A duck rimer's bacillus bacteriophage high-density culture method

Abstract

This invention discloses a high-density phage culture method for *R. anatidae*, belonging to the technical field of high-density phage culture of *R. anatidae*. First, a standard strain of prevalent *R. anatidae* serotype is selected as the host bacterium. After activation, it is inoculated into an optimized culture medium supplemented with divalent cations. When the host bacterium is in the logarithmic growth phase, it is pre-amplified by inoculating the original phage lysate at a low multiplicity of infection, and high-lytic-activity phage strains are screened. Then, the screened phages are inoculated into an expansion culture system. During the culture process, nutrients are dynamically supplemented to maintain the activity of the host bacterium. After complete lysis of the host bacterium, a specific nuclease is added to the culture system to digest the free nucleic acid. The phage prepared by this invention has a high titer and can be directly used in formulation production without additional concentration. The culture system has low free nucleic acid residue, the phage loss rate during purification is low, and the obtained phage lysis spectrum covers a wide range.

Description

Technical Field

[0001] This invention relates to the field of high-density culture technology of Riemerella anatipestifer bacteriophage, and particularly to a method for high-density culture of Riemerella anatipestifer bacteriophage. Background Technology

[0002] Riemerebrionidosis of ducks is a highly prevalent bacterial infectious disease that threatens the waterfowl farming industry. Also known as duck infectious serositis, it affects commercial ducks of all ages, with morbidity rates exceeding 30% and mortality rates reaching as high as 80%, severely impacting the economic benefits of farming. Currently, the control of this disease in farming relies heavily on antibiotics. However, long-term excessive use of antibiotics induces continuous increases in antibiotic resistance in strains, leading to a decline in control effectiveness year by year. Furthermore, it poses risks such as drug residues and disruption of the microecological environment in farming. Therefore, phage biocontrol technology has become a core research direction for replacing antibiotics in the control of this disease. The industry has an urgent need for low-cost, high-titer, broad-spectrum phage preparation technology for Riemerebrionidosis of ducks.

[0003] The current mainstream method for preparing Riemerella anatipestifer bacteriophage is the conventional shake-flask batch culture method. Its working principle is to inoculate the activated host bacteria into ordinary liquid culture medium, then add the original bacteriophage lysis buffer and shake and culture together. After the host bacteria are completely lysed, the bacteriophage is filtered and harvested. This method is simple to operate, requires no special equipment, and is suitable for small-batch preparation in the laboratory. It is currently widely used in the pilot-scale research and development stage. However, this method has several technical defects: First, the ordinary culture medium does not contain the divalent cations required for phage adsorption, so the adsorption efficiency of phages on the host bacteria is only about 50%, resulting in poor synchronization of replication initiation. Second, in the single-feed culture mode, the system's nutrients are depleted after 3 hours of culture, the host bacteria die prematurely, and the phages cannot continue to complete the replication cycle. The final phage titer can only reach the level of 1×10^9 PFU / mL. To meet the standards for formulation use, additional ultracentrifugation concentration is required, which increases the production cost by more than 3 times. In addition, the lack of a free nucleic acid removal step means that the large amount of nucleic acid released by the lysis of the host bacteria increases the viscosity of the system, resulting in a phage retention loss rate of more than 50% during subsequent purification.

[0004] The second mainstream technology is the fermenter chemostat culture method, which extends the logarithmic growth phase of the host bacteria by real-time control of parameters such as dissolved oxygen and pH in the fermenter. Its production scale can reach the ton level, which is suitable for preliminary large-scale production. However, this method does not include a phage pre-amplification and screening step. A large number of weak strains with low lytic activity contained in the original lysate will be amplified simultaneously, resulting in uneven overall lytic activity of the finally harvested phages. The lytic spectrum coverage of the prevalent duck plague Riemerella anatipestifer strain is only about 50%, and the actual field control effect is unstable. At the same time, no targeted feeding strategy is set up, which cannot balance the contradiction between nutrient supply and metabolic waste accumulation. The increase in phage titer is limited, and it is still difficult to meet the needs of low-cost large-scale production.

[0005] In summary, existing phage culture technologies for Riemerella anatipestifer suffer from problems such as low titer, high purification loss rate, insufficient lysis spectrum coverage, and high production cost, which cannot meet the needs of large-scale promotion of phage biocontrol agents in aquaculture. There is an urgent need to develop new high-density culture methods to solve the above-mentioned technical bottlenecks. Summary of the Invention

[0006] To address the problems of low titer, high purification loss, narrow lysis spectrum, and high production cost in existing Riemerella anatipestifer phage culture methods, this invention provides a high-density culture method for Riemerella anatipestifer phage.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a method for high-density culture of *Riemerella anatipestifer* bacteriophage, characterized by comprising the following steps: S1. Preparation of host bacteria: Riemerella anatipestifer was selected as the host strain and inoculated into tryptic soybean liquid medium (TSB liquid medium). The medium was placed in a constant temperature shaking incubator at 37°C and shaken at 180 rpm. During this period, the host bacteria had the highest metabolic activity and the cell wall structure was intact, making it most suitable for the adsorption and infection of bacteriophages. The culture was stopped when the strain grew to the mid-logarithmic growth phase, and finally a host bacteria suspension with an OD600 value that was stable in the range of 0.4 to 0.6 was obtained. S2. Phage pre-amplification: Take the original Riemerella anatipestifer phage lysate preserved at low temperature and inoculate it into the prepared host bacterial suspension at a multiplicity of infection of 0.001~0.01. The low multiplicity of infection setting can screen out the progeny phage with the strongest lysis activity and eliminate weak strains. After shaking culture at 37℃ and 180rpm until the culture system is completely clear by visual observation and the OD600 value drops below 0.05, centrifuge at 10000rpm and 4℃ for 10min to remove the precipitate and obtain the crude phage lysate without intact host bacterial residue. S3. High-density fed-batch co-culture: Optimized LB medium was pre-prepared, with 3-5 g / L yeast extract powder added to the standard LB medium to supplement nitrogen and growth factors, and 0.8-1.5 g / L magnesium chloride added as a divalent cation to promote phage adsorption. The logarithmic-phase host bacterial suspension was inoculated into the optimized LB medium, adjusting the initial host bacterial concentration to 1×10^8~5×10^8 CFU / mL. Then, crude phage lysate was inoculated into the system at a multiplicity of infection of 0.05-0.2. After standing at 37℃ for 10 min to ensure sufficient phage adsorption, shaking culture was started. During the culture process, filtered and sterilized glucose-tryptone mixture was added every 30 min to dynamically maintain the glucose concentration in the culture system within the range of 0.8-1.2 g / L to avoid nutrient depletion or accumulation of metabolic waste. Co-culture was carried out for 4-6 h. S4. Phage Harvesting and Purification: After co-culture, DNase I and RNase A were added to the culture system to digest the free nucleic acids released by the host bacteria lysis, reducing the viscosity of the system to facilitate subsequent purification. After sterilization filtration through a 0.22μm microporous membrane, phage particles were enriched by polyethylene glycol precipitation. The precipitate was resuspended in SM buffer to finally obtain a high-titer, highly infectious Riemerella anatipestifer phage product.

[0008] Preferably, the host bacterium used in step S1 is the standard strain CVCC1532 of Riemerella anatipestifer serotype 1. This strain is the most widely prevalent pathogenic serotype standard strain in duck farming in my country. It is deposited in the China Veterinary Microbial Culture Collection Center. Its surface phage receptor expression level is stable, and the corresponding phage lysis spectrum can cover more than 90% of the field-prevalent Riemerella anatipestifer strains, which has extremely strong practical value.

[0009] Preferably, the optimized LB medium used in step S3 comprises 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 1 g / L magnesium chloride. After preparation, the pH of the medium is adjusted to the optimal growth range of 7.2-7.4 for Riemerella anatipestifer. After autoclaving at 121°C for 20 min, it is cooled to room temperature for later use. These sterilization parameters can completely kill all bacteria in the medium without destroying the nutrients and active substances in the medium.

[0010] Preferably, step S1 includes a host bacteria activation step before the preparation of the host bacteria. The *Riemerella anatipestifer* strain, which is cryopreserved at -80°C in glycerol tubes, is streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37°C for 18-24 hours. Uniform, typical single colonies that are round, smooth, translucent, and 1-2 mm in diameter are picked and inoculated into TSB liquid medium at a 1% inoculum for expansion culture. This activation step ensures that the host bacteria cultured subsequently is a pure culture free from contamination by other microorganisms.

[0011] Preferably, in the pre-amplification process of step S2, the culture system volume is set to a small volume of 10~50mL to facilitate real-time observation of the lysis state. The shaking culture speed is set to 180rpm to ensure sufficient dissolved oxygen supply. The culture time is controlled to 3~4h, which can satisfy the phage to complete 2~3 complete replication cycles to completely lyse all host bacteria. The centrifugation parameters are set to 10000rpm, centrifugation at 4℃ for 10min, which can completely remove host bacteria fragments and unly lysed intact cells. At the same time, the low temperature environment can avoid denaturation and inactivation of the phage protein coat.

[0012] Preferably, in the high-density co-culture initial system of step S3, the concentration of host bacteria is controlled at 1×10^8~5×10^8 CFU / mL. At this concentration, the host bacteria are all in the mid-logarithmic growth phase and have the best activity. After inoculating the crude phage lysate, the culture is first allowed to stand at 37°C for 10 min to ensure that the phage tail fimbriae protein fully binds to the receptors on the surface of the host bacteria. This standing step can increase the adsorption efficiency of the phage by more than 30%. Then, the shaking culture and feeding program are started.

[0013] Preferably, in step S3, the glucose-tryptone mixture added has a glucose concentration of 250 g / L and a tryptone concentration of 150 g / L. The mixture is sterilized by filtration through a 0.22 μm filter membrane to avoid nutrient degradation caused by autoclaving. 1.5 mL of the mixture is added to each 1 L of initial culture system. This addition amount, after calculation, can directly maintain the glucose concentration in the system within the preset range of 0.8~1.2 g / L, eliminating the need for additional glucose concentration testing and simplifying the operation process.

[0014] Preferably, in step S4, the final concentrations of both DNase I and RNase A are set to 1 μg / mL. This concentration can completely digest all free host bacterial nucleic acids in the system without damaging the genome encapsulated inside the phage capsid. The digestion conditions are set to incubation in a water bath at 37°C for 30 min, which are the optimal conditions for the two nucleases. The sterilization filtration is performed using a 0.22 μm hydrophilic microporous membrane, which can completely retain residual live bacteria and other bacteria while ensuring that all phage particles pass through.

[0015] Preferably, the polyethylene glycol precipitation method in step S4 specifically involves: adding PEG6000 to the filtered lysis buffer to a final mass concentration of 10%, and simultaneously adding sodium chloride to a final concentration of 0.6 mol / L. Sodium chloride can neutralize the negative charge on the surface of the phage and promote particle aggregation. After stirring evenly, the mixture is allowed to stand at 4°C for 12 hours to ensure complete phage sedimentation. The precipitate is collected by centrifugation at 4°C and 11,000 rpm for 20 minutes. These centrifugation parameters can collect more than 95% of the phage particles. The precipitate is resuspended in SM buffer to obtain the finished phage product. SM buffer can maintain the osmotic pressure and pH stability of the phage and extend its shelf life.

[0016] Preferably, the titer of the final prepared Riemerella anatipestifer phage is not less than 1×10^11 PFU / mL. This titer is much higher than the 1×10^9 PFU / mL titer obtained by conventional batch culture methods. It can be directly used for the preparation of phage antibacterial agents without additional ultracentrifugation concentration, which greatly reduces production and usage costs.

[0017] The present invention has the following beneficial effects: This invention significantly improves the overall amplification efficiency of phages through a combination of techniques: pre-amplification screening with low infection multiples, optimization of the culture medium by adding divalent cations to enhance phage adsorption efficiency, and dynamic feeding to maintain the nutrient supply of the culture system. The resulting phage titers are far higher than those of conventional batch culture methods. They can be directly used for the preparation of phage antibacterial agents without additional ultracentrifugation concentration, effectively solving the defects of low phage amplification efficiency, insufficient titers, and high production costs caused by additional concentration in existing technologies.

[0018] This invention effectively reduces the viscosity of the culture system and minimizes phage retention loss in subsequent filtration and enrichment steps by adding a specific nuclease to digest the free nucleic acid released by the host bacteria during the phage harvesting stage. This improves the phage purification and recovery rate and effectively solves the defect of high phage loss rate during the purification process caused by residual free nucleic acid in the prior art.

[0019] This invention utilizes the technique of selecting standard strains of prevalent serotypes of Riemerella anatipestifer as host bacteria. The resulting phage lysis spectrum can cover the vast majority of prevalent Riemerella anatipestifer strains in the field, and the actual field control effect is stable. This effectively solves the defects of the existing technology, such as the narrow phage lysis spectrum and poor adaptability to prevalent strains in the field.

[0020] This method does not require specialized high-end fermentation equipment and is suitable for conventional laboratory shake flask and large-scale fermenter production scenarios. It has a wide range of adjustable process parameters, high batch stability, and does not require the addition of expensive reagents. It can be directly integrated with existing large-scale phage production processes without significant modifications to existing production lines. It can fully meet the bulk demand of waterfowl farming for low-cost Riemerella duck plague phage biocontrol agents and has high industry promotion value. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the process for a high-density culture method of Riemerella anatipestifer bacteriophage proposed in this invention; Figure 2 This is a flowchart of the host bacteria preparation and phage pre-amplification process in this invention; Figure 3 This is a flowchart of the high-density co-culture and harvesting purification process in this invention. Detailed Implementation

[0022] The following will refer to the appendices in the embodiments of the present invention. Figure 1-3 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 This embodiment uses the intermediate range values ​​of each process parameter. The technical details of each step and the corresponding defect resolution logic are as follows: S1. Host strain preparation: Clinical isolates of *Riemerella anatipestifer* were selected as the host strain. The strain, preserved in glycerol at -80℃, was streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37℃ for 22 hours. Uniform, typical single colonies with a morphology of round, smooth, semi-transparent, and a diameter of 1.5 mm were picked and inoculated into tryptic soy broth (TSB) at a 1% inoculation rate. The culture was then placed in a constant temperature shaking incubator at 37℃ and shaken at 180 rpm. During this period, the expression level of phage receptors on the surface of the host strain is the highest and the metabolic activity is the strongest, which can effectively improve the phage adsorption efficiency and solve the defect of low adsorption efficiency in the existing technology. The culture was stopped when the strain grew to the mid-logarithmic growth stage to obtain a host strain suspension with an OD600 value of 0.5.

[0024] S2. Phage pre-amplification: Take the original Riemerella anatipestifer phage lysate stored at 4℃ and inoculate it into the prepared host bacterial suspension at a multiplicity of infection (MOI) of 0.005. The low MOI setting can screen out the progeny phages with the strongest lysis activity and eliminate weak strains, thus solving the defect of low phage titer in the existing technology. Incubate at 37℃ and 180 rpm with shaking until the culture system is completely clear to the naked eye and the OD600 value drops to 0.04. Centrifuge at 10000 rpm and 4℃ for 10 min to remove the precipitate and obtain crude phage lysate without intact host bacterial residue.

[0025] S3. High-density fed-batch co-culture: Optimized LB medium was pre-prepared. This medium, based on conventional LB medium, had an additional 4 g / L yeast extract powder added to supplement nitrogen and growth factors, and 1 g / L magnesium chloride added as a divalent cation to promote phage adsorption, further addressing the low adsorption efficiency. The logarithmic-phase host bacterial suspension was inoculated into the optimized LB medium, adjusting the initial host bacterial concentration to 3 × 10^8 CFU / mL. Then, crude phage lysate was inoculated into the system at a multiplicity of infection of 0.1. After standing at 37°C for 10 min to ensure sufficient phage adsorption, shaking culture was started. During the culture process, a glucose-tryptone mixture sterilized through a 0.22 μm filter was added every 30 min to dynamically maintain the glucose concentration in the culture system at 1 g / L, avoiding nutrient depletion and premature apoptosis of the host bacteria, thus solving the nutrient deficiency in the later stages of culture in existing technologies. Co-culture was carried out for 5 hours.

[0026] S4. Phage Harvesting and Purification: After co-culturing, DNase I and RNase A were added to the culture system to a final concentration of 1 μg / mL. The mixture was incubated in a 37°C water bath for 30 min to digest the free nucleic acid released by the host bacteria lysis, reducing the viscosity of the system and overcoming the defect of high purification loss due to residual free nucleic acid in existing technologies. After sterilization filtration through a 0.22 μm microporous membrane, PEG6000 was added to the filtered lysate to a final mass concentration of 10%, and sodium chloride was added to a final concentration of 0.6 mol / L. After stirring evenly, the mixture was allowed to stand at 4°C for 12 h to precipitate. The precipitate was collected by centrifugation at 11000 rpm for 20 min at 4°C and resuspended in SM buffer to finally obtain high-titer Riemerella anatipestifer phage product.

[0027] Example 2 This embodiment uses the lower limit values ​​of each process parameter to verify the process stability of the lower limit of the parameter range: S1. Preparation of host bacteria: Clinical isolates of Riemerella anatipestifer were selected as host bacteria. The strain, preserved in glycerol at -80℃, was streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37℃ for 18 hours. Uniform, typical single colonies with round, smooth, translucent shapes and a diameter of 1 mm were picked and inoculated into tryptic soy liquid medium (TSB) at a 1% inoculation rate. The medium was then placed in a constant temperature shaking incubator at 37℃ and shaken at 180 rpm. The culture was stopped when the strain reached the mid-logarithmic growth phase to obtain a host bacterial suspension with an OD600 value of 0.4.

[0028] S2. Phage pre-amplification: Take the original Riemerella anatipestifer phage lysate stored at 4℃ and inoculate it into the prepared host bacterial suspension at a multiplicity of infection of 0.001. Incubate at 37℃ and 180rpm with shaking until the culture system is completely clear to the naked eye and the OD600 value drops to 0.05. Centrifuge at 10000rpm and 4℃ for 10min to remove the precipitate and obtain crude phage lysate without intact host bacteria residue.

[0029] S3. High-density fed-batch co-culture: Optimized LB medium was pre-prepared, with 3 g / L yeast extract powder added to the standard LB medium to supplement nitrogen and growth factors, and 0.8 g / L magnesium chloride added as a divalent cation to promote phage adsorption. The logarithmic phase host bacterial suspension was inoculated into the optimized LB medium, and the initial host bacterial concentration was adjusted to 1×10^8 CFU / mL. Then, crude phage lysate was inoculated into the system at a multiplicity of infection of 0.05. After standing at 37℃ for 10 min to ensure sufficient phage adsorption, shaking culture was started. During the culture, glucose-tryptone mixture sterilized through a 0.22 μm filter was added every 30 min to dynamically maintain the glucose concentration in the culture system at 0.8 g / L, and the culture was co-cultured for 4 h.

[0030] S4. Phage Harvesting and Purification: After co-culture, DNase I and RNase A were added to the culture system to a final concentration of 1 μg / mL. The mixture was incubated in a 37°C water bath for 30 min to digest the free nucleic acid released by the host bacteria lysis. After sterilization filtration through a 0.22 μm microporous membrane, PEG6000 was added to the filtered lysate to a final mass concentration of 10%, and sodium chloride was added to a final concentration of 0.6 mol / L. After stirring evenly, the mixture was allowed to stand at 4°C for 12 h to precipitate. The precipitate was collected by centrifugation at 11,000 rpm for 20 min at 4°C. The precipitate was resuspended in SM buffer to obtain the high-titer Riemerella anatipestifer phage product.

[0031] Example 3 This embodiment uses the upper limit values ​​of each process parameter to verify the process stability of the upper limit of the parameter range: S1. Host strain preparation: Clinical isolates of Riemerella anatipestifer were selected as the host strain. The strain, preserved in glycerol at -80℃, was streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37℃ for 24 hours. Uniform, typical single colonies with round, smooth, translucent shapes and a diameter of 2 mm were picked and inoculated into tryptic soy liquid medium (TSB) at a 1% inoculation rate. The medium was then placed in a constant temperature shaking incubator at 37℃ and shaken at 180 rpm. The culture was stopped when the strain reached the mid-logarithmic growth phase to obtain a host strain suspension with an OD600 value of 0.6.

[0032] S2. Phage pre-amplification: Take the original duck plague Riemerella phage lysate stored at 4℃ and inoculate it into the prepared host bacterial suspension at a multiplicity of infection of 0.01. Incubate at 37℃ and 180rpm with shaking until the culture system is completely clear to the naked eye and the OD600 value drops to 0.03. Centrifuge at 10000rpm and 4℃ for 10min to remove the precipitate and obtain crude phage lysate without intact host bacteria residue.

[0033] S3. High-density fed-batch co-culture: Optimized LB medium was pre-prepared, with 5 g / L yeast extract added to the standard LB medium to supplement nitrogen and growth factors, and 1.5 g / L magnesium chloride added as a divalent cation to promote phage adsorption. The logarithmic-phase host bacterial suspension was inoculated into the optimized LB medium, adjusting the initial host bacterial concentration to 5 × 10^8 CFU / mL. Then, crude phage lysate was inoculated into the system at a multiplicity of infection of 0.2. After standing at 37°C for 10 min to ensure sufficient phage adsorption, shaking culture was started. During the culture process, glucose-tryptone mixture sterilized through a 0.22 μm filter was added every 30 min to dynamically maintain the glucose concentration in the culture system at 1.2 g / L for 6 h.

[0034] S4. Phage Harvesting and Purification: After co-culture, DNase I and RNase A were added to the culture system to a final concentration of 1 μg / mL. The mixture was incubated in a 37°C water bath for 30 min to digest the free nucleic acid released by the host bacteria lysis. After sterilization filtration through a 0.22 μm microporous membrane, PEG6000 was added to the filtered lysate to a final mass concentration of 10%, and sodium chloride was added to a final concentration of 0.6 mol / L. After stirring evenly, the mixture was allowed to stand at 4°C for 12 h to precipitate. The precipitate was collected by centrifugation at 11,000 rpm for 20 min at 4°C. The precipitate was resuspended in SM buffer to obtain the high-titer Riemerella anatipestifer phage product.

[0035] Example 4 This embodiment uses the duck plague Riemerella anatipestifer serotype 1 standard strain CVCC1532 as the host as defined in claim 2 to verify the phage lysis profile adaptability. Other parameters are the same as in Example 1. S1. Host strain preparation: The standard strain CVCC1532 of Riemerella anatipestifer serotype 1 was selected as the host strain. The strain, preserved in glycerol at -80℃, was streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37℃ for 22 hours. Uniform, typical single colonies with a morphology of round, smooth, semi-transparent and a diameter of 1.5 mm were picked and inoculated into tryptic soy liquid medium (TSB) at a 1% inoculation rate. The medium was placed in a constant temperature shaking incubator at 37℃ and shaken at 180 rpm. The culture was stopped when the strain reached the mid-logarithmic growth stage to obtain a host strain suspension with an OD600 value of 0.5.

[0036] S2. Phage pre-amplification: Take the original duck plague Riemerella phage lysate stored at 4℃ and inoculate it into the prepared host bacterial suspension at a multiplicity of infection of 0.005. Incubate at 37℃ and 180rpm with shaking until the culture system is completely clear to the naked eye and the OD600 value drops to 0.04. Centrifuge at 10000rpm and 4℃ for 10min to remove the precipitate and obtain crude phage lysate without intact host bacteria residue.

[0037] S3. High-density fed-batch co-culture: Optimized LB medium was pre-prepared, with 4 g / L yeast extract powder added to the standard LB medium to supplement nitrogen and growth factors, and 1 g / L magnesium chloride added as a divalent cation to promote phage adsorption. The logarithmic-phase host bacterial suspension was inoculated into the optimized LB medium, and the initial host bacterial concentration was adjusted to 3 × 10^8 CFU / mL. Then, crude phage lysate was inoculated into the system at a multiplicity of infection of 0.1. After standing at 37°C for 10 min to ensure sufficient phage adsorption, shaking culture was started. During the culture process, glucose-tryptone mixture sterilized through a 0.22 μm filter was added every 30 min to dynamically maintain the glucose concentration in the culture system at 1 g / L for 5 h.

[0038] S4. Phage Harvesting and Purification: After co-culture, DNase I and RNase A were added to the culture system to a final concentration of 1 μg / mL. The mixture was incubated in a 37°C water bath for 30 min to digest the free nucleic acid released by the host bacteria lysis. After sterilization filtration through a 0.22 μm microporous membrane, PEG6000 was added to the filtered lysate to a final mass concentration of 10%, and sodium chloride was added to a final concentration of 0.6 mol / L. After stirring evenly, the mixture was allowed to stand at 4°C for 12 h to precipitate. The precipitate was collected by centrifugation at 11,000 rpm for 20 min at 4°C. The precipitate was resuspended in SM buffer to obtain the high-titer Riemerella anatipestifer phage product.

[0039] Comparative Example This comparative example uses a conventional batch culture process from existing technologies, without the improved features of pre-amplification, optimized culture medium, and feeding as described in this invention. It is used to compare and verify the effectiveness of this patent in addressing its shortcomings. S1. Host strain preparation: Clinical isolates of Riemerella anatipestifer were selected as the host strain. The strain, preserved in glycerol at -80℃, was streaked onto tryptic soy agar (TSA) solid plates and incubated upside down at 37℃ for 22 hours. Uniform, typical single colonies with a round, smooth, translucent shape and a diameter of 1.5 mm were picked and inoculated into tryptic soy liquid medium (TSB) at a 1% inoculation rate. The medium was placed in a constant temperature shaking incubator at 37℃ and shaken at 180 rpm. The culture was stopped when the strain reached the mid-logarithmic growth stage to obtain a host strain suspension with an OD600 value of 0.5.

[0040] S2, Phage Co-culture: The original phage lysate was inoculated into the host bacterial suspension in ordinary LB medium at a multiplicity of infection of 0.1, and cultured at 37°C and 180 rpm for 6 h with shaking. There was no pre-amplification, feeding, or optimization of the medium settings.

[0041] S3. Phage harvesting and purification: After co-culture, the phages were directly filtered through a 0.22μm microporous membrane for sterilization and then enriched by PEG precipitation without nuclease digestion to obtain the finished phage product.

[0042] The nuclease digestion reaction involved in step S4 of this invention is a DNase I-catalyzed hydrolysis reaction of DNA phosphodiester bonds, and the reaction formula is as follows: ; In the formula This refers to host free genomic DNA containing n nucleotides. This reaction can specifically degrade free host nucleic acids in the system without destroying the genome wrapped by the phage capsid. It effectively reduces the viscosity of the system and reduces phage retention loss during subsequent filtration and precipitation processes, thus addressing the shortcomings of existing technologies where high purification loss rates are caused by residual free nucleic acids.

[0043] Table 1. Core process parameters for each embodiment and comparative example This table clearly records the core process parameters of all experimental groups. Examples 1-3 cover the upper and lower limits and intermediate values ​​of each parameter range recorded in this invention, verifying the stability within the range of process parameters. Example 4 uses the serum type 1 standard strain CVCC1532 as the host as defined in this invention. The comparative example completely adopts the conventional batch culture process of existing technology and has no improvement features of this invention.

[0044] Table 2 Comparison of experimental results for each embodiment and comparative example This table records the core detection results of all experimental groups. All data are the average of three parallel experiments, with an error rate of less than 5%. The phage titers of Examples 1-4 all reached above 1×10^11 PFU / mL, which is two orders of magnitude higher than the comparative example. This eliminates the need for additional ultracentrifugation concentration to meet the requirements of formulation production, thus addressing the defect of low titer. The adsorption efficiency was increased by more than 30% compared to the comparative example, thus addressing the defect of low adsorption efficiency. The purification recovery rate was increased by more than 25% compared to the comparative example, thus addressing the defect of high purification loss due to residual free nucleic acids. The cleavage spectrum coverage of Example 4 reached 92%, far exceeding the 48% of the comparative example, thus addressing the defect of narrow cleavage spectrum.

[0045] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for high-density culture of a bacteriophage of Riemerella anatipestifer, characterized by, Includes the following steps: S1. Preparation of host bacteria: Inoculate Riemerella anatipestifer into TSB liquid medium and culture at 37°C with shaking until mid-log growth to obtain a host bacterial suspension with an OD600 value of 0.4~0.6; S2. Phage pre-amplification: The original duck plague Riemerella phage lysate was inoculated into the host bacterial suspension at a multiplicity of infection of 0.001~0.

01. After shaking culture at 37°C until the system was completely clear, the precipitate was removed by centrifugation to obtain the crude phage lysate. S3, High-density fed-batch co-culture: The crude phage lysate was inoculated into the logarithmic-phase host bacterial suspension in optimized LB medium containing 3-5 g / L of additional yeast extract and 0.8-1.5 g / L of magnesium chloride at an infection multiple of 0.05-0.

2. During the shaking culture at 37°C, sterile glucose-tryptone mixture was added every 30 min to maintain the glucose concentration in the culture system at 0.8-1.2 g / L, and the culture was co-cultured for 4-6 h. S4. Phage harvesting and purification: After co-culture, DNase I and RNase A are added to the system to digest free nucleic acids. After sterilization and filtration, the phages are enriched by polyethylene glycol precipitation. After resuspending, high-titer Riemerella anatipestifer phage products are obtained.

2. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, The host bacterium used in step S1 is the standard strain CVCC1532 of Riemerella anatipestifer serotype 1.

3. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, The optimized LB medium used in step S3 comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 1 g / L magnesium chloride. The pH of the medium is adjusted to 7.2-7.4 and then autoclaved at 121°C for 20 min before use.

4. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, Before preparing the host bacteria in step S1, an activation step is also included: streaking the cryopreserved Riemerella anatipestifer onto a TSA solid plate, incubating it upside down at 37°C for 18-24 hours, and picking out morphologically uniform single colonies to inoculate them into TSB liquid medium for expansion culture.

5. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, In the pre-amplification process of step S2, the culture system volume is 10~50mL, the shaking culture speed is 180rpm, the culture time is 3~4h, and the centrifugation parameters are 10000rpm, 4℃ centrifugation for 10min to remove host bacterial fragments and unlysed bacterial cells.

6. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, In the initial high-density co-culture system of step S3, the concentration of the host bacteria is 1×10^8~5×10^8 CFU / mL. After inoculating the crude phage lysis solution, the system is first allowed to stand at 37℃ for 10 min for adsorption, and then the shaking culture and feeding program is started.

7. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, In step S3, the glucose-tryptone mixture added has a glucose concentration of 250 g / L and a tryptone concentration of 150 g / L. 1.5 mL of the mixture is added to each 1 L of the initial culture system each time.

8. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, In step S4, the final concentrations of DNase I and RNase A are both 1 μg / mL. The digestion conditions are incubation in a water bath at 37°C for 30 min, and sterilization filtration is performed using a 0.22 μm microporous membrane.

9. The method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, The polyethylene glycol precipitation method in step S4 is as follows: PEG6000 is added to the filtered lysis buffer until the final mass concentration is 10%, and sodium chloride is added to the final concentration of 0.6 mol / L. After stirring evenly, the mixture is allowed to stand at 4°C for 12 h to precipitate. The precipitate is collected by centrifugation at 11000 rpm for 20 min at 4°C. The precipitate is then resuspended in SM buffer to obtain the phage product.

10. A method for high-density culture of *Riemerella anatipestifer* bacteriophage according to claim 1, characterized in that, The titer of the final prepared Riemerella anatipestifer bacteriophage is not less than 1×10^11 PFU / mL.

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