Method for improving stability and antibacterial persistence of coliphage preparation
By adding chitosan and combining it with a stabilizer during the phage proliferation process, the instability of phage preparations during storage was solved, achieving high efficiency, stability, and long-term antibacterial effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO RUNDA BIOTECH
- Filing Date
- 2026-01-23
- Publication Date
- 2026-05-01
AI Technical Summary
Phage preparations are susceptible to temperature changes, shear forces, and impurities during storage, which can lead to a decrease in potency and the development of tolerance in the host bacteria, affecting the duration of their antibacterial activity.
Chitosan was added during the phage proliferation process, and a stable phage solution was formed through a combination of low-shear centrifugation, membrane filtration, membrane concentration, and the addition of phage stabilizers. Finally, multi-stage filtration was performed to remove bacteria.
It significantly improves the storage stability of phage preparations, prolongs their effective antibacterial duration against host bacteria, and demonstrates superior performance in practical applications.
Smart Images
Figure CN121943979A_ABST
Abstract
Description
A method for improving the stability and antibacterial persistence of Escherichia coli bacteriophage preparations Technical Field
[0001] This invention belongs to the field of biological agent preparation technology, specifically relating to a method for improving the stability and antibacterial persistence of Escherichia coli bacteriophage preparations. Background Technology
[0002] Bacteriophages are a type of virus that can specifically lyse bacteria. Compared with traditional broad-spectrum antibiotics, bacteriophages are highly specific to host bacteria and have less impact on non-target microbial communities during application. Therefore, they have good application prospects in livestock and poultry breeding, biocontrol, and microbial regulation.
[0003] However, in actual preparation and application, bacteriophages are usually in liquid form, and their structure and activity are easily affected by factors such as temperature changes, shear force, and residual impurities, leading to a rapid decline in potency during storage. Furthermore, during bacteriophage proliferation and application, host bacteria can easily develop tolerance, shortening the effective inhibition period of the bacteriophage against the host bacteria, thus affecting its practical application effectiveness.
[0004] Existing research reports that encapsulating bacteriophages in chitosan-based materials can improve their stability in adverse environments to some extent. However, these methods mostly focus on phage delivery or encapsulation protection, typically requiring complex preparation steps. Furthermore, their technical focus is primarily on protective effects under specific application scenarios. Systematic process design and validation are still lacking for maintaining the stability of liquid phage formulations under long-term storage conditions and improving their antibacterial persistence.
[0005] Therefore, there is an urgent need for a new method for preparing phage formulations that can effectively improve their storage stability and prolong their effective antibacterial duration against host bacteria without affecting their activity.
[0006] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a method for improving the stability and antibacterial persistence of Escherichia coli phage preparations. By jointly regulating the phage proliferation stage and post-processing, the stability of phage preparations under normal and low temperature conditions is significantly improved without affecting the phage lysis activity, and the effective antibacterial duration against the host bacteria is extended.
[0008] This invention proposes a method to improve the stability and antibacterial persistence of Escherichia coli phage preparations, which includes the following steps: (1) Phage proliferation: chitosan is added during the cultivation of Escherichia coli host bacteria, and Escherichia coli phage is inoculated when the host bacteria are in the logarithmic growth phase. The bacteria are cultured until the phage lyses and releases, and a phage proliferation solution is obtained; (2) Post-treatment: the phage proliferation solution in step (1) is subjected to low-shear centrifugation, membrane filtration and membrane concentration treatment in sequence to obtain a concentrated phage solution; (3) Stabilization treatment: phage stabilizer is added to the concentrated phage solution in step (2) to obtain a stabilized phage solution; (4) Sterilization: the stabilized phage solution in step (3) is subjected to multi-stage filtration sterilization to obtain an Escherichia coli phage preparation.
[0009] Preferably, the final concentration of chitosan in the culture system in step (1) is 0.01% to 0.05%, and more preferably, the final concentration is 0.02%.
[0010] Preferably, the chitosan in step (1) is added before phage inoculation. The chitosan, as a regulatory component in the culture system, participates in the phage proliferation process, providing a process basis for obtaining a phage preparation with high stability in the future.
[0011] Preferably, the centrifugation in step (2) is carried out using a tubular centrifuge with a rotation speed of 17,000 rpm and a processing capacity of 50 L / h.
[0012] Preferably, the membrane filtration in step (2) uses a hollow fiber membrane with a pore size of 800 nm and a filtration pressure of less than 0.4 MPa.
[0013] Preferably, the membrane concentration in step (2) uses a hollow fiber membrane with a pore size of 10 nm.
[0014] Preferably, the phage stabilizer in step (3) is a composite stabilizer, comprising 0.01 w / v % ethylenediaminetetraacetic acid, 0.01 w / v % glycine, 2 w / v % glycerol, 1 w / v % trehalose and 0.1 w / v % polyethylene glycol.
[0015] Preferably, the multi-stage filtration in step (4) uses polypropylene 1.00 μm, polyethersulfone 0.45 μm and polyethersulfone 0.22 μm filter membranes in sequence.
[0016] Furthermore, the Escherichia coli phage preparation obtained by the above method showed a potency decrease of no more than two orders of magnitude after 12 months of storage at room temperature.
[0017] Furthermore, the titer of the Escherichia coli phage preparation obtained by the above method decreased by no more than one order of magnitude after being stored at 4 °C for 12 months.
[0018] Furthermore, compared with conventional preparation methods, the Escherichia coli phage preparation obtained by the above method has an antibacterial duration of at least 300% longer in the in vitro antibacterial system.
[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention introduces chitosan during the phage proliferation process and adds phage stabilizer in the post-treatment stage, which effectively improves the microenvironmental conditions of the phage preparation and reduces the risk of inactivation of phage due to shearing, oxidation or impurity interference during preparation and storage. The results of the examples show that the phage preparation prepared by the present invention has a titer decrease of no more than two orders of magnitude after 12 months of storage at room temperature and no more than one order of magnitude after 12 months of storage at 4 ℃, which shows significantly enhanced storage stability compared with conventional methods without the addition of chitosan and stabilizer; (2) Under the conditions of using the proliferation process described in the present invention and combined with the stabilizer system, the prepared phage preparation can maintain a high activity level for a long time in the in vitro antibacterial system, thereby prolonging the duration of inhibition against host Escherichia coli. The results of the examples show that, under the same culture conditions and inoculation amount, compared with the conventional method, the phage preparation of the present invention has a longer inhibition time on the host bacteria to 540 min, and the final OD600 value of the host bacteria is significantly reduced, indicating that it has a more durable and stable antibacterial effect during use; (3) Based on the above-mentioned improvement in stability and antibacterial persistence, the phage preparation prepared by the present invention shows a better effect under actual application conditions. The results of large-scale animal experiments show that the phage preparation prepared by the process of the present invention can significantly reduce the average mortality rate of the experimental chicken flock, and its 7-day average mortality rate can be reduced to as low as 0.031%, which is significantly better than the phage preparation prepared by the conventional process, indicating that the preparation method has good application feasibility and promotion value. Attached Figure Description
[0020] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the following description is only a part of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 shows a comparison of the potency changes of phage preparations obtained by different preparation methods under storage conditions at 4 °C.
[0022] Figure 2 shows a comparison of the antibacterial function of bacteriophages under different treatment conditions. Detailed Implementation
[0023] This invention proposes a method to improve the stability and antibacterial persistence of Escherichia coli bacteriophage preparations. To facilitate understanding of this invention by those skilled in the art, the specific embodiments of this invention are described below with reference to the accompanying drawings.
[0024] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0025] The bacteriophage used in this example is Escherichia coli Bacteriophage, named RDP-EC-22156, which was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.45432.
[0026] Example 1: Phage propagation process. 2% *E. coli* host bacteria BE-20310 were inoculated into a 5 L seed tank and cultured for 2 h. Then, sterilized chitosan solution was added to a final concentration of 0.02%, and the culture was continued for another 1 h. At this point, the host bacteria had grown to the logarithmic growth phase, with an OD600 (optical density value of the bacterial culture measured at 600 nm) of 0.6–0.8. Then, 1% of the phage seed RDP-EC-22156 was inoculated, and the culture continued for 6–8 h. The propagation process was terminated when dissolved oxygen levels rebounded and pH stabilized.
[0027] Throughout the process, the temperature of the seed tank was set at 37 ℃, a paddle agitator was used, the agitation speed was 250 rpm (200~300 rpm is also acceptable), and sterile air was ventilated to maintain dissolved oxygen, with an aeration rate of 0.6~1 vvm. During the culture process, the pH of the system was maintained at around 7, and the aeration rate was adjusted according to the real-time pH situation.
[0028] Culture medium composition: yeast extract 1.0%, peptone 0.5%, beef extract 0.1%, glycerol 0.5%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, calcium chloride 20 ppm and magnesium chloride 20 ppm.
[0029] Example 2: Post-processing method for bacteriophages. During the proliferation of bacteriophages, cell debris is generated, and some hosts develop tolerance to bacteriophages, allowing them to grow and multiply. Residual host bacteria have an adverse effect on the product. To reduce the adverse effects of impurities on bacteriophage activity, physical methods are needed to remove cell debris and residual host bacteria. This invention, through research and exploration, adopts the following process for sterilization and concentration, and further optimizes the composition and ratio of stabilizers to improve the storage stability of the product.
[0030] (1) Centrifugation: A tubular centrifuge was used to centrifuge at a speed of 17,000 rpm and a throughput of 50-60 L / h to remove unlysed cells and bacterial fragments.
[0031] (2) Filtration: Hollow fiber membrane with a pore size of 800 nm is used for filtration to further remove cell debris and unlysed host bacteria. The filtration pressure is less than 0.4 MPa.
[0032] (3) Concentration: Concentrate to 1 / 4 to 1 / 3 of the original volume using a hollow fiber membrane with a pore size of 10 nm.
[0033] (4) Add phage stabilizer: The phage stabilizer consists of 0.01% (w / v) ethylenediaminetetraacetic acid (EDTA), 0.01% (w / v) glycine, 2% (w / v) glycerol, 1% (w / v) trehalose and 0.1% (w / v) polyethylene glycol.
[0034] (5) Sterilization: Three-stage filtration is used for sterilization, with the filter membranes used for filtration being 1.00 μm polypropylene (PP), 0.45 μm polyethersulfone and 0.22 μm polyethersulfone in sequence.
[0035] (6) Store the filtered and sterilized bacteriophage liquid at 4 °C.
[0036] Example 3: Storage stability verification of phage preparation. The verification experiment was divided into two groups. One group was the conventional method, in which chitosan was not added during the proliferation process and phage stabilizer was not added during the post-processing process. The other group was the method of this patent, in which chitosan was added during the proliferation process and phage stabilizer was added during the post-processing process.
[0037] 3.1 Room temperature storage experiment: The two groups of bacteriophage preparations were stored at room temperature and their activity was tested periodically.
[0038] The results are shown in Table 1. The titer changes of liquid phage samples prepared using the two methods after one year of storage at room temperature showed significant differences. The titer of the patented method group was 9.12 log(PFU / mL), a decrease of approximately two orders of magnitude compared to the initial titer, while the titer of the conventional method group was 7.66 log(PFU / mL), a decrease of 3.6 orders of magnitude compared to the initial titer. The titer of phage produced using the patented method and stored for 12 months was approximately 1.5 orders of magnitude higher than that of the conventional method. These results indicate that by introducing chitosan during the phage proliferation stage and adding a composite stabilizer during the post-processing stage, the rate of decrease in phage titer can be effectively slowed down during the overall preparation and storage process, thereby significantly improving the storage stability of phage preparations under room temperature conditions.
[0039] Table 1. Relationship between room temperature storage time and potency of phage reagents
[0040] 3.2 Low-temperature storage experiment: The two groups of bacteriophage preparations were stored in a 4 ℃ refrigerator and their activity was tested periodically.
[0041] As shown in Figure 1, the phage samples prepared using the patented method showed a titer decrease of only one order of magnitude after being stored at 4 °C for 360 days. In contrast, the samples treated using the conventional method (control group) showed a titer decrease of 2.5 orders of magnitude. This indicates that the samples prepared using this process can effectively extend the storage period of phages at 4 °C, providing reliable process support for the preparation and application of phage formulations.
[0042] Example 4: In vitro antibacterial persistence verification. Two processes were used to prepare bacteriophages in BE-20310 host suspension. Due to the lysis effect of bacteriophages on the host, the value of OD600 changed, thus obtaining the antibacterial line of the bacteriophages on the host.
[0043] 4.1 Experimental Methods The host bacterial suspension cultured for 6 h was inoculated into 100 mL LB medium at a ratio of 1:100, and then 1% bacteriophage was added. The medium was cultured at 37 ℃ with shaking at 200 rpm, and OD600 was detected at regular intervals.
[0044] 4.2 Experimental Results As shown in Figure 2, without the influence of bacteriophages, the host bacteria entered the exponential growth phase within approximately 30-60 minutes, with a significant increase in OD600. Under the influence of bacteriophages, the host bacteria proliferated slowly in the early stages due to phage lysis, but the proliferation rate increased in the later stages due to the emergence of tolerant bacteria, and OD600 gradually increased. The inhibitory duration of the bacteriophage prepared by conventional processes on host bacteria growth was approximately 120 minutes. Compared to bacteriophage prepared by conventional processes, the bacteriophage prepared using the process of this invention extended the inhibitory duration on host bacteria growth to 540 minutes, an increase of 350%, and the corresponding OD600 value was lower at the same culture time point. This indicates that the bacteriophage prepared using the process of this invention can prolong the inhibitory duration on host bacteria and maintain a lower host bacteria growth level under the same culture conditions, which is beneficial for improving the activity retention capacity of bacteriophages during application.
[0045] Example 5: Application Validation Experiment 5.1 Experimental Methods Two processes were used to prepare a batch of phage samples for large-scale comparative experiments. Seven-day-old broiler chickens were used as test subjects, and application validation was conducted under natural rearing conditions. The phage preparation was provided to the test subjects via feeding at a dosage of 10... 8 PFU / bird was administered for 3 consecutive days, with 30,000 chickens per shed. The flock's condition was observed for 7 consecutive days after administration. The experiment was divided into 4 groups, and the survival performance of the flocks during the experiment was statistically analyzed. The specific groupings are shown in Table 2.
[0046] Table 2 Grouping of phage preparations in large-scale experiments
[0047] 5.2 The experimental results are shown in Table 3. The mortality rates of the four experimental groups differed significantly. The phage prepared using the patented process can effectively reduce the mortality rate, indicating that the phage preparation prepared using the process of this invention can maintain a more stable potency level and more consistent application performance under the same application conditions.
[0048] Table 3. Statistical analysis of large-scale phage experiments.
[0049] In summary, based on the storage stability, in vitro antibacterial experiments, and large-scale experiments of phages prepared by different processes, it is demonstrated that the method used in this embodiment can effectively improve the stability of phage products and prolong the antibacterial time. Under practical application conditions, it exhibits good stability and feasibility, providing a technical basis for the development and application of phage preparations.
[0050] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for improving the stability and antibacterial persistence of Escherichia coli bacteriophage preparations, characterized in that, The process includes the following steps: (1) Phage proliferation: Chitosan is added during the cultivation of Escherichia coli host bacteria, and Escherichia coli phage is inoculated when the host bacteria are in the logarithmic growth phase. The phage is cultured until it lyses and releases, and a phage proliferation solution is obtained; (2) Post-treatment: The phage proliferation solution in step (1) is subjected to low-shear centrifugation, membrane filtration and membrane concentration in sequence to obtain a concentrated phage solution; (3) Stabilization treatment: A phage stabilizer is added to the concentrated phage solution in step (2) to obtain a stabilized phage solution; (4) Sterilization: The stabilized phage solution in step (3) is subjected to multi-stage filtration sterilization to obtain an Escherichia coli phage preparation.
2. The method according to claim 1, characterized in that: The final concentration of chitosan in the culture system in step (1) is 0.01% to 0.05%.
3. The method according to claim 2, characterized in that: The final concentration is 0.02%.
4. The method according to claim 1, characterized in that: The chitosan mentioned in step (1) is added before phage inoculation.
5. The method according to claim 1, characterized in that: The centrifugation in step (2) uses a tubular centrifuge, the membrane filtration in step (2) uses a hollow fiber membrane with a pore size of 800 nm and a filtration pressure of less than 0.4 MPa, and the membrane concentration in step (2) uses a hollow fiber membrane with a pore size of 10 nm.
6. The method according to claim 1, characterized in that: The phage stabilizer mentioned in step (3) is a composite stabilizer, comprising 0.01 w / v % ethylenediaminetetraacetic acid, 0.01 w / v % glycine, 2 w / v % glycerol, 1 w / v % trehalose and 0.1 w / v % polyethylene glycol.
7. The method according to claim 1, characterized in that: The multi-stage filtration in step (4) uses polypropylene 1.00 μm, polyethersulfone 0.45 μm and polyethersulfone 0.22 μm filter membranes in sequence.
8. The method according to claim 1, characterized in that: The potency of the Escherichia coli phage preparation decreased by no more than two orders of magnitude after 12 months of storage at room temperature.
9. The method according to claim 1, characterized in that: The potency of the Escherichia coli phage preparation decreased by no more than one order of magnitude after being stored at 4 °C for 12 months.
10. The method according to claim 1, characterized in that: Compared with preparations obtained by conventional methods, the Escherichia coli phage preparation has an antibacterial duration that is extended by at least 300% in the in vitro antibacterial system.