Method for improving titer of clostridium perfringens bacteriophage

Abstract

The invention relates to the technical field of microorganisms. The invention discloses a method for improving the titer of clostridium perfringens bacteriophage, which is characterized by comprising the following steps: S1, seed activation; s2, seed amplification; s3, expanded production of a fermentation tank: inoculating the host bacteria solution prepared in the step S2 into a fermentation medium, carrying out anaerobic culture for 0-3 hours at the temperature of 40-45 DEG C and the rotating speed of 10-30 rpm, then inoculating the bacteriophage proliferation liquid prepared in the step S2, carrying out anaerobic condition culture for 0.5-2 hours at the temperature of 35-40 DEG C and the rotating speed of 40-60 rpm, then raising the temperature to 40-45 DEG C and the rotating speed of 10-20 rpm, maintaining the tank pressure to 0.5-1.0 MPa, and carrying out fermentation for 2-3 hours at the temperature of 35-40 DEG C and the rotating speed of 40-60 rpm. Performing anaerobic culture for 4-6 hours, ending fermentation, and regulating the pH value on line. The invention also provides a bacteriophage product. According to the method for improving the titer of the clostridium perfringens phage provided by the invention, by improving the problem of pH drop in the production process, the production efficiency is improved, the stability of the clostridium perfringens phage in the finished product fermentation liquor is improved, and the storage period of the clostridium perfringens phage product in a normal-temperature environment is prolonged.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for improving the titer of Clostridium perfringens bacteriophage. Background Art

[0002] The treatment of diseases caused by Clostridium perfringens infection with Clostridium perfringens phage is widely recognized by the public. The method is fast-acting, has no antibiotic residues, and does not cause bacterial resistance. Since Clostridium perfringens is an anaerobic bacterium, it will undergo anaerobic respiration during the culture process to produce acid using glucose to reduce the pH of the fermentation broth. The lower pH value will affect the titer of the phage in the fermentation broth and have a great impact on the stability of the phage. At present, the preparation of Clostridium perfringens phage mostly uses finished culture media such as brain heart infusion medium, TSB medium, fortified Clostridium medium, anaerobic meat liver soup medium, etc. Although the titer of the fermentation broth is relatively stable, the cost is high when industrialized. In view of the above-mentioned related technologies, the inventors found that in order to solve the problem of low titer and instability of the fermentation broth, the specific solutions are: screening high titer phages. Although this method is simple, it is not friendly to some phages with excellent performance but low titer; optimizing the culture medium and fermentation process. For example, CN117431219A is for the culture medium and fermentation process of phage RDP-CP-21008, but the technical solution still has the problem of low pH value of the fermentation broth, resulting in low titer and instability of the final fermentation broth phage; the culture medium in the CN117925441A patent is complex in composition and has a high production cost. The existence of these problems is not conducive to the industrial production of Clostridium perfringens phage, and ultimately hinders its large-scale application in the aquaculture industry. Summary of the invention

[0003] The present invention aims to provide a method for improving the titer of Clostridium perfringens phage in view of the deficiencies of the prior art. The purpose of the present invention is to provide a method for improving the titer of Clostridium perfringens phage, which method enables the produced phage to maintain a stable titer for a long time. The method improves the problem of pH drop during the production process and maintains the pH at 6.8-7.4. On the one hand, the titer of the industrial production of Clostridium perfringens phage fermentation liquid is improved, and the production efficiency is improved. On the other hand, the method can improve the stability of Clostridium perfringens phage in the finished fermentation liquid and extend the storage period of the Clostridium perfringens phage product under normal temperature.

[0004] To solve the above problems, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a method for increasing the titer of Clostridium perfringens phage, comprising the following steps:

[0006] S1. Seed activation: activating the host Clostridium perfringens seeds and the Clostridium perfringens phage seeds to obtain the host bacterial solution and phage proliferation solution after seed activation, respectively; the step S1 uses a test tube to activate the strain, which is a primary seed;

[0007] S2. Seed amplification: performing seed amplification on the host bacterial solution and the phage proliferation solution described in step S1 to obtain the host bacterial solution and the phage proliferation solution after seed amplification, respectively; the step S2 belongs to amplification, amplification to an inoculation amount suitable for fermentation tank production, specifically, stepwise expansion culture, each stage is expanded by 10 to 100 times the volume, and the inoculation amount of the fermentation tank can be reached after two to three stages of amplification;

[0008] S3. Expanding production in a fermentation tank: inoculate the host bacteria liquid prepared in step S2 into the fermentation medium, at a temperature of 40-45°C, a rotation speed of 10-30rpm, a tank pressure of 0.5-1.0MPa, and culture anaerobically for 0-3h, then inoculate the phage proliferation liquid prepared in step S2, at a temperature of 35-40°C, a rotation speed of 40-60rpm, a tank pressure of 0.5-1.0MPa, and culture under anaerobic conditions for 0.5-2h, then increase the temperature to 40-45°C, the rotation speed of 10-20rpm, maintain the tank pressure at 0.5-1.0MPa, culture anaerobicly for 4-6h to end the fermentation, and adjust the pH online.

[0009] In the method for improving the titer of Clostridium perfringens phage as described above, the fermentation medium in step S3 is any one of industrial fermentation medium, TSB broth, anaerobic liver broth, and FTG broth.

[0010] Preferably, the fermentation medium in step S3 is an industrial fermentation medium, and the industrial production fermentation medium includes the following components: by mass percentage, 1-2% anaerobic liver soup, 1-5% yeast extract powder, 0.2-1.5% peptone, 1-3% carbon source, 0.5-0.9% sodium chloride, 0.002-0.01% magnesium chloride, 0.002-0.01% calcium chloride, 0.001-0.01% calcium carbonate, and the initial pH is 8.0-8.5. A method for improving the titer of Clostridium perfringens phage as described above, the carbon source in the industrial production fermentation medium is any one of glucose, sucrose or soluble starch. Preferably, the carbon source in the industrial production fermentation medium is sucrose.

[0011] In the above method for increasing the titer of Clostridium perfringens bacteriophage, the initial pH of the fermentation medium in step S3 is 6.0-10.0. Preferably, the initial pH of the fermentation medium in step S3 is 8.0.

[0012] A method for improving the titer of Clostridium perfringens phage as described above, the online pH control in step S3 specifically includes the following steps: monitor the pH change at all times during the culture process after the phage proliferation liquid prepared in step S2 is inoculated, and when the pH drops to 7.0, add a pH fermentation regulator in time to adjust the pH, add 10mL each time, and detect whether the pH reaches 7.5 after 5 minutes. If it reaches, stop adding, and continue to add if it does not reach, and maintain the pH value at 6.5-7.5 during the entire fermentation process. Preferably, the pH value is maintained at 6.8-7.4 during the entire fermentation process. Preferably, the pH fermentation regulator is 1mol / L sodium hydroxide solution. Preferably, when the pH drops to 7, the pH fermentation regulator sodium hydroxide solution is added, and the final fermentation liquid titer is higher.

[0013] In the above method for improving the titer of Clostridium perfringens phage, in step S3, the optimal inoculation time interval between the host bacteria solution and the phage proliferation solution is 0.5-2 hours. Preferably, in step S3, the optimal inoculation time interval between the host bacteria solution and the phage proliferation solution is 0.5 hours.

[0014] A method for improving the titer of Clostridium perfringens phage as described above, wherein the Clostridium perfringens phage is selected from any one of Clostridium perfringens phage RDP-CP-22001, Clostridium perfringens phage RDP-CP-21008, Clostridium perfringens phage RDPCP23006, and Clostridium perfringens phage RDPCP23040; wherein the Clostridium perfringens phage RDP-CP-22001 was deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on March 23, 2023, with a deposit number of CGMCC NO.45448; the Clostridium perfringens phage RDP-CP-21008 was deposited in the General Microbiology Center of China National Microorganism Culture Collection on April 20, 2022, with a deposit number of CGMCC No.45091; the Clostridium perfringens phage RDPCP23006 was deposited in the General Microbiology Center of China National Microorganism Culture Collection on June 12, 2023, with a deposit number of CGMCC NO.45573; the Clostridium perfringens phage RDPCP23040 was deposited in the General Microbiology Center of China National Microorganism Culture Collection on June 12, 2023, with a deposit number of CGMCC No.45583.

[0015] In the second aspect, based on the same inventive concept, the present invention provides a phage fermentation product obtained by a method for improving the titer of Clostridium perfringens phage. The phage product is stored at 4°C for 35 days, and the titer is basically maintained at the same order of magnitude. The phage product is stored at 37°C for 35 days, and the titer decreases by less than two orders of magnitude. It can be seen that the production process of online pH control of the present invention not only improves the production titer of phage fermentation liquid, but also improves the stability of phage, producing unexpected technical effects.

[0016] The phage fermentation product as described above is characterized in that the titer of the phage fermentation product is ≥10 7 pfu / mL.

[0017] Compared with the existing technology, the effects and advantages of the present invention are:

[0018] 1. The present invention provides a method for increasing the titer of Clostridium perfringens phage, which uses industrial fermentation medium and stabilizes the titer at 10 7 pfu / mL or above, and compared with several other culture media, this culture medium has lower cost and is more suitable for industrial production.

[0019] 2. The present invention provides a method for improving the titer of Clostridium perfringens phage, wherein the optimal inoculation time interval between the final host bacteria and the phage is 0.5h, and the titer of the phage produced by the fermentation method is optimal. The concentration of Clostridium perfringens in the fermentation broth is low, which reduces the difficulty of the subsequent sterilization process. If the inoculation time interval is too long, the host bacteria grow vigorously, and there are many impurities in the final fermentation broth, which makes the subsequent sterilization process difficult.

[0020] 3. The present invention provides a method for improving the titer of Clostridium perfringens phage, wherein the titer of the phage produced by fermentation is optimal when the initial pH of the industrial fermentation medium is 8. When the initial pH is 8, Clostridium perfringens can quickly enter the logarithmic phase, shortening the phage inoculation time.

[0021] 4. The present invention provides a method for improving the titer of Clostridium perfringens phage, which uses a pH fermentation regulator to improve the titer of Clostridium perfringens phage and maintain the titer stable for a long time. The method improves the problem of pH drop during the production process, maintains the pH at 6.8-7.4, improves the titer of Clostridium perfringens phage fermentation liquid, and improves production efficiency.

[0022] 5. The method for improving the titer of Clostridium perfringens phage provided by the present invention can improve the stability of Clostridium perfringens phage and prolong the storage period of Clostridium perfringens phage at room temperature. The phage product is stored at 4°C for 35 days, and the titer basically remains at the same order of magnitude. The phage product is stored at 37°C for 35 days, and the titer decreases by less than two orders of magnitude. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The pH change curves of different phage fermentations. DETAILED DESCRIPTION

[0024] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] The TSB broth, anaerobic liver broth and FTG broth in the following examples and comparative examples were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0029] The sources of Clostridium perfringens phages used in the following examples and comparative examples are as follows:

[0030] Clostridium perfringens phage RDP-CP-22001 is the phage disclosed in Chinese patent CN202410159859.0; Clostridium perfringens phage RDP-CP-21008 is the phage disclosed in Chinese patent CN202311341257.9; Clostridium perfringens phage RDPCP23006 is the phage disclosed in Chinese patent CN202410180552.9; Clostridium perfringens phage RDPCP23040 is the phage disclosed in Chinese patent CN202410389937.6; of course, it should be noted that the method for improving the titer of Clostridium perfringens phage provided by the present invention is not only applicable to the above Clostridium perfringens phage, but also applicable to the industrial production of other Clostridium perfringens phages.

[0031] Example 1: Optimization of carbon source

[0032] This embodiment provides a method for increasing the titer of Clostridium perfringens phage, comprising the following steps:

[0033] S1. Seed activation

[0034] Take the Clostridium perfringens and its phage seeds stored at -80℃ and put them in a 37℃ water bath and stir them constantly to melt quickly. Then take two test tubes containing 5mL of sterile anaerobic liver soup, inoculate 1mL of host bacteria in one tube and 1mL of host bacteria and 1mL of phage in the other tube. The concentration of host bacteria is 10 7 cfu / mL, phage concentration is 10 6 The cells were cultured in an anaerobic incubator at 43 °C for 18 h to obtain the host bacterial solution and phage proliferation solution after seed activation.

[0035] S2. Seed amplification

[0036] Take two conical flasks containing 1L of sterile anaerobic liver soup culture medium, inoculate one with 5mL of the host bacteria prepared in step S1, and inoculate the other with 5mL of the host bacteria prepared in step S1 and 5mL of the phage proliferation liquid prepared in step S1. After inoculation, place them in a 43°C anaerobic incubator for static culture for 6h to obtain the host bacteria liquid and phage proliferation liquid after seed amplification, respectively.

[0037] S3.200L fermentation tank expansion production

[0038] First, 3L of the host bacteria prepared in step S2 is inoculated into 150L of industrial production fermentation medium, the temperature is 43°C, the speed is 20rpm, the tank pressure is 0.5MPa, and the anaerobic culture is 0.5h, and then 3L of the phage proliferation liquid prepared in step S2 is inoculated, the temperature is 37°C, the speed is 50rpm, the tank pressure is 0.5MPa, and the anaerobic conditions are cultured for 1h, and then the temperature is raised to 43°C, the speed is 15rpm, the tank pressure is maintained at 0.5MPa, and the anaerobic culture is 5h to end the fermentation. During the culture process after inoculating the phage, the pH change is monitored at all times. When the pH drops to 7.0, the pH fermentation regulator is added in time to adjust the pH, and 10mL is added each time. After 5min, it is detected whether the pH reaches 7.5. If it reaches, stop adding, and continue to add if it does not reach, and the pH value is maintained at 6.8-7.4 during the entire fermentation process. The pH fermentation regulator is 1mol / L sodium hydroxide solution.

[0039] In this embodiment, the industrial production fermentation medium is composed of, by mass percentage: 1.5% anaerobic liver broth, 2% yeast extract powder, 0.5% peptone, 1.5% carbon source, 0.5% sodium chloride, 0.002% magnesium chloride, 0.002% calcium chloride, 0.001% calcium carbonate, and an initial pH of 8.0.

[0040] First, the other components in the industrial production medium remain unchanged, the carbon source (glucose, sucrose, soluble starch) is changed, and the Clostridium perfringens phage is prepared according to the industrial production process. After the fermentation is completed, the titer of the fermentation liquid and the final pH of the fermentation liquid are determined. Then, the other components in the medium remain unchanged, the addition of sucrose is changed, and Clostridium perfringens phage RDP-CP-21008 (hereinafter referred to as "RDP-CP-21008"), Clostridium perfringens phage RDP-CP-22001 (hereinafter referred to as "RDP-CP-22001"), Clostridium perfringens phage RDPCP23006 (hereinafter referred to as "RDPCP23006"), Clostridium perfringens phage RDPCP23040 (hereinafter referred to as "RDPCP23040") are prepared according to the industrial production process, and the phage titer is determined after the fermentation is completed.

[0041] Table 1 Optimization results of carbon source

[0042]

[0043] As can be seen from Table 1, under different carbon sources and the same other culture conditions, the titers of the fermentation broths of the four phage strains in the sucrose group were significantly higher than those in the other two groups, so sucrose was the best carbon source.

[0044] Example 2: Effects of different culture media on Clostridium perfringens phage

[0045] Take the host bacteria (viable count 10) prepared in step S2 8 cfu / mL) and phage proliferation fluid (titer 10 8 pfu / mL), host bacteria and phages were inoculated into four culture media, including industrial fermentation medium, TSB broth, anaerobic meat liver broth and FTG broth, at a volume ratio of 2%, and cultured in an anaerobic incubator at 43°C for 6 h. Then samples were taken and spread on double-layer plates to determine the phage titer. The effects of fermenting Clostridium perfringens phages with four different culture media on the phage titer were compared.

[0046] Table 2 Effects of different culture media on phage titer

[0047]

[0048] As can be seen from Table 2, under the same culture conditions, when using industrial fermentation medium in laboratory tests, the titer of Clostridium perfringens phage is not much different from that of most commonly used phages, and the titer is stable at 1 7 pfu / mL, and this culture medium has lower cost than other culture media and is more suitable for industrial production. Therefore, this culture medium can be used as an industrial fermentation medium.

[0049] Example 3: Effects of different inoculation times on Clostridium perfringens phage

[0050] Take the host bacteria (viable count 10 8 cfu / mL) and phage proliferation fluid (titer 10 8 pfu / mL), first, the host bacteria were inoculated into the industrial production fermentation medium (carbon source is sucrose) of Example 1 at a volume ratio of 2%, the inoculated conical flask was placed in an anaerobic incubator at 43°C for static culture, the conical flask was taken out at 0h, 0.5h, 1h, 1.5h, 2h, and 3h of culture, and 2% of the phage proliferation liquid was inoculated, and the fermentation was terminated by further culture for 6h after inoculation of the phage proliferation liquid, and the phage titer was determined. The specific experimental results are shown in Table 3.

[0051] Table 3 Effects of different inoculation times on Clostridium perfringens phage

[0052]

[0053] It can be seen from Table 3 that when four different phages are used, the optimal inoculation time interval between the final host bacteria and the phage is 0.5 h, which saves time and improves production efficiency.

[0054] Example 4: Effect of initial medium pH on titer of Clostridium perfringens phage

[0055] The medium of the present invention (1.5% anaerobic liver soup, 2% yeast extract powder, 0.5% peptone, 1.5% sucrose, 0.5% sodium chloride, 0.002% magnesium chloride, 0.002% calcium chloride, 0.001% calcium carbonate) was adjusted to 6-10 using 1 mol / L sodium hydroxide solution, and then the host bacteria (viable count 10 8 cfu / mL) and phage proliferation fluid (titer 10 8 pfu / mL), were inoculated into culture media with different pH values ​​at a volume ratio of 2%, and then placed in an anaerobic incubator at 43°C for 6 hours. After the culture was completed, the phage titer was determined by the double-layer plate method. The results are shown in Table 4.

[0056] Table 4 Effect of initial pH of culture medium on titer of Clostridium perfringens phage

[0057]

[0058] It can be seen from Table 4 that the initial pH value has a great influence on the phage titer. When the four phage strains are produced under different initial pH conditions, the phage titer of the fermentation broth is optimal when the pH is 8. Therefore, the initial pH value is set to 8.0 during production.

[0059] Example 4: Effect of adding pH regulator at different times on the titer of fermentation broth

[0060] An industrial production fermentation medium was used and sucrose was used as the carbon source, and other culture conditions were the same as those in Example 1. The difference from Example 1 was that the timing of adding sodium hydroxide solution in step S3 was when the pH dropped to 5, 6, and 7, respectively, and the adding method was the same as in Example 1.

[0061] Table 5 Effect of adding pH regulator at different time on the titer of Clostridium perfringens phage

[0062]

[0063] From the above experimental data, it can be seen that when the pH drops to 7 and sodium hydroxide solution is added, the final fermentation liquid has a higher titer.

[0064] Comparative Example 1:

[0065] The difference from the method for improving the titer of Clostridium perfringens phage provided in Example 1 is that the process of online pH control in step S3 is removed, and an industrial production fermentation medium is used with sucrose as the carbon source, and other culture conditions are kept the same to produce a batch of Clostridium perfringens phage, and the titers of the fermentation broths prepared by the two processes are compared. Then, 1L of fermentation broth is taken from each fermentation tank and stored at 37°C. The titer is measured every 1 day in the early stage, and the titer is measured every 7 days after one week, and the stability of the phage liquids prepared by the two processes is compared.

[0066] According to the above operation, the results are as follows: the titer of the fermentation broth in Example 1 of the present invention (online pH control) is 1.67×10 10 pfu / mL, and the titer of the fermentation broth of comparative example 1 (without online pH control) was 7.2×10 7 It can be seen that the titer of the in-line pH value control process of the present invention is higher and more stable.

[0067] Comparative Example 2:

[0068] Take two batches of phage produced in Example 1 and Comparative Example 1, and then take 40 fermentation broth samples from each batch, each sample is 5 mL, 20 of which are stored at 4°C, and the other 20 are stored at 37°C. Take one sample every 7 days to determine the phage titer. Specific experimental results are shown in Tables 6 and 7 below.

[0069] Table 6 Changes in potency of samples stored at 4°C

[0070]

[0071] Table 7 Changes in potency of samples stored at 37°C

[0072]

[0073] It can be seen from Table 6 and Table 7 that the production process of online pH control of the present invention not only improves the production titer of phage fermentation broth, but also improves the stability of phage, producing unexpected technical effects.

[0074] Comparative Example 3:

[0075] This experiment compares the pH changes of Escherichia coli phage, Salmonella phage, Riemerella anatipestifer phage and Clostridium perfringens phage under the same culture conditions. Take three 250mL conical flasks, add 100mL of culture medium (2% yeast extract powder, 0.5% sodium chloride, 0.5% glucose, 0.02% calcium chloride, pH7.0) to each, and inoculate the host bacteria and phage at a volume ratio of 2%. Then, Escherichia coli phage, Salmonella phage and Riemerella anatipestifer phage are cultured in a 37℃ constant temperature incubator, and Clostridium perfringens is cultured in an anaerobic incubator at 37℃. Take 10mL samples every 1h to measure the pH. The pH change curves during fermentation of different phages are shown below. Figure 1 shown.

[0076] Depend on Figure 1 It can be seen that under the same culture conditions, only the pH of the fermentation liquid of Clostridium perfringens phage decreases rapidly, and the pH value is finally stabilized at about 4.5. The titer stability of most phages will be affected after the pH value is lower than 5. The reason is that Clostridium perfringens phage will produce acid during the production process to reduce the pH value, which eventually affects the stability of the phage. The present invention can maintain a suitable fermentation environment, increase the phage yield, and extend its storage time by real-time monitoring and controlling the pH value, thereby solving the industrialization problem that the industrial production of Clostridium perfringens phage and its large-scale application in the aquaculture industry are hindered.

[0077] It should be pointed out that the specific implementation methods are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-mentioned embodiments, and there are many variations. Those skilled in the art who can obtain the invention clearly disclosed in the present invention or without objection based on the written description of the document should be considered as the scope of protection of this patent.

Claims

1. A method for increasing the titer of Clostridium perfringens phage, characterized in that: The following steps are involved: S1. Seed activation: activating the host Clostridium perfringens seeds and the Clostridium perfringens phage seeds to obtain the host bacterial liquid and phage proliferation liquid after seed activation, respectively; S2. Seed amplification: performing seed amplification on the host bacterial solution and the phage proliferation solution described in step S1 to obtain the host bacterial solution and the phage proliferation solution after seed amplification, respectively; S3. Expanding production in a fermentation tank: inoculate the host bacteria liquid prepared in step S2 into the fermentation medium, at a temperature of 40-45°C, a rotation speed of 10-30rpm, a tank pressure of 0.5-1.0MPa, and culture anaerobically for 0-3h, then inoculate the phage proliferation liquid prepared in step S2, at a temperature of 35-40°C, a rotation speed of 40-60rpm, a tank pressure of 0.5-1.0MPa, and culture under anaerobic conditions for 0.5-2h, then increase the temperature to 40-45°C, the rotation speed of 10-20rpm, maintain the tank pressure at 0.5-1.0MPa, culture anaerobicly for 4-6h to end the fermentation, and adjust the pH online.

2. A method for increasing the titer of Clostridium perfringens phage according to claim 1, characterized in that: The fermentation medium in step S3 is any one of industrial fermentation medium, TSB broth, anaerobic liver broth, and FTG broth.

3. A method for increasing the titer of Clostridium perfringens phage according to claim 2, characterized in that: The fermentation medium in step S3 is an industrial fermentation medium, and the industrial production fermentation medium comprises the following components: in mass percentage, 1-2% anaerobic liver soup, 1-5% yeast extract powder, 0.2-1.5% peptone, 1-3% carbon source, 0.5-0.9% sodium chloride, 0.002-0.01% magnesium chloride, 0.002-0.01% calcium chloride, 0.001-0.01% calcium carbonate, and the initial pH is 8.0-8.

5.

4. A method for increasing the titer of Clostridium perfringens phage according to claim 3, characterized in that: The carbon source in the industrial production fermentation medium is any one of glucose, sucrose or soluble starch.

5. A method for increasing the titer of Clostridium perfringens phage according to claim 2, characterized in that: The initial pH of the fermentation medium in step S3 is 6.0-10.

0.

6. A method for increasing the titer of Clostridium perfringens phage according to claim 3, characterized in that: The online pH control in step S3 specifically includes the following steps: during the culture process after inoculating the phage proliferation liquid prepared in step S2, the pH change is constantly monitored, and when the pH drops below 7.0, a pH fermentation regulator is added in time to adjust the pH, so as to maintain the pH value at 6.5-7.5 during the entire fermentation process.

7. A method for increasing the titer of Clostridium perfringens phage according to claim 3, characterized in that: In step S3, the optimal time interval for inoculating the host bacterial solution and the phage proliferation solution is 0.5-2 hours.

8. The method for increasing the titer of Clostridium perfringens phage according to claim 1, characterized in that: The Clostridium perfringens phage is selected from any one of Clostridium perfringens phage RDP-CP-22001, Clostridium perfringens phage RDP-CP-21008, Clostridium perfringens phage RDPCP23006, and Clostridium perfringens phage RDPCP23040; wherein the Clostridium perfringens phage RDP-CP-22001 was deposited in the General Microbiological Center of the China National Microbiological Culture Collection on March 23, 2023, with a deposit number of CGMCC NO.45448; the Clostridium perfringens phage RDP-CP-21008 was deposited in the General Microbiological Center of the China National Microbiological Culture Collection on April 20, 2022, with a deposit number of CGMCC No.45091; the Clostridium perfringens phage RDPCP23006 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 12, 2023, with a deposit number of CGMCC NO.45573; the Clostridium perfringens phage RDPCP23040 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 12, 2023, with a deposit number of CGMCC No.45583.

9. A phage fermentation product obtained by the method for increasing the titer of Clostridium perfringens phage according to any one of claims 1 to 8.

10. The bacteriophage fermentation product according to claim 9, characterized in that: The titer of the phage fermentation product is ≥10 7 pfu / mL.

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