Method for culturing low mortality rate fluorescent pseudomonas bacteriophage and application thereof
By employing low-temperature domestication and evolutionary culture methods, the problem of high mortality rate in bacteriophage culture has been solved, achieving low mortality rate and stability of bacteriophages, which are applicable to agriculture, animal husbandry, and aquaculture.
Patent Information
- Application Number
- CN202511596379.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-02-13
- Estimated Expiration
- 2045-11-04
AI Technical Summary
Existing phage culture methods result in a high phage mortality rate, which affects their application in agriculture, animal husbandry, and aquaculture, as well as transportation costs.
A low-temperature domestication and evolutionary culture method was adopted. Ancestor-type Pseudomonas fluorescens was inoculated into the culture medium at 8-12℃ for domestication and evolutionary culture. The isolation and evolutionary culture steps were repeated. The low-temperature domesticated ancestral bacteria were used as the host to avoid the co-evolution of bacteria and bacteriophages.
It significantly reduces the death rate of bacteriophages and improves their stability, making it suitable for preparing low-death-rate bacteriophage products, reducing transportation costs and improving therapeutic effects.
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Figure CN121065110B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] 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
[0002] Bacteriophages can infect and lyse bacteria, and therefore, 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.
[0003] 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 mode. 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
[0004] 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.
[0005] Another purpose of the present application is to provide a bacteriophage obtained by the culture method.
[0006] 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.
[0007] In order to achieve the above-mentioned purposes of the application, the present application provides the following technical solutions:
[0008] 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 domesticating and culturing at 8-12 DEG C to obtain low-temperature domesticated ancestral type bacterial culture solution; inoculating bacteriophage and the low-temperature domesticated ancestral type bacterial culture solution into culture medium, and evolutionally culturing at 8-12 DEG C to obtain bacteriophage solution; and inoculating the bacteriophage solution and the low-temperature domesticated ancestral type bacterial culture solution into culture medium, and evolutionally culturing at 8-12 DEG C, and repeating the steps of separation and evolutional culturing.
[0009] Preferably, the culture medium comprises KB liquid medium.
[0010] Preferably, the domestication culture time is 36-60 h.
[0011] Preferably, the evolutional culture time is 36-60 h.
[0012] Preferably, the volume ratio of the low-temperature domesticated 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 evolutional culture, vortexing and oscillating, standing, centrifuging, and taking 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:
[0019] This invention provides a method for culturing low-death-rate fluorescent Pseudomonas phage. The phage is passaged at low temperatures, with each passage consistently providing the phage with ancestral host bacteria that have undergone low-temperature acclimation. This results in phage cultures with low mortality rates. This invention utilizes a low-temperature environment for phage passage. Because the bacterial population size is smaller and the virus-bacteria ratio is higher in a low-temperature environment, the phage needs to remain outside the host bacteria for a longer period. Only phages with low mortality rates can survive the long-term culture process. Compared to high-temperature evolutionary culture environments, the phages obtained in this invention have a lower mortality rate. Furthermore, this invention uses ancestral bacteria that have undergone low-temperature acclimation as the host source for the phage during the cultivation process. This ensures that the bacteria provided to the phage during passage are always those from the initial time point, avoiding co-evolution between bacteria and phage. The resulting low mortality rate of the phage is more stable, laying the foundation for the development of low-death-rate phage formulations. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the phage evolution culture process in Example 1;
[0021] Figure 2 This is a schematic diagram of the phage evolution culture process in Comparative Example 1;
[0022] Figure 3 This is a schematic diagram illustrating the co-evolution process of bacteriophages and fluorescent Pseudomonas in Comparative Example 2;
[0023] Figure 4 The relative mortality rate of bacteriophages obtained by the culture method in Example 4 of Experiment 1 was measured at different temperatures;
[0024] Figure 5 The relative mortality rate of bacteriophages obtained by the culture method of Comparative Example 1 in Experiment 1 was measured at different temperatures.
[0025] Figure 6 The relative mortality rate of bacteriophages obtained by the culture method of Comparative Example 2 in Experiment 1 was measured at different temperatures. Detailed Implementation
[0026] This invention provides a method for culturing low-death-rate fluorescent Pseudomonas phage. The method includes: inoculating ancestral fluorescent Pseudomonas into a culture medium and acclimating it at 8-12°C to obtain a low-temperature acclimated ancestral bacterial culture; inoculating the phage and the low-temperature acclimated ancestral bacterial culture into the culture medium and culturing and isolating the phage fluid at 8-12°C; and inoculating the phage fluid and the low-temperature acclimated ancestral bacterial culture into the culture medium and culturing and isolating them at 8-12°C, repeating the isolation and culturing steps.
[0027] 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 comprises Pseudomonas fluorescens (Pf0-2) SBW25, and the bacteriophage preferably comprises Pseudomonas fluorescens bacteriophage SBW25Ф2. The Pseudomonas 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 )SBW25, the bacteriophage preferably comprises Pseudomonas fluorescens bacteriophage SBW25Ф2. The Pseudomonas 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.
[0028] In the present application, the culture medium preferably comprises KB liquid medium. In some embodiments, the KB liquid medium preferably comprises 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; the sterilization time is preferably 15-30 min, more preferably 20 min.
[0029] 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; the time of the domesticated culture is preferably 36-60 h, further preferably 42-54 h, and more preferably 48 h.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] In the present application, the method for separating the bacteriophage liquid is not particularly limited and can be routinely selected according to actual needs. In some embodiments, the method for 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.
[0035] 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.
[0036] 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.
[0037] In the present application, the number of repetitions is preferably ≥20, further preferably 20-30, and more preferably 20-25.
[0038] The present application also provides a bacteriophage obtained by the culture method.
[0039] 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.
[0040] The present application also provides a use of the culture method in the preparation of a low-mortality bacteriophage product.
[0041] 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.
[0042] In the following examples, the conventional methods are used unless otherwise specified.
[0043] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.
[0044] In the following examples, the culture medium and reagents used are as follows:
[0045] 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.
[0046] 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.
[0047] 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.
[0048] Example 1
[0049] A culture method of a low mortality rate Pseudomonas fluorescens bacteriophage, the culture method comprising the following steps:
[0050] (1) Obtaining a low-temperature acclimated ancestral type bacterium culture solution:
[0051] Using a inoculation loop, the frozen ancestral type Pseudomonas fluorescens is picked and inoculated into 6 mL of fresh KB liquid medium, and cultured at 28℃ for 48 h. 60 µL of the bacterial culture solution is taken and inoculated into 6 mL of fresh KB liquid medium, and cultured at 10℃ for 48 h to obtain a low-temperature acclimated ancestral type bacterium culture solution.
[0052] (2) Evolutionary culture of the bacteriophage at low temperature
[0053] Using a inoculation loop, the frozen ancestral type Pseudomonas fluorescens is picked and inoculated into 6 mL of fresh KB liquid medium, and cultured at 28℃ for 48 h. 60 µL of the bacterial culture solution is taken and inoculated into 6 mL of fresh KB liquid medium, and cultured at 10℃ for 48 h to obtain a low-temperature acclimated ancestral type bacterium culture solution. Figure 1
[0054] Example 2
[0055] A culture method of a low mortality rate Pseudomonas fluorescens bacteriophage, the culture method comprising the following steps:
[0056] (1) Obtaining a low-temperature acclimated ancestral type bacterium culture solution:
[0057] The frozen ancestral Pseudomonas fluorescens was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium, and incubated at 25°C for 60 h. 40 μL of the bacterial culture was inoculated into 6 mL of fresh KB liquid medium, and incubated at 8°C for 60 h to obtain the low-temperature acclimated ancestral bacterial culture.
[0058] (2) Evolutionary culture of phage at low temperature
[0059] The frozen phage was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium, and 40 μL of the low-temperature acclimated ancestral bacterial culture was added, mixed, and 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, and then allowed to stand for 8 min. Centrifugation was performed at 10,000 rpm for 5 min, and 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 the low-temperature acclimated ancestral bacterial culture was added, mixed, and incubated at 8°C for 60 h. The above process was repeated 25 times.
[0060] Example 3
[0061] A culture method of a low-mortality Pseudomonas fluorescens phage, the culture method comprising the following steps:
[0062] (1) Obtaining a low-temperature acclimated ancestral bacterial culture:
[0063] The frozen ancestral Pseudomonas fluorescens was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium, and 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 bacterial culture.
[0064] (2) Evolutionary culture of phage at low temperature
[0065] The frozen phage was picked up with a loop and inoculated into 6 mL of fresh KB liquid medium, and 120 μL of the low-temperature acclimated ancestral bacterial culture was added, mixed, and 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, and then allowed to stand for 3 min. Centrifugation was performed at 15,000 rpm for 1 min, and 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 the low-temperature acclimated ancestral bacterial culture was added, mixed, and incubated at 12°C for 36 h. The above process was repeated 30 times.
[0066] Example 4
[0067] 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 steps of the method being as in Example 1.
[0068] Example 5
[0069] 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 steps of the method being as in Example 2.
[0070] Example 6
[0071] 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 steps of the method being as in Example 3.
[0072] Comparative Example 1
[0073] 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 steps of the method being as follows:
[0074] (1) Obtaining a high-temperature acclimated ancestral bacterium culture solution:
[0075] The ancestral Pseudomonas fluorescens bacterium was thawed and inoculated into 6 mL of fresh KB liquid medium using a loop, 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.
[0076] (2) Evolutionary culture of the bacteriophage at high temperature
[0077] The bacteriophage was thawed and inoculated into 6 mL of fresh KB liquid medium using a loop, and 60 µL of the high-temperature acclimated ancestral bacterium culture solution was inoculated, mixed, and incubated 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 shaken for 5 min, and centrifuged at 13000 rpm for 2 min. The supernatant was the bacteriophage solution. 60 µL of the bacteriophage solution was taken and inoculated into 6 mL of fresh KB liquid medium, and 60 µL of the high-temperature acclimated ancestral bacterium culture solution was inoculated, mixed, and incubated at 28°C for 48 h. The above process was repeated 20 times, and the evolutionary culture process is shown in Figure 2 .
[0078] Comparative Example 2
[0079] A culture method of a Pseudomonas fluorescens bacteriophage, using an ancestral Pseudomonas fluorescens of Pseudomonas fluorescens SBW25 and a bacteriophage of Pseudomonas fluorescens bacteriophage SBW25Ф2, the culture method comprising the following steps:
[0080] (1) Obtaining a low-temperature acclimated ancestral bacterial culture solution: same as Example 1.
[0081] (2) Co-evolution of the bacteriophage and the host bacteria at low temperature
[0082] The frozen bacteriophage was picked up using an inoculation loop, inoculated into 6 mL of fresh KB liquid medium, and inoculated with 60 µL of the low-temperature acclimated ancestral bacterial culture solution. After mixing, the mixture was incubated at 10°C for 48 h. After the incubation, 60 µL of the culture solution was taken and inoculated into 6 mL of fresh KB liquid medium. After mixing, the mixture was incubated at 10°C for 48 h. The above process was repeated for 20 times, and the co-evolution process is shown in Figure 3 .
[0083] Test Example 1
[0084] The Pseudomonas fluorescens bacteriophage SBW25Ф2 was cultured according to the culture method of Example 4, Comparative Example 1, and Comparative Example 2, respectively.
[0085] 1. Virus-bacteria ratio determination
[0086] (1) Bacteriophage density determination
[0087] The bacteriophage density was measured using a double-layer agar plate method every 4 passages. Specifically, the bacteriophage 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 medium was added, the mixture was mixed and poured onto KB solid medium, and the mixture was incubated at 28°C for 24 h. The bacteriophage density was calculated.
[0088] (2) Bacterial density determination
[0089] 200 µL of the corresponding acclimated bacterial culture solution after 48 h of incubation 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 treatment 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, when the absorbance value is 1, the number of Pseudomonas fluorescens in each milliliter of the liquid is about 3 billion.
[0090] (3) Virus-bacteria ratio calculation during evolution
[0091] Based on the measured density of phage during the process of subculturing and the measured density of bacteria, the virus-bacteria ratio was calculated every 4 subcultures, and the mean value was calculated as the virus-bacteria ratio during the whole evolution process. The results are shown in Table 1.
[0092] Table 1 Virus-bacteria ratio of different culture methods
[0093]
[0094] The data analysis combined with the relative death rate showed that with the increase of the virus-bacteria ratio, 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).
[0095] 2. Measurement of phage death rate
[0096] After the end of the culture, the death rate of the ancestral phage and the phage after the culture was measured. The specific method is as follows: 800 μL of phage liquid was taken into a 1.5 mL centrifuge tube, at which time the time T = 0 was counted. The phage liquid was placed in a culture box at 10°C and 28°C respectively, and was placed statically for 48 h, at which time the time T = 48 h was counted. The double-layer agar culture method was used to measure the phage density N0 at T = 0 and the phage density N48 at T = 48 h. t Based on the density data, the death rate of the phage at 10°C and 28°C was calculated.
[0097] The calculation formula of the death rate of the phage is: .
[0098] Based on the death rate of the ancestral phage and the death rate of the phage after the culture , the relative death rate of the phage was calculated.
[0099] The relative death rate of the phage = .
[0100] The value greater than 1 means that the death rate of the phage after the culture is increased, and less than 1 means that the death rate of the phage after the culture is decreased.
[0101] In order to ensure the normality of the data, the death rate was logarithmically converted before the statistical test, and then the death rate of the phage after the culture was compared with the death rate of the ancestral phage using a one-tailed t-test. The results are shown in Table 2. Figures 4-6The results are shown in Table 1.
[0102] It can be seen that at the measurement temperature of 28℃, the phages cultured by the culture method of Example 4 all evolved lower death rates than the ancestral phages, and the statistical test result was significant (one-tailed t-test: t=-5.835, df=5, P=0.001), and the death rates of 4 groups of the phages cultured at the measurement temperature of 10℃ were also lower than those of the ancestral phages, and the statistical test result was marginally significant (one-tailed t-test: t=-1.57, df=5, P=0.088).
[0103] The phages of Pseudomonas fluorescens were cultured at high temperature of 28℃ in Comparative Example 1, and at the measurement temperature of 28℃, only the phages in one group of treatment showed lower death rates than the ancestral phages, and the statistical test result was not significant (one-tailed t-test: t=1.416, df=5, P=0.892); at the measurement temperature of 10℃, only 2 groups of the phages showed lower death rates than the ancestral phages at 10℃, and the statistical test result was not significant (one-tailed t-test: t=1.326, df=5, P=0.879).
[0104] The phages of Pseudomonas fluorescens and host bacteria were co-evolutionarily cultured at 10℃ in Comparative Example 2, and at the measurement temperature of 28℃, the phages in 6 groups of treatment all showed higher death rates than the ancestral phages (one-tailed t-test: t=10.249, df=5, P=0.999); at the measurement temperature of 10℃, 4 groups of the phages showed lower death rates than the ancestral phages, but the statistical test result was not significant (one-tailed t-test: t=-0.959, df=5, P=0.191).
[0105] It can be seen that compared with the high-temperature evolutionary culture environment, the phages obtained by the low-temperature evolutionary culture environment of the present application have lower death rates; compared with the low-temperature co-evolutionary culture method, the low death rates of the phages obtained by the low-temperature evolutionary culture scheme of the present application are more stable. It is shown that the culture method of the present application can significantly reduce the death rate of the phages and improve the stability thereof.
[0106] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for culturing low-death-rate fluorescent Pseudomonas phage, characterized in that, The cultivation method comprises the following steps: ancestral type *Pseudomonas fluorescens* is inoculated into a culture medium and acclimatized at 8-12°C to obtain a low-temperature acclimatized ancestral bacterial culture; bacteriophages and the low-temperature acclimatized ancestral bacterial culture are inoculated into a culture medium and cultured and isolated at 8-12°C to obtain bacteriophage fluid; the bacteriophage fluid and the low-temperature acclimatized ancestral bacterial culture are inoculated into a culture medium and cultured and isolated at 8-12°C, repeating the isolation and culture steps. The ancestral type of *Pseudomonas fluorescens* is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens )SBW25, wherein the bacteriophage is fluorescent Pseudomonas bacteriophage SBW25Ф2; The method for separating the phage fluid includes: adding chloroform to the culture medium after evolution culture, vortexing and allowing it to stand, centrifuging, and taking the supernatant to obtain the phage fluid; The repetition is repeated 20 to 30 times.
2. The cultivation method according to claim 1, characterized in that, The culture medium includes KB liquid culture medium.
3. The cultivation method according to claim 1, characterized in that, The acclimatization and cultivation period is 36-60 hours.
4. The cultivation method according to claim 1, characterized in that, The evolution culture time is 36-60 hours.
5. The cultivation method according to claim 1, characterized in that, The volume ratio of the ancestral bacterial culture medium to the culture medium after low-temperature domestication is 1:(50~150).
6. The cultivation method according to claim 1, characterized in that, The volume ratio of the phage fluid to the culture medium is 1:(50~150).
7. Bacteriophages obtained by the culture method according to any one of claims 1 to 6.
8. The application of the culture method according to any one of claims 1 to 6 in the preparation of bacteriophage products containing low-death-rate bacteriophages.