Culture method and application of pseudomonas fluorescens phage with low death rate

Abstract

The invention discloses a culture method and application of pseudomonas fluorescens bacteriophage with a low death rate, and belongs to the technical field of microorganisms. The culture method uses a low-temperature environment as a culture means, and comprises the following steps: inoculating ancestral pseudomonas fluorescens into a culture medium, and carrying out domestication culture at 8-12 DEG C to obtain an ancestral bacterial culture solution subjected to low-temperature domestication; the method comprises the following steps: inoculating a phage and a low-temperature domesticated ancestral bacteria culture solution into a culture medium, carrying out evolution culture at 8-12 DEG C, and separating to obtain a phage solution; and inoculating the phage liquid and the low-temperature domesticated ancestral bacteria culture solution into a culture medium, carrying out evolution culture at 8-12 DEG C, and repeating the steps of separation and evolution culture. According to the invention, a low-temperature environment for limiting the growth of bacteria is used for subculturing the bacteriophage, and the ancestral bacteria subjected to low-temperature domestication are used as a host source in the culture process, so that the death rate of the bacteriophage can be reduced, and meanwhile, the low death rate of the obtained bacteriophage is more stable.

Description

TECHNICAL FIELD

[0002] The application belongs to the technical field of microorganisms, and particularly relates to a culture method of a low mortality rate Pseudomonas fluorescens bacteriophage and application thereof. BACKGROUND

[0003] Bacteriophages can infect and lyse bacteria, and thus, biological control of bacterial diseases and prevention of bacterial diseases by using virulent bacteriophages has become a safe and effective method. Due to the special biological characteristics of bacteriophages, bacteriophages will quickly inactivate in an environment away from the host. This will reduce the prevention and control effect of bacteriophages on bacteria and also bring challenges to the transportation of bacteriophage preparations. Since the use of bacteriophages to treat bacterial diseases has a wide application prospect in various scenes such as agriculture, animal husbandry and aquaculture, the preparation of bacteriophages with a low mortality rate is of great significance to improve the treatment effect of bacteriophages and reduce transportation costs.

[0004] However, at present, the main purpose of the culture and screening of bacteriophages is to obtain bacteriophages with higher virulence, and the culture method of bacteriophages with a low mortality rate is not paid enough attention. The existing acquisition of bacteriophages usually includes isolating lytic bacteriophages from the environment and expanding the culture in the laboratory. The temperature used in the culture process is the suitable growth temperature of the host bacteria, and the bacteriophages and host bacteria are cultured in a co-evolution manner. However, the obtained bacteriophages still have the defect of a high mortality rate. Therefore, it is urgent to provide a culture method capable of reducing the mortality rate of bacteriophages and improving the stability of the low mortality rate, so as to lay a foundation for the development of bacteriophage preparations with a low mortality rate. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a culture method of a low mortality rate Pseudomonas fluorescens bacteriophage, which can reduce the mortality rate of bacteriophages and improve the stability of the low mortality rate of bacteriophages.

[0006] Another purpose of the present application is to provide a bacteriophage obtained by the culture method.

[0007] Another purpose of the present application is to provide an application of the culture method in the preparation of a product containing a low mortality rate bacteriophage.

[0008] In order to achieve the above-mentioned purposes of the application, the present application provides the following technical solutions: The application provides a culture method of low mortality rate fluorescent pseudomonas bacteriophage, and the culture method comprises the following steps: inoculating ancestral type fluorescent pseudomonas into culture medium, and carrying out acclimatization culture under the condition of 8-12 DEG C, so as to obtain low-temperature acclimatized ancestral type bacterial culture solution; inoculating the bacteriophage and the low-temperature acclimatized ancestral type bacterial culture solution into culture medium, and carrying out evolution culture under the condition of 8-12 DEG C, so as to obtain bacteriophage solution; and inoculating the bacteriophage solution and the low-temperature acclimatized ancestral type bacterial culture solution into culture medium, and carrying out evolution culture under the condition of 8-12 DEG C, and repeating the steps of separation and evolution culture.

[0009] Preferably, the culture medium comprises KB liquid culture medium.

[0010] Preferably, the acclimatization culture time is 36-60 h.

[0011] Preferably, the evolution culture time is 36-60 h.

[0012] Preferably, the volume ratio of the low-temperature acclimatized ancestral type bacterial culture solution and the culture medium is 1: (50-150).

[0013] Preferably, the separation method of the bacteriophage solution comprises the following steps: adding chloroform into the culture solution after evolution culture, carrying out vortex oscillation, standing, centrifugation, and taking the supernatant to obtain the bacteriophage solution.

[0014] Preferably, the volume ratio of the bacteriophage solution and the culture medium is 1: (50-150).

[0015] Preferably, the repeating times are greater than or equal to 20.

[0016] The application further provides a bacteriophage obtained by the culture method.

[0017] The application further provides an application of the culture method in preparing a low mortality rate bacteriophage product.

[0018] The application has the following beneficial effects: The application provides a culture method of low mortality rate fluorescent pseudomonas bacteriophage, the bacteriophage is subcultured in a low temperature environment, and the ancestral type host bacteria after low temperature acclimation culture are always provided to the bacteriophage in each subculture process, so that the bacteriophage culture with a lower mortality rate is obtained. The bacteriophage is subcultured in a low temperature environment, the population size of the bacteria is lower, the virus-bacteria ratio is higher, and the bacteriophage needs to stay in the host bacteria for a longer time in the low temperature environment, only the bacteriophage with a lower mortality rate can survive in the long-term culture process, and the mortality rate of the bacteriophage obtained in the low temperature evolution culture environment is lower compared with that in a high temperature evolution culture environment. Meanwhile, the ancestral type bacteria after low temperature acclimation are used as the host source of the bacteriophage in the culture process, so that the bacteria provided for the bacteriophage in the bacteriophage subculture process are always the bacteria at the initial time point, the co-evolution of the bacteria and the bacteriophage is avoided, the low mortality rate of the obtained bacteriophage is more stable, and the development of the low mortality rate bacteriophage preparation is facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a schematic diagram of the bacteriophage evolution culture process in Example 1; Figure 2 It is a schematic diagram of the bacteriophage evolution culture process in Comparative Example 1; Figure 3 It is a schematic diagram of the co-evolution process of the bacteriophage and the fluorescent pseudomonas in Comparative Example 2; Figure 4 It is the relative mortality rate of the bacteriophage obtained by the culture method of Example 4 in Test Example 1 when measured at different temperatures; Figure 5 It is the relative mortality rate of the bacteriophage obtained by the culture method of Comparative Example 1 in Test Example 1 when measured at different temperatures; Figure 6 It is the relative mortality rate of the bacteriophage obtained by the culture method of Comparative Example 2 in Test Example 1 when measured at different temperatures. DETAILED DESCRIPTION

[0020] The application provides a culture method of low mortality rate fluorescent pseudomonas bacteriophage, the culture method comprises the following steps: inoculating ancestral type fluorescent pseudomonas into a culture medium and acclimating and culturing under the condition of 8-12 DEG C, to obtain a low-temperature-acclimated ancestral type bacterial culture solution; inoculating the bacteriophage and the low-temperature-acclimated ancestral type bacterial culture solution into a culture medium and evolution culturing under the condition of 8-12 DEG C, to obtain a bacteriophage solution; inoculating the bacteriophage solution and the low-temperature-acclimated ancestral type bacterial culture solution into a culture medium and evolution culturing under the condition of 8-12 DEG C, and repeating the steps of separation and evolution culturing.

[0021] In the present application, the species of the ancestral Pseudomonas fluorescens and the bacteriophage are not particularly limited and can be routinely selected according to actual needs. In some embodiments, the ancestral Pseudomonas fluorescens preferably includes Pseudomonas fluorescens (P. fluorescens) SBW25, and the bacteriophage preferably includes Pseudomonas fluorescens bacteriophage SBW25Ф2. The P. fluorescens SBW25 and the Pseudomonas fluorescens bacteriophage SBW25Ф2 are disclosed in the literature “Zhang Q.-G., Chu X.-L., Buckling A., Overcoming the growth-infectivity trade-off in a bacteriophage slows bacterial resistance evolution. Evolutionary Applications. 2021. 14, 2055-2063” and can be obtained from Beijing Normal University. Pseudomonas fluorescens

[0022] In the present application, the culture medium preferably includes KB liquid medium. In some embodiments, the KB liquid medium preferably includes the following components: glycerol 5-15 g / L, proteose peptone No. 3 10-30 g / L, K2HPO4·3H2O 0.5-3 g / L, and MgSO4·7H2O 0.5-3 g / L; further preferably glycerol 7-12 g / L, proteose peptone No. 3 15-25 g / L, K2HPO4·3H2O 0.7-2 g / L, and MgSO4·7H2O 0.7-2 g / L; more preferably glycerol 10 g / L, proteose peptone No. 3 20 g / L, K2HPO4·3H2O 1.5 g / L, and MgSO4·7H2O 1.5 g / L. The KB liquid medium is preferably used after sterilization; the sterilization temperature is preferably 110-130°C, more preferably 121°C; and the sterilization time is preferably 15-30 min, more preferably 20 min.

[0023] In the present application, the temperature of the domesticated culture is preferably 8-12°C, further preferably 9-11°C, and more preferably 10°C; and the time of the domesticated culture is preferably 36-60 h, further preferably 42-54 h, and more preferably 48 h.

[0024] ​In the present application, the ancestral Pseudomonas fluorescens is preferably activated before acclimatization culture. In some embodiments, the method of activation culture preferably comprises: using a loop to pick up the frozen ancestral Pseudomonas fluorescens, inoculating into KB liquid medium for culture, and obtaining the activated ancestral Pseudomonas fluorescens culture solution; the culture temperature is preferably 25-30℃, further preferably 26-29℃, and more preferably 28℃; the culture time is preferably 36-60h, further preferably 42-54h, and more preferably 48h.

[0025] The activated ancestral Pseudomonas fluorescens culture solution is inoculated into the culture medium, and the volume ratio of the activated ancestral Pseudomonas fluorescens culture solution to the culture medium is preferably 1: (50-150), further preferably 1: (75-125), and more preferably 1:100.

[0026] In the present application, the volume ratio of the low-temperature acclimated ancestral bacterial culture solution to the culture medium is preferably 1: (50-150), further preferably 1: (75-125), and more preferably 1:100.

[0027] In the present application, the evolution culture temperature is preferably 8-12℃, further preferably 9-11℃, and more preferably 10℃; the evolution culture time is preferably 36-60h, further preferably 42-54h, and more preferably 48h. The temperature condition of 8-12℃ adopted in the present application is a temperature condition that significantly limits the growth of Pseudomonas fluorescens, i.e. a temperature lower than the suitable growth temperature of Pseudomonas fluorescens. Under this temperature condition, the growth of Pseudomonas fluorescens is significantly inhibited, the virus-bacteria ratio in the evolution culture process is higher than that at the suitable growth temperature of Pseudomonas fluorescens, the bacteriophages need to stay in Pseudomonas fluorescens for a longer time in vitro, and only bacteriophages with a lower mortality rate can survive in the long-term culture process, thereby obtaining bacteriophages with a low mortality rate.

[0028] In the present application, the method of separating the bacteriophage liquid is not particularly limited and can be routinely selected according to actual needs. In some embodiments, the method of separating the bacteriophage liquid preferably comprises: adding chloroform to the culture solution after evolution culture, vortexing and standing, centrifuging, and obtaining the bacteriophage liquid by taking the supernatant.

[0029] The volume ratio of the culture solution to chloroform is preferably (5-15):1, further preferably (7-12):1, and more preferably 10:1. The standing time is preferably 3-8min, further preferably 4-6min, and more preferably 5min. The centrifugation speed is preferably 10000-15000rpm, further preferably 11000-14000rpm, and more preferably 13000rpm; the centrifugation time is preferably 1-5min, and more preferably 2min.

[0030] In the present application, the volume ratio of the bacteriophage solution and the culture medium is preferably 1: (50-150), further preferably 1: (75-125), and more preferably 1:100.

[0031] In the present application, the number of repetitions is preferably ≥20, further preferably 20-30, and more preferably 20-25.

[0032] The present application also provides a bacteriophage obtained by the culture method.

[0033] The existing bacteriophage is usually obtained by separating the lytic bacteriophage from the environment and culturing in the laboratory. The temperature used in the culture process is the suitable growth temperature of the host bacteria, and the obtained bacteriophage usually has a high mortality rate. In the present application, the bacteriophage is subcultured in a low-temperature environment. In the low-temperature environment, the population size of the bacteria is lower, the virus-bacteria ratio is higher, and the bacteriophage needs to stay outside the host bacteria for a longer time. Only the bacteriophage with a lower mortality rate can survive in the long-term culture process. Compared with the high-temperature evolution culture environment, the mortality rate of the bacteriophage obtained in the low-temperature evolution culture environment of the present application is lower. At the same time, the ancestor bacteria after low-temperature acclimation are used as the host source of the bacteriophage in the culture process, so that the bacteria provided for the bacteriophage in the bacteriophage subculture process are always the bacteria at the initial time point, avoiding the co-evolution of the bacteria and the bacteriophage, and the low mortality rate of the obtained bacteriophage is more stable.

[0034] The present application also provides a use of the culture method in the preparation of a low-mortality bacteriophage product.

[0035] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0036] In the following examples, the conventional methods are used unless otherwise specified.

[0037] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0038] In the following examples, the culture medium and reagents used are as follows: KB liquid culture medium: glycerol 10 g / L, proteose peptone No. 3 20 g / L, K2HPO4·3H2O 1.5 g / L, MgSO4·7H2O 1.5 g / L; sterilized at 121℃ for 20 min.

[0039] KB solid medium: glycerol 10 g / L, proteose peptone No. 3 20 g / L, K2HPO4·3H2O 1.5 g / L, MgSO4·7H2O 1.5 g / L, agar 15 g / L; sterilized at 121℃ for 20 min.

[0040] KB semi-solid medium: glycerol 10 g / L, proteose peptone No. 3 20 g / L, K2HPO4·3H2O 1.5 g / L, MgSO4·7H2O 1.5 g / L, agar 7.5 g / L; sterilized at 121℃ for 20 min.

[0041] Example 1 A culture method of a low mortality rate Pseudomonas fluorescens bacteriophage, the culture method comprising the following steps: (1) Obtaining a low-temperature acclimated ancestral type bacterium culture solution: Using a inoculation loop to pick up the frozen ancestral type Pseudomonas fluorescens, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 28℃ for 48 h. Taking 60 µL of the bacterial culture solution, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 10℃ for 48 h to obtain a low-temperature acclimated ancestral type bacterium culture solution.

[0042] (2) Evolutionary culture of the bacteriophage at low temperature Using a inoculation loop to pick up the frozen ancestral type Pseudomonas fluorescens, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 28℃ for 48 h. Taking 60 µL of the bacterial culture solution, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 10℃ for 48 h to obtain a low-temperature acclimated ancestral type bacterium culture solution. Figure 1

[0043] Example 2 A culture method of a low mortality rate Pseudomonas fluorescens bacteriophage, the culture method comprising the following steps: (1) Obtaining a low-temperature acclimated ancestral type bacterium culture solution: Using a inoculation loop to pick up the frozen ancestral type Pseudomonas fluorescens, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 28℃ for 48 h. Taking 60 µL of the bacterial culture solution, and inoculating into 6 mL of fresh KB liquid medium, and culturing at 10℃ for 48 h to obtain a low-temperature acclimated ancestral type bacterium culture solution.

[0044] (2) Evolutionary culture of the bacteriophage at low temperature ​The frozen phage was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium and 40 μL of low-temperature acclimated ancestral bacteria culture, mixed, and then incubated at 8°C for 60 h. 1 mL of the culture was taken and placed in a 1.5 mL centrifuge tube, 70 μL of chloroform was added, vortexed and shaken for 8 min, and then centrifuged at 10,000 rpm for 5 min. The supernatant was the phage solution. 40 μL of the phage solution was inoculated into 6 mL of fresh KB liquid medium and 40 μL of low-temperature acclimated ancestral bacteria culture, mixed, and then incubated at 8°C for 60 h. The above process was repeated 25 times.

[0045] Example 3 A culture method of a low mortality rate Pseudomonas fluorescens phage, the steps of which are as follows: (1) Low-temperature acclimated ancestral bacteria culture acquisition: The frozen ancestral Pseudomonas fluorescens was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium, and then incubated at 30°C for 36 h. 120 μL of the bacterial culture was inoculated into 6 mL of fresh KB liquid medium and incubated at 12°C for 36 h to obtain the low-temperature acclimated ancestral bacteria culture.

[0046] (2) Evolutionary culture of phage at low temperature The frozen phage was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium and 120 μL of low-temperature acclimated ancestral bacteria culture, mixed, and then incubated at 12°C for 36 h. 1 mL of the culture was taken and placed in a 1.5 mL centrifuge tube, 200 μL of chloroform was added, vortexed and shaken for 3 min, and then centrifuged at 15,000 rpm for 1 min. The supernatant was the phage solution. 120 μL of the phage solution was inoculated into 6 mL of fresh KB liquid medium and 120 μL of low-temperature acclimated ancestral bacteria culture, mixed, and then incubated at 12°C for 36 h. The above process was repeated 30 times.

[0047] Example 4 A culture method of a low mortality rate Pseudomonas fluorescens phage, the ancestral Pseudomonas fluorescens used was Pseudomonas fluorescens SBW25, and the phage used was Pseudomonas fluorescens phage SBW25Ф2, and the steps of the culture method were the same as in Example 1.

[0048] Example 5 A culture method of a low mortality rate Pseudomonas fluorescens phage, the ancestral Pseudomonas fluorescens used was Pseudomonas fluorescens SBW25, and the phage used was Pseudomonas fluorescens phage SBW25Ф2, and the steps of the culture method were the same as in Example 2.

[0049] Example 6 A method for culturing a low mortality rate Pseudomonas fluorescens bacteriophage, using an ancestral Pseudomonas fluorescens bacterium of Pseudomonas fluorescens SBW25 and a bacteriophage of Pseudomonas fluorescens bacteriophage SBW25Ф2, the culturing method comprising the following steps:

[0050] Comparative Example 1 A method for culturing a Pseudomonas fluorescens bacteriophage, using an ancestral Pseudomonas fluorescens bacterium of Pseudomonas fluorescens SBW25 and a bacteriophage of Pseudomonas fluorescens bacteriophage SBW25Ф2, the culturing method comprising the following steps: (1) Obtaining a high-temperature acclimated ancestral bacterium culture solution: The ancestral Pseudomonas fluorescens bacterium was taken out using a loop, inoculated into 6 mL of fresh KB liquid medium, and incubated at 28°C for 48 h. 60 µL of the bacterial culture solution was taken and inoculated into 6 mL of fresh KB liquid medium, and incubated at 28°C for 48 h to obtain a high-temperature acclimated ancestral bacterium culture solution.

[0051] (2) Evolutionary culture of the bacteriophage at high temperature The bacteriophage was taken out using a loop, inoculated into 6 mL of fresh KB liquid medium, and mixed with 60 µL of the high-temperature acclimated ancestral bacterium culture solution. After incubation at 28°C for 48 h, 1 mL of the culture solution was taken and placed in a 1.5 mL centrifuge tube, 100 µL of chloroform was added, vortexed, and allowed to stand for 5 min. The supernatant was obtained by centrifugation at 13,000 rpm for 2 min. 60 µL of the bacteriophage solution was taken and inoculated into 6 mL of fresh KB liquid medium, and mixed with 60 µL of the high-temperature acclimated ancestral bacterium culture solution. After incubation at 28°C for 48 h, the above process was repeated 20 times. The evolutionary culture process is shown in Figure 2 .

[0052] Comparative Example 2 A method for culturing a Pseudomonas fluorescens bacteriophage, using an ancestral Pseudomonas fluorescens bacterium of Pseudomonas fluorescens SBW25 and a bacteriophage of Pseudomonas fluorescens bacteriophage SBW25Ф2, the culturing method comprising the following steps: (1) Obtaining a low-temperature acclimated ancestral bacterium culture solution: same as Example 1.

[0053] (2) Co-evolution of the bacteriophage and the host bacterium at low temperature The bacteriophage was taken out using a loop, inoculated into 6 mL of fresh KB liquid medium, and mixed with 60 µL of the low-temperature acclimated ancestral bacterium culture solution. After incubation at 10°C for 48 h, 60 µL of the culture solution was taken and inoculated into 6 mL of fresh KB liquid medium, and mixed. After incubation at 10°C for 48 h, the above process was repeated 20 times. The co-evolution process is shown in Figure 3 .

[0054] Test Example 1 Pseudomonas fluorescens bacteriophage SBW25 Φ2 was cultured according to the culture method of Example 4, Comparative Example 1, and Comparative Example 2, respectively.

[0055] 1. Virus-bacteria ratio measurement (1) Measurement of phage density The phage density was measured using a double-layer agar plate method every 4 passages. Specifically, the phage solution was diluted 10 times with sterile water, 100 μL of the diluted solution was mixed with 100 μL of the corresponding acclimated ancestral bacterial culture solution, 5 mL of preheated KB semi-solid culture medium was added, and after mixing, it was poured onto KB solid culture medium and incubated at 28°C for 24 h, and the phage density was calculated.

[0056] (2) Measurement of bacterial density 200 μL of the corresponding acclimated bacterial culture solution after 48 h of cultivation was placed in a 96-well plate. In addition, 200 μL of fresh KB culture solution was placed in a 96-well plate as a blank control. The absorbance value at 600 nm of the culture solution and the control was measured. Based on the linear relationship between the absorbance value and the cell density, the density of the bacterial population was calculated. For Pseudomonas fluorescens, the number of Pseudomonas fluorescens per milliliter of liquid was about 3 billion when the absorbance value was 1.

[0057] (3) Calculation of virus-bacteria ratio during evolution Based on the measured phage density during the passage and the measured bacterial density, the virus-bacteria ratio was calculated every 4 passages, and the average value was calculated as the virus-bacteria ratio during the entire evolution. Each culture method was repeated 6 times, and the results are shown in Table 1.

[0058] Table 1 Virus-bacteria ratio of different culture methods

[0059] Based on the data analysis of the relative death rate, it was found that as the virus-bacteria ratio increased, the relative death rate of the phage showed a downward trend (linear regression analysis, the statistical quantities measured at 10°C and 28°C were: F 1,10 = 6.446, P = 0.029; F 1,10 = 12.060, P = 0.006).

[0060] 2. Measurement of phage death rate After the end of the incubation, the death rates of the ancestral phage and the post-incubation phage were measured. The method is as follows: 800 μL of the phage solution was taken in a 1.5 mL centrifuge tube, and at this time, the time T = 0 was counted. The phage solution was placed in an incubator at 10°C and 28°C, respectively, and was left to stand for 48 h, and the time T = 48 h was counted. The double-layer agar culture method was used to measure the phage density N0at T = 0 and the phage density N48h at T = 48 h t , and the death rates of the phage at 10°C and 28°C were calculated based on the density data.

[0061] The formula for calculating the death rate of the phage is: .

[0062] Based on the death rate of the ancestral phage and the death rate of the post-incubation phage , the relative death rate of the phage was calculated.

[0063] The relative death rate of the phage = .

[0064] The value greater than 1 means that the death rate of the post-incubation phage is increased, and the value less than 1 means that the death rate of the post-incubation phage is decreased.

[0065] In order to ensure the normality of the data, the death rate was logarithmically converted before the statistical test was performed, and then the death rates of the post-incubation phage and the ancestral phage were compared using a one-tailed t-test. There were 6 groups of repeats for each incubation method, and the results are shown in Figures 4-6 .

[0066] It can be seen that at the measurement temperature of 28°C, the phage cultured by the incubation method of Example 4 all evolved a lower death rate than the ancestral phage, and the statistical test result was significant (one-tailed t-test: t = -5.835, df = 5, P = 0.001), and among them, the death rates of 4 groups of post-incubation phage at the measurement temperature of 10°C were also lower than those of the ancestral phage, and the statistical test result was marginally significant (one-tailed t-test: t = -1.57, df = 5, P = 0.088).

[0067] The comparative example 1 incubated the Pseudomonas fluorescens phage at a high temperature of 28°C, and at the measurement of 28°C, only 1 group of phage in the treatment showed a lower death rate than the ancestral phage, and the statistical test result was not significant (one-tailed t-test: t = 1.416, df = 5, P = 0.892); at the measurement of 10°C, only 2 groups of phage showed a lower death rate than the ancestral phage at 10°C, and the statistical test result was not significant (one-tailed t-test: t = 1.326, df = 5, P = 0.879).

[0068] The Pseudomonas fluorescens phage and host bacteria were subjected to co-evolution culture at 10℃, and the phages in the 6 groups of treatments all showed higher death rates than the ancestral phage when measured at 28℃ (one-tailed t-test: t=10.249, df=5, P=0.999); the phages in the 4 groups of repeated treatments showed lower death rates than the ancestral phage when measured at 10℃, but the statistical test result was not significant (one-tailed t-test: t=-0.959, df=5, P=0.191).

[0069] It can be seen that the death rate of the phage obtained in the low-temperature evolution culture environment of the application is lower than that in the high-temperature evolution culture environment; and the low death rate of the phage obtained in the low-temperature evolution culture scheme of the application is more stable than that in the low-temperature co-evolution culture method. It is shown that the culture method of the application can significantly reduce the death rate of the phage and improve the stability thereof.

[0070] The above only describes the preferred embodiments of the application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the application, and these improvements and refinements should also be considered as falling within the protection scope of the application.

Claims

1. A method for culturing a low mortality rate Pseudomonas fluorescens bacteriophage, characterized by, The culture method comprises: inoculating the ancestral Pseudomonas fluorescens into a culture medium, acclimating and culturing at 8-12 DEG C to obtain a low-temperature acclimated ancestral bacterial culture solution; inoculating the phage and the low-temperature acclimated ancestral bacterial culture solution into a culture medium, evolutionarily culturing at 8-12 DEG C, and isolating to obtain a phage solution; inoculating the phage solution and the low-temperature acclimated ancestral bacterial culture solution into a culture medium, evolutionarily culturing at 8-12 DEG C, and repeating the isolating and evolutionarily culturing steps.

2. The culture method according to claim 1, characterized by, The culture medium comprises a KB liquid culture medium.

3. The culturing method according to claim 1, wherein, The acclimating and culturing time is 36-60 h.

4. The culturing method according to claim 1, wherein The evolutionarily culturing time is 36-60 h.

5. The culturing method according to claim 1, wherein The volume ratio of the low-temperature acclimated ancestral bacterial culture solution to the culture medium is 1: (50-150).

6. The culturing method according to claim 1, wherein The isolating method of the phage solution comprises: adding chloroform to the culture solution after evolutionarily culturing, vortexing and shaking, standing, centrifuging, and taking the supernatant to obtain the phage solution.

7. The culturing method according to claim 1, wherein The volume ratio of the phage solution to the culture medium is 1: (50-150).

8. The culturing method according to claim 1, wherein The number of repetitions is ≥20 times.

9. The phage obtained by the culture method in any one of claims 1-8.

10. The use of the culture method in any one of claims 1-8 in the preparation of a product containing low mortality rate phage.

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