratio of 1:100, and cultured at 37°C and 180 r / min for 2 hours. Then, the highly pathogenic Vibrio phage hBP04 was inoculated at an inoculation multiplicity of 0.01 MOI and cultured for another 4 hours.

[0008] After the phage was inoculated and initially cultured, the culture temperature was adjusted to 32°C, and the culture was continued at 200 r / min for 6 hours to harvest the phage fermentation liquid;

[0009] After the culture is completed, the host bacteria and impurities are removed by centrifugation and filtration steps to obtain a purified phage suspension;

[0010] The double-layer plate method was used to determine the titer of the purified phage, and the concentration and activity of the phage were evaluated by counting the number of plaques to ensure the quality of the phage;

[0011] Based on the preliminary fermentation results, the culture conditions were fine-tuned to further improve the titer and yield of the phage, and the effects of different culture media on the phage fermentation effect were evaluated;

[0012] The optimized phage fermentation process was applied to the Vibrio prevention and control experiment, and the actual application effect of the phage was evaluated by counting the mortality rate of juvenile shrimp.

[0013] Among them, in “preparing ZWHV liquid culture medium under sterile conditions for fermentation culture of highly pathogenic Vibrio phage”, the method comprises the following steps:

[0014] The formula of the ZWHV liquid culture medium is as follows: per liter, it contains 10 g of yeast powder, 8 g of peptone, 5 mL of glycerol, 5 g of corn starch, 2 g of magnesium sulfate, 30 g of sodium chloride, and 0.5 g of L-arginine.

[0015] Among them, in “after the cultivation is completed, the host bacteria and impurities are removed by centrifugation and filtration steps to obtain a purified phage suspension”, the method includes:

[0016] Collecting fermentation broth from a highly pathogenic Vibrio phage hBP04 culture that has completed fermentation, wherein the fermentation broth is in the optimized ZWHV liquid culture medium;

[0017] The collected phage fermentation broth was subjected to preliminary centrifugation treatment, and centrifuged at a speed of 5000 r / min using a centrifuge for 8-10 minutes to remove large particle impurities and incompletely lysed host bacterial cells in the fermentation broth, and the supernatant was collected after centrifugation;

[0018] The supernatant obtained by the initial centrifugation was filtered through a 0.22 μm filter membrane to further remove residual host bacterial cell fragments, proteins, and other fine impurities;

[0019] Verify the purity and titer of the phage suspension obtained after centrifugation and filtration steps to ensure that the phage suspension meets the requirements of subsequent applications.

[0020] Among them, in “using the double-layer plate method to determine the titer of the purified phage, and evaluating the concentration and activity of the phage by counting the number of plaques to ensure the quality of the phage”, the following steps are included:

[0021] The purified phage suspension was taken and serially diluted 10 times with a phage buffer solution to obtain a series of phage dilution solutions with different concentrations;

[0022] Take 100 μl of the diluted phage suspension from each tube, add 100 μl of the host bacteria suspension respectively, mix well, add 0.7% water agar, pour on the LBS solid plate, shake the plate to allow the phage to fully contact with the host bacteria;

[0023] The prepared double-layer plate was placed in a 37°C incubator for overnight culture;

[0024] Observe and record the formation of plaques, and count the number of plaques on each plate;

[0025] The phage concentration in the original phage suspension was calculated based on the dilution multiple and the number of plaques;

[0026] By comparing the number of plaques at different dilutions, the phage titer, that is, the number of infectious phage particles contained in each milliliter of phage suspension, is determined.

[0027] Among them, in “applying the optimized phage fermentation process to the Vibrio prevention and control test, and evaluating the actual application effect of the phage by counting the mortality rate of young shrimp”, the method also includes the following steps:

[0028] 200 samples were taken and randomly divided into 5 groups, including healthy control group, infection control group, LBS liquid group, Vibrio liquid group and ZWHV liquid group;

[0029] Except for the healthy control group, other experimental groups were added with highly pathogenic Vibrio hV009 to make the final concentration of highly pathogenic Vibrio reach 5×10 5 CFU / mL, the total culture volume was 5 L, and the liquid level was 20 cm;

[0030] In the LBS liquid group, Vibrio liquid group and ZWHV liquid group, the final concentration was 2×10 3 PFU / mL was added to the hBP04 phage obtained by fermentation in the corresponding liquid culture medium;

[0031] The experimental group was cultured under the same culture conditions for 7 days, and the mortality of young shrimp was counted daily.

[0032] An application of a broad-spectrum and highly pathogenic Vibrio phage is applicable to the fermentation and cultivation process of the broad-spectrum and highly pathogenic Vibrio phage. The highly pathogenic Vibrio phage hBP04 is applied to the prevention and control of vibriosis.

[0033] The invention discloses a broad-spectrum highly pathogenic Vibrio phage fermentation and cultivation process and application, comprising the following steps: preparing a ZWHV liquid culture medium under sterile conditions for fermentation and cultivation of highly pathogenic Vibrio phage; inoculating a logarithmic phase host bacterium into the ZWHV liquid culture medium at a ratio of 1:100, culturing at 37°C and 180 r / min for 2 hours, inoculating a highly pathogenic Vibrio phage hBP04 at an inoculation multiplicity of 0.01 MOI, and continuing to culture for 4 hours; after the phage is inoculated and initially cultured, adjusting the culture temperature to 32°C, continuing to culture at a speed of 200 r / min for 6 hours, and harvesting the phage fermentation liquid; after the culture is completed, removing the host bacteria and impurities through centrifugation and filtration steps to obtain to a purified phage suspension; the titer of the purified phage was determined by the double-layer plate method, and the concentration and activity of the phage were evaluated by counting the number of plaques to ensure the quality of the phage; according to the preliminary fermentation results, the culture conditions were fine-tuned to further improve the titer and yield of the phage, and the effects of different culture media on the phage fermentation effect were evaluated; the optimized phage fermentation process was applied to the Vibrio prevention and control test, and the actual application effect of the phage was evaluated by counting the mortality rate of young shrimps. This patent uses the highly pathogenic Vibrio isolated in the previous laboratory as the host bacteria, and isolates and screens a phage (hBP04) that can efficiently lyse highly pathogenic Vibrio, and optimizes the culture process to increase its titer from the original 10 8 pfu / mL increased to 10 11～12 pfu / mL, which greatly improves the titer of the phage and reduces the production cost, and will be beneficial to promote the application of the highly pathogenic Vibrio phage hBP04 in the prevention and control of Vibrio disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0035] Figure 1 These are plaques formed by the bacteriophage hBP04 of the present invention on a double-layer plate.

[0036] Figure 2 This is the morphological characterization of the bacteriophage hBP04 of the present invention.

[0037] Figure 3 This is the one-step growth curve of the highly pathogenic Vibrio phage hBP04 of the present invention.

[0038] Figure 4 These are the results of measuring the pH and temperature tolerance of the highly pathogenic Vibrio phage hBP04 of the present invention.

[0039] Figure 5This is the optimal MOI determination of the highly pathogenic Vibrio phage hBP04 of the present invention.

[0040] Figure 6 The present invention compares the potency of highly pathogenic Vibrio phage hBP04 after fermentation in different culture media.

[0041] Figure 7 The present invention compares the titer of hBP04 phage after conventional culture and optimization using ZWHV liquid culture medium.

[0042] Figure 8 The figure is a comparison of the amplification of the hBP04 phage of the present invention on LBS and ZWHV solid culture media.

[0043] Fig. 9 The present invention is a flow chart of the fermentation and cultivation process of the broad-spectrum and highly pathogenic Vibrio phage.

[0044] Fig.10 The present invention is a flow chart of obtaining a purified phage suspension by removing host bacteria and impurities through centrifugation and filtration steps after the cultivation is completed.

[0045] Fig.11 The present invention adopts the double-layer plate method to determine the titer of the purified phage, evaluates the concentration and activity of the phage by counting the number of plaques, and ensures the quality of the phage.

[0046] Fig.12 The present invention is a flow chart of applying the optimized phage fermentation process to a Vibrio prevention and control test, and evaluating the actual application effect of the phage by counting the mortality rate of young shrimps. DETAILED DESCRIPTION

[0047] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0048] See also Figures 9 to 12 , Fig. 9 This is a flow chart of the fermentation and cultivation process of the broad-spectrum and highly pathogenic Vibrio phage of the present invention. Fig.10 This is a flow chart of obtaining a purified phage suspension by removing host bacteria and impurities through centrifugation and filtration steps after the cultivation of the present invention is completed. Fig.11 This is a flow chart of the present invention that uses a double-layer plate method to determine the titer of purified phages, evaluates the concentration and activity of phages by counting the number of plaques, and ensures the quality of phages. Fig.12 The present invention is a flow chart of applying the optimized phage fermentation process to a Vibrio prevention and control test, and evaluating the actual application effect of the phage by counting the mortality rate of young shrimps.

[0049] A broad-spectrum and highly pathogenic Vibrio phage fermentation and cultivation process comprises the following steps:

[0050] S1: Prepare ZWHV liquid culture medium under sterile conditions for fermentation culture of highly pathogenic Vibrio phages. The formula of the ZWHV liquid culture medium is 10 g yeast powder, 8 g peptone, 5 mL glycerol, 5 g corn starch, 2 g magnesium sulfate, 30 g sodium chloride, and 0.5 g L-arginine per liter.

[0051] In this embodiment: After screening and optimization, the present invention obtains a culture medium formula suitable for culturing highly pathogenic Vibrio phages, wherein each liter of liquid culture medium contains the following ingredients: 10 g yeast powder, 8 g peptone, 5 mL glycerol, 5 g corn starch, 2 g magnesium sulfate, 30 g sodium chloride, and 0.5 g L-arginine. The liquid culture medium is named ZWHV liquid culture medium, and the solid culture medium is based on the liquid culture medium, and agar powder is added at a ratio of 1.5%.

[0052] S2: The host bacteria in the logarithmic phase were inoculated into ZWHV liquid culture medium at a ratio of 1:100. After culturing at 37°C and 180 r / min for 2 hours, the highly pathogenic Vibrio phage hBP04 was inoculated at an inoculation multiplicity of 0.01 MOI and the culture was continued for 4 hours.

[0053] In this embodiment: the logarithmic phase host bacteria are inoculated into liquid culture at a ratio of 1:100, and cultured at 37°C and 180 r / min for 2 hours, and then the highly pathogenic Vibrio phage hBP04 is inoculated at an inoculation multiplicity of 0.01 MOI, and cultured at 37°C and 200 r / min for 4 hours, and then the temperature is adjusted to 32°C, and the culture is continued for 6 hours, and then the phage fermentation liquid is harvested.

[0054] S3: After the phage is inoculated and initially cultured, the culture temperature is adjusted to 32°C, and the culture is continued at a speed of 200 r / min for 6 hours to harvest the phage fermentation liquid.

[0055] S4: After the culture is completed, the host bacteria and impurities are removed by centrifugation and filtration steps to obtain a purified phage suspension;

[0056] S41: collecting fermentation broth from the highly pathogenic Vibrio phage hBP04 culture that has completed fermentation, wherein the fermentation broth is in the optimized ZWHV liquid culture medium;

[0057] S42: performing preliminary centrifugation on the collected phage fermentation broth, using a centrifuge to centrifuge at a speed of 5000 r / min, and setting the centrifugation time to 8-10 minutes to remove large particle impurities and incompletely lysed host bacterial cells in the fermentation broth, and collecting the supernatant after centrifugation;

[0058] S43: filtering the supernatant obtained by the preliminary centrifugation through a 0.22 μm filter membrane to further remove residual host bacterial cell fragments, proteins and other fine impurities;

[0059] S44: Verify the purity and titer of the phage suspension obtained after centrifugation and filtration steps to ensure that the phage suspension meets the requirements of subsequent applications.

[0060] S5: The titer of the purified phage was determined by the double-layer plate method, and the concentration and activity of the phage were evaluated by counting the number of plaques to ensure the quality of the phage.

[0061] S51: taking the purified phage suspension, and diluting the phage suspension 10 times in series with a phage buffer solution to obtain a series of phage dilution solutions with different concentrations;

[0062] S52: Take 100 μl of the diluted phage suspension in each tube, add 100 μl of the host bacteria suspension respectively, mix well, add 0.7% water agar, pour on the LBS solid plate, shake the plate to make the phage and host bacteria fully contact;

[0063] S53: Place the prepared double-layer plate in a 37°C incubator for overnight culture;

[0064] S54: Observe and record the formation of plaques, and count the number of plaques on each plate;

[0065] S55: Calculate the phage concentration in the original phage suspension based on the dilution factor and the number of plaques;

[0066] S56: By comparing the number of plaques at different dilutions, the phage titer, that is, the number of infectious phage particles contained in each milliliter of phage suspension, is determined.

[0067] S6: Based on the preliminary fermentation results, fine-tune the culture conditions to further improve the titer and yield of the phage, and evaluate the effects of different culture media on the phage fermentation effect.

[0068] S7: The optimized phage fermentation process was applied to the Vibrio control test, and the actual application effect of the phage was evaluated by counting the mortality rate of juvenile shrimp.

[0069] S71: 200 samples were collected and randomly divided into 5 groups, including healthy control group, infection control group, LBS liquid group, Vibrio liquid group and ZWHV liquid group;

[0070] S72: In addition to the healthy control group, other experimental groups were added with highly pathogenic Vibrio hV009 to make the final concentration of highly pathogenic Vibrio reach 5×10 5CFU / mL, the total culture volume was 5 L, and the liquid level was 20 cm;

[0071] S73: In the LBS liquid group, Vibrio liquid group and ZWHV liquid group, the final concentration was 2×10 3 PFU / mL was added to the hBP04 phage obtained by fermentation in the corresponding liquid culture medium;

[0072] S74: The experimental group was cultured under the same culture conditions for 7 days, and the mortality of young shrimps was counted daily.

[0073] In this embodiment:

[0074] 1. Isolation, purification and characterization of bacteriophage

[0075] Take 10mL of water sample from a white shrimp farm in Guangdong, dilute it with 10mL of phage diluent (patent: phage protective agent, preparation method and application in phage preparation, application number: CN202411088234.6), centrifuge it at 5000r / min for 8 minutes at room temperature, and filter the supernatant with a 0.22μm filter membrane. Take 5mL of the filtrate and mix it with 5mL 2×LBS (each liter of liquid culture medium contains 20g of yeast powder, 20g of peptone, and 70g of sodium chloride), add 100μL of a highly pathogenic Vibrio in the logarithmic phase (isolated in the laboratory in the early stage, hV009) and co-culture at 37℃ overnight. Repeat the co-culture three times, take 500μL of the filtrate and mix it with 100μL of highly pathogenic Vibrio in the logarithmic phase (OD600≈0.8), mix it with 4mL of water agar (about 55℃), and pour it into the LBS solid plate. Incubate at 37℃ for 5-8 hours, pick a single plaque, dilute with phage buffer and repeat the above purification operation 3 times, and isolate a phage from it, which is named hBP04. Phage hBP04 forms clear and transparent plaques on the double-layer plate, with a diameter of 0.5-1mm and no halo ( Figure 1 ).

[0076] We then observed the morphological characteristics of bacteriophage hBP04 using a transmission electron microscope. First, the copper mesh was treated with glow discharge to increase its hydrophilicity. Then 20 μL of phage solution was placed on a sealing film, and the edge of the copper mesh was gently clamped with tweezers and inserted into the droplet for adsorption for 10 minutes. Finally, the copper mesh was removed from the droplet and moved to 20 μL of PTA negative staining solution. After negative staining for about 5 minutes, the copper mesh was removed and gently placed on filter paper. After the liquid was completely absorbed, the phage morphology was observed using a transmission electron microscope. The results showed that the head of the phage was a regular hexagon with a diameter of about 110 nm and a tail length of about 150 nm ( Figure 2 ).

[0077] 2. Determination of the host spectrum of the highly pathogenic Vibrio phage hBP04

[0078] Based on the results of our laboratory's previous analysis of Vibrio communities in diseased ponds in various places, the three most common Vibrio species in the Vibrio community and stored in our laboratory's Vibrio library were selected for experiments, including 26 highly pathogenic Vibrio parahaemolyticus, 13 strains of Vibrio alginolyticus and 10 strains of Vibrio harveyi (including one highly pathogenic bacterium). The host spectrum of phages was determined using the double-layer plate method.

[0079] The double-layer plate method for determining the host spectrum is as follows: 10 μl of phage suspension is taken, and 100 μl of host bacterial suspension (bacterial concentration is 1×10 9 CFU / mL), add 0.7% water agar after mixing, pour on LBS solid plate, culture at 37°C overnight, and determine whether the phage can infect the host bacteria by observing whether there are plaques.

[0080] The results showed that hBP04 had a significant lysis effect on a variety of highly pathogenic Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio harveyi, and was an efficient and broad-spectrum highly pathogenic Vibrio phage.

[0081] Table 1 Host spectrum of bacteriophage hBP04

[0082]

[0083] 3. One-step growth curve of highly pathogenic Vibrio phage hBP04

[0084] Phage hBP04 was mixed with logarithmic bacterial solution at a ratio of MOI = 1, and co-cultured at 37°C for 10 minutes. 10 mL of the mixture was centrifuged at 4°C and 5000 r / min for 8 minutes, the supernatant was discarded, and the precipitate was resuspended and washed with 10 mL of LBS liquid culture medium. After repeated washing twice, 2 mL of the resuspension was added to 100 mL of LBS liquid culture medium and cultured at 37°C and 200 r / min. Samples were taken every 20 minutes to determine the phage titer. The one-step growth curve was drawn using GraphPadPrism 8.

[0085] like Figure 3 As shown in the figure, the titer of hBP04 remained basically unchanged in the first 20 minutes of growth, and increased rapidly by 3 orders of magnitude from 20 to 140 minutes. After 140 minutes, the curve became stable. It can be seen that the first 20 minutes is the incubation period of bacteriophage hBP04, 20 to 140 minutes is its lysis period, and after 140 minutes it enters the stable period. The titer of bacteriophage hBP04 at 140 minutes, i.e. the end of the lysis period, can reach 1.20×10 8 PFU / mL.

[0086] 4. Stability test of highly pathogenic Vibrio phage hBP04

[0087] Phage hBP04 was mixed with 900 μL of phage buffer of different pH values, placed in a 37°C incubator for incubation for 1.5 h, and then the phage titer was measured to investigate the stability of the phage at different pH values. 500 μL of phage solution was treated in a water bath at 4°C, 37°C, 45°C, 55°C, 65°C, 75°C, and 85°C for 30 minutes, and then the phage titer was measured to investigate the thermal stability of the phage.

[0088] according to Figure 4 The results showed that the highly pathogenic Vibrio phage hBP04 can tolerate a pH range of 2 to 11 and a high temperature of 55°C, which shows that the phage has good pH and temperature tolerance and can be better applied in actual clinical environments.

[0089] 5. Selection of host bacteria for highly pathogenic Vibrio phage hBP04

[0090] For more than 100 strains of Vibrio isolated in the laboratory, including more than 30 highly pathogenic Vibrio, the double-layer plate method was first used to determine the host bacteria that can be lysed by highly pathogenic Vibrio phages. Based on the size of the lysis plaques, the top 10 host bacteria with larger plaques were selected to further determine the titer of the phage.

[0091] The double-layer plate method for determining phage titer is as follows: 100 μl of phage suspension is taken, and 10 times serial dilutions are made with phage buffer solution. 100 μl of dilution solution in each tube is taken, and 100 μl of host bacterial suspension (bacterial concentration is 1×10 9 CFU / mL), add 0.7% water agar after mixing, pour on LBS solid plate, culture at 37℃ overnight, and count the number of plaques. Repeat three times for each sample.

[0092] The process of culturing phages with the selected 10 host Vibrio strains is as follows: culturing the host bacteria to OD 600 The value was 0.6, and then LBS liquid culture medium was inoculated at a ratio of 1:100, and then phage was inoculated at an infection multiplicity of 0.01 MOI. The culture was carried out at 37°C and 180 r / min for 8 hours, and then the phage titer was measured after harvesting.

[0093] The formula of the LBS liquid culture medium is: each liter of the liquid culture medium contains 10 g of yeast powder, 10 g of peptone, and 35 g of sodium chloride. The test results are shown in Table 2.

[0094] Table 2 Selection of host bacteria for highly pathogenic Vibrio phage hBP04

[0095]

[0096] According to the data in Table 2, host bacterium hV028 can be used to amplify the highly pathogenic Vibrio phage hBP04, and the titer obtained is relatively high.

[0097] 6. Determination of the Optimal MOI of Highly Pathogenic Vibrio phage hBP04

[0098] The highly pathogenic Vibrio phage hBP04 phage was mixed with the logarithmic host bacterium hV028 bacterial solution at a multiplicity of infection (MOI) of 0.0001, 0.001, 0.01, 0.1, 1 and 5, inoculated into LBS medium, and cultured at 37°C, 180r / min shaking for 10 hours. The phage titer was measured, and the MOI corresponding to the highest titer group was the optimal MOI. The results showed that the titer of phage hBP04 was the highest at an MOI of 0.01, reaching 1.61×10 8 PFU / mL Figure 5 ), indicating that the optimal MOI of this phage is 0.01.

[0099] 7. Determination of the effect of culture medium on the growth of host Vibrio

[0100] Five highly pathogenic Vibrio isolated in the laboratory, hV001, hV009, hV021, hV026, hV028, and hV031, were inoculated into LBS liquid culture medium, Vibrio liquid culture medium (see patent application number CN202210908481.0), and ZWHV liquid culture medium, respectively;

[0101] The formula of LBS liquid culture medium is as follows: each liter of liquid culture medium contains 10g yeast powder, 10g peptone, and 35g sodium chloride;

[0102] The formula of Vibrio liquid culture medium is as follows: each liter of the liquid culture medium contains 10g yeast extract powder, 10g peptone, 10g potassium sulfate, 0.1g ferric citrate, 10g sodium chloride, and 20g sucrose.

[0103] The formula of ZWHV liquid culture medium is: yeast powder 10g, peptone 8g, glycerol 5mL, corn starch 5g, magnesium sulfate 2g, sodium chloride 30g, L-arginine 0.5g.

[0104] Each highly pathogenic Vibrio was inoculated into three different culture media at the same amount of bacteria, cultured at 37°C, 180r / min, for 12 hours, and the bacterial growth density of each Vibrio under different culture media was observed and recorded. The specific results are shown in Table 3.

[0105] Table 3 Determination of the effect of different culture media on the growth of 5 highly pathogenic Vibrio strains

[0106]

[0107] It can be seen from the results in the table that there is no significant difference in the growth of 5 highly pathogenic Vibrio phages in different culture media, indicating that different culture media can well meet the growth requirements of the above highly pathogenic Vibrio.

[0108] 8. Culture medium screening of highly pathogenic Vibrio phage hBP04

[0109] The highly pathogenic Vibrio phage hBP04 was inoculated into the logarithmic phase hV028 host bacteria, with an inoculation MOI of 0.01. The culture media were LBS liquid culture medium, Vibrio liquid culture medium, and ZWHV liquid culture medium, respectively. Three replicates were set up, and the culture conditions were 37°C and 200r / min. The fermentation liquid was harvested after 8 hours of culture, and then the phage titer was determined by the double-layer plate method. The results of the determination are shown in Figure 5 .

[0110] See also Figure 6 According to the results, the titers of the phages obtained after fermentation based on LBS and Vibrio liquid culture medium were low, which were 1.08×10 8 PFU / mL, 3.25×10 8 PFU / mL, all of which did not reach the ninth order of magnitude, while after fermentation with ZWHV, the average titer of hBP04 could reach 7.37×10 10 PFU / mL. Therefore, the use of ZWHV can significantly improve the fermentation titer of pathogenic Vibrio phage.

[0111] 9. Further optimization of the fermentation process of highly pathogenic Vibrio phage hBP04

[0112] Considering that the host bacteria need a period of adaptation to the new culture environment, and that the phage needs a certain amount of time to assemble in the later stage, in order to improve the titer of hBP04, the hBP04 fermentation process was further optimized. The logarithmic phase host bacteria were inoculated into the liquid culture medium at a ratio of 1:100, and cultured at 37°C at 180r / min for 2 hours. The highly pathogenic Vibrio phage hBP04 was inoculated at an inoculation multiplicity of 0.01 MOI, and cultured at 37°C at 200r / min for 4 hours. The temperature was then adjusted to 32°C and cultured for another 6 hours. The phage fermentation liquid was then harvested and compared with the conventional culture method. The specific results are shown in Figure 7 .

[0113] 10. Comparison of hBP04 phage amplification in ZWHV solid medium and LBS solid medium

[0114] Using the double-layer plate method, dilute hBP04 phage to 1×10 4PFU / mL titer, then take 10μL and drop it onto LBS solid plate and ZWHV solid plate containing the same density and number of hV028 host bacteria, then culture it in a 5% CO2 incubator at 37℃ for 16 hours, and then observe the plaque formation. Figure 8 .

[0115] according to Figure 8 The results showed that the number of plaques formed by amplifying the highly pathogenic Vibrio phage hBP04 using ZWHV solid medium was significantly greater than that formed when cultured on LBS solid plates, indicating that ZWHV solid medium is more suitable for the amplification of hBP04 phage.

[0116] 11. Study on the effect of ZWHV liquid culture of highly pathogenic Vibrio phage hBP04 for Vibrio prevention and control

[0117] The highly pathogenic Vibrio hBP04 amplified by LBS liquid medium, Vibrio liquid medium and ZWHV liquid medium was used for the Vibrio prevention and control test of infecting white shrimp. The specific method was as follows: 200 healthy white shrimps of 15 days old were randomly divided into 5 groups, including healthy control group, infection control group, LBS liquid group, Vibrio liquid group and ZWHV liquid group. In addition to the healthy control group, highly pathogenic Vibrio hV009 was added to other experimental groups to make the final concentration of highly pathogenic Vibrio reach 5×10 5 CFU / mL, the total culture volume was 5L, the liquid level was 20cm, and then the final concentration of 2×10 3 PFU / mL was added to the corresponding liquid culture medium to obtain hBP04 phage fermentation. The above test groups were cultured under the same culture conditions for 7 days, and the mortality of young shrimp was counted every day. The results are shown in Table 4.

[0118] Table 4 Comparison of the effects of hBP04 phage obtained by fermentation in different culture media on the prevention and control of highly pathogenic Vibrio

[0119]

[0120] According to the test results in Table 4, the highly pathogenic Vibrio phage hBP04 prepared using different culture media can effectively inhibit the occurrence of highly pathogenic Vibrio disease and reduce the mortality of white shrimp. This shows that the high titer of highly pathogenic Vibrio phage hBP04 obtained by amplification using ZWHV culture medium did not change the characteristics of its host bacteria for infection and lysis, and can be used for the prevention and control of clinical highly pathogenic Vibrio disease.

[0121] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A broad-spectrum and highly pathogenic Vibrio phage fermentation and culture process, characterized in that: The steps include: Prepare ZWHV liquid culture medium under sterile conditions for fermentation culture of highly pathogenic Vibrio phages; The host bacteria in the logarithmic phase were inoculated into ZWHV liquid culture medium at a ratio of 1:100, and cultured at 37°C and 180 r / min for 2 hours. Then, the highly pathogenic Vibrio phage hBP04 was inoculated at an inoculation multiplicity of 0.01 MOI and cultured for another 4 hours. After the phage was inoculated and initially cultured, the culture temperature was adjusted to 32°C, and the culture was continued at 200 r / min for 6 hours to harvest the phage fermentation liquid; After the culture is completed, the host bacteria and impurities are removed by centrifugation and filtration steps to obtain a purified phage suspension; The double-layer plate method was used to determine the titer of the purified phage, and the concentration and activity of the phage were evaluated by counting the number of plaques to ensure the quality of the phage; Based on the preliminary fermentation results, the culture conditions were fine-tuned to further improve the titer and yield of the phage, and the effects of different culture media on the phage fermentation effect were evaluated; The optimized phage fermentation process was applied to the Vibrio prevention and control experiment, and the actual application effect of the phage was evaluated by counting the mortality rate of juvenile shrimp.

2. The broad-spectrum and highly pathogenic Vibrio phage fermentation and culture process according to claim 1, characterized in that: In "Preparing ZWHV liquid culture medium under sterile conditions for fermentation culture of highly pathogenic Vibrio phage", the method comprises the following steps: The formula of the ZWHV liquid culture medium is as follows: per liter, it contains 10 g of yeast powder, 8 g of peptone, 5 mL of glycerol, 5 g of corn starch, 2 g of magnesium sulfate, 30 g of sodium chloride, and 0.5 g of L-arginine.

3. The broad-spectrum and highly pathogenic Vibrio phage fermentation and culture process according to claim 2, characterized in that: In “after the culture is completed, the host bacteria and impurities are removed by centrifugation and filtration steps to obtain a purified phage suspension”, the method includes: Collecting fermentation broth from a highly pathogenic Vibrio phage hBP04 culture that has completed fermentation, wherein the fermentation broth is in the optimized ZWHV liquid culture medium; The collected phage fermentation broth was subjected to preliminary centrifugation treatment, and centrifuged at a speed of 5000 r / min using a centrifuge for 8-10 minutes to remove large particle impurities and incompletely lysed host bacterial cells in the fermentation broth, and the supernatant was collected after centrifugation; The supernatant obtained by the initial centrifugation was filtered through a 0.22 μm filter membrane to further remove residual host bacterial cell fragments, proteins, and other fine impurities; Verify the purity and titer of the phage suspension obtained after centrifugation and filtration steps to ensure that the phage suspension meets the requirements of subsequent applications.

4. The fermentation and cultivation process of broad-spectrum and highly pathogenic Vibrio phage according to claim 1, characterized in that: In "the double-layer plate method is used to determine the titer of the purified phage, and the concentration and activity of the phage are evaluated by counting the number of plaques to ensure the quality of the phage", the steps include: The purified phage suspension was taken and serially diluted 10 times with a phage buffer solution to obtain a series of phage dilution solutions with different concentrations; Take 100 μl of the diluted phage suspension from each tube, add 100 μl of the host bacteria suspension respectively, mix well, add 0.7% water agar, pour on the LBS solid plate, shake the plate to allow the phage to fully contact with the host bacteria; The prepared double-layer plate was placed in a 37°C incubator for overnight culture; Observe and record the formation of plaques, and count the number of plaques on each plate; The phage concentration in the original phage suspension was calculated based on the dilution multiple and the number of plaques; By comparing the number of plaques at different dilutions, the phage titer, that is, the number of infectious phage particles contained in each milliliter of phage suspension, is determined.

5. The fermentation and cultivation process of broad-spectrum and highly pathogenic Vibrio phage according to claim 1, characterized in that: In "Applying the optimized phage fermentation process to the Vibrio control test, and evaluating the actual application effect of the phage by counting the mortality rate of young shrimp", the method also includes the following steps: 200 samples were taken and randomly divided into 5 groups, including healthy control group, infection control group, LBS liquid group, Vibrio liquid group and ZWHV liquid group; Except for the healthy control group, other experimental groups were added with highly pathogenic Vibrio hV009 to make the final concentration of highly pathogenic Vibrio reach 5×10 5 CFU / mL, the total culture volume was 5 L, and the liquid level was 20 cm; In the LBS liquid group, Vibrio liquid group and ZWHV liquid group, the final concentration was 2×10 3 PFU / mL was added to the hBP04 phage obtained by fermentation in the corresponding liquid culture medium; The experimental group was cultured under the same culture conditions for 7 days, and the mortality of young shrimp was counted daily.

6. An application of a broad-spectrum highly pathogenic Vibrio phage, suitable for the broad-spectrum highly pathogenic Vibrio phage fermentation and culture process as claimed in any one of claims 1 to 5, characterized in that: The highly pathogenic Vibrio phage hBP04 is used in the prevention and control of Vibrio disease.

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