High-activity clostridium perfringens bacteriophage RDP-CP-2310009, bacteriophage preparation and application thereof

Abstract

The invention belongs to the technical field of microorganisms. The invention provides a clostridium perfringens bacteriophage RDP-CP-2310009, the preservation number of the clostridium perfringens bacteriophage RDP-CP-2310009 is CGMCC (China General Microbiological Culture Collection Center) No.45779, and the clostridium perfringens bacteriophage RDP-CP-2310009 is preserved in the China General Microbiological Culture Collection Center on November 29, 2023. The invention also provides a phage composition. The invention further provides an application of the clostridium perfringens bacteriophage RDP-CP-2310009 or the bacteriophage composition. The invention also provides a bacteriophage preparation. The invention also provides a preparation method of the bacteriophage water-in-oil liquid preparation. The invention also provides a preparation method of the phage coated solid preparation. The invention also provides application of the phage preparation. According to the invention, the titer of the bacteriophage is greatly improved by optimizing the proliferation process of the bacteriophage; according to the bacteriophage preparation provided by the invention, the activity of the bacteriophage can be preserved for a long time; and the preparation of the phage preparation is simple to operate, high in efficiency, low in production cost and beneficial to large-scale popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a highly active Clostridium perfringens bacteriophage RDP-CP-2310009, a bacteriophage preparation and application thereof. Background Art

[0002] With the rapid scale development of poultry farming, bacterial diseases have become increasingly prominent, restricting the sustainable and healthy development of China's poultry industry. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that is widely distributed in soil, dust, animal production environment and feed. It mainly causes necrotic enteritis in poultry. The intestines of infected chickens are fragile and full of gas, and necrosis occurs in the jejunum and ileum, forming pseudomembranes. The colonization rate of Clostridium perfringens in the intestine is as high as 95%, causing clinical or subclinical infection of necrotic enteritis, increasing the cost of chicken breeding and causing serious economic losses. "Growth-promoting" antibiotics such as bacitracin, lincomycin, tylosin and penicillin are usually added to feed to prevent Clostridium perfringens infection in chickens. However, with the rapid spread of antibiotic resistance, antibiotics are banned. The prohibition of antibiotics may increase the rapid proliferation of Clostridium perfringens in the poultry environment, resulting in reduced feed digestibility and absorption rate, slow weight gain, increased breeding costs, and ultimately increased consumer burden. According to relevant estimates, poultry necrotic enteritis causes huge losses of more than $6 billion to the poultry industry worldwide each year. It can be seen that a safe, effective and sustainable program to control Clostridium perfringens infection will effectively improve the economic benefits of the poultry industry.

[0003] Bacteriophage is a bacterial virus that can kill bacteria through a specific lytic cycle. The specificity of bacteriophage allows it to attack only the target host without affecting the normal intestinal flora. Therefore, bacteriophage has good safety, which also makes it possible to treat bacterial infections in a targeted manner. Based on this biological characteristic of bacteriophage, bacteriophage can be used as an antibacterial agent for treatment and as an alternative to antibiotic treatment.

[0004] However, the current application of Clostridium perfringens phage is limited by the following two aspects: first, Clostridium perfringens is a strictly anaerobic Gram-positive bacterium, which ferments nitrogen sources into acid during its metabolism, causing the pH of the proliferation liquid to decrease, and the growth characteristics of Clostridium perfringens will affect the proliferation of phages; second, the effectiveness and practicality of phage preparations depend on the maintenance of their stability, but during logistics transportation and long-term storage, the phage titer decreases. Therefore, the prior art lacks Clostridium perfringens phages that can both efficiently proliferate and ensure the stability of phage preparations.

[0005] Therefore, the prior art needs to be solved urgently. Summary of the invention

[0006] The present invention aims to provide a highly active Clostridium perfringens phage RDP-CP-2310009, a phage preparation and its application in view of the deficiencies in the prior art. The present invention can greatly improve the titer of the phage by optimizing the phage proliferation process, and according to the requirements of the use scenario, provides two phage preparation production processes of phage oil-in-water liquid preparation and phage-coated solid preparation, which can preserve the activity of the phage for a long time. The phage preparation production process provided by the present invention is simple to operate, high in efficiency, and low in production cost, which is conducive to the large-scale promotion of the application of Clostridium perfringens phage.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention provides a Clostridium perfringens bacteriophage RDP-CP-2310009, which was obtained from fresh feces collected from a laying hen farm in Qingdao, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 29, 2023. The storage address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the storage number is CGMCC No. 45779. The Clostridium perfringens CP-9.6 used in the experiment of the present invention was isolated from the cecum of enteritis chickens in a laying hen farm in Qingdao, and was preserved together with the Clostridium perfringens phage RDP-CP-2310009 of the present invention.

[0009] Through electron microscopic observation: the bacteriophage RDP-CP-2310009 has a polyhedral three-dimensional symmetrical head wrapped with nucleic acid, with a diameter of 40nm, a 30nm long tail, a tail sheath, and a neck connecting the head and tail. It belongs to the family Breviviridae of the order Caudovirales.

[0010] After the bacteriophage RDP-CP-2310009 was placed at 60°C for 90 minutes, its titer remained at 10 5 pfu / mL, and the titer remained at 10 after being placed at 80°C for 90 min. 4 pfu / mL, indicating that the Clostridium perfringens phage RDP-CP-2310009 has good high temperature resistance; the lysis rate of the phage RDP-CP-2310009 is 93.66%, and it has a good lysis effect on the host bacteria; through the lysis experiment of the phage RDP-CP-2310009 on the host bacteria, it can be seen that within 450 minutes, the phage can effectively inhibit the growth of the host bacteria, and the inhibition time is long.

[0011] In a second aspect, based on the same inventive concept, the present invention provides a phage composition, comprising the Clostridium perfringens phage RDP-CP-2310009 as described above.

[0012] In a third aspect, based on the same inventive concept, the present invention provides the use of the Clostridium perfringens phage RDP-CP-2310009 or the phage composition as described above in at least one of the following (1) to (4):

[0013] (1) Preparing a product for killing Clostridium perfringens;

[0014] (2) preparing products that inhibit Clostridium perfringens;

[0015] (3) Preparation of products for preventing and / or treating poultry diseases caused by Clostridium perfringens;

[0016] (4) Preparation of products for preventing and / or treating inflammatory responses caused by Clostridium perfringens.

[0017] In a fourth aspect, based on the same inventive concept, the present invention provides an industrial production process of Clostridium perfringens bacteriophage RDP-CP-2310009, comprising the following steps:

[0018] Step 1: Seed preparation: host bacteria seed preparation; phage seed preparation;

[0019] Step 2, phage proliferation: the host bacteria seeds prepared in step 1 are inoculated in a fermenter, cultured anaerobically, the pH is controlled at 7.0-7.5, then the phage seeds are inoculated, and then feeding is performed. After the feeding is completed, the culture is continued for 2-3 hours to terminate the proliferation; preferably, the host bacteria seeds prepared in step 1 are inoculated in a fermenter, cultured anaerobically, the pH is controlled at 7.2, then the phage seeds are inoculated, and then feeding is performed. After the feeding is completed, the culture is continued for 2-3 hours, during which the pH is controlled above 7.2 to terminate the proliferation; the most Preferably, the prepared host bacteria seeds are inoculated in a 200L fermenter at a volume ratio of 2%, and anaerobically cultured at 40°C, during which the pH is controlled at 7.2 using a 40% sodium hydroxide solution. After culturing for about 80 minutes, when the OD600nm is between 0.8 and 0.9, phage seeds are inoculated at a volume ratio of 2%, and then fed at a rate of 50 mL / min, and the temperature is raised to 42°C. After the feeding is completed, the culture is continued for 2 to 3 hours, during which the pH is controlled at 7.2 to 7.5 to terminate the proliferation.

[0020] Step 3, phage post-treatment process: centrifuge the phages proliferated in step 2, filter out bacteria and bacterial fragments using a glass fiber membrane, add a deoxidizer, and then use three-stage filtration for sterilization to obtain a phage proliferation liquid after filtration and sterilization. Preferably, the glass fiber membrane uses an 800nm ​​glass fiber membrane. Preferably, the deoxidizer is vitamin C. Preferably, the three-stage filtration is performed by filtering through polypropylene 0.45μm, polyethersulfone 0.45μm, and polyethersulfone 0.22μm in sequence.

[0021] In a fifth aspect, based on the same inventive concept, the present invention provides a phage preparation, the active ingredients of which include the Clostridium perfringens phage RDP-CP-2310009 as described above or the phage proliferation liquid prepared by the industrial production process of the Clostridium perfringens phage RDP-CP-2310009 as described above.

[0022] The phage preparation as described above is in the form of a phage water-in-oil liquid preparation, a phage coated solid preparation or a phage freeze-dried preparation.

[0023] In the sixth aspect, based on the same inventive concept, the present invention provides a method for preparing the phage oil-in-water dosage liquid preparation as described above, comprising the following steps: mixing the sterilized phage proliferation liquid with oregano oil and cinnamaldehyde, and then adding sodium dodecyl sulfate, placing the mixed material in an emulsifier, and performing high-pressure homogenization and emulsification to obtain the phage oil-in-water dosage liquid preparation.

[0024] In the preparation method of the bacteriophage water-in-oil liquid preparation as described above, the amount of oregano oil added is 0.2% to 0.5% (v / v), the amount of cinnamaldehyde added is 0.1% to 0.3% (v / v), and the amount of sodium dodecyl sulfate added is 0.4% to 0.8% (w / v). Preferably, the amount of oregano oil added is 0.3% (v / v), the amount of cinnamaldehyde added is 0.2% (v / v), and the amount of sodium dodecyl sulfate added is 0.5% (w / v).

[0025] In a seventh aspect, based on the same inventive concept, the present invention provides a method for preparing the phage-coated solid preparation as described above, comprising the following steps:

[0026] (1) Adding a carrier: adding acidified starch and pyrrolidone K-30 to the phage proliferation solution after filtration and sterilization, and stirring until completely dissolved; preferably, the amount of acidified starch added is 8% (w / w), and the amount of pyrrolidone K-30 added is 2% (w / w);

[0027] (2) Concentration: The above materials are concentrated using a vacuum ultra-low temperature concentrator to a solid content of about 60% at a concentration temperature of ≤45°C;

[0028] (3) Drying: Drying the concentrated material in step (2) using a vacuum belt dryer, wherein the temperature of the material during the drying process is ≤45°C and the material is concentrated to a moisture content of ≤6%;

[0029] (4) Crushing: After the material is dried, crush it and pass it through a 60-mesh sieve to collect the bacteriophage dry powder;

[0030] (5) Dry granulation: weigh the bacteriophage dry powder, corn starch, carboxymethyl cellulose and chitosan materials in proportion, place them in a mixer and mix them, place the mixed materials in a dry granulator, extrusion granulate, and screen pore size is 20 mesh to obtain bacteriophage particles; preferably, according to the mass fraction of 20% bacteriophage dry powder (w / w), 77% (w / w) corn starch, 2% (w / w) carboxymethyl cellulose, 1% (w / w) chitosan, weigh the materials, place them in a mixer, mix for 5 minutes, place the mixed materials in a dry granulator, extrusion granulate, and screen pore size is 20 mesh to obtain bacteriophage particles;

[0031] (6) Coating: The phage particles obtained in step (5) are placed in a coating machine and coated with stearic acid to obtain a phage-coated solid preparation; preferably, the amount of stearic acid used is 3-4%.

[0032] In an eighth aspect, based on the same inventive concept, the present invention provides the use of the phage preparation as described above in the following (1) or (2):

[0033] (1) Preparation of products for preventing and / or treating poultry diseases caused by Clostridium perfringens;

[0034] (2) Preparation of products for preventing and / or treating inflammatory reactions caused by Clostridium perfringens.

[0035] The beneficial effects of the present invention are:

[0036] 1. The Clostridium perfringens phage RDP-CP-2310009 provided by the present invention has excellent biological properties, and has the advantages of high titer, good thermal stability and high lysis rate. It can effectively treat enteritis caused by Clostridium perfringens in livestock and poultry breeding, and has potential application value.

[0037] 2. The Clostridium capsulatum phage RDP-CP-2310009 provided by the present invention can increase the titer of the phage by 100-1000 times by controlling the pH, feeding supplementary culture medium, temperature control and inoculation timing during the proliferation process.

[0038] 3. In the respective preparation processes of the phage water-in-oil liquid preparation (i.e., emulsion) and the phage coated solid preparation provided by the present invention, by adding a carrier and a protective agent, the two prepared phage preparations have good stability during storage.

[0039] 4. The phage oil-in-water dosage liquid preparation prepared by the Clostridium phage RDP-CP-2310009 of the present invention can effectively improve the stability of the phage at room temperature through an emulsification process. In particular, the phage preparation prepared by the Clostridium phage RDP-CP-2310009 has better storage stability than the phage preparation prepared by the phage RDP-CP-2310007, and can effectively ensure the activity of the phage preparation during transportation or storage.

[0040] 5. The phage-coated solid preparation prepared by the Clostridium capsular phage RDP-CP-2310009 of the present invention can increase the tolerance to gastric acid, choline and digestive enzymes through drying, granulation and coating, so that it can smoothly reach the small intestine to exert its effect and maximize the function of the phage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 An electron microscope photo of the Clostridium perfringens phage RDP-CP-2310009 provided by the present invention;

[0042] Figure 2 The thermal stability curve of Clostridium perfringens phage RDP-CP-2310009 provided by the present invention;

[0043] Figure 3 A one-step growth curve of the Clostridium perfringens phage RDP-CP-2310009 provided by the present invention;

[0044] Figure 4 The lysis curve of the Clostridium perfringens phage RDP-CP-2310009 provided by the present invention against the host bacteria CP-9.6;

[0045] Figure 5 The antibacterial curve of the Clostridium perfringens phage RDP-CP-2310009 provided by the present invention;

[0046] Figure 6 Comparison of stability curves of the Clostridium perfringens phage RDP-CP-2310009 and the Clostridium perfringens phage RDP-CP-2310007 provided by the present invention and the emulsions prepared therefrom;

[0047] Figure 7The invention discloses a storage stability curve of the Clostridium perfringens phage RDP-CP-2310009 and the Clostridium perfringens phage RDP-CP-2310007 provided by the invention and the freeze-dried powder and coated solid preparation prepared therefrom. DETAILED DESCRIPTION

[0048] 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.

[0049] In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by technicians in the technical field of the present invention. In the present invention, Clostridium perfringens CP-9.6 may be referred to as "host CP-9.6" or "CP-9.6", Clostridium perfringens phage RDP-CP-2310009 may be referred to as "phage RDP-CP-2310009" or "RDP-CP-2310009", Clostridium perfringens phage RDP-CP-2310007 may be referred to as "phage RDP-CP-2310007" or "RDP-CP-2310007".

[0050] Example 1 Isolation and identification of bacteriophage RDP-CP-2310009

[0051] 1. Isolation and identification of pathogenic Clostridium perfringens CP-9.6

[0052] The cecum of chickens with enteritis in a laying hen farm in Qingdao was collected, the cecum was cut into pieces on a clean bench, placed in FTG culture medium, and the enrichment solution was placed in an anaerobic incubator at 42°C for 8 to 10 hours. After enrichment, about 20 μL of fresh bacterial culture solution was aspirated and inoculated on TSC culture medium by streaking method, and then the black colonies on TSC were purified using 5% sheep blood plate. Finally, the strain that can produce double hemolytic rings on the blood plate, is a Gram-positive coarse and short bacillus under a microscope, and can produce the phenomenon of "violent fermentation" of milk was preliminarily identified as suspected Clostridium perfringens. PCR amplification was performed using 16SRNA universal primers. After successful amplification, the PCR product was purified and sequenced, and it was finally determined to be Clostridium perfringens. One of the strains was named "CP-9.6" and stored in a -80°C refrigerator with 20% glycerol.

[0053] 2. Isolation and identification of Clostridium perfringens phage RDP-CP-2310009

[0054] (1) Feces treatment: Fresh feces were collected from a laying hen farm in Qingdao. About 5 g of feces was mixed with 30 mL of physiological saline and placed in an anaerobic incubator at 42 °C for 4 to 6 hours. The feces was then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter and the filtered supernatant was collected for later use.

[0055] (2) Preparation of mixed bacterial suspension: 0.2 mL of bacterial suspension and 0.3 mL of filtered supernatant were added to 5 mL of liquid culture medium, cultured at 43°C for 12 to 16 h under anaerobic conditions, and then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter and the filtered supernatant was collected for later use;

[0056] The liquid culture medium of the present invention comprises the following components: 3.0% yeast extract powder, 0.5% yeast peptone, 1.0% glucose, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20ppm calcium chloride, 20ppm magnesium chloride, pH 8.0, 121°C, and sterilization for 30min.

[0057] (3) Phage isolation: Phages were isolated by double plate method. 0.2 mL of the filtered supernatant of the mixed bacterial suspension was mixed with 0.2 mL of the host Clostridium perfringens CP-9.6 bacterial suspension, and then the mixture was evenly mixed. The mixture was incubated at 43°C for 10 min, and then spread on double plates. The mixture was incubated at 43°C for 12 to 16 h under anaerobic conditions, and the results were observed. If there were phages, there would be transparent plaques on the plates. A single plaque was picked up and placed in 1 mL of physiological saline, and then incubated at 43°C for 30 min. The plaque was centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter. 0.1 mL of the filtrate and 0.2 mL of the CP-9.6 bacterial suspension were taken and bathed at 43°C for 10 min. The mixture was spread on double plates. The plaques were incubated at 43°C for 12 to 16 h under anaerobic conditions. The plaques were purified 3 to 4 times according to the above steps until the plaques were uniform in size. A phage strain was obtained, which was named RDP-CP-2310009. The diameter of the plaques formed on the double plates was 2mm-3mm.

[0058] 3. Preservation of bacteriophages

[0059] The product is preserved by vacuum freeze-drying, wherein the protective agent is 20% (w / v) skimmed milk powder and 2% (w / v) mannitol, 78% purified water, which are sterilized and mixed with the phage proliferation liquid at a ratio of 1:1 (v / v), and then vacuum freeze-dried. The dried ampoule is evacuated and sealed.

[0060] The Clostridium perfringens bacteriophage RDP-CP-2310009 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 29, 2023. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45779.

[0061] Example 2 Morphological observation of bacteriophage RDP-CP-2310009

[0062] 1. Experimental methods:

[0063] Using the phosphotungstic acid negative staining method: drop phage solution (titer 10) on the paraffin slide. 9 pfu / mL), place the copper mesh with the membrane on the phage droplet, remove it after 10 minutes, dry it in the air for 2-3 minutes, then drop a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper mesh for staining, remove it after 10 minutes, dry it in the air for 10-15 minutes, observe it with an electron microscope, and select a clear phage image to take a picture. The results are as follows Figure 1 shown.

[0064] 2. Experimental results:

[0065] from Figure 1 It can be seen that the bacteriophage has a polyhedral, three-dimensionally symmetrical head that wraps nucleic acid, has a diameter of 40nm, a 30nm long tail, and a tail sheath. The neck connects the head and the tail, and belongs to the family Breviviridae of the order Caudovirales.

[0066] Example 3 Bacteriophage RDP-CP-2310009 genome sequencing

[0067] Through the whole gene analysis, the gene size is about 18.7kp, contains 3 lytic enzyme genes, and the phage does not contain virulence genes, drug resistance genes and lysogeny, which proves the safety of the phage at the molecular level. The sequence of the phage is shown in SEQ ID No. 1.

[0068] Example 4 Thermal stability experiment of bacteriophage RDP-CP-2310009

[0069] 1. Experimental methods:

[0070] The phage stock solution was divided into 50 mL sterile conical bottles and incubated at 60°C and 80°C, respectively. Samples were taken every 10 minutes, and then diluted 10 times with normal saline, and double plates were spread to determine the titer. The experiment was performed in parallel for 3 times, and the results were averaged. The results are shown in Figure 2 shown.

[0071] 2. Experimental results:

[0072] from Figure 2 It can be seen that after the bacteriophage RDP-CP-2310009 was placed in a 60℃ environment for 90 minutes, its titer remained at 10 5 pfu / mL, and the titer remained at 10 after being placed at 80°C for 90 min. 4 pfu / mL, indicating that the Clostridium perfringens phage RDP-CP-2310009 has good high temperature resistance, which is of great reference value in the production, dosage form preparation, transportation and storage of phages.

[0073] Example 5 Optimal multiplicity of infection of bacteriophage RDP-CP-2310009

[0074] 1. Experimental methods:

[0075] The phage proliferation solution and the host were added to the liquid culture medium at a multiplicity of infection of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, and the total volume of the culture system was ensured to be the same. Under anaerobic conditions, the culture was carried out at 43°C for 6 hours, and then centrifuged at 12000r / min for 5 minutes at room temperature. The supernatant was spread on two plates to determine its titer. The results are shown in Table 1.

[0076] 2. Experimental results:

[0077] As shown in Table 1, when the infection multiplicity was 0.1, after 6 h of culture, the proliferation fluid was relatively clear and the titer was the highest, indicating that the optimal infection multiplicity of bacteriophage RDP-CP-2310009 was 0.1, and the corresponding highest titer was 6.93×10 10 pfu / mL.

[0078] Table 1 Optimal infection multiplicity results of bacteriophage RDP-CP-2310009

[0079]

[0080] Example 6 Determination of the one-step growth curve of bacteriophage RDP-CP-2310009

[0081] 1. Experimental methods:

[0082] Take a suspension of Clostridium perfringens in the logarithmic growth phase, inoculate Clostridium perfringens CP-9.6 and bacteriophage RDP-CP-2310009 according to the ratio of the optimal infection multiplicity, place in a water bath at 43°C for 10 minutes, and then centrifuge at 12000r / min at room temperature for 5 minutes, discard the supernatant, remove the free phage that is not adsorbed on the host, and wash twice with liquid culture medium. Resuspend the precipitate in a liquid culture medium at 43°C, quickly place it at 43°C, culture it anaerobically and start timing. Take samples at regular intervals to determine its titer, and draw a one-step growth curve with time as the horizontal axis and the logarithm of the phage titer as the vertical axis. The incubation period and lysis period of the phage are thus obtained, and the average lysis amount is calculated. The average lysis amount is calculated as follows: Average lysis amount = phage titer at the end of the outbreak / host bacteria concentration at the beginning of infection. The results are as follows: Figure 3 shown.

[0083] 2. Experimental results:

[0084] Depend on Figure 3 It can be seen that the titer of the phage did not change significantly within 50 minutes after the phage infected the host bacteria, indicating that its incubation period was about 50 minutes. The titer of the phage increased significantly within 50 to 110 minutes after infection, and then stabilized. By calculation, the phage lysis amount was about 196 PFU / infected cell, indicating that the phage RDP-CP-2310009 has the characteristics of short incubation period, strong lysis ability and high replication.

[0085] Example 7 Lysis experiment of bacteriophage RDP-CP-2310009 on host bacteria

[0086] 1. Experimental methods:

[0087] The host Clostridium perfringens CP-9.6 bacterial suspension cultured for 6 hours was inoculated into 100 mL of liquid culture medium at a ratio of 2:100 (v / v) and cultured anaerobically at 43°C for 2 hours to obtain a bacterial suspension of about 5.0×10 8 cfu / mL. The bacterial suspension was divided into 4 sterilized conical flasks, and 0.2 mL of phage proliferation solution was added to 3 of them at the optimal multiplicity of infection. Figure 4 The host cells were recorded as "CP-9.6 and phage RDP-CP-2310009" and the cells without phage proliferation solution were used as controls. Figure 4 The results are as follows: Figure 4 shown.

[0088] 2. Experimental results:

[0089] Depend on Figure 4It can be seen that when the Clostridium perfringens phage RDP-CP-2310009 was added to the host bacterial suspension of CP-9.6, because the phage has a strong lysis effect on the host, the OD600 absorbance gradually increased after 450 minutes. This shows that within 450 minutes, the phage can effectively inhibit the growth of the host bacteria, which has a very important application value in the treatment of animal diseases.

[0090] Example 8 Lysis rate experiment of bacteriophage RDP-CP-2310009

[0091] 1. Experimental methods:

[0092] Under sterile conditions, take 1 mL of sample and 1 mL of host bacterial solution (1×10 5 cfu / mL), incubated at 43°C for 15 min, mixed and diluted to 10 with saline. -1 ~10 -4 Each gradient was plated with 100 μL of each gradient on LB agar plate and incubated at 43°C for 12 to 16 hours. Each gradient was repeated twice. At the same time, 1 mL of saline and 1 mL of host bacterial solution (1×10 5 cfu / mL) as a blank control, repeat the above steps. Select a plate with 30 to 300 colonies for counting. Repeat the test 3 times and take the average value.

[0093] Phage lysis rate = (1-number of colonies in the treatment group / number of colonies in the control group) × 100%

[0094] 2. Experimental results:

[0095] Experiments show that the lysis rate of bacteriophage RDP-CP-2310009 is as high as 93.66%, which has a good lysis effect on host bacteria and is suitable for use in aquaculture.

[0096] Example 9 Industrial production process of bacteriophage RDP-CP-2310009

[0097] This embodiment is an industrial production process of Clostridium perfringens phage RDP-CP-2310009, comprising the following steps:

[0098] Step 1: Seed preparation

[0099] (1) Preparation of host bacteria seeds: a single colony was picked up under a sterile environment and inoculated into 5 mL of liquid culture medium. After culturing at 43°C for 4 to 6 hours under anaerobic conditions, the cultured host was inoculated into 100 mL of liquid culture medium at a 5% inoculum. After culturing at 43°C for 4 to 6 hours under anaerobic conditions, the cultured host was inoculated into 1000 mL of liquid culture medium at a 5% inoculum. After culturing at 43°C for 4 to 6 hours under anaerobic conditions, the cultured host seed solution was placed in a 4°C refrigerator for standby use;

[0100] (2) Preparation of phage seeds: The stored phage RDP-CP-2310009 seeds were appropriately diluted and the dilution was 10 5-8 200 μL of the double dilution solution and 200 μL of the host bacteria were placed in the upper culture medium, poured into a double-layer plate, and cultured at 43°C under anaerobic conditions for 12 to 16 hours. The plate with cloudy plaques was selected, and the upper culture medium was washed with 5 mL of liquid culture medium, then centrifuged at 12000 rpm for 10 min, filtered through a 0.22 μm filter membrane, and the clear liquid was collected.

[0101] The clear liquid and host bacteria seeds were inoculated into 50 mL of liquid culture medium at a ratio of 2%, respectively. Under anaerobic conditions, the cultured phage and host were cultured at 43°C for 6 to 8 hours. Then, the cultured phage and host were inoculated into 1000 mL of liquid culture medium at a 2% inoculation rate. Under anaerobic conditions, the cultured phage seeds were placed in a 4°C refrigerator for standby use after being cultured at 43°C for 4 to 6 hours.

[0102] Liquid culture medium ingredients: yeast extract powder 3.0%, yeast peptone 0.5%, glucose 1.0%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.2%, dipotassium hydrogen phosphate 0.2%, calcium chloride 20ppm, magnesium chloride 20ppm, pH 8.0, 121℃, sterilization for 30min.

[0103] Step 2: Phage proliferation:

[0104] The prepared host bacteria seeds were inoculated in a 200L fermentation tank at a volume ratio of 2%, and anaerobically cultured at 40°C. During the culture, a 40% sodium hydroxide solution was used to control the pH at 7.2. After culturing for about 80 minutes, when the OD600nm was 0.8-0.9, phage seeds were inoculated at a volume ratio of 2%, and then fed at a rate of 50mL / min, and the temperature was raised to 42°C. After the feeding was completed, the culture was continued for 2-3 hours, during which the pH was controlled at 7.2-7.5 to terminate the proliferation.

[0105] The fermentation tank culture medium components in this step are: yeast extract powder 1.0%, yeast peptone 0.5%, glucose 0.5%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.2%, dipotassium hydrogen phosphate 0.2%, calcium chloride 20ppm, magnesium chloride 20ppm, pH 8.0, 121°C, 30min sterilization, liquid volume 150L.

[0106] The alkali solution in this step: 40% sodium hydroxide solution by mass, 121°C, sterilization for 30 minutes, about 10L.

[0107] In this step, the feed was supplemented with 10% yeast extract powder, 2% peptone, sterilized at 121°C for 30 min, a total of 10 L.

[0108] Step 3: Phage post-processing process

[0109] (1) Centrifugation: The proliferated phages are centrifuged at 6000 rpm in a butterfly centrifuge to remove unlysed host and bacterial debris.

[0110] (2) Use 800nm ​​glass fiber membrane to filter out bacteria and bacterial fragments.

[0111] (3) Add vitamin C at a mass ratio of 0.2 to 0.3% as a deoxidizing agent.

[0112] (4) Filtration and sterilization: The phage is sterilized by three-stage filtration, i.e., filtering through polypropylene 0.45 μm, polyethersulfone 0.45 μm, and polyethersulfone 0.22 μm in sequence to obtain a phage proliferation liquid after filtration and sterilization.

[0113] Example 10 Preparation of liquid preparation of bacteriophage RDP-CP-2310009

[0114] 1. Preparation of bacteriophage liquid preparation:

[0115] The phage proliferation liquid after filtration and sterilization in Example 9 was mixed with 0.3% (v / v) of oregano oil and 0.2% (v / v) of cinnamaldehyde, and then 0.5% (w / v) of sodium dodecyl sulfate was added. The mixed material was placed in an emulsifier and emulsified by high-pressure homogenization for 5 minutes to obtain a phage liquid preparation in an oil-in-water dosage form.

[0116] The preparation method of the bacteriophage liquid preparation of the RDP-CP-2310007 water-in-oil dosage form is the same as that of the bacteriophage liquid preparation of the RDP-CP-2310009 water-in-oil dosage form.

[0117] 2. Antibacterial experiment of bacteriophage liquid preparation:

[0118] The host strain CP-9.6 was used as the control ( Figure 5) and compared the phage RDP-CP-2310009 proliferation solution after filtration and sterilization ( Figure 5 "phage RDP-CP-2310009 + host CP-9.6"), oregano oil and cinnamaldehyde (mixed in a volume ratio of 3:2) Figure 5 in the form of “oregano oil + cinnamaldehyde + host CP-9.6”), a water-in-oil formulation of bacteriophage liquid preparation ( Figure 5 The inhibitory effect of phage RDP-CP-2310009 + oregano oil + cinnamaldehyde + host CP-9.6) on host bacteria CP-9.6 was recorded in the figure. After the above samples were diluted 100 times, 100 μL of the sample and 100 μL of host bacteria CP-9.6 were added to 5 mL of anaerobic liver broth medium, and 1 mL of sterilized paraffin was added on top. The colorimetric tube with the sample was placed in a microbial growth instrument at 43°C and 50 rpm, and the changes in OD600 were continuously recorded. The results are shown in the figure. Figure 5 shown.

[0119] from Figure 5 It can be seen that through the four findings, in the oil-in-water formulation of phage liquid preparations, phage RDP-CP-2310009, oregano oil and cinnamaldehyde play a synergistic antibacterial role. The oil-in-water formulation of phage liquid preparations has a longer inhibition time on the host bacteria and can effectively reduce the concentration of the host bacteria, which has a better effect in the treatment of animal diseases.

[0120] 3. Stability test of bacteriophage liquid preparation in water-in-oil dosage form:

[0121] The liquid phage (titer ≥ 10 10 pfu / mL) and the oil-in-water formulation of phage liquid preparations were compared for stability. After the samples were prepared, they were placed at room temperature and their titers were measured regularly. Figure 6 shown. Figure 6 In the present invention, the proliferation fluid of bacteriophage RDP-CP-2310007 after filtration and sterilization is recorded as "bacteriophage RDP-CP-2310007"; the phage liquid preparation in oil-in-water dosage form prepared from bacteriophage RDP-CP-2310007 is recorded as "emulsion: bacteriophage RDP-CP-2310007"; the proliferation fluid of bacteriophage RDP-CP-2310009 after filtration and sterilization is recorded as "bacteriophage RDP-CP-2310009"; the phage liquid preparation in oil-in-water dosage form prepared from bacteriophage RDP-CP-2310009 is recorded as "emulsion: bacteriophage RDP-CP-2310009".

[0122] In this example, the Clostridium perfringens bacteriophage RDP-CP-2310007 used was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 29, 2023. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45777.

[0123] from Figure 6 It can be seen that within 240 days, the potency of the phage liquid preparation in oil-in-water dosage form prepared by phage RDP-CP-2310007 decreased from the initial 10.29 lgpfu / mL to 7.39 lgpfu / mL, but the potency of the phage liquid preparation in oil-in-water dosage form prepared by phage RDP-CP-2310009 decreased from the initial 10.38 lgpfu / mL to only 9.76 lgpfu / mL. Through comparison, it was found that the phage liquid preparation in oil-in-water dosage form, that is, the phage after emulsification, has better stability than the phage; compared with the phage liquid preparation in oil-in-water dosage form RDP-CP-2310009 of Clostridium perfringens phage and the phage liquid preparation in oil-in-water dosage form RDP-CP-2310007, the phage liquid preparation in oil-in-water dosage form RDP-CP-2310009 has better storage stability, has high stability, high activity and easy storage during the application and use of the product, which is more conducive to the use and promotion of the product.

[0124] Example 11 Preparation of solid preparation of bacteriophage RDP-CP-2310009

[0125] 1. Preparation of phage-coated solid preparations:

[0126] (1) Adding a carrier: Add 8.0% (w / w) acidified starch and 2.0% (w / w) pyrrolidone K-30 to the phage proliferation solution after filtration and sterilization in Example 9, and stir until completely dissolved;

[0127] (2) Concentration: Use a vacuum ultra-low temperature concentrator to concentrate the above materials to a solid content of about 60.0% and a concentration temperature of ≤45.0°C;

[0128] (3) Drying: The concentrated material is dried using a vacuum belt dryer. During the drying process, the material temperature is ≤45.0°C and the moisture content is ≤6.0%;

[0129] (4) Crushing: After the material is dried, crush it and pass it through a 60-mesh sieve to collect the bacteriophage dry powder.

[0130] (5) Dry granulation:

[0131] According to the mass fraction of 20% bacteriophage dry powder (w / w), 77% (w / w) corn starch, 2% (w / w) carboxymethyl cellulose, and 1% (w / w) chitosan, the materials are weighed and placed in a mixer and mixed for 5 minutes. The mixed materials are placed in a dry granulator and extruded and granulated with a sieve aperture of 20 mesh to obtain bacteriophage particles.

[0132] (6) Coating:

[0133] The phage particles obtained by extrusion and screening were placed in a coating machine and coated with stearic acid at a dosage of 4%, thereby obtaining a coated solid preparation of bacteriophage RDP-CP-2310009.

[0134] The preparation method of the bacteriophage RDP-CP-2310007 coated solid preparation is the same as the preparation method of the bacteriophage RDP-CP-2310009 coated solid preparation.

[0135] 2. The permeability of bacteriophage solid preparations in the digestive tract

[0136] Take 0.45g of each of the above two phage-coated solid preparation samples and place them in 45mL of gastric juice and intestinal juice respectively, place them at 37°C, and stir them continuously during the period. Take samples every 10 minutes, adjust the pH to neutral with buffer immediately after sampling, and detect the phage titer. After the sample is diluted 10 times in a gradient, spread on a double-layer plate and measure its titer. The gastric juice and intestinal juice of this example were purchased from Sinopharm Chemical Reagent Co., Ltd. The results are shown in Tables 2 and 3.

[0137] Table 2 Results of potency determination of two phage-coated solid preparations in gastric juice

[0138]

[0139] As can be seen from Table 2, the phage RDP-CP-2310009 coated solid preparation was placed in gastric juice for 10 minutes and slightly swollen. After the clear liquid was neutralized and tested, no phage was detected, indicating that the coated solid preparation did not release phage after coating. The coating material on the surface of the particles can effectively protect the phage and prevent damage from gastric juice. The phage RDP-CP-2310007 coated solid preparation is rapidly released after swelling and exposed to gastric juice. As the action time increases, the potency gradually decreases. After 60 minutes, the phage activity is completely lost. By comparing the above two phages, it can be shown that phage RDP-CP-2310009 is more suitable for coating than phage RDP-CP-2310007, and can effectively avoid the influence of gastric acid on phage activity.

[0140] As can be seen from Table 3, due to the presence of digestive enzymes and choline in the intestinal fluid, the coated solid preparation swelled after 30 minutes of contact with the intestinal fluid, and continuous sampling was performed to detect its potency, and the results are shown in Table 3. The phage RDP-CP-2310009 coated solid preparation can effectively avoid the destruction of the phage by the digestive fluid when passing through the digestive tract, and the phage reaches the small intestine to exert its function, maximizing the function of the phage. Compared with the phage RDP-CP-2310007 coated solid preparation, the phage RDP-CP-2310009 coated solid preparation has better stability in the intestine and can better maintain its activity.

[0141] Table 3 Potency test results of two phage-coated solid preparations in intestinal fluid

[0142]

[0143] 3. Storage stability experiment of bacteriophage solid preparation and lyophilized powder:

[0144] The preparation method of phage freeze-dried powder is as follows: using conventional freeze-drying process, adding 10% (w / v) skim milk powder, 3% (w / v) trehalose, 3% (w / v) pyrrolidone k-30 and 0.5% (w / v) Vc as protective agents, and freeze-drying to obtain phage freeze-dried powder.

[0145] The above obtained phage coated solid preparation and phage freeze-dried powder were subjected to storage stability test. The samples were placed at room temperature and their potency was measured regularly. The results were as follows: Figure 7 shown. Figure 7 In the present invention, the lyophilized powder prepared from bacteriophage RDP-CP-2310007 is recorded as “lyophilized powder: RDP-CP-2310007”, and the phage-coated solid preparation prepared from bacteriophage RDP-CP-2310007 is recorded as “coating: RDP-CP-2310007”; the lyophilized powder prepared from bacteriophage RDP-CP-2310009 is recorded as “lyophilized powder: RDP-CP-2310009”, and the phage-coated solid preparation prepared from bacteriophage RDP-CP-2310009 is recorded as “coating: RDP-CP-2310009”.

[0146] from Figure 7 It can be seen that within 240 days, the potency of the lyophilized powder prepared by phage RDP-CP-2310007 increased from the initial 10 9.62 pfu / g decreased to 10 8.36 pfu / g, the potency of the phage-coated solid preparation prepared by phage RDP-CP-2310007 increased from the initial 10 9.36 pfu / g decreased to 10 8.59pfu / g, but the titer of the lyophilized powder prepared by bacteriophage RDP-CP-2310009 increased from the initial 10 9.96 pfu / g only dropped to 10 9.19 pfu / g, the potency of the phage-coated solid preparation prepared by phage RDP-CP-2310009 increased from the initial 10 9.79 pfu / g only dropped to 10 9.49 pfu / g. Through both, whether it is lyophilized powder or phage-coated solid preparation, bacteriophage RDP-CP-2310009 has better stability, and the activity of RDP-CP-2310009 is better maintained during storage at room temperature. Moreover, the coated solid preparation prepared by bacteriophage RDP-CP-2310009 has better storage stability than the lyophilized powder prepared by bacteriophage RDP-CP-2310009. This is because the coated solid preparation can isolate the air due to the coating on the surface, preventing the particles from absorbing water and moisture and air oxidation, making the bacteriophage relatively stable. Because the coating process is carried out at a relatively low temperature, and the bacteriophage itself has a good tolerance to temperature, there is a layer of oil on the surface of the embedded phage, which can effectively isolate moisture and oxygen in the air, making the product more stable. And the cost of coated solid preparations is lower, which is more conducive to the promotion and application of products.

[0147] Comparative Example 1: Industrial production process of bacteriophage RDP-CP-2310009 without pH control and feed supplement

[0148] The only difference from Example 9 is that the steps in "Step 2, phage proliferation" are as follows: the prepared host bacteria seeds are inoculated in a 200L fermenter at a volume ratio of 2%, and cultured anaerobically at 40°C. After culturing for about 60 minutes, when the OD600nm is 0.8-0.9, phage seeds are inoculated at a volume ratio of 2%, and cultured for 3-4 hours to end the proliferation.

[0149] The titers of the phage proliferation fluids obtained in Comparative Example 1 and Example 9 were measured. The results showed that the titer of the phage proliferation fluid prepared in Comparative Example 1 without controlling pH and without feeding was 6.6×10 7 pfu / mL, while the phage titer of Example 9 reached 3.6×10 10 This indicates that during the phage proliferation process, controlling pH and supplementing nutrients in a timely manner are crucial to improving the titer of phages.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Clostridium perfringens bacteriophage RDP-CP-2310009, characterized in that: Its deposit number is CGMCC No.45779, and it was deposited in the General Microbiology Center of China Culture Collection Administration on November 29, 2023.

2. A bacteriophage composition, characterized in that: It comprises the Clostridium perfringens phage RDP-CP-2310009 as claimed in claim 1.

3. Use of the Clostridium perfringens phage RDP-CP-2310009 according to claim 1 or the phage composition according to claim 2 in at least one of the following (1) to (4): (1) Preparing a product for killing Clostridium perfringens; (2) preparing products that inhibit Clostridium perfringens; (3) Preparation of products for preventing and / or treating poultry diseases caused by Clostridium perfringens; (4) Preparation of products for preventing and / or treating inflammatory responses caused by Clostridium perfringens.

4. The industrial production process of Clostridium perfringens bacteriophage RDP-CP-2310009 according to claim 1, characterized in that: The following steps are involved: Step 1: Seed preparation: host bacteria seed preparation; phage seed preparation; Step 2, phage proliferation: inoculate the host bacteria seeds prepared in step 1 into a fermentation tank, culture anaerobically, control the pH at 7.0-7.5, then inoculate the phage seeds, and then feed, and culture for another 2-3 hours after the feeding is completed to end the proliferation; Step 3, phage post-processing process: centrifuge the phages proliferated in step 2, filter out bacteria and bacterial fragments using a glass fiber membrane, add a deoxidizer, and then perform three-stage filtration for sterilization to obtain a phage proliferation solution after filtration and sterilization.

5. A bacteriophage preparation, characterized in that: The effective ingredients include the Clostridium perfringens phage RDP-CP-2310009 as claimed in claim 1 or the phage proliferation liquid prepared by the industrial production process of the Clostridium perfringens phage RDP-CP-2310009 as claimed in claim 4.

6. The bacteriophage preparation according to claim 5, characterized in that The dosage form of the bacteriophage preparation is a bacteriophage water-in-oil liquid preparation, a bacteriophage coated solid preparation or a bacteriophage freeze-dried preparation.

7. A method for preparing the bacteriophage water-in-oil liquid preparation according to claim 6, characterized in that: The following steps are involved: The sterilized phage proliferation liquid is mixed with oregano oil and cinnamaldehyde, and then sodium dodecyl sulfate is added. The mixed materials are placed in an emulsifier and homogenized and emulsified under high pressure to obtain a phage oil-in-water dosage liquid preparation.

8. The method for preparing the bacteriophage water-in-oil liquid preparation according to claim 7, characterized in that: The added amount of the oregano oil is 0.2% to 0.5% (v / v), the added amount of the cinnamaldehyde is 0.1% to 0.3% (v / v), and the added amount of the sodium dodecyl sulfate is 0.4% to 0.8% (w / v).

9. A method for preparing a phage-coated solid preparation as claimed in claim 6, characterized in that: The following steps are involved: (1) Adding carrier: Add acidified starch and pyrrolidone K-30 to the phage proliferation solution after filtration and sterilization, and stir until completely dissolved; (2) Concentration: The above materials are concentrated using a vacuum ultra-low temperature concentrator to a solid content of about 60% at a concentration temperature of ≤45°C; (3) Drying: Drying the concentrated material in step (2) using a vacuum belt dryer, wherein the temperature of the material during the drying process is ≤45°C and the material is concentrated to a moisture content of ≤6%; (4) Crushing: After the material is dried, crush it and pass it through a 60-mesh sieve to collect the bacteriophage dry powder; (5) Dry granulation: weigh bacteriophage dry powder, corn starch, carboxymethyl cellulose and chitosan materials in proportion, place them in a mixer, mix them, place the mixed materials in a dry granulator, extrude and granulate them, and use a sieve with a pore size of 20 mesh to obtain bacteriophage particles; (6) Coating: The phage particles obtained in step (5) are placed in a coating machine and coated with stearic acid to obtain a phage-coated solid preparation.

10. Use of the bacteriophage preparation according to any one of claims 5 to 6 in the following (1) or (2): (1) Preparation of products for preventing and / or treating poultry diseases caused by Clostridium perfringens; (2) Preparation of products for preventing and / or treating inflammatory reactions caused by Clostridium perfringens.

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