Freeze-drying protective agent of bacteriophage, freeze-dried powder of bacteriophage, preparation method and application

Abstract

The invention provides a bacteriophage freeze-drying protective agent, a bacteriophage freeze-drying powder, a preparation method and application, and belongs to the technical field of biological medicine preparations. The freeze-drying protective agent comprises the following components: a cryoprotectant, an antioxidant, an acid-base regulator and a filler. The freeze-drying protective agent disclosed by the invention is reasonably compounded, and the freeze-drying protective agent, the antioxidant, the pH regulator and the filler have a good protective effect on the bacteriophage through a synergistic effect, so that the inactivation rate or the death rate of the bacteriophage in a freeze-drying process can be effectively reduced, and the biological activity and the titer of the bacteriophage are improved. Moreover, the freeze-drying protective agent disclosed by the invention is relatively long in protective effect acting time, can improve the stability of the freeze-dried powder of the bacteriophage, improves the transportation and storage convenience of the freeze-dried powder of the bacteriophage, and effectively solves the problem that the freeze-dried powder of the bacteriophage is difficult to transport and store for a long time in practical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicine preparations, and in particular relates to a phage freeze-dried protective agent, a phage freeze-dried powder, and a preparation method and application thereof. Background Art

[0002] In livestock and poultry farming, common pathogenic bacteria include Escherichia coli, Salmonella, Staphylococcus, Pasteurella, and Clostridium perfringens, etc. The main pathogenic bacteria in aquaculture include Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Aeromonas veseri, and Edwardsiella tarda, etc.

[0003] Bacteriophages can infect and lyse bacteria, so the use of virulent phages to control bacterial diseases has become a safe and effective method. At present, the phage preparations used in animal clinics include liquid preparations and phage powders. Among them, the preparation processes of phage powders include freeze-drying and spray-drying. Due to the special biological characteristics of phages, they will be quickly inactivated in an environment above 60°C, so the spray-drying process will greatly destroy the biological activity of phages and is not suitable for large-scale production. Therefore, freeze-drying is generally used to prepare phage freeze-dried powders. However, the phage titer of phage freeze-dried powders still needs to be further improved. Summary of the invention

[0004] The purpose of the present invention is to provide a phage freeze-dried protective agent, a phage freeze-dried powder, a preparation method and an application thereof. The phage freeze-dried powder prepared by using the freeze-dried protective agent of the present invention has a high phage titer.

[0005] The invention provides a phage freeze-drying protective agent, which uses water as a solvent and comprises the following components in mass concentrations: 5% to 10% of a cryoprotectant, 0.5% to 1.5% of an antioxidant, 0.5% to 1.5% of an acid-base regulator and 5% to 10% of a filler.

[0006] Preferably, the cryoprotectant includes at least one of trehalose, glycerol and sucrose; the antioxidant includes sodium thiosulfate and / or vitamin E; the acid-base regulator includes sorbitol and / or phosphoric acid; and the filler includes at least one of mannitol, lactose and gelatin.

[0007] Preferably, the lyoprotectant comprises the following components in mass concentrations: 10% cryoprotectant, 1% antioxidant, 1.5% acid-base regulator and 10% filler.

[0008] The present invention also provides a phage freeze-dried powder, comprising a phage and the freeze-dried protective agent described in the above scheme.

[0009] Preferably, the phage titer of the phage lyophilized powder is ≥10 10 PFU / ml.

[0010] Preferably, the bacteriophage includes poultry pathogenic bacteriophage, livestock pathogenic bacteriophage or aquatic pathogenic bacteriophage.

[0011] Preferably, the bacteriophage includes at least one of avian Escherichia coli phage, Salmonella phage, Vibrio alginolyticus phage, Vibrio parahaemolyticus phage, Vibrio vulnificus phage, Vibrio harveyi phage, Vibrio erwinia phage, Pasteurella phage, Bordetella bronchiseptica phage, porcine Escherichia coli phage and Riemerella anatipestifer phage.

[0012] The present invention also provides a method for preparing the phage freeze-dried powder described in the above scheme, comprising the following steps:

[0013] The phage solution is mixed with the lyophilization protective agent described in the above scheme to obtain a mixed solution; the mixed solution is freeze-dried to obtain a freeze-dried mixture; the freeze-dried mixture is crushed to obtain a phage freeze-dried powder.

[0014] Preferably, the freeze-drying procedure includes: -30 to -40°C, 8 to 10 h; 5 to 10°C, 15 to 18 h; 18 to 20°C, 2 to 4 h; 25 to 30°C, 8 to 10 h; the vacuum degree of the freeze-drying is 0.04 to 0.10 mbar.

[0015] The present invention also provides the use of the lyoprotectant described in the above scheme, the phage lyophylic powder or the phage lyophylic powder prepared by the preparation method in the preparation of livestock and poultry drugs, aquatic drugs or feed additives.

[0016] The present invention provides a phage freeze-dried protective agent, which uses water as a solvent and includes the following components in mass concentrations: 5% to 10% cryoprotectant, 0.5% to 1.5% antioxidant, 0.5% to 1.5% acid-base regulator and 5% to 10% filler. The freeze-dried protective agent of the present invention is reasonably compounded, wherein the cryoprotectant, antioxidant, acid-base regulator and filler have a good protective effect on the bacteriophage through synergistic action, and can effectively reduce the inactivation rate or mortality rate of the bacteriophage during the freeze-drying process, thereby improving the biological activity and titer of the bacteriophage. In addition, the freeze-dried protective agent of the present invention has a long protective effect time, can improve the stability of the phage freeze-dried powder, improve the convenience of transportation and storage of the phage freeze-dried powder, and effectively solve the problem of transportation and long-term storage difficulties in the actual application of the phage freeze-dried powder.

[0017] Biological Deposit Description

[0018] Salmonella phage SD-SE-001 was deposited at the General Microbiology Center of China Culture Collection on September 10, 2021. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No.23090.

[0019] Vibrio parahaemolyticus phage (VibrioParahaemolyticus phage) SD-VP1 was deposited in the China Center for Type Culture Collection on January 6, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M 2023042.

[0020] Vibrio owensii phage SD-VP2 was deposited in the China Center for Type Culture Collection on January 6, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M2023043.

[0021] Vibrio harveyi phage SD-VP3 was deposited in the China Center for Type Culture Collection on January 6, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M2023044.

[0022] Vibrio vulnificus phage SD-VP4 was deposited in the China Center for Type Culture Collection on January 6, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M2023045.

[0023] Pasteurella suis phage SD-PM1 was deposited in the China Center for Type Culture Collection on December 18, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M20232589.

[0024] Brodetella bronchiseptica phage SD-Bb1 was deposited in the China Center for Type Culture Collection on December 19, 2023. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M 20232599.

[0025] Riemerella anatipestiferphage SD-RA1 was deposited in the China Center for Type Culture Collection on August 23, 2021. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: M 20211061. DETAILED DESCRIPTION

[0026] The invention provides a phage freeze-drying protective agent, which uses water as a solvent and comprises the following components in mass concentrations: 5% to 10% of a cryoprotectant, 0.5% to 1.5% of an antioxidant, 0.5% to 1.5% of an acid-base regulator and 5% to 10% of a filler.

[0027] In the specific implementation process of the present invention, the cryoprotectant includes at least one of trehalose, glycerol and sucrose; the antioxidant includes sodium thiosulfate and / or vitamin E; the acid-base regulator includes sorbitol and / or phosphoric acid; the filler includes at least one of mannitol, lactose and gelatin.

[0028] In one embodiment of the present invention, the lyoprotectant is trehalose, which is a non-reducing sugar with a special disaccharide molecular structure. When the cells are in freeze-drying conditions, it can form a protective film on the cell surface, which can better control the moisture in freeze-drying and maintain the stability of the cell membrane, thereby reducing the death of phage cells and improving the biological activity rate of phages; the antioxidant is sodium thiosulfate, which can well protect the biological activity of phages. The antioxidant effect of sodium thiosulfate is provided by self-oxidation. When the oxygen in the freezing environment and the freeze-drying After the oxygen inside the product is consumed, the oxygen content that the freeze-dried product is exposed to will also decrease, so that the oxidation reaction will also be reduced, thereby achieving an antioxidant effect; the acid-base regulator is sorbitol, which has a good protective effect on the biological activity of the bacteriophage. Sorbitol is a polyol, which can not only adjust the pH value, but also provide a supporting structure for the active components, and has a dual protective effect on the bacteriophage; the filler is gelatin, which plays a solubilizing and stabilizing role. Gelatin not only has a good solubilizing effect, but also has a low cost, is easy to prepare, and is convenient to store, and has a small ratio with the bacteriophage, which can reduce the cost of freeze-drying.

[0029] In the specific implementation process of the present invention, the lyophilized protective agent includes the following components in mass concentrations: 10% cryoprotectant, 1% antioxidant, 1.5% acid-base regulator and 10% filler.

[0030] In one embodiment of the present invention, the lyoprotectant is composed of a solvent and the following components in mass concentrations: 10% cryoprotectant, 1% antioxidant, 1.5% acid-base regulator and 10% filler.

[0031] The lyophilization protectant of the present invention has the advantage of low cost.

[0032] The present invention also provides a phage freeze-dried powder, comprising a phage and the freeze-dried protective agent described in the above scheme.

[0033] In the specific implementation process of the present invention, the phage titer of the phage lyophilized powder is ≥10 10 PFU / ml, preferably 10 10 ~10 11 PFU / ml.

[0034] In the specific implementation process of the present invention, the bacteriophage includes one or more strains of biologically active bacteriophages, and the types of bacteriophages can be flexibly controlled according to the types of clinical pathogens to achieve targeted bacterial disease prevention and control without destroying other flora in the environment or causing flora imbalance. It is green, safe, and has no residue.

[0035] In the specific implementation process of the present invention, the bacteriophage includes poultry pathogenic bacteria phage, livestock pathogenic bacteria phage or aquatic pathogenic bacteria phage, and further includes: at least one of avian Escherichia coli phage, Salmonella phage, Vibrio alginolyticus phage, Vibrio parahaemolyticus phage, Vibrio vulnificus phage, Vibrio harveyi phage, Vibrio erwinia phage, Pasteurella phage, Bordetella bronchiseptica phage, porcine Escherichia coli phage and Riemerella anatipestifer phage; the Salmonella phage is avian Salmonella phage; the Pasteurella phage is porcine Pasteurella phage; the Bordetella bronchiseptica phage is porcine Bordetella phage.

[0036] In one embodiment of the present invention, the Salmonella phage includes Salmonella phage SD-SE-001, and the preservation number of the Salmonella phage SD-SE-001 is CGMCC 23090; the Vibrio parahaemolyticus phage includes Vibrio parahaemolyticus phage SD-VP1; the Vibrio erwinia phage includes Vibrio erwinia phage SD-VP2; the Vibrio harveyi phage includes Vibrio harveyi phage SD-VP3; the Vibrio vulnificus phage includes Vibrio vulnificus phage SD-VP4; the preservation numbers of the Vibrio parahaemolyticus phage SD-VP1, the Vibrio erwinia phage SD-VP2, the Vibrio harveyi phage SD-VP3 and the Vibrio vulnificus phage SD-VP4 are CCTCC NO: M 2023042, CCTCC NO: M 2023043, CCTCC NO: M 2023044, respectively. 2023044, CCTCC NO: M 2023045; the Pasteurella phage includes Pasteurella phage SD-PM1; the Bordetella bronchiseptica phage includes Bordetella bronchiseptica phage SD-Bb1; the preservation numbers of the Pasteurella phage SD-PM1 and the Bordetella bronchiseptica phage SD-Bb1 are CCTCC NO: M20232589 and CCTCC NO: M 20232599, respectively; the Riemerella anatipestifer phage includes the Riemerella anatipestifer phage SD-RA1, whose preservation number is CCTCC NO: M 20211061.

[0037] The present invention also provides a method for preparing the phage freeze-dried powder described in the above scheme, comprising the following steps:

[0038] The phage solution is mixed with the lyophilization protective agent described in the above scheme to obtain a mixed solution; the mixed solution is freeze-dried to obtain a freeze-dried mixture; the freeze-dried mixture is crushed to obtain a phage freeze-dried powder.

[0039] In the present invention, the phage solution is preferably a phage fermentation stock solution; in the specific implementation process of the present invention, the preparation method of the phage fermentation stock solution includes the following steps: inoculating the host bacteria seed solution into a fermentation tank for expansion culture, and after the host bacteria grows to the logarithmic phase, inoculating the phage seed solution, fermenting and culturing for 4 to 10 hours to obtain the phage fermentation liquid; centrifuging and filtering the phage fermentation liquid in turn to obtain a sterile phage stock solution. In the present invention, the host bacteria seed solution is inoculated into the fermentation tank at a volume ratio of 1% to 2%. In the present invention, the concentration of the host bacteria in the host bacteria solution that has grown to the logarithmic phase is 10 8 ~10 10 CFU / ml; the phage seed solution is inoculated into the host bacterial solution at a MOI of 0.0001-1.

[0040] In the specific implementation process of the present invention, the freeze-drying procedure includes: -30 to -40°C, 8 to 10 hours; 5 to 10°C, 15 to 18 hours; 18 to 20°C, 2 to 4 hours; 25 to 30°C, 8 to 10 hours; the vacuum degree of the freeze-drying is 0.04 to 0.10 mbar.

[0041] In the specific implementation process of the present invention, the phage titer of the lyophilized mixture is ≥10 10 PFU / ml, preferably 10 10 ~10 11 PFU / ml.

[0042] The preparation method of the bacteriophage freeze-dried powder of the present invention is simple, the conditions are easy to control, and the practical application is convenient.

[0043] In the specific implementation of the present invention, the storage temperature of the phage freeze-dried powder is -20°C to 30°C.

[0044] Compared with the phage stock solution, the phage freeze-dried powder of the present invention has better acid resistance and heat resistance. In an environment with pH < 3.0, the phage stock solution is inactivated, while the phage freeze-dried powder can still survive and can withstand a high temperature of 80°C for more than 10 minutes, thus overcoming the defect that liquid phage preparations must be stored and transported at 2 to 8°C or below. Therefore, the phage freeze-dried powder of the present invention can have a good application effect in clinical applications, not only can it reduce the damage of gastric acid in animal bodies to phages, but also can enhance clinical prevention or treatment effects; at the same time, the phage freeze-dried powder of the present invention can withstand high temperatures, so that the phage freeze-dried powder has better stability and is convenient for storage and transportation.

[0045] The phage freeze-dried powder of the present invention can be stored at room temperature, has good water solubility, does not absorb moisture and agglomerate, has stable phage activity, has low requirements for storage conditions, and can be stored stably for more than 3 years. The phage freeze-dried powder of the present invention has good stability, greatly improves the convenience of transportation and storage, the cost of the freeze-drying protective agent used is low, the freeze-drying process is simple, the conditions are easy to control, and the practical application is convenient.

[0046] The present invention also provides the use of the lyoprotectant described in the above scheme, the phage lyophylic powder or the phage lyophylic powder prepared by the preparation method in the preparation of livestock and poultry drugs, aquatic drugs or feed additives.

[0047] In the specific implementation process of the present invention, the phage freeze-dried powder can be used alone or in combination with livestock and poultry and aquaculture drugs.

[0048] In one embodiment of the present invention, the phage freeze-dried powder is the only active ingredient of the livestock and poultry medicine, aquatic medicine or feed additive.

[0049] In order to further illustrate the present invention, a phage freeze-dried protectant, phage freeze-dried powder, preparation method and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1 Preparation and selection of freeze-dried protective materials

[0051] 1. Resuscitation of host bacteria

[0052] Take the bacterial strain from the -80℃ refrigerator, streak it on LB agar medium, and culture it in a constant temperature incubator at 37℃ for 18-24h. Pick a single colony and inoculate it into LB broth medium. Culture it in a shaker at 37℃ and 180rpm for 12-16h to obtain the proliferated bacterial solution for the test.

[0053] 2. Phage propagation

[0054] Take 100 μl of host bacteria proliferation liquid and 100 μl of phage stock solution in 5 ml LB broth medium, culture at 37°C, 180 rpm shaking for 8-10 hours, centrifuge the above culture solution at 8000-10000 rpm for 5-10 minutes, take the supernatant, filter it with a 0.22 μm filter, and obtain the phage proliferation liquid for the experiment.

[0055] 3. Freeze drying The prepared phage proliferation liquid was freeze-dried under the following conditions: -30°C for 10 h, 8°C for 15 h, 18°C ​​for 4 h, 25°C for 8 h. The vacuum degree was 0.10 mbar.

[0056] 4. Preparation and selection of single type protective agent material

[0057] A single factor experiment was used to investigate the effects of different types of protective agents on the titer of phages after freeze-drying. The experimental conditions of precooling and freeze-drying were kept unchanged. The survival rate of phage titer after freeze-drying was used as an indicator to screen cryoprotectants, antioxidants, acid-base regulators and fillers. The materials with the highest survival rate of phages after freeze-drying among various protective agents were screened out. Each material of cryoprotectants (trehalose, sucrose, glycerol), antioxidants (sodium thiosulfate, vitamin E), acid-base regulators (amino acids, sorbitol, phosphoric acid), and fillers (mannitol, lactose, gelatin) was mixed with purified water to form a single-component protective agent, wherein the cryoprotectant and acid-base regulator were mixed at a concentration of 10% to 15%, and the antioxidant and acid-base regulator were mixed at a concentration of 1% to 1.5%. After mixing, they were contacted with the phages and mixed evenly, freeze-dried, and the phage titer was detected.

[0058] 5. Screening of the optimal ratio of composite protective agent

[0059] As shown in Table 1, trehalose, sodium thiosulfate, sorbitol and gelatin selected by single factor test were used as raw materials, and the phage titer after freeze-drying was used as an indicator. A three-level four-factor test was used to perform compound optimization to determine the optimal ratio of the composite protective agent.

[0060] Table 1 Level selection of various factors of composite freeze-drying protective agent

[0061] level A Trehalose (%) B Sodium thiosulfate (%) C Gelatin (%) D Sorbitol (%) 1 5 0.5 5 0.5 0 10 1.0 10 1.0 -1 15 1.5 15 1.5

[0062] 6. Phage titer detection and survival rate

[0063] Aseptically take 1 ml of the above phage proliferation solution and add it to a test tube containing 9 ml of SM buffer. After thorough mixing, take 1 ml of the diluted phage suspension and add it to another centrifuge tube containing 9 ml of SM buffer. Repeat the above steps to dilute the sample to a concentration of 10. -2 ~10 -8 8 gradients. Select 2 to 3 appropriate sample dilution gradients, take 100μl of each with a sterile pipette tip, add to 100μl bacterial solution, mix, and incubate at 37℃ for 5min. Add 5ml of the upper semi-solid medium (LB broth medium + 0.7% agar powder), mix, and pour on the lower LB solid medium. After solidification, invert and culture at 37℃ for 10 to 15h. Observe the morphology of the individual and uniform plaques formed, and count the number of plaques. Select plates with 30 to 300 plaques for counting, calculate the phage titer according to the formula, and retain one significant digit in the result. Repeat 3 times and take the average result. Phage titer (PFU / ml) = number of plaques on a plate with appropriate plaque density × dilution factor × 10.

[0064] Phage survival rate (%) = (m / m0) × 100

[0065] Where: m = phage titer measured after freeze drying (PFU / ml)

[0066] m0 = phage titer measured before freeze-drying (PFU / ml)

[0067] 7. Results

[0068] (1) Determination of single protective agent

[0069] In order to improve the survival rate of phages during the freeze-drying process, trehalose, glycerol, and sucrose were used as cryoprotectants for experiments. The titer of phages before freeze-drying was 3.6×10 9 PFU / ml, the results are shown in Table 2. Trehalose has the best protective effect on phages, which is significantly higher than glycerol and sucrose. Sucrose has the worst protective effect on phages. The phage titer of the trehalose test group was detected to be 1.3×10 9PFU / ml, the phage titer of the glycerol test group was 2.3×10 7 PFU / ml, the phage titer of the sucrose test group was 2.8×10 5 PFU / ml. Therefore, trehalose was selected as the cryoprotectant.

[0070] In order to improve the survival rate of phages during freeze-drying, two antioxidants, sodium thiosulfate and vitamin E, were tested respectively. The results are shown in Table 2. The protective effect of sodium thiosulfate on phages is better than that of vitamin E. The titer of phages in the sodium thiosulfate test group was detected to be 1.8×10 6 PFU / ml, and the phage titer of the vitamin E test group was 2.2×10 3 PFU / ml. Therefore, sodium thiosulfate was selected as the antioxidant.

[0071] In order to improve the survival rate of phages during the freeze-drying process, mannitol, lactose and gelatin were used as fillers for experiments. The results are shown in Table 2. Gelatin has the best protective effect on phages, while lactose has the worst effect. The phage titer of the gelatin test group was detected to be 2.1×10 7 PFU / ml, the phage titer of the mannitol test group was 3.2×10 5 PFU / ml, the phage titer of the lactose test group was 2.6×10 3 PFU / ml. Therefore, gelatin was selected as the filler of lyoprotectant.

[0072] In order to improve the survival rate of phages during freeze-drying, amino acids, sorbitol, and phosphoric acid were used as acid-base regulators. The results are shown in Table 2. Sorbitol has the best protective effect on phages, while amino acids have the worst effect. The phage titer of the sorbitol test group was 1.6×10 3 PFU / ml, the phage titer of the phosphate test group was 2.8×10 PFU / ml, and the phage titer of the amino acid test group was 0. Therefore, sorbitol was selected as the acid-base regulator.

[0073] Table 2 Effect of lyophilization protectant on phage titer

[0074] Group Protective agent Phage titer after freezing 1 Trehalose <![CDATA[3.5×10 9 ]]> 2 glycerin <![CDATA[2.3×10 7 ]]> 3 sucrose <![CDATA[2.8×10 5 ]]> 4 Sodium Thiosulfate <![CDATA[1.8×10 6 ]]> 5 Vitamin E <![CDATA[2.2×10 3 ]]> 6 gelatin <![CDATA[2.1×10 7 ]]> 7 Mannitol <![CDATA[3.2×10 5 ]]> 8 lactose <![CDATA[2.6×10 3 ]]> 9 Sorbitol <![CDATA[1.6×10 3 ]]> 10 Phosphoric acid 2.8×10 11 Amino Acids 0

[0075] (2) Determination of the optimal ratio of composite protective agent

[0076] The trehalose, sodium thiosulfate, sorbitol and gelatin selected by the single factor test were used as raw materials, and the phage titer after freeze drying was used as an indicator. The three-level four-factor test was used to optimize the compounding to determine the optimal ratio of the composite protective agent. The optimal ratio of the composite freeze-dried protective agent was 10% trehalose, 10% gelatin, 1.0% sodium thiosulfate and 1.5% sorbitol. Taking the phage titer as an indicator, the ratio was compared with 4.5×10 9 PFU / ml of phage were mixed for testing. The test results are shown in Table 3. As can be seen from Table 3, after 36 hours of freezing, the phage titer measured by the composite protective agent prepared with this ratio was 2.2×10 10 PFU / ml; after refrozen for 42-48h, the phage titer decreased and the measured phage titer was 3.1×10 7 PFU / ml, indicating that the composite freeze-dried protective agent can achieve a relatively ideal protective effect.

[0077] Table 3 Experimental verification of the optimal ratio of composite protective agent

[0078] Time (h) 36 40 44 48 Phage titer (PFU / ml) <![CDATA[2.2×10 9 ]]> <![CDATA[1.1×10 9 ]]> <![CDATA[3.6×10 8 ]]> <![CDATA[3.1×10 7 ]]>

[0079] Example 2 Preparation method of Vibrio phage freeze-dried powder

[0080] 1. Resuscitation of host bacteria

[0081] Clinically isolated Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus, and Vibrio erwinia were taken from a -80°C refrigerator, streaked on TCBS agar medium, cultured overnight in a constant temperature incubator at 37°C, single colonies were picked, inoculated into LB liquid culture medium with a salinity of 2.5% to 3.5%, and cultured on a shaker at 37°C and 180rpm to 220rpm for 8 to 12h to obtain a proliferated bacterial solution for the experiment.

[0082] 2. Fermentation culture of bacteria

[0083] The seeds of the different host bacteria cultured above were inoculated into different 100L fermentation tanks at a ratio of 1% to 2%, and cultured. After the host bacteria grew to the logarithmic phase, the inoculation titers were 10 at MOI = 1 to 0.001. 10 PFU / ml of Vibrio parahaemolyticus phage SD-VP1, Vibrio Erwinia phage SD-VP2, Vibrio harveyi phage SD-VP3, and Vibrio vulnificus phage SD-VP4 were cultured in each fermenter at 37°C and 220rpm for 6h to obtain culture fluid of Vibrio parahaemolyticus phage SD-VP1, Vibrio Erwinia phage SD-VP2, Vibrio harveyi phage SD-VP3, and Vibrio vulnificus phage SD-VP4, respectively.

[0084] 3. Post-treatment of phage culture fluid

[0085] The fermented Vibrio parahaemolyticus phage SD-VP1, Vibrio Erwinia phage SD-VP2, Vibrio harveyi phage SD-VP3, and Vibrio vulnificus phage SD-VP4 were centrifuged at 6000 g in a disc centrifuge, and the supernatants were filtered through a 0.22 μm filter membrane to obtain sterile Vibrio parahaemolyticus phage SD-VP1, Vibrio Erwinia phage SD-VP2, Vibrio harveyi phage SD-VP3, and Vibrio vulnificus phage SD-VP4 stock solutions. The phage titers were all 10 10 PFU / ml

[0086] 4. Preparation of lyoprotectant and freeze drying

[0087] Sterile stock solutions of Vibrio parahaemolyticus phage SD-VP1, Vibrio Erwinia phage SD-VP2, Vibrio harveyi phage SD-VP3, and Vibrio vulnificus phage SD-VP4 were mixed to prepare a phage mixture solution.

[0088] The freeze-drying protective agent is compounded according to 10% trehalose, 8% gelatin, 0.5% sodium thiosulfate and 1.5% sorbitol.

[0089] The above phage mixture and lyophilization protective agent mixture were mixed in a ratio of 1:1 and freeze-dried. The freeze-drying conditions were set at -35℃ for 8h, 10℃ for 16h, 20℃ for 2h, and 28℃ for 8h, and the vacuum degree was 0.06mbar. The Vibrio phage freeze-dried powder was obtained, and the titers of the freeze-dried powder were as follows: SD-VP1 of Vibrio parahaemolyticus was 8.2×10 10 PFU / ml, Erwinia phage SD-VP2 is 3.2×10 10 PFU / ml, Vibrio harveyi bacteriophage SD-VP3 is 2.6×10 10 PFU / ml, Vibrio vulnificus phage SD-VP4 is 5.4×10 10 PFU / ml.

[0090] Example 3 Preparation method of a pasteurella phage and bronchiseptica Bordetella phage freeze-dried powder

[0091] 1. Resuscitation of host bacteria

[0092] Clinically isolated Pasteurella and Bordetella suis strains were taken from a -80°C refrigerator, streaked onto TSA agar medium respectively, cultured in a constant temperature incubator at 37°C overnight, single colonies were picked, inoculated into TSB liquid culture medium, and cultured in a shaker at 37°C and 180rpm-220rpm for 16-20h to obtain proliferated bacterial liquid for use in the experiment.

[0093] 2. Fermentation culture of bacteria

[0094] The seeds of the different host bacteria cultured above were inoculated into different 100L fermentation tanks at a ratio of 1.5%, and the culture was expanded. After the host bacteria grew to the logarithmic phase, the inoculation titers were 10 at MOI = 0.01-0.001. 10 PFU / ml of clinically isolated Pasteurella phage SD-PM1 and Bordetella bronchiseptica phage SD-Bb1 were cultured in each fermenter at 37°C and 220rpm for 12h to obtain culture fluids of Pasteurella phage SD-PM1 and Bordetella bronchiseptica phage SD-Bb1, respectively.

[0095] 3. Post-treatment of phage culture fluid

[0096] The fermented Pasteurella phage SD-PM1 and Bordetella bronchiseptica phage SD-Bb1 were centrifuged at 6000 g in a disc centrifuge, and the supernatants were filtered through a 0.22 μm filter membrane to obtain sterile Pasteurella phage SD-PM1 and Bordetella bronchiseptica phage SD-Bb1. The phage titer was 3.6×10 10 PFU / ml and 4.5×10 10 PFU / ml.

[0097] 4. Preparation of lyoprotectant and freeze drying

[0098] The sterile stock solutions of Pasteurella phage SD-PM1 and Bordetella bronchiseptica phage SD-Bb1 were mixed to prepare a phage mixture solution.

[0099] The freeze-drying protective agent is compounded according to 10% trehalose, 10% gelatin, 1.0% sodium thiosulfate and 1.5% sorbitol.

[0100] The above phage mixture and lyophilization protective agent mixture were mixed in a ratio of 1:1 and freeze-dried. The freeze-drying conditions were set at -30℃ for 10h, 8℃ for 16h, 18℃ for 3h, 25℃ for 10h, and the vacuum degree was 0.08mbar. The phage freeze-dried powders of swine Pasteurella and Bordetella were obtained, and the titers of the freeze-dried powders were as follows: Pasteurella phage SD-PM1 was 2.8×10 10 PFU / ml, Bordetella bronchiseptica phage SD-Bb1 is 4.0×10 10 PFU / ml.

[0101] Example 4 Preparation method of Salmonella phage freeze-dried powder

[0102] 1. Resuscitation of host bacteria

[0103] Take the clinically isolated Salmonella strains from the -80℃ refrigerator, streak them on SS agar medium, culture them in a constant temperature incubator at 37℃ overnight, pick a single colony, inoculate it into LB liquid culture medium, and culture it in a shaker at 37℃ and 180rpm~220rpm for 16~20h to obtain the proliferated bacterial solution for the test.

[0104] 2. Fermentation culture of bacteria

[0105] The above-cultured host bacteria seeds were inoculated into a 100L fermentation tank at a ratio of 1.5%, and the culture was expanded. After the host bacteria grew to the logarithmic phase, the inoculation titer was 10 at an MOI of 0.001. 10 The clinically isolated Salmonella phage SD-SE-001 with a PFU / ml was cultured in a fermenter at 37°C and 220 rpm for 4 hours to obtain the Salmonella phage SD-SE-001 culture fluid.

[0106] 3. Post-treatment of phage culture fluid

[0107] The fermented Salmonella phage SD-SE-001 was centrifuged at 6000 g in a disc centrifuge, and the supernatant was filtered through a 0.22 μm filter membrane to obtain sterile Salmonella phage SD-SE-001. The phage titer was 2.5×10 10 PFU / ml.

[0108] 4. Preparation of lyoprotectant and freeze drying

[0109] The freeze-drying protective agent is compounded according to 10% trehalose, 8% gelatin, 1.0% sodium thiosulfate and 1.0% sorbitol.

[0110] The above phage culture solution and lyophilization protective agent mixture were mixed in a ratio of 1:1 and freeze-dried. The freeze-drying conditions were set at -40℃ for 8h, 5℃ for 15h, 18℃ for 4h, 28℃ for 8h, and the vacuum degree was 0.05mbar. The lyophilized powder of Salmonella phage was obtained, and the titer of the lyophilized powder was 1.8×10 10 PFU / ml.

[0111] Example 5 Preparation method of Riemerella anatipestifer phage freeze-dried powder

[0112] 1. Resuscitation of host bacteria

[0113] Take the clinically isolated Riemerella anatipestifer from a -80°C refrigerator, streak it on TSA agar medium, culture it in a constant temperature incubator at 37°C overnight, pick a single colony, inoculate it in TSB liquid culture medium, and culture it in a shaker at 37°C and 180rpm-220rpm for 16-20h to obtain the proliferated bacterial solution for the test.

[0114] 2. Fermentation culture of bacteria

[0115] The above-cultured host bacteria seeds were inoculated into a 100L fermentation tank at a ratio of 2.0%, and the culture was expanded. After the host bacteria grew to the logarithmic phase, the inoculation titer was 10 at an MOI of 0.001. 10 The clinically isolated Riemerella anatipestifer phage SD-RA1 with a PFU / ml was cultured in a fermenter at 37°C and 220 rpm for 12 hours to obtain the Riemerella anatipestifer phage SD-RA1 culture fluid.

[0116] 3. Post-treatment of phage culture fluid

[0117] The fermented Riemerella anatipestifer phage SD-RA1 was centrifuged at 6000 g in a disc centrifuge, and the supernatant was filtered through a 0.22 μm filter membrane to obtain sterile Riemerella anatipestifer phage SD-RA1. The phage titer was 5.6×10 10 PFU / ml.

[0118] 4. Preparation of lyoprotectant and freeze drying

[0119] The freeze-drying protective agent is compounded according to 10% trehalose, 10% gelatin, 0.5% sodium thiosulfate and 0.5% sorbitol.

[0120] The above phage culture solution and lyophilization protective agent mixture were mixed in a ratio of 1:1 and freeze-dried. The freeze-drying conditions were set at -38℃ for 9h, 10℃ for 15h, 20℃ for 2h, and 25℃ for 10h, and the vacuum degree was 0.08mbar. The Riemerella anatipestifer phage freeze-dried powder was obtained, and the titer of the freeze-dried powder was 4.5×10 10 PFU / ml.

[0121] Example 6 Temperature stability and acid-base stability test of bacteriophage freeze-dried powder

[0122] 1. Stability of phage stock solution in acidic environment

[0123] 1.0 ml of Salmonella phage stock solution, Riemerella anatipestifer phage stock solution, Vibrio parahaemolyticus phage stock solution and Pasteurella phage stock solution were taken respectively, and mixed with artificial gastric juice at a volume ratio of 1:1. Samples were taken at 30 minutes, 60 minutes, 90 minutes and 120 minutes to detect the phage titer. Detailed results are shown in Table 4. As can be seen from Table 4, the titer of the phage stock solutions of different pathogens began to decrease after 10 minutes of action in an acidic environment of pH = 2.0, and was basically inactivated after 40 minutes of action. This shows that the phage stock solution is not resistant to acidic environment.

[0124] Table 4 Results of stability test of phage stock solution in acidic environment

[0125]

[0126]

[0127] 2. Stability of phage freeze-dried powder in acidic environment

[0128] 1 g each of the lyophilized powder of Salmonella phage prepared in Example 4, the lyophilized powder of Riemerella anatipestifer phage prepared in Example 5, the lyophilized powder of Vibrio phage prepared in Example 2, and the lyophilized powder of Pasteurella phage prepared in Example 3 were taken and dissolved in artificial gastric juice (pH=2) to prepare 0.1 g / ml working solution.

[0129] The above working solution was sampled and tested for phage titer at 30min, 60min, 90min and 120min. Detailed results are shown in Table 5. As can be seen from Table 5, the titer of phage freeze-dried powder of different pathogens remained basically unchanged (all in the same order of magnitude) after 120min in an acidic environment of pH = 2.0. This shows that phage freeze-dried powder has better acid resistance than phage stock solution. The acid resistance of phage stock solution is significantly improved by adding a composite freeze-dried protective agent. This result further shows that after phage freeze-dried powder has experienced the gastric acid environment in the animal body, it can still maintain good activity and enter other organs such as the intestine to exert its effect, and the clinical prevention and control effect is more certain.

[0130] Table 5 Results of stability test of bacteriophage freeze-dried powder in acidic environment

[0131]

[0132] 3. Thermal stability of phage stock solution

[0133] Take 100 μl of the original Salmonella phage solution and add it to the EP tube containing 900 μl of SM solution. Place each EP tube in a water bath at 50°C, 60°C, 70°C, and 80°C, with 3 replicate tubes for each temperature group. At the action time of 10 min, 30 min, 60 min, 90 min, and 120 min, the current phage titer was determined using the double-layer agar plate method.

[0134] The results are shown in Table 6. As can be seen from Table 6, after the phage stock solution was exposed to 50°C for 10 minutes, the titer began to decrease, and after 30 minutes, the titer decreased significantly, and it was inactivated after 90 minutes. The titer of the phage stock solution was significantly decreased after 10 minutes at 60°C, and it was inactivated after 60 minutes. The titer of the phage stock solution was rapidly decreased after 10 minutes at 70°C, and it was inactivated after 30 minutes. At 80°C, the phage was completely inactivated after 10 minutes. It can be seen that the phage stock solution is not resistant to high temperatures.

[0135] Table 6 Results of thermal stability test of phage stock solution

[0136]

[0137] 4. Thermal stability of phage freeze-dried powder

[0138] Take 1g of Salmonella phage freeze-dried powder, dissolve it in purified water, and adjust the titer to 1.5×10 9 PFU / ml, take the above 100μl phage solution and add it to the EP tube containing 900μl SM solution. Place each EP tube in a water bath at 50℃, 60℃, 70℃, and 80℃, and set up 3 replicate tubes for each temperature group. At the action time of 10min, 30min, 60min, 90min, and 120min, the current phage titer was determined by the double-layer agar plate method.

[0139] The results are shown in Table 7. As can be seen from Table 7, the titer of phage freeze-dried powder did not change after 120 minutes at 50°C and 60°C. The titer of phage freeze-dried powder did not change after 60 minutes at 70°C, and the titer decreased by 1 gradient after 90 minutes, and the titer decreased by 2 gradients after 120 minutes. The titer of phage freeze-dried powder did not change at 80°C for 10 minutes, and the titer decreased by 1 gradient after 30 minutes, 2 gradients after 60 minutes, 4 gradients after 90 minutes, and 5 gradients after 120 minutes. It can be seen that phage freeze-dried powder can have better activity at a temperature of 50-70°C. The stability of phage freeze-dried powder is better, which is more conducive to transportation and storage.

[0140] Table 7 Thermal stability test results of phage freeze-dried powder

[0141]

[0142]

[0143] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lyophilization protectant for bacteriophage, characterized in that: The invention uses water as solvent and comprises the following components in mass concentrations: 5% to 10% of a cryoprotectant, 0.5% to 1.5% of an antioxidant, 0.5% to 1.5% of an acid-base regulator and 5% to 10% of a filler.

2. The lyoprotectant according to claim 1, characterized in that The cryoprotectant includes at least one of trehalose, glycerol and sucrose; the antioxidant includes sodium thiosulfate and / or vitamin E; the acid-base regulator includes sorbitol and / or phosphoric acid; and the filler includes at least one of mannitol, lactose and gelatin.

3. The lyoprotectant according to claim 1 or 2, characterized in that The freeze-drying protective agent comprises the following components in mass concentrations: 10% of a cryoprotectant, 1% of an antioxidant, 1.5% of an acid-base regulator and 10% of a filler.

4. A bacteriophage freeze-dried powder, characterized in that: The invention comprises a bacteriophage and the lyophilization protectant according to any one of claims 1 to 3.

5. The phage freeze-dried powder according to claim 4, characterized in that: The phage titer of the phage freeze-dried powder is ≥10 10 PFU / ml.

6. The phage freeze-dried powder according to claim 4, characterized in that: The bacteriophages include poultry pathogenic bacteria phages, livestock pathogenic bacteria phages or aquatic pathogenic bacteria phages.

7. The phage freeze-dried powder according to claim 6, characterized in that: The bacteriophage includes at least one of avian Escherichia coli phage, Salmonella phage, Vibrio alginolyticus phage, Vibrio parahaemolyticus phage, Vibrio vulnificus phage, Vibrio harveyi phage, Vibrio erwinia phage, Pasteurella phage, Bordetella bronchiseptica phage, porcine Escherichia coli phage and Riemerella anatipestifer phage.

8. The method for preparing the phage freeze-dried powder according to any one of claims 4 to 7, characterized in that: The following steps are involved: Mixing the phage solution with the lyophilization protective agent according to any one of claims 1 to 3 to obtain a mixed solution; freeze-drying the mixed solution to obtain a freeze-dried mixture; The freeze-dried mixture is crushed to obtain a phage freeze-dried powder.

9. The preparation method according to claim 8, characterized in that: The freeze-drying procedure includes: -30 to -40°C, 8 to 10 hours; 5 to 10°C, 15 to 18 hours; 18 to 20°C, 2 to 4 hours; 25 to 30°C, 8 to 10 hours; the vacuum degree of the freeze-drying is 0.04 to 0.10 mbar.

10. Use of the lyoprotectant according to any one of claims 1 to 3, the phage lyophilized powder according to any one of claims 4 to 7, or the phage lyophilized powder prepared by the preparation method according to claim 8 or 9 in the preparation of livestock and poultry drugs, aquatic drugs or feed additives.

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