Clostridium perfringens bacteriophage RDP-CP-2310009 with high activity, bacteriophage preparation and application thereof

Abstract

The application belongs to the technical field of microorganisms. The application provides a Clostridium perfringens bacteriophage RDP-CP-2310009, the preservation number of which is CGMCC No. 45779, and the application was preserved in the China General Microbiological Culture Collection Center on November 29, 2023. The application further provides a bacteriophage composition. The application further provides application of the Clostridium perfringens bacteriophage RDP-CP-2310009 or the bacteriophage composition. The application further provides a bacteriophage preparation. The application further provides a preparation method of a water-in-oil type liquid preparation of the bacteriophage. The application further provides a preparation method of a bacteriophage coated solid preparation. The application further provides application of the bacteriophage preparation. The application greatly improves the titer of the bacteriophage by optimizing the proliferation process of the bacteriophage. The bacteriophage preparation provided by the application can preserve the activity of the bacteriophage for a long time. Moreover, the bacteriophage preparation is simple to operate, efficient, low in production cost and conducive to large-scale promotion.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a high-activity Clostridium perfringens bacteriophage RDP-CP-2310009, a bacteriophage preparation and application thereof. BACKGROUND

[0002] With the rapid scale development of poultry industry, bacterial diseases are increasingly prominent, which restricts the sustainable and healthy development of China's poultry industry. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacteria, which is widely distributed in soil, dust, animal production environment and feed, and mainly causes necrotic enteritis in poultry. The intestinal tract of infected chickens is fragile and full of gas, and the jejunum and ileum appear necrosis and form a pseudomembrane. The colonization rate of Clostridium perfringens in the intestinal tract is as high as 95%, thereby causing clinical or subclinical infection of necrotic enteritis, increasing the cost of chicken feeding, and causing serious economic losses. Usually, "growth-promoting" antibiotics such as bacitracin, lincomycin, tylosin and penicillin are added to feed to prevent Clostridium perfringens infection in chickens. However, with the rapid spread of antibiotic resistance, the use of antibiotics is prohibited. The prohibition of antibiotics may increase the rapid proliferation of Clostridium perfringens in poultry environment, resulting in reduced feed digestibility and absorption rate of chickens, slow weight gain, increased breeding cost, and ultimately increased burden on consumers. According to relevant estimates, poultry necrotic enteritis will cause more than 6 billion US dollars in huge losses to the world's poultry industry every year. Therefore, a safe, effective and sustainable solution to control Clostridium perfringens infection will effectively improve the economic benefits of the poultry industry.

[0003] Bacteriophages are bacterial viruses that can kill bacteria through specific lysis cycles. The specificity of bacteriophages makes them only attack target hosts without affecting the normal intestinal flora. Therefore, bacteriophages have good safety, and also make it possible to target bacterial infections. According to this biological characteristic of bacteriophages, bacteriophages can be used as antibacterial agents for treatment, and as one of the alternative options for antibiotic treatment.

[0004] However, the application of Clostridium perfringens bacteriophage is limited by the following two aspects: first, Clostridium perfringens is a strict anaerobic gram-positive bacterium, which can ferment nitrogen source to acid in its metabolic process, so that the pH of the proliferation solution decreases, and the growth characteristics of Clostridium perfringens affect the proliferation of bacteriophages; second, the effectiveness and practicability of bacteriophage preparation depend on the maintenance of its stability, but the titer of bacteriophages decreases during the process of logistics transportation and long-term storage. Therefore, there is a lack of Clostridium perfringens bacteriophage which can be efficiently proliferated and ensure the stability of bacteriophage preparation in the prior art.

[0005] Therefore, the prior art needs to be solved. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides a high-activity Clostridium perfringens bacteriophage RDP-CP-2310009, a bacteriophage preparation and application thereof. The present application can greatly improve the titer of the bacteriophage by optimizing the proliferation process of the bacteriophage, and according to the requirements of the use scene, the production process of the bacteriophage water-in-oil type liquid preparation and the bacteriophage coated solid preparation is provided, the activity of the bacteriophage can be preserved for a long time, the bacteriophage preparation production process provided by the present application is simple in operation, high in efficiency and low in production cost, and is conducive to large-scale promotion of the application of the Clostridium perfringens bacteriophage.

[0007] The above-mentioned purposes of the present application are realized by the following technical solutions.

[0008] In a first aspect, the present application provides a Clostridium perfringens bacteriophage RDP-CP-2310009, which is obtained from fresh manure collected from a certain egg farm in Qingdao, and is preserved in the China General Microbiological Culture Collection Center on November 29, 2023. The preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 45779. The Clostridium perfringens CP-9.6 used in the experiment of the present application is isolated from the cecum of enteritis chickens in a certain egg farm in Qingdao, and is preserved together with the Clostridium perfringens bacteriophage RDP-CP-2310009 of the present application.

[0009] Through electron microscope observation, the bacteriophage RDP-CP-2310009 has a polyhedral stereoscopic head, which wraps nucleic acid, has a diameter of 40 nm, has a tail of 30 nm, has a tail sheath, and has a neck connecting the head and the tail, which belongs to the short tail virus family of the Caudovirales order.

[0010] The titer of the bacteriophage RDP-CP-2310009 remains above 10 5 pfu / mL after being placed at 60℃ for 90 min, and the titer remains above 10 4 pfu / mL after being placed at 80℃ for 90 min, indicating that the Clostridium perfringens bacteriophage RDP-CP-2310009 has good high-temperature resistance; the lysis rate of the bacteriophage RDP-CP-2310009 reaches 93.66%, and the host bacteria have good lysis effect; through the lysis experiment of the host bacteria by the bacteriophage RDP-CP-2310009, it can be seen that the bacteriophage can effectively inhibit the growth of the host bacteria within 450 min, and the antibacterial time is long.

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

[0012] In a third aspect, based on the same inventive concept, the present application provides use of the Clostridium perfringens bacteriophage RDP-CP-2310009 as described above or the bacteriophage 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 a product for inhibiting Clostridium perfringens;

[0015] (3) preparing a product for preventing and / or treating avian diseases caused by Clostridium perfringens;

[0016] (4) preparing a product for preventing and / or treating inflammatory reactions caused by Clostridium perfringens.

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

[0018] Step one, seed preparation: host bacteria seed preparation; bacteriophage seed preparation;

[0019] Step two, bacteriophage propagation: inoculate the host bacteria seed prepared in step one into a fermenter, anaerobically culture, control the pH at 7.0 to 7.5, then inoculate the bacteriophage seed, then perform feeding, after the feeding is completed, culture for 2 to 3 hours, and end the propagation; preferably, inoculate the host bacteria seed prepared in step one into a fermenter, anaerobically culture, control the pH at 7.2, then inoculate the bacteriophage seed, then perform feeding, after the feeding is completed, culture for 2 to 3 hours, during which the pH is controlled to be above 7.2, and end the propagation; most preferably, inoculate the host bacteria seed prepared into a 200L fermenter at a volume ratio of 2%, anaerobically culture at 40°C, during which 40% sodium hydroxide solution is used to control the pH at 7.2, after about 80 minutes of culture, when the OD600nm is at 0.8 to 0.9, inoculate the bacteriophage seed at a volume ratio of 2%, then feed at a speed of 50mL / min, and increase the temperature to 42°C, after the feeding is completed, culture for 2 to 3 hours, during which the pH is controlled to be at 7.2 to 7.5, and end the propagation.

[0020] Step three, phage post-treatment process: centrifugation is performed on the phage proliferation completed in step two, glass fiber membrane is used to filter bacteria and bacterial fragments, a deoxidizing agent is added, and three-stage filtration is performed to remove bacteria, thereby obtaining the filtered phage proliferation liquid. Preferably, the glass fiber membrane is an 800 nm glass fiber membrane. Preferably, the deoxidizing agent is vitamin C. Preferably, the three-stage filtration is filtration through polypropylene 0.45 μm, polyether sulfone 0.45 μm, and polyether sulfone 0.22 μm in sequence.

[0021] In a fifth aspect, based on the same inventive concept, the present application provides a phage preparation, wherein the effective component comprises the Clostridium perfringens phage RDP-CP-2310009 or the phage proliferation liquid prepared by the industrial production process of the Clostridium perfringens phage RDP-CP-2310009.

[0022] The phage preparation is a phage oil-in-water type liquid preparation, a phage coated solid preparation, or a phage lyophilized preparation.

[0023] In a sixth aspect, based on the same inventive concept, the present application provides a preparation method of the phage oil-in-water type liquid preparation, comprising the following steps: mixing the filtered phage proliferation liquid, oregano oil, and cinnamyl aldehyde, then adding sodium dodecyl sulfonate, and placing the mixed material in an emulsifier for high-pressure homogenization and emulsification, thereby obtaining the phage oil-in-water type liquid preparation.

[0024] The preparation method of the phage oil-in-water type liquid preparation is as described above, wherein the oregano oil is added in an amount of 0.2% to 0.5% (v / v), the cinnamyl aldehyde is added in an amount of 0.1% to 0.3% (v / v), and the sodium dodecyl sulfonate is added in an amount of 0.4% to 0.8% (w / v). Preferably, the oregano oil is added in an amount of 0.3% (v / v), the cinnamyl aldehyde is added in an amount of 0.2% (v / v), and the sodium dodecyl sulfonate is added in an amount of 0.5% (w / v).

[0025] In a seventh aspect, based on the same inventive concept, the present application provides a preparation method of the phage coated solid preparation, comprising the following steps:

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

[0027] (2) concentration: using a vacuum ultra-low temperature concentrator to concentrate the above material to a solid content of about 60%, and the concentration temperature is ≤45℃;

[0028] (3) drying: the concentrated material in step (2) is dried by a vacuum belt dryer, the temperature of the material during the drying process is ≤45℃, and the concentration is performed until the moisture content is ≤6%;

[0029] (4) crushing: after drying, the material is crushed and then passed through a 60-mesh sieve, and the bacteriophage dry powder is collected;

[0030] (5) dry granulation: the bacteriophage dry powder, corn starch, carboxymethyl cellulose and chitosan material are weighed according to the proportion and then sequentially placed in a mixer, the mixed material is placed in a dry granulator, extrusion granulation is performed, and a 20-mesh sieve aperture is used to obtain bacteriophage granules; preferably, the bacteriophage dry powder is 20% (w / w) by mass fraction, the corn starch is 77% (w / w), the carboxymethyl cellulose is 2% (w / w), and the chitosan is 1% (w / w), the materials are weighed and placed in a mixer, mixed for 5 minutes, the mixed material is placed in a dry granulator, extrusion granulation is performed, and a 20-mesh sieve aperture is used to obtain bacteriophage granules;

[0031] (6) coating: the bacteriophage granules obtained in step (5) are placed in a coating machine, and stearic acid is used for coating, so as to obtain the bacteriophage coated solid preparation; preferably, the amount of stearic acid is 3-4%.

[0032] In an eighth aspect, based on the same inventive concept, the present application provides the bacteriophage preparation as described above for use in (1) or (2) as follows:

[0033] (1) preparing a product for preventing and / or treating poultry diseases caused by Clostridium perfringens;

[0034] (2) preparing a product for preventing and / or treating inflammatory reactions caused by Clostridium perfringens.

[0035] The present application has the following beneficial effects:

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

[0037] 2. The bacteriophage RDP-CP-2310009 provided by the present application can increase the titer of the bacteriophage by 100-1000 times by controlling the pH, flow supplementing medium, temperature control and inoculation timing during the proliferation process.

[0038] 3. The bacteriophage oil-in-water emulsion liquid preparation and the bacteriophage coated solid preparation provided by the present application have good stability during storage by adding carriers and protective agents in the preparation process of each of them.

[0039] 4. The bacteriophage oil-in-water emulsion liquid preparation prepared by the Clostridium perfringens bacteriophage RDP-CP-2310009 can effectively improve the stability of the bacteriophage at room temperature through the emulsification process, and the bacteriophage preparation prepared by the Clostridium perfringens bacteriophage RDP-CP-2310009 has better storage stability than the bacteriophage preparation prepared by the Clostridium perfringens bacteriophage RDP-CP-2310007, which can effectively ensure the activity of the bacteriophage preparation during transportation or storage.

[0040] 5. The bacteriophage coated solid preparation prepared by the Clostridium perfringens bacteriophage RDP-CP-2310009 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 play a role and maximize the function of the bacteriophage. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 An electron micrograph of the Clostridium perfringens bacteriophage RDP-CP-2310009 provided by the present application is provided.

[0042] Figure 2 A thermal stability curve of the Clostridium perfringens bacteriophage RDP-CP-2310009 provided by the present application is provided.

[0043] Figure 3 A one-step growth curve of the Clostridium perfringens bacteriophage RDP-CP-2310009 provided by the present application is provided.

[0044] Figure 4 A lysis curve of the Clostridium perfringens bacteriophage RDP-CP-2310009 provided by the present application on host bacteria CP-9.6 is provided.

[0045] Figure 5 A bacteriostatic curve of the Clostridium perfringens bacteriophage RDP-CP-2310009 provided by the present application is provided.

[0046] Figure 6 A stability curve comparison of the Clostridium perfringens bacteriophage RDP-CP-2310009 and the Clostridium perfringens bacteriophage RDP-CP-2310007 and the emulsion prepared therefrom is provided.

[0047] Figure 7The storage stability curve of Clostridium perfringens bacteriophage RDP-CP-2310009 and Clostridium perfringens bacteriophage RDP-CP-2310007 provided by the present application and the freeze-dried powder and coated solid preparation prepared by the present application. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the contents in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0049] In the present application, the equipment and raw materials used, unless specified, can be purchased from the market or commonly used in the art. The methods in the following examples, unless specified, are the conventional methods in the art. Unless otherwise defined, all the technical and scientific terms used in the present application have the same meanings as those commonly understood by the person skilled in the art to which the present application belongs. In the present application, Clostridium perfringens CP-9.6 is referred to as “host CP-9.6” or “CP-9.6” for short, Clostridium perfringens bacteriophage RDP-CP-2310009 is referred to as “bacteriophage RDP-CP-2310009” or “RDP-CP-2310009” for short, and Clostridium perfringens bacteriophage RDP-CP-2310007 is referred to as “bacteriophage RDP-CP-2310007” or “RDP-CP-2310007” for short.

[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 a chicken farm in Qingdao suffering from enteritis was collected, and the cecum was cut into pieces on a clean bench and placed in FTG medium. The enrichment liquid was placed in an anaerobic incubator and cultured at 42°C for 8-10h. After enrichment, about 20μL of fresh bacterial culture was taken and inoculated on TSC medium by streaking method, and then the black colonies on TSC were purified by 5% sheep blood plate. Finally, the strain that could produce double hemolytic rings on the blood plate, was gram-positive short rod-shaped bacteria under a microscope, and could produce the phenomenon of “violent fermentation” of cow milk was preliminarily identified as suspected Clostridium perfringens. The universal primer of 16S RNA was used for PCR amplification, and after successful amplification, the PCR product was purified and sequenced, and finally determined as Clostridium perfringens. One of them was named “CP-9.6” and stored in a-80°C refrigerator with 20% glycerol.

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

[0054] (1) Feces treatment: Fresh feces was collected from a certain egg chicken farm in Qingdao, about 5g was mixed with 30mL normal saline, and was soaked in an anaerobic incubator at 42℃ for 4-6 hours, then was centrifuged at 10000 rpm for 5 minutes, the supernatant was filtered through a 0.22 μm filter, and the filtered supernatant was collected for use;

[0055] (2) Preparation of mixed bacteria suspension: 0.2 mL of bacteria suspension and 0.3 mL of filtered supernatant were added into 5 mL of liquid medium, and were cultured under anaerobic conditions at 43℃ for 12-16 hours, then were centrifuged at 10000 rpm for 5 minutes, the supernatant was filtered through a 0.22 μm filter, and the filtered supernatant was collected for use;

[0056] In the present application, the composition of the liquid medium is as follows: 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 20 ppm, magnesium chloride 20 ppm, pH 8.0, sterilized at 121℃ for 30 minutes.

[0057] (3) Bacteriophage isolation: the double plate method was used to isolate the bacteriophage, 0.2 mL of mixed bacteria suspension filtered supernatant and 0.2 mL of host Clostridium perfringens CP-9.6 bacteria suspension were mixed uniformly, then were placed in a water bath at 43℃ for 10 minutes, then were plated, and were cultured under anaerobic conditions at 43℃ for 12-16 hours, then the results were observed. If there is bacteriophage, there will be transparent plaque on the plate. A single plaque was picked up in 1 mL of normal saline, and was placed in a water bath at 43℃ for 30 minutes, then was centrifuged at 10000 rpm for 5 minutes, 0.1 mL of filtered supernatant and 0.2 mL of CP-9.6 bacteria suspension were placed in a water bath at 43℃ for 10 minutes, then were plated, and were cultured under anaerobic conditions at 43℃ for 12-16 hours, then the results were observed. The above step was repeated for 3-4 times until the plaque size was uniform, and a bacteriophage was obtained, which was named RDP-CP-2310009. The diameter of the plaque formed by the double plate was 2mm-3mm.

[0058] 3. Preservation of the bacteriophage

[0059] The bacteriophage was preserved by vacuum freeze-drying, the protective agent was 20% (w / v) skimmed milk powder and 2% (w / v) mannitol, 78% purified water, and after sterilization, the bacteriophage was mixed with the proliferation liquid at 1:1 (v / v), then was vacuum freeze-dried, and the dried ampoule bottle was sealed after vacuumizing.

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

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

[0062] 1. Experimental method:

[0063] Phosphotungstic acid negative staining method: 100 μL of bacteriophage liquid (titer 10 9 pfu / mL) was dropped on a paraffin section, the membrane side of the copper mesh was placed on the bacteriophage liquid drop, removed after 10 min, naturally dried in air for 2-3 min, then a drop of 2% phosphotungstic acid (PTA) aqueous solution was dropped on the copper mesh for staining, removed after 10 min, dried in air for 10-15 min, observed with an electron microscope, and clear bacteriophage images were selected for photography. The results are shown in Figure 1

[0064] 2. Experimental results:

[0065] It can be seen from Figure 1 that the bacteriophage has a polyhedral stereometric head that encapsulates nucleic acid, a diameter of 40 nm, a tail of 30 nm long, a tail sheath, and a neck connecting the head and tail, which is a Caudovirales family Short-tailed virus family.

[0066] Example 3 Genome sequencing of bacteriophage RDP-CP-2310009

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

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

[0069] 1. Experimental method:

[0070] The bacteriophage stock solution was divided into 50 mL sterile conical flasks, incubated at 60°C and 80°C respectively, sampled every 10 min, then diluted 10 times with physiological saline, then double plates were prepared to determine the titer, 3 parallel experiments were performed, and the average value was taken, and the results are shown in Figure 2

[0071] ​​2. Experimental results:

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

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

[0074] 1. Experimental method:

[0075] The bacteriophage propagation liquid and the host liquid medium were added according to the multiplicity of infection of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and the total volume of the culture system was ensured to be the same. After 6h of culture under anaerobic conditions at 43℃, centrifugation was performed at 12000r / min for 5min at room temperature, and the supernatant was plated to determine the titer, and the results are shown in Table 1.

[0076] 2. Experimental results:

[0077] As can be seen from Table 1, when the multiplicity of infection is 0.1, the propagation liquid is relatively clear after 6h of culture, and the titer is the highest, indicating that the optimal multiplicity of infection of the bacteriophage RDP-CP-2310009 is 0.1, and the corresponding highest titer is 6.93×10 10 pfu / mL.

[0078] Table 1 Results of the optimal multiplicity of infection of the bacteriophage RDP-CP-2310009

[0079]

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

[0081] 1. Experimental method:

[0082] The logarithmic growth phase of Clostridium perfringens suspension was inoculated with Clostridium perfringens CP-9.6 and bacteriophage RDP-CP-2310009 at the optimal multiplicity of infection ratio, and then incubated at 43°C for 10 min in a water bath, followed by centrifugation at 12,000 r / min for 5 min at room temperature. The supernatant was discarded to remove the free phage that was not adsorbed on the host. The precipitate was resuspended in liquid medium at 43°C, and then incubated at 43°C under anaerobic conditions. The titer was determined at timed intervals, and the one-step growth curve was plotted with time as the abscissa and the logarithmic value of the phage titer as the ordinate. The latent period and the lysis period of the phage were determined, and the average burst size was calculated according to the formula: average burst size = phage titer at the end of the burst / host cell concentration at the beginning of the infection. The results are shown in Figure 3

[0083] 2. Experimental results

[0084] As can be seen from Figure 3 the phage titer did not change significantly within 50 min after infection of the host bacteria, indicating that the latent period was about 50 min. The phage titer increased significantly within 50-110 min after infection, and then stabilized. The phage burst size was about 196 PFU per infected cell, indicating that the phage RDP-CP-2310009 has the characteristics of short latent period, strong lysis, and high replication.

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

[0086] 1. Experimental method

[0087] The host Clostridium perfringens CP-9.6 bacterial suspension cultured for 6 h was inoculated into 100 mL of liquid medium at a ratio of 2:100 (v / v), and then incubated at 43°C under anaerobic conditions for 2 h to obtain a bacterial suspension with a concentration of about 5.0 x 10 8 cfu / mL. The bacterial suspension was divided into four sterilized conical flasks, and the optimal multiplicity of infection was used to add 0.2 mL of phage propagation liquid to three of the flasks, Figure 4 which were designated as "host CP-9.6 and phage RDP-CP-2310009". The other flask without the addition of phage propagation liquid was used as a control, Figure 4 which was designated as "host CP-9.6". The results are shown in Figure 4

[0088] 2. Experimental results

[0089] As can be seen from Figure 4 ​​It can be seen that the Clostridium perfringens bacteriophage RDP-CP-2310009 was added to the host bacteria CP-9.6 bacterial suspension. Because the bacteriophage has strong lysis effect on the host, after the bacteriophage was added, the OD600 absorbance gradually increased after 450 min. This indicates that within 450 min, the bacteriophage can effectively inhibit the growth of the host bacteria, which has very important application value in animal disease treatment.

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

[0091] 1. Experimental method:

[0092] Under sterile conditions, 1 mL of sample and 1 mL of host bacteria broth (1 x 10 5 cfu / mL) were taken, incubated at 43°C for 15 min, mixed, and then diluted with physiological saline to 10 -1 ~ 10 -4 gradients, 100 μL of each gradient was spread on LB agar plates, which were incubated at 43°C under anaerobic conditions for 12-16 h, and each gradient was repeated twice. At the same time, 1 mL of physiological saline and 1 mL of host bacteria broth (1 x 10 5 cfu / mL) were taken as a blank control, and the above steps were repeated. Plates with 30-300 colonies were selected for counting. The experiment was repeated 3 times, and the average value was taken.

[0093] Bacteriophage lysis rate = (1 - treated group colony number / control group colony number) x 100%

[0094] 2. Experimental results:

[0095] The experiment showed that the lysis rate of bacteriophage RDP-CP-2310009 was 93.66%, which had good lysis effect on the host bacteria and was suitable for use in the breeding process.

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

[0097] This example is the industrial production process of Clostridium perfringens bacteriophage RDP-CP-2310009, which includes the following steps:

[0098] Step one, seed preparation

[0099] (1) Host seed preparation: under sterile environment, single colony was inoculated in 5 mL of liquid medium, under anaerobic condition, 43°C for 4-6 h, then the culture was inoculated in 100 mL of liquid medium at 5% inoculation, under anaerobic condition, 43°C for 4-6 h, then the culture was inoculated in 1000 mL of liquid medium at 5% inoculation, under anaerobic condition, 43°C for 4-6 h, then the culture was stored in 4°C refrigerator for standby;

[0100] (2) Bacteriophage seed preparation: the stored bacteriophage RDP-CP-2310009 seed was diluted appropriately, 200 μL of 10 5-8 fold dilution was mixed with 200 μL of host bacteria in upper medium, double-layer plate was inverted, under anaerobic condition, 43°C for 12-16 h, then the plate with cloud-like bacteriophage plaque was selected, the upper medium was washed with 5 mL of liquid medium, then centrifuged at 12000 rpm for 10 min, the clear liquid was collected by passing through 0.22 μm filter membrane.

[0101] The clear liquid and host seed were inoculated in 50 mL of liquid medium at 2% proportion, under anaerobic condition, 43°C for 6-8 h, then the culture was inoculated in 1000 mL of liquid medium at 2% proportion, under anaerobic condition, 43°C for 4-6 h, then the culture was stored in 4°C refrigerator for standby.

[0102] Liquid medium composition: 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 20 ppm, magnesium chloride 20 ppm, pH 8.0, sterilized at 121°C for 30 min.

[0103] Step two, bacteriophage propagation:

[0104] The prepared host seed was inoculated in 200 L fermenter at 2% proportion, under anaerobic condition, 40°C for culture, during which 40% sodium hydroxide solution was used to control pH at 7.2, after about 80 min of culture, OD600nm was at 0.8-0.9, then the bacteriophage seed was inoculated at 2% proportion, then the temperature was increased to 42°C at a feeding rate of 50 mL / min, after the end of feeding, the culture was further incubated for 2-3 h, during which pH was controlled at 7.2-7.5, and the propagation was ended.

[0105] The fermentation tank medium composition in this step: 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 20 ppm, magnesium chloride 20 ppm, pH 8.0, 121°C, 30 min sterilization, liquid volume 150L.

[0106] The lye in this step: 40% sodium hydroxide solution with a mass fraction, 121°C, 30 min sterilization, about 10L.

[0107] The feed in this step: 10% yeast extract powder, 2% peptone, 121°C, 30 min sterilization, a total of 10L.

[0108] Step three, phage post-processing technology

[0109] (1) Centrifugation: the proliferation of phage, with butterfly centrifuge, 6000 rpm, to remove unlysed host and bacterial debris, etc.

[0110] (2) 800 nm glass fiber membrane filter bacteria and bacterial debris.

[0111] (3) Add 0.2-0.3% vitamin C as deoxidizer.

[0112] (4) Filter bacteria: phage is filtered by three levels, that is, polypropylene 0.45 μm, polyether sulfone 0.45 μm, polyether sulfone 0.22 μm, to get the filtered phage proliferation liquid.

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

[0114] 1. Preparation of phage liquid preparation:

[0115] The filtered phage proliferation liquid in Example 9 was mixed with 0.3% (v / v) oregano oil and 0.2% (v / v) cinnamyl aldehyde, and then 0.5% (w / v) sodium dodecyl sulfonate was added. The mixed material was placed in an emulsifier and homogenized for 5 min to obtain a water-in-oil type phage liquid preparation.

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

[0117] 2. Bacteriostatic experiment of phage liquid preparation:

[0118] The host bacteria CP-9.6 was used as a control Figure 5"Host CP-9.6"), the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization (marked as "Bacteriophage RDP-CP-2310009"), oregano oil and cinnamaldehyde mixed in a volume ratio of 3:2 (marked as "Oregano oil + cinnamaldehyde"), and the bacteriophage liquid preparation in the form of water-in-oil (marked as "Bacteriophage RDP-CP-2310009 + oregano oil + cinnamaldehyde + Host CP-9.6"). Figure 5 "Host CP-9.6"), the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization (marked as "Bacteriophage RDP-CP-2310009"), oregano oil and cinnamaldehyde mixed in a volume ratio of 3:2 (marked as "Oregano oil + cinnamaldehyde"), and the bacteriophage liquid preparation in the form of water-in-oil (marked as "Bacteriophage RDP-CP-2310009 + oregano oil + cinnamaldehyde + Host CP-9.6"). Figure 5 "Host CP-9.6"), the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization (marked as "Bacteriophage RDP-CP-2310009"), oregano oil and cinnamaldehyde mixed in a volume ratio of 3:2 (marked as "Oregano oil + cinnamaldehyde"), and the bacteriophage liquid preparation in the form of water-in-oil (marked as "Bacteriophage RDP-CP-2310009 + oregano oil + cinnamaldehyde + Host CP-9.6"). Figure 5 "Host CP-9.6"), the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization (marked as "Bacteriophage RDP-CP-2310009"), oregano oil and cinnamaldehyde mixed in a volume ratio of 3:2 (marked as "Oregano oil + cinnamaldehyde"), and the bacteriophage liquid preparation in the form of water-in-oil (marked as "Bacteriophage RDP-CP-2310009 + oregano oil + cinnamaldehyde + Host CP-9.6"). Figure 5 "Host CP-9.6"), the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization (marked as "Bacteriophage RDP-CP-2310009"), oregano oil and cinnamaldehyde mixed in a volume ratio of 3:2 (marked as "Oregano oil + cinnamaldehyde"), and the bacteriophage liquid preparation in the form of water-in-oil (marked as "Bacteriophage RDP-CP-2310009 + oregano oil + cinnamaldehyde + Host CP-9.6").

[0119] Figure 5 It can be seen from the above that the bacteriophage RDP-CP-2310009 and the oregano oil and cinnamaldehyde in the bacteriophage liquid preparation in the form of water-in-oil have a synergistic bacteriostatic effect, the bacteriophage liquid preparation in the form of water-in-oil has a longer bacteriostatic 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 experiment of the bacteriophage liquid preparation in the form of water-in-oil

[0121] The stability of the liquid bacteriophage (titer ≥ 10 10 pfu / mL) after filtration and sterilization and the bacteriophage liquid preparation in the form of water-in-oil was compared. After the samples were prepared, they were placed at room temperature and their titers were determined regularly. The results are shown in Table 3. Figure 6 Figure 6 In the experiment, the bacteriophage RDP-CP-2310007 proliferation liquid after filtration and sterilization was marked as "Bacteriophage RDP-CP-2310007", the bacteriophage liquid preparation in the form of water-in-oil prepared from the bacteriophage RDP-CP-2310007 was marked as "Emulsion: Bacteriophage RDP-CP-2310007", the bacteriophage RDP-CP-2310009 proliferation liquid after filtration and sterilization was marked as "Bacteriophage RDP-CP-2310009", and the bacteriophage liquid preparation in the form of water-in-oil prepared from the bacteriophage RDP-CP-2310009 was marked as "Emulsion: Bacteriophage RDP-CP-2310009".

[0122] ​​In this embodiment, the gas-producing Clostridium perfringens bacteriophage RDP-CP-2310007 used was deposited with the China General Microbiological Culture Collection Center on November 29, 2023. The deposit address is No. 3, 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 titer of the bacteriophage liquid preparation in the water-in-oil form prepared by the bacteriophage RDP-CP-2310007 decreased from the initial 10.29 lgpfu / mL to 7.39 lgpfu / mL, but the titer of the bacteriophage liquid preparation in the water-in-oil form prepared by the bacteriophage RDP-CP-2310009 only decreased from the initial 10.38 lgpfu / mL to 9.76 lgpfu / mL. By comparison, it is found that the bacteriophage liquid preparation in the water-in-oil form, i.e., the emulsified bacteriophage, has better stability than the bacteriophage; in comparison of the bacteriophage liquid preparation in the water-in-oil form of the gas-producing Clostridium perfringens bacteriophage RDP-CP-2310009 with the bacteriophage liquid preparation in the water-in-oil form of RDP-CP-2310007, the bacteriophage liquid preparation in the water-in-oil form of RDP-CP-2310009 has better storage stability, has high stability, high activity and convenient storage in the process of product application and use, and is more conducive to the use and promotion of the product.

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

[0125] 1. Preparation of a bacteriophage coated solid preparation:

[0126] (1) Adding a carrier: adding 8.0% (w / w) acidified starch and 2.0% (w / w) pyrrolidone K-30 to the sterilized bacteriophage propagation liquid in Example 9, and stirring until completely dissolved;

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

[0128] (3) Drying: using a vacuum belt dryer to dry the concentrated material, and the temperature of the material during drying is ≤45.0°C, and the concentration is carried out to a moisture content of ≤6.0%;

[0129] (4) Grinding: after drying, the material is ground and passed through a 60-mesh sieve, and the bacteriophage dry powder is collected.

[0130] (5) Dry granulation:

[0131] According to the mass fraction of 20% of the bacteriophage dry powder (w / w), 77% (w / w) of corn starch, 2% (w / w) of carboxymethyl cellulose, 1% (w / w) of chitosan, the materials are weighed and placed in a mixer for mixing for 5 min, and then the mixed materials are placed in a dry granulator for extrusion and granulation with a screen aperture of 20 meshes to obtain bacteriophage granules.

[0132] (6) Coating:

[0133] The bacteriophage granules obtained by screening after extrusion are placed in a coating machine, and coated with stearic acid, and the amount of stearic acid is 4%, to obtain the bacteriophage RDP-CP-2310009 coated solid preparation.

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

[0135] 2. Passage of bacteriophage solid preparation in the digestive tract

[0136] Each 0.45 g of the two kinds of bacteriophage coated solid preparation samples is respectively placed in 45 mL of gastric juice and intestinal juice, and placed at 37°C with continuous stirring. Every 10 min, sample is taken, and the pH is immediately adjusted to neutral with a buffer solution, and the titer of the bacteriophage is detected. After 10-fold gradient dilution of the sample, double-layer plates are prepared, and the titer is determined. The gastric juice and intestinal juice of this example are purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. The results are shown in Tables 2 and 3.

[0137] Table 2 Titer determination results of two kinds of bacteriophage coated solid preparations in gastric juice

[0138]

[0139] As can be seen from Table 2, the bacteriophage RDP-CP-2310009 coated solid preparation is slightly swollen in the gastric juice for 10 min, and no bacteriophage is detected after neutralization of the supernatant, indicating that the coated solid preparation after coating does not release bacteriophage. The coating material on the surface of the granules can effectively protect the bacteriophage and prevent the destruction of the gastric juice. The bacteriophage RDP-CP-2310007 coated solid preparation releases bacteriophage rapidly after swelling, and is exposed to the gastric juice. With the extension of the action time, the titer gradually decreases, and the bacteriophage activity is completely lost after 60 min. Through the comparison of the above two bacteriophages, it is shown that the bacteriophage RDP-CP-2310009 is more suitable for coating than the bacteriophage RDP-CP-2310007, and can effectively avoid the influence of gastric acid on the activity of the bacteriophage.

[0140] As shown in Table 3, due to the presence of digestive enzymes and choline in intestinal fluid, the coated solid dosage form swelled after 30 minutes of contact with intestinal fluid. Continuous sampling and titer testing were conducted, and the results are shown in Table 3. The phage RDP-CP-2310009 coated solid dosage form effectively avoids damage to the phage by digestive juices during passage through the digestive tract, allowing the phage to reach the small intestine and exert its function to the maximum extent. Compared to the phage RDP-CP-2310007 coated solid dosage form, the phage RDP-CP-2310009 coated solid dosage form exhibits better stability in the intestine and better maintains its activity.

[0141] Table 3. Potency determination results of two phage-coated solid formulations in intestinal fluid.

[0142]

[0143] 3. Storage stability test of bacteriophage solid formulation and lyophilized powder:

[0144] The preparation method of phage lyophilized powder is as follows: using conventional freeze-drying process, 10% (w / v) skim milk powder, 3% (w / v) trehalose, 3% (w / v) pyrrolidone K-30 and 0.5% (w / v) vitamin C are added as protectants, and the phage lyophilized powder is obtained by freeze-drying.

[0145] The storage stability of the phage-coated solid formulations and phage lyophilized powders obtained above was tested. The samples were kept at room temperature, and their titers were determined periodically using a double-layer plate method. The results are as follows: Figure 7 As shown. Figure 7 In this document, the lyophilized powder prepared from phage RDP-CP-2310007 is designated as "lyophilized powder: RDP-CP-2310007", and the phage-coated solid dosage form prepared from phage RDP-CP-2310007 is designated as "coating: RDP-CP-2310007"; the lyophilized powder prepared from phage RDP-CP-2310009 is designated as "lyophilized powder: RDP-CP-2310009", and the phage-coated solid dosage form prepared from phage RDP-CP-2310009 is designated as "coating: RDP-CP-2310009".

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

[0147] The industrial production process of bacteriophage RDP-CP-2310009 without controlling pH and without feeding

[0148] The difference between Example 9 and Comparative Example 1 is only that in the step of "Step two, bacteriophage propagation", the prepared host seed is inoculated in a 200L fermenter at a volume ratio of 2%, and anaerobic culture is carried out at 40℃, and after about 60min, when the OD600nm is 0.8-0.9, the bacteriophage seed is inoculated at a volume ratio of 2%, and the culture is carried out for 3-4h, and the propagation is ended.

[0149] The titer of the obtained bacteriophage propagation liquid of Comparative Example 1 and Example 9 is determined, and the results show that the titer of the bacteriophage propagation liquid prepared by Comparative Example 1 without controlling pH and without feeding is 6.6x10 7 pfu / mL, while the titer of the bacteriophage of Example 9 reaches 3.6x10 10 pfu / mL. This shows that during the propagation of the bacteriophage, it is crucial to control the pH and supplement the nutrients in time to improve the titer of the bacteriophage.

[0150] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Clostridium perfringens bacteriophage ( Clostridium perfringens bacteriophage RDP-CP-2310009, characterized in that, The preservation number of which is CGMCC No. 45779, preserved in China General Microbiological Culture Collection Center on November 29, 2023.

2. A bacteriophage composition, characterized in that, The Clostridium perfringens bacteriophage RDP-CP-2310009 as claimed in claim 1.

3. The Clostridium perfringens bacteriophage RDP-CP-2310009 of claim 1 or the bacteriophage composition of claim 2 is used in at least one of the following (1) to (3): (1) preparing a product for killing Clostridium perfringens; (2) preparing a product for inhibiting Clostridium perfringens; (3) preparing a product for preventing and / or treating poultry diseases caused by Clostridium perfringens.

4. The industrial production process of Clostridium perfringens bacteriophage RDP-CP- 2310009 according to claim 1, characterized in that, The method comprises the following steps: Step one, seed preparation: host bacteria seed preparation; bacteriophage seed preparation; Step two, bacteriophage propagation: inoculate the host bacteria seed prepared in step one into a fermenter, anaerobically culture, control the pH at 7.0-7.5, then inoculate the bacteriophage seed, then perform feeding, after the feeding is completed, culture for 2-3 h, and end the propagation; Step three, bacteriophage post-treatment process: centrifuge the bacteriophage propagated in step two, filter bacteria and bacterial fragments using a glass fiber membrane, add a deoxidizing agent, and then filter bacteria using three-stage filtration, so as to obtain the bacteriophage propagation liquid after filtering and removing bacteria.

5. A bacteriophage preparation, characterized in that, The effective component comprises the bacteriophage propagation liquid prepared by the industrial production method of the Clostridium perfringens bacteriophage RDP-CP-2310009 as claimed in claim 1 or the Clostridium perfringens bacteriophage RDP-CP-2310009 as claimed in claim 4.

6. The bacteriophage preparation of claim 5, wherein, The dosage form of the bacteriophage preparation is a bacteriophage water-in-oil dosage form liquid preparation, a bacteriophage coated solid preparation, or a bacteriophage lyophilized preparation.

7. A method of preparing a bacteriophage preparation as claimed in claim 6, characterized in that, In the bacteriophage water-in-oil dosage form liquid preparation, The preparation method of the bacteriophage water-in-oil dosage form liquid preparation comprises the following steps: mixing the bacteriophage propagation liquid after filtering and removing bacteria, oregano oil, and cinnamyl aldehyde, then adding sodium dodecyl sulfate, placing the mixed material in an emulsifying machine, and performing high-pressure homogenization emulsification, so as to obtain the bacteriophage water-in-oil dosage form liquid preparation.

8. The method of claim 7, wherein the phage preparation is prepared by, The addition amount of the oregano oil is 0.2%-0.5% v / v, the addition amount of the cinnamyl aldehyde is 0.1%-0.3% v / v, and the addition amount of the sodium dodecyl sulfate is 0.4%-0.8% w / v.

9. A method of preparing a bacteriophage preparation as claimed in claim 6, characterized in that, In the bacteriophage coated solid preparation, The preparation method of the bacteriophage coated solid preparation comprises the following steps: (1) adding a carrier: adding the bacteriophage propagation liquid after filtering and removing bacteria, acidified starch, and pyrrolidone K-30, and stirring until completely dissolved; (2) concentrating: concentrating the above material using a vacuum ultra-low temperature concentrator, the concentration temperature is ≤45℃, and the concentration is performed until the solid content is 60%; (3) drying: drying the concentrated material in step (2) using a vacuum belt dryer, the temperature of the material during the drying process is ≤45℃, and the concentration is performed until the moisture content is ≤6%; (4) crushing: after the material is dried, crushing the material through a 60-mesh sieve, and collecting the bacteriophage dry powder; (5) Dry granulation: bacteriophage dry powder, corn starch, carboxymethyl cellulose and chitosan materials are weighed according to the proportion and placed in a mixer, mixed, and then the mixed materials are placed in a dry granulator for extrusion and granulation, with a sieve aperture of 20 meshes, to obtain bacteriophage granules; (6) Coating: the bacteriophage granules obtained in step (5) are placed in a coating machine and coated with stearic acid to obtain a bacteriophage coated solid preparation.

10. Use of the bacteriophage preparation according to any one of claims 5-6 in the preparation of a product for preventing and / or treating avian diseases caused by Clostridium perfringens.

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