Phage protective agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a bacteriophage protective agent, a preparation method and a bacteriophage biological preparation. The bacteriophage protective agent comprises lactose, glycine, magnesium sulfate, sodium chloride, dipotassium phosphate and the balance of water. Compared with the traditional bacteriophage protective agent, the bacteriophage protective agent provided by the invention has the advantages that the titer stability of a bacteriophage preparation in short-term storage at room temperature can be improved, the validity period of low-temperature storage can be prolonged, the cost is low, and the bacteriophage protective agent is more suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a bacteriophage protectant, its preparation method, and its application. Background Technology

[0002] The overuse of antibiotics has led to drug resistance, ecological environment, drug residues in food, and biosafety issues, which have attracted widespread attention worldwide. Bacteriophages are predators of bacteria, and in recent years, research on using bacteriophages as antibiotic alternatives has received widespread attention, especially in the fields of medicine, aquaculture, and food safety, which has become one of the current research hotspots.

[0003] However, in the application of bacteriophages, due to their large quantities, they are mostly in liquid form, often requiring the addition of preservatives to maintain their viability. Existing preservatives are ineffective at preserving bacteriophages, resulting in short preservation times, low survival rates, susceptibility to contamination, and the need for preservation at low and ultra-low temperatures. This significantly limits the development of bacteriophages.

[0004] Currently, commonly used bioprotectants for biological products in my country generally require storage at -15°C, and only 3-6 months at 2-8°C, with the remaining product needing to be used as soon as possible at room temperature. Therefore, in environments with inconvenient transportation and inadequate refrigeration facilities, bacteriophages are prone to reduced potency or even inactivation due to transportation or storage issues, leading to disease outbreaks. This not only hinders livestock development but also causes significant waste and financial losses. Therefore, the development of novel bacteriophage protectants is imperative. Summary of the Invention

[0005] In view of this, the present invention at least partially solves the aforementioned existing technical problems. Therefore, the present invention provides a phage protectant, which, through the synergistic effect between its components, improves the shelf life of liquid phage preparations during low-temperature storage, increases the stability of phage preparations, and ensures the large-scale application of phage preparations in the pig farming sector.

[0006] To solve the above-mentioned technical problems, the present invention is implemented as follows: According to one aspect of the present invention, a bacteriophage protectant is provided, the bacteriophage protectant comprising 3 wt% to 7 wt% lactose, 0.5 wt% to 1.5 wt% glycine, 1 wt% to 3 wt% magnesium sulfate, 0.5 wt% to 1.5 wt% sodium chloride, 0.5 wt% to 2 wt% dipotassium hydrogen phosphate, and water as the balance.

[0007] In some embodiments, the phage protectant further includes at least one of histidine (0.5 wt% to 1.5 wt%) and trehalose (1 wt% to 5 wt%).

[0008] According to another aspect of this application, this application provides a method for preparing a bacteriophage protectant, comprising the following steps: A) Dissolve lactose and glycine in the first part of water, and filter the solution through a filter membrane to remove bacteria to obtain solution A for later use; B) Dissolve sodium chloride, magnesium sulfate, and dipotassium hydrogen phosphate in the second part of water, sterilize, and cool to room temperature to obtain solution B for later use. C) Mix the above solutions A and B thoroughly to obtain the phage protectant, and store at 2℃~8℃ for later use.

[0009] In some embodiments, step A) further includes dissolving at least one of histidine or trehalose in the first portion of water.

[0010] In some of these embodiments, in step A), the pore size of the filter membrane ranges from 0.1 μm to 0.4 μm.

[0011] In some of these embodiments, in step B), the sterilization temperature is 110°C to 135°C and the sterilization time is 15 min to 30 min.

[0012] In some of these embodiments, the volume ratio of the first portion of water to the second portion of water in steps A) and B) is (1~3):(1~3).

[0013] According to a third aspect of this application, this application provides a bacteriophage biological agent, including a bacteriophage and a bacteriophage protectant, wherein the bacteriophage protectant is the bacteriophage protectant mentioned above or a bacteriophage protectant prepared by the preparation method mentioned above.

[0014] In some of these embodiments, the volume ratio of the phage protectant to the phage is 1:(1~4).

[0015] In some embodiments, the bacteriophage includes at least one of Pasteurella multocida bacteriophage, Escherichia coli bacteriophage, Staphylococcus aureus bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage. Preferably, the bacteriophage includes a cocktail bacteriophage, which includes Escherichia coli bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage.

[0016] According to the fourth aspect of this application, this application provides a method for preparing a bacteriophage biological agent, comprising the following steps: under aseptic conditions, filtering the bacteriophage fermentation stock solution through a three-stage filter of 0.7~0.9μm, 0.4~0.5μm, and 0.2~0.3μm to obtain a bacteriophage stock solution; adding a bacteriophage protectant to the bacteriophage stock solution and mixing evenly to obtain a bacteriophage biological agent.

[0017] In some of these embodiments, the bacteriophage biological agent can be stored under refrigerated conditions, room temperature conditions, and accelerated stability testing conditions, wherein the refrigerated conditions are 2°C to 8°C, the room temperature conditions are 20°C to 30°C, and the accelerated stability testing conditions are 36°C to 38°C.

[0018] Implementing the technical solution of the present invention has at least the following beneficial effects: The phage protectant provided by this invention has few components, a simple preparation process, readily available raw materials, and low cost. It can maintain the activity of phage preparations during room temperature transport and extend their shelf life to more than 12 months when stored at 2℃~8℃.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0021] Figure 1 The graph shows the protection rate of the new protective agent described in this invention against Pasteurella phage at 2℃~8℃.

[0022] Figure 2 The graph shows the protection rate of the new protective agent described in this invention against Escherichia coli phage at 2℃~8℃.

[0023] Figure 3 The graph shows the protection rate of the new protective agent described in this invention against Staphylococcus aureus phage at 2℃~8℃.

[0024] Figure 4 The graph shows the protection rate of the new protective agent described in this invention against Clostridium perfringens phage at 2℃~8℃.

[0025] Figure 5 The graph shows the protection rate of the new protective agent described in this invention against Salmonella phage at 2℃~8℃.

[0026] Figure 6 The graph shows the protection rate of the new protective agent described in this invention against cocktail phage at 2℃~8℃.

[0027] The accompanying drawings have illustrated specific embodiments of the invention, which will be described in more detail below. These drawings and descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art through reference to particular embodiments. Detailed Implementation

[0028] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0029] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges or individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0030] In the description of this application, "same chemical composition" should be interpreted broadly, that is, the main components of the two have the same chemical composition, or the two have substantially the same chemical composition, but may have errors or impurities within the acceptable range that can be understood by those skilled in the art.

[0031] In the description of this application, "A and / or B" can include any of the cases of A alone, B alone, or A and B, where A and B are merely examples and can be any technical feature connected by "and / or" in this application.

[0032] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0033] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0034] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0035] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0036] Currently, bacteriophage preparations are typically used by sprinkling, spraying, or pouring, and rarely by injection. Due to their large dosage, they are usually not preserved by freeze-drying and are mostly stored in liquid form at 2℃~8℃. However, they also face the technical drawback of being difficult to maintain at low temperatures for long periods due to the relatively remote nature of aquaculture sites and poor transportation and storage conditions, leading to inactivation of the bacteriophage preparations. Currently reported bacteriophage liquid preservation agents are mostly used in aquaculture. Chinese patent document application number 201711200242.5 discloses a bacteriophage liquid preservation agent that can preserve bacteriophage preparations in liquid form at 4℃~37℃. However, this preserver has seven components, and the volume ratio between the preserver and the bacteriophage is 1:1, which increases the amount of preserver used and raises the cost of the bacteriophage preparation. Therefore, developing a bacteriophage preservation biological agent with lower cost, longer protective effect, and better stability is very meaningful and necessary.

[0037] [Phage protectant] According to a first aspect of this application, this application provides a bacteriophage protectant comprising 3 wt% to 7 wt% lactose, 0.5 wt% to 1.5 wt% glycine, 1 wt% to 3 wt% magnesium sulfate, 0.5 wt% to 1.5 wt% sodium chloride, 0.5 wt% to 2 wt% dipotassium hydrogen phosphate, and water as the balance.

[0038] In this application, the components of the phage protectant exhibit excellent synergistic effects. Lactose, by binding with water molecules, can alleviate osmotic stress in phages under low-temperature conditions to a certain extent, maintaining the uniform suspension of the phage preparation. The addition of glycine aims to maintain the neutral environment of the preservation solution and stabilize the protein function of the phage. Sodium chloride and magnesium sulfate work synergistically to maintain the isotonic environment of the phage protectant, while magnesium ions (Mg²⁺) contribute to its stability. + ( ) is a cofactor for the activity of phage tail enzymes and plays a crucial role in maintaining phage infectivity.

[0039] In this application, the phage protectant further includes at least one of histidine (0.5wt%~1.5wt%) and trehalose (1wt%~5wt%). It should be further noted that, in the above-mentioned phage protectant, the addition of histidine can form hydrogen bonds with free water molecules in the aqueous solution, reducing the "impact" of water molecules on the hydrophobic groups on the protein surface, thereby ensuring the stability of the phage protein structure and further improving the survival and infectivity of the phage. The addition of trehalose can form a protective film on the surface of the phage cells, which has the functions of resisting high temperature, maintaining osmotic pressure, and inhibiting bacteria.

[0040] In this application, the water includes at least one of deionized water and distilled water. In a preferred embodiment of this application, deionized water is selected. In this application, there are no special requirements for the source of water. Commercially available products or homemade products well known to those skilled in the art can be used. The main purpose of the water is to dissolve other components in the carrier protective agent.

[0041] [Preparation of bacteriophage protectants] In this application, the method for preparing the phage protectant includes the following steps: A) Dissolve lactose and glycine in the first part of water, and filter the solution through a filter membrane to remove bacteria to obtain solution A for later use; B) Dissolve sodium chloride, magnesium sulfate, and dipotassium hydrogen phosphate in the second part of water, sterilize, and cool to room temperature to obtain solution B for later use. C) Mix the above solutions A and B thoroughly to obtain the phage protectant, and store at 2℃~8℃ for later use.

[0042] In this application, the components of the phage protectant are divided into two groups, A and B, for separate processing because different components in the phage have different thermal stability. By grouping the components, the activity of the components in group A is ensured by filtration, while the components in group B are ensured by high temperature. This approach does not sacrifice the core function of protecting the phage and achieves sterility assurance through a simple process.

[0043] In this application, in step A), the pore size of the filter membrane is in the range of 0.1μm to 0.4μm, for example, 0.1μm, 0.2μm, 0.3μm, 0.4μm or any two of them. By controlling the pore size of the filter membrane within the above range, most harmful microorganisms can be intercepted, ensuring sterility.

[0044] In this application, in step B, the sterilization temperature is 110℃~135℃, and the sterilization time is 15min~30min. By controlling the sterilization temperature and sterilization time within the above range, it can be ensured that most harmful microorganisms are killed, thus ensuring the sterility of the solution.

[0045] In this application, in step B, the volume ratio of the first part of water to the second part of water is (1~3):(1~3), for example, 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, 3:2 or any two of these ratios. By controlling the first part of water and the second part of water within the above volume ratio range, the full dissolution of the solute is ensured, and the flexibility of the process is improved to adapt to the production needs of different batches.

[0046] [Bacteriophage Biological Agents] In another aspect of this application, a bacteriophage biological agent is provided, which is composed of a bacteriophage and the aforementioned bacteriophage protectant or a bacteriophage protectant prepared by the aforementioned preparation method, which will not be described in detail here.

[0047] In this application, the volume ratio of the phage protectant to the phage is 1:(1~4), for example, 1:1, 1:2, 1:3, 1:4, or any ratio of two of them. It should be further noted that the phage protectant of this application has a good protective effect. When the volume ratio of the phage protectant to the phage is 1:4, the activity of the phage can still be ensured, which can greatly reduce the cost of phage biological agents.

[0048] In this application, the bacteriophage includes at least one of Pasteurella multocida bacteriophage, Escherichia coli bacteriophage, Staphylococcus aureus bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage. In a preferred embodiment of this application, the bacteriophage includes a cocktail bacteriophage, which includes Escherichia coli bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage. It should be further noted that the bacteriophage protection solution of this application can be used to mix and protect different types of bacteriophages, greatly enriching the application scenarios of bacteriophage biological agents.

[0049] [Preparation of bacteriophage biological agents] According to a fourth aspect of this application, this application provides a method for preparing a bacteriophage biological agent, comprising the following steps: Under aseptic conditions, the phage fermentation broth was filtered through a three-stage filter with a filter thickness of 0.7~0.9μm, 0.4~0.5μm, and 0.2~0.3μm to obtain the phage stock solution. The phage protectant was then added to the phage stock solution and mixed thoroughly to obtain the phage biological agent.

[0050] In the above experimental steps, the phage stock solution was subjected to three-stage filtration at 0.7~0.9μm, 0.4~0.5μm, and 0.2~0.3μm. This step-by-step "from coarse to fine" retention method achieved efficient removal of impurities while preserving the phage activity to the greatest extent, laying the foundation for the subsequent preparation of safe and stable phage biological agents.

[0051] In this application, the bacteriophage biological agent can be stored under refrigerated storage conditions, room temperature storage conditions, and accelerated stability testing conditions. The refrigerated storage conditions are 2℃~8℃, the room temperature storage conditions are 20℃~30℃, and the accelerated stability testing conditions are 36℃~38℃. It should be further noted that, through long-term experimental verification, the applicant has found that the bacteriophage biological agent prepared by the bacteriophage protectant provided in this application has achieved good preservation effects under different storage conditions, including refrigerated storage conditions, room temperature storage conditions, and accelerated stability testing conditions. This provides data support for the subsequent transportation of bacteriophage biological agents and greatly expands the applicable scenarios of bacteriophage biological agents.

[0052] The present application will be described in detail below with reference to the accompanying drawings and embodiments. However, the implementation and protection of the present invention are not limited thereto. The following embodiments are only some embodiments of the present application and are not intended to limit the present application.

[0053] Example 1 The seven protective agent components of this application were prepared at different concentrations, and single-component phage protective agents were prepared according to the concentrations shown in Table 1. Using pure water as a control group, the single-component phage protective agents were prepared by either filtration through a 0.22 μm filter under aseptic conditions or sterilization at 121℃ for 15 min followed by cooling to room temperature. The Pasteurella phage fermentation broth was filtered through three stages to obtain Pasteurella phage stock solution, which was then mixed with the aforementioned single-component phage protective agents to obtain a phage biological preparation. The Pasteurella phage biological preparation was stored at 37℃, and its potency was measured at 0, 3, 7, 15, and 30 days to observe the protective effect of each protective agent component on Pasteurella phage. The results are shown in Table 2.

[0054] Table 1. Concentration of phage protectant components Table 2. Potency of Pasteurella multocida phage in different protective agents at 37℃ The above experimental results show that, under 37 ℃ conditions, after 7 days of storage, the titer of Pasteurella multocida phage decreased by approximately 2.1 times in the seven protective agents and by approximately 6.6 times in the control group; after 15 days of storage, the titer of Pasteurella multocida phage decreased by 5 to 15 times in the seven protective agents and by 30 to 45 times in the control group. Furthermore, the results from days 15 and 30 show that the titer of Pasteurella multocida phage was higher in all seven protective agents than in the control group, indicating that all seven protective agents at three different concentrations provided good protection for Pasteurella multocida phage.

[0055] Example 2: Formulation combinations of different protective agents Following the method of Example 1, phage protectant combinations with different components were prepared. The components and concentrations of the different phage protectants are shown in Table 3. The aforementioned phage protectants were mixed with PmS01 Pasteurella multocida phage and PmS02 Pasteurella multocida phage to prepare phage biological agents. The control group was selected from the protectant formulation of Chinese Patent 201711200242.5, which consists of the following components in mass percentage: 1.5% sodium chloride, 2.5% magnesium sulfate, 1.6% calcium chloride, 2% histidine, 2% gelatin, 6% lactose, 5% trehalose, and the balance being deionized water. The phage protectants were prepared according to the patent method. The phage protectants of the control group were mixed with PmS01 Pasteurella multocida phage and PmS02 Pasteurella multocida phage to prepare phage biological agents. Stability tests were conducted at 37 °C, and the experimental results are shown in Tables 4 and 5.

[0056] Table 3. Combinations and concentrations of different protective agents Table 4. Titer of Pasteurella multocida PmS01 bacteriophage in different protective agents at 37℃ (PFU / mL) Table 5. Titer of Pasteurella multocida PmS02 bacteriophage in different protective agents at 37℃ (PFU / mL) The experimental results show that, after 7 days of storage, the phage protectants 1-4 provided by this invention exhibited a 2-2.4 times higher titer in protecting *Pasteurella multocida* phage *PmS01* compared to the control group, and a 1.85-2.14 times higher titer in protecting *Pasteurella multocida* phage *PmS02* compared to the control group. After 30 days of storage, the phage protectants 1-4 provided by this invention exhibited a 12-30 times higher titer in protecting *Pasteurella multocida* phage *PmS01* compared to the control group, and a 16-35 times higher titer in protecting *Pasteurella multocida* phage *PmS02* compared to the control group. These results demonstrate that the phage protectants 1-4 provided by this invention significantly protect the activity of the phages.

[0057] Example 3: Protective titer against Staphylococcus aureus bacteriophage at 37°C The bacteriophage protectant from Example 2 was used to prepare Staphylococcus aureus bacteriophage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental results are shown in Table 6.

[0058] Table 6. Phage titers of Staphylococcus aureus at 37℃ (PFU / mL) Example 4: Protective titer against Salmonella bacteriophage at 37°C The bacteriophage protectant from Example 2 was used to prepare Salmonella bacteriophage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental results are shown in Table 7.

[0059] Table 7. Salmonella phage titers (PFU / mL) at 37℃ Example 5: Protective titer against Clostridium perfringens bacteriophage at 37°C The bacteriophage protectant from Example 2 was used to prepare Clostridium perfringens bacteriophage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental results are shown in Table 8.

[0060] Table 8. Phage titer of Clostridium perfringens at 37℃ (PFU / mL) Example 6: Protective titer against two types of Escherichia coli at 37°C The phage protectant from Example 2 was used to prepare EcS01 and EcS02 Escherichia coli phage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental results are shown in Table 9.

[0061] Table 9. Phage titers (PFU / mL) of two Escherichia coli strains at 37℃ The experimental results from Examples 2 to 6 show that, under 37°C conditions, after 15 days of storage, the phage titers in the phage biological agents provided by this invention are 3.5 to 12 times higher than those in the control group; after 30 days of storage, the phage titers in the phage biological agents provided by this invention are 16 to 40 times higher than those in the control group. This indicates that the phage biological agents provided by this invention have good protective effects against Pasteurella multocida phage, Escherichia coli phage, Staphylococcus aureus phage, Clostridium perfringens phage, and Salmonella phage.

[0062] Example 7: Protective titer against Staphylococcus aureus bacteriophage at 25°C The bacteriophage protectant from Example 2 was used to prepare Staphylococcus aureus bacteriophage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental conditions were that it was stored at room temperature. The experimental results are shown in Table 10.

[0063] Table 10. Potency of Staphylococcus aureus bacteriophage biological agents at 25℃ (PFU / mL) Example 8: Protective titer against two Pasteurella multocida phages at 25°C Using the phage protectant from Example 2, PmS01 and PmS02 Pasteurella phage biological agents were prepared according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at room temperature. The experimental results are shown in Table 11. Table 11. Potency of Pasteurella multocida phage biological agents at 25℃ (PFU / mL) Example 9: Protective titer against two Escherichia coli bacteriophages at 25°C The phage protectant in Example 2 was used to prepare EcS01 Escherichia coli phage and EcS01 Escherichia coli phage biological agent according to the formula of protectant 2 in Example 2. The control protectant in Example 2 was used for the control experiment. The experimental conditions were that it was stored at room temperature. The experimental results are shown in Table 12.

[0064] Table 12. Potency (PFU / mL) of two Escherichia coli bacteriophage biological agents at 25℃ Example 10: Protective titer against Clostridium perfringens bacteriophage at 25°C The bacteriophage protectant in Example 2 was selected, and the Clostridium perfringens bacteriophage biological agent was prepared according to the formula of protectant 2 in Example 2. The control protectant in Example 2 was selected for the control experiment. The experimental conditions were that it was stored at room temperature. The experimental results are shown in Table 13.

[0065] Table 13. Potency of Clostridium perfringens phage biological agents at 25℃ (PFU / mL) Example 11 Protective titer against Salmonella bacteriophage at 25°C The bacteriophage protectant in Example 2 was selected, and Salmonella bacteriophage biological agents were prepared according to the formula of protectant 2 in Example 2. The control protectant in Example 2 was selected for the control experiment. The experimental conditions were that it was stored at room temperature. The experimental results are shown in Table 14.

[0066] Table 14. Potency of Salmonella bacteriophage biological agents at 25℃ (PFU / mL) The test results from Examples 7 to 11, conducted at room temperature, show that when stored at 25°C for 30 days, the new protectant maintained 92-99% of the titers of each phage, while the control protectant maintained 66-84%. The new protectant demonstrates superior protection against Pasteurella multocida phage, Escherichia coli phage, Staphylococcus aureus phage, Clostridium perfringens phage, and Salmonella phage. The new protectant maintains stable activity after one month of storage at room temperature, eliminating the need for cold chain transportation, reducing transportation costs, and facilitating the widespread application of phage preparations.

[0067] Example 12: Protective titer against two Pasteurella multocida phages at 2℃~8℃ Using the phage protectant from Example 2, PmS01 and PmS02 Pasteurella phage biological agents were prepared according to the method of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 15. Figure 1 As shown.

[0068] Table 15 Potency of two Pasteurella multocida phage biological agents at 2℃~8℃ Example 13: Protective titer against two Escherichia coli bacteriophages at 2℃~8℃ Using the phage protectant from Example 2, EcS01 Escherichia coli phage and EcS01 Escherichia coli phage biological agents were prepared according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 16 and... Figure 2 As shown.

[0069] Table 16 Potency of two Escherichia coli bacteriophage biological agents at 2℃~8℃ Example 14: Protective titer against Staphylococcus aureus bacteriophage at 2℃~8℃ Using the phage protectant from Example 2, Staphylococcus aureus phage biological agents were prepared according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 17 and... Figure 3 As shown.

[0070] Table 17 Potency of Staphylococcus aureus bacteriophage biological agents at 2℃~8℃ Example 15: Protective titer against Clostridium perfringens bacteriophage at 2℃~8℃ The bacteriophage protectant from Example 2 was used to prepare Clostridium perfringens bacteriophage biological agents according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for the control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 18 and 1999. Figure 4 As shown.

[0071] Table 18 Potency of Clostridium perfringens phage biological agents at 2℃~8℃ Example 16: Protective titer against Salmonella bacteriophage at 2℃~8℃ Using the phage protectant from Example 2, Salmonella phage biological agents were prepared according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 19. Figure 5 As shown.

[0072] Table 19 Potency of Clostridium perfringens phage biological agents at 2℃~8℃ Example 17 Protective titer against cocktail phage at 2℃~8℃ Using the phage protectant from Example 2, a cocktail phage biological agent was prepared according to the formula of protectant 2 in Example 2. The control protectant from Example 2 was used for a control experiment. The experimental conditions were storage at 2℃~8℃. The experimental results are shown in Table 20 and... Figure 6 As shown.

[0073] Table 20. Potency of cocktail phage biological agents at 2℃~8℃ Based on the results of Examples 12 to 17, it can be seen from Table 15 and... Figure 1 The results showed that, after being stored at 2℃~8℃ for 18 months, the protective titer of the Pasteurella multocida phage protectant provided by this invention showed a steady decreasing trend against both PmS01 and PmS02 phages, and the protection rate remained above 80% in the 18th month. In contrast, the protective titer of the control group protectant against Pasteurella multocida phages PmS01 and PmS02 decreased to about 80% in the 4th month, and then slowly decreased to about 40% in the 18th month.

[0074] According to Table 16 and Figure 2 The results show that the phage protectant provided by this invention exhibits a steady decline in protective titer against Escherichia coli phages EcS01 and EcS02, with a protection rate of approximately 84% at 18 months. In contrast, the control group protectant showed relatively stable titer against Escherichia coli phages for the first 6 months, followed by a slow decline, with protection rates of 92%, 67%, and 53% at 6, 12, and 18 months, respectively.

[0075] According to Table 17 and Figure 3 The results showed that, after being stored at 2℃~8℃ for 18 months, the protective titer of the phage protectant provided by this invention against Staphylococcus aureus phage showed a steady decreasing trend, with the protection rate remaining above 70% in the 18th month. In contrast, the protective titer of the control group protectant against Staphylococcus aureus phage was relatively stable in the first 3 months, with a protection rate of 84%, and then slowly decreased, dropping to 55%, 29%, and 14% in the 6th, 12th, and 18th months, respectively.

[0076] According to Table 18 and Figure 4 The results showed that when stored at 2℃~8℃ for 18 months, the protective titer of Clostridium perfringens phage provided by the present invention showed a steady decline trend, with the protection rate remaining above 80% in the 18th month. In contrast, the protective titer of the control group protectant against Clostridium perfringens phage was relatively stable in the first 6 months, and then slowly declined, with protection rates of 85%, 56%, and 35% in the 6th, 12th, and 18th months, respectively.

[0077] According to Table 19 and Figure 5 The results showed that when stored at 2℃~8℃ for 18 months, the protective titer of the protectant provided by this invention against Salmonella phage showed a steady decreasing trend, with the protection rate remaining above 77% in the 18th month. In contrast, the protective titer of the control group protectant against Clostridium perfringens phage was relatively stable in the first 6 months, and then slowly decreased, with protection rates of 93%, 52%, and 33% in the 6th, 12th, and 18th months, respectively.

[0078] According to Table 20 and Figure 6 The results showed that, after being stored at 2℃~8℃ for 18 months, the protective titer of the phage protectant provided by this invention against cocktail phages showed a steady decreasing trend. In the 18th month, the protection rate against Escherichia coli phage, Clostridium perfringens phage, and Salmonella phage remained above 70%. In contrast, the protective titer against cocktail phages in the control group was relatively stable for the first 6 months, and then slowly decreased, with protection rates of 78%~88%, 51%~63%, and 30%~48% in the 6th, 12th, and 18th months, respectively.

[0079] In summary, the phage protectant provided by this invention exhibits superior protective effects against Pasteurella multocida phage, Escherichia coli phage, Staphylococcus aureus phage, Clostridium perfringens phage, and Salmonella phage compared to the control protectant. Furthermore, the phages stored at 2℃~8℃ show good stability and a shelf life exceeding 12 months when the protectant provided by this invention is added.

[0080] The parts of this invention not described in detail are techniques known to those skilled in the art.

[0081] The basic principles of the present invention have been described above with reference to specific embodiments. However, it should be noted that the advantages, benefits, and effects mentioned in the present invention are merely examples and not limitations, and should not be considered as essential features of each embodiment of the present invention. Furthermore, the specific details disclosed above are for illustrative and facilitative purposes only, and are not limitations. These details do not limit the present invention to the necessity of employing the aforementioned specific details.

[0082] In the foregoing description of this specification, references to terms such as "one embodiment," "another embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples, without contradiction. Additionally, it should be noted that in this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bacteriophage protectant, characterized in that, The protective agent comprises 3wt%~7wt% lactose, 0.5wt%~1.5wt% glycine, 1wt%~3wt% magnesium sulfate, 0.5wt%~1.5wt% sodium chloride, 0.5wt%~2wt% dipotassium hydrogen phosphate, and water as the balance.

2. The phage protectant according to claim 1, characterized in that, The phage protectant also includes at least one of histidine (0.5wt%~1.5wt%) and trehalose (1wt%~5wt%).

3. A method for preparing a bacteriophage protectant, characterized in that, Includes the following steps: A) Dissolve lactose and glycine in the first part of water, and filter the solution through a filter membrane to remove bacteria to obtain solution A for later use; B) Dissolve sodium chloride, magnesium sulfate, and dipotassium hydrogen phosphate in the second part of water, sterilize, and cool to room temperature to obtain solution B for later use. C) Mix the above solutions A and B thoroughly to obtain the phage protectant, and store at 2℃~8℃ for later use.

4. The preparation method according to claim 3, characterized in that, In step A), at least one of histidine or trehalose is dissolved in the first portion of water.

5. The preparation method according to claim 3, characterized in that, The pore size range of the filter membrane in step A) is 0.1 μm to 0.4 μm; And / or, in step B, the sterilization temperature is 110℃~135℃, and the sterilization time is 15min~30min; And / or, in steps A) and B), the volume ratio of the first portion of water to the second portion of water is (1~3):(1~3).

6. A bacteriophage biological agent, characterized in that, It includes a phage protectant and a phage, wherein the phage protectant is the phage protectant according to any one of claims 1 to 3 or the phage protectant prepared by the preparation method according to any one of claims 4 to 5.

7. The bacteriophage biological agent according to claim 6, characterized in that, The volume ratio of the bacteriophage protectant to the bacteriophage is 1:(1~4).

8. The bacteriophage biological agent according to claim 6, characterized in that, The bacteriophage includes at least one of Pasteurella multocida bacteriophage, Escherichia coli bacteriophage, Staphylococcus aureus bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage. Preferably, the bacteriophage includes a cocktail bacteriophage, which includes Escherichia coli bacteriophage, Clostridium perfringens bacteriophage, and Salmonella bacteriophage.

9. A method for preparing a bacteriophage biological agent, characterized in that, Includes the following steps: Under aseptic conditions, the phage fermentation broth was filtered through a three-stage filter with a filter thickness of 0.7~0.9μm, 0.4~0.5μm, and 0.2~0.3μm to obtain the phage stock solution. The phage protectant was then added to the phage stock solution and mixed thoroughly to obtain the phage biological agent.

10. The preparation method according to claim 9, characterized in that, The bacteriophage biological agent can be stored under refrigerated conditions, room temperature conditions, and accelerated stability test conditions. The refrigerated conditions are 2℃~8℃, the room temperature conditions are 20℃~30℃, and the accelerated stability test conditions are 36℃~38℃.

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