Preparation method of phage compound preparation of Chinese softshell turtles

Abstract

The invention relates to the technical field of aquaculture, in particular to a preparation method of a phage compound preparation of Chinese softshell turtles. The method comprises the following steps: S1, respectively separating bacillus cereus, vibrio parahaemolyticus and aeromonas hydrophila from soft-shelled turtle bodies suffering from head shaking disease, nail rot disease and hemorrhagic disease, and mixing to prepare a multi-host bacterial suspension, mixing the multi-host bacterium suspension with an environmental bacteriophage sample, performing enrichment induction, screening out a target bacteriophage, and performing purification culture to obtain a bacteriophage proliferation solution; s2, purifying the bacteriophage proliferation liquid to remove endotoxin to obtain a high-purity bacteriophage solution; and S3, preparing the high-purity bacteriophage liquid and the embedding material into the micro-capsule oral preparation through an electrostatic spraying device. The preparation method comprises the following steps: directionally screening high-cracking bacteriophages aiming at main pathogenic bacteria of soft-shelled turtles, constructing a bacteriophage combination, preparing a targeted microcapsule preparation by combining an electrostatic spraying technology through a constructed composite embedding structure, and cooperating with a bacteriophage synergist, so that spectral antibiosis can be realized, and the stability of the preparation is improved.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a method for preparing a phage complex preparation of Chinese soft-shelled turtle. Background Technology

[0002] As a distinctive and advantageous aquaculture species in my country, the Chinese soft-shelled turtle (Typha orientalis) has seen its aquaculture industry expand year by year. However, frequent bacterial diseases severely restrict the industry's development. Besides the traditional Bacillus cereus-induced head-shaking disease, the incidence of diseases such as shell rot caused by Vibrio parahaemolyticus and hemorrhagic septicemia caused by Aeromonas hydrophila is increasing annually, and single-disease control is no longer sufficient to meet aquaculture needs. While traditional antibiotics are effective in the short term, long-term use leads to the proliferation of multidrug-resistant bacteria (MDRs). Studies show that 36 common antibiotics used in aquaculture have developed resistant strains to varying degrees. Furthermore, drug residues harm consumer health through the food chain and pollute the aquaculture environment.

[0003] Bacteriophages, as natural bacterial viruses, possess advantages such as strong host specificity, no risk of drug resistance, and environmental friendliness, making them an ideal alternative for the prevention and control of aquatic diseases. However, existing technologies have the following problems: 1. Bacteriophage screening is mostly targeted at single pathogens, lacking broad-spectrum coverage and making it difficult to address the actual scenario of mixed bacterial infections in turtle farming; 2. Bacteriophages have poor environmental tolerance, a short half-life at room temperature (only 3-5 days), and are easily degraded by turtle stomach acid and digestive enzymes after oral administration, with an activity retention rate of less than 20%; 3. Some bacteriophages may carry toxin genes or induce bacterial drug resistance through horizontal gene transfer, posing a biosafety risk.

[0004] Therefore, there is an urgent need to develop bacteriophage agents that are effective against a variety of aquatic diseases and have good stability, suitable for Chinese soft-shelled turtles. Summary of the Invention

[0005] In view of the above problems, the present invention provides a method for preparing a phage complex preparation of Chinese soft-shelled turtle. By selectively screening highly lytic phages targeting the main pathogenic bacteria of soft-shelled turtles, a phage combination is constructed. By constructing a composite encapsulation structure of "phage-sodium alginate-nano silica", a targeted microcapsule preparation is prepared by combining electrostatic spraying technology. With the addition of a phage synergist, a broad-spectrum antibacterial effect can be achieved and the stability of the preparation can be improved.

[0006] To achieve the above and other related objectives, the present invention provides the following technical solution: A method for preparing a phage complex preparation of Chinese soft-shelled turtle, the method comprising the following steps: S1. Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease, respectively. They were mixed to prepare a multi-host bacterial suspension. The multi-host bacterial suspension was mixed with environmental bacteriophage samples for enrichment and induction. After screening out the target bacteriophages, the bacteriophages were purified and cultured to obtain the bacteriophage proliferation solution. S2. After purifying the phage proliferation broth to remove endotoxins, a high-purity phage solution was obtained; S3. High-purity bacteriophage fluid and embedding material are prepared into microcapsule oral formulations using an electrostatic spraying device.

[0007] Furthermore, step S1 includes the following steps: S110. Isolation and Purification of Multi-Host Bacteria: Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease, respectively. After culture and purification, the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, mixed at a volume ratio of 1:1:1 to prepare a multi-host bacterial suspension; S120. Phage sample collection and induction enrichment: Collect water samples, bottom mud and intestinal contents of healthy turtles from turtle farming ponds, prepare mixed samples, add multi-host bacterial suspension to induce enrichment for 6-8 hours, and add penicillin to inhibit host bacterial proliferation. S130. Phage screening and purification: The double-layer plate method was used to screen phage plaques. Clear phage plaques with a diameter >2mm were selected, and after proliferation, filtration and purification, target phages with a lysis rate >95% against pathogenic bacteria were screened. S140. Phage amplification culture: Inoculate the target phage into a multi-host bacterial suspension, incubate at 37°C with shaking for 8-10 hours, centrifuge and collect the supernatant to obtain a titer ≥1×10¹. 0 Phage proliferation medium with PFU / mL.

[0008] Furthermore, step S2 includes the following steps: S210, Tangential flow filtration concentration: Use a polyethersulfone membrane with a molecular weight cutoff of 300-500kDa to perform tangential flow filtration on the phage proliferation solution to concentrate it to 5%-10% of the original volume, and wash with SM buffer. S220. Membrane chromatography purification and endotoxin removal: Purification was performed using an anion exchange membrane chromatography column, followed by elution with SM buffer containing 0.3M NaCl, and then treatment with an endotoxin removal column to achieve an endotoxin content of <0.5 EU / mL, resulting in a high-purity phage solution.

[0009] Furthermore, step S3 includes the following steps: S310. Electrostatic spraying preparation of microcapsules: 1-3% w / v sodium alginate solution and high-purity bacteriophage solution are mixed at a ratio of 4-8:1. After stirring evenly to obtain a mixture, 0.5-1% glycine-EDTA composite synergist is added to the total mass of the mixture. Microcapsules are prepared using a coaxial electrostatic spraying device. The receiving liquid is 0.2M CaCl2 solution. The sodium alginate solution contains 0.1% w / v nano silica. S320. Microcapsule drying and shaping: The microcapsules are pre-frozen and then freeze-dried under vacuum to obtain oral phage microcapsule formulations.

[0010] Furthermore, in step S110, Bacillus cereus is cultured on BHI medium, Vibrio parahaemolyticus on TCBS medium, and Aeromonas hydrophila on LB medium, with a culture temperature of 30-37℃ and a culture time of 18-24h.

[0011] Furthermore, in step S210, the tangential flow filtration operates at a pressure of 0.1-0.2 MPa, a temperature of 25-30°C, and a flow rate of 15-20 mL / min; the SM buffer solution consists of 100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris-HCl, and a pH of 7.5.

[0012] Furthermore, in step S310, the nozzle diameter of the electrostatic spray device is 0.2 mm, the voltage is 18 kV, the receiving distance is 15 cm, and the flow rate is 0.3 mL / h.

[0013] Furthermore, in step S320, the vacuum freeze-drying conditions are a vacuum degree of 10-100 Pa, a freeze-drying temperature of -40 to -50°C, and a freeze-drying time of 12-24 h.

[0014] The present invention has the following positive effects: 1. This invention simultaneously screens bacteriophages targeting the main pathogens of turtle head-shaking disease, shell rot, and hemorrhagic disease, solving the problem of narrow antibacterial spectrum of single bacteriophages. It is suitable for the actual scenario of mixed bacterial infections in aquaculture and has inhibitory effects on multiple homologous strains, making it widely applicable. Furthermore, the preparation process employs a composite encapsulation structure of "bacteriophage-sodium alginate-nano silica," combined with vacuum freeze-drying technology, enabling the bacteriophage preparation to maintain an activity retention rate of 75% after 60 days of storage at room temperature and >90% under gastric acid conditions, significantly superior to existing technologies. It can be directly mixed into feed without the need for special equipment.

[0015] 2. This invention employs a three-stage purification method in the purification step, effectively removing endotoxins (<0.5 EU / mL) and impurity proteins, reducing the risk of immune stress in turtles. The phage originates from the turtle farming environment, preventing biological invasion and leaving no drug residues. The innovative combination of glycine-EDTA synergist in the preparation inhibits bacterial anti-phage mechanisms, reducing the emergence of drug-resistant strains. The agent maintains stable efficacy even after three consecutive farming cycles, resolving drug resistance issues in phage therapy. Detailed Implementation

[0016] The following description, in conjunction with examples, illustrates exemplary embodiments of the present disclosure, including various details to aid understanding. These should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present disclosure. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description. Example

[0017] This embodiment provides a method for preparing a phage complex preparation of Chinese soft-shelled turtle, including the following steps: S1. Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease, respectively. They were mixed to prepare a multi-host bacterial suspension. The multi-host bacterial suspension was mixed with environmental bacteriophage samples for enrichment and induction. After screening out the target bacteriophages, the bacteriophages were purified and cultured to obtain the bacteriophage proliferation solution. Specifically: S110. Pathogenic bacteria were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease. For head-shaking disease, 0.5g each of liver and spleen tissue were collected from turtles, ground, and homogenized with sterile physiological saline to a 1:10 ratio. This homogenate was spread onto BHI agar plates and incubated at 37°C for 24 hours. Milky-white, rough-surfaced colonies were picked. Gram staining, spore staining, and biochemical identification confirmed *Bacillus cereus*. For shell rot, samples were taken from the ulcerated areas of the shell using sterile swabs and inoculated onto TCBS agar plates. Incubation was at 37°C for 24 hours. Green colonies were picked, and positive oxidase test and negative sucrose fermentation test identified *Vibrio parahaemolyticus*. For hemorrhagic disease, 0.2mL of blood was collected from the heart of turtles, inoculated onto LB agar, and incubated at 37°C with shaking for 18 hours. Colorless, transparent colonies were picked, and positive oxidase test and glucose fermentation acid production test identified *Aeromonas hydrophila*. The isolated bacteria were inoculated onto their respective culture media: Bacillus cereus on BHI medium, Vibrio parahaemolyticus on TCBS medium, and Aeromonas hydrophila on LB medium. They were incubated statically at 30-37°C for 18-24 hours. Single colonies were picked and purified for three generations to ensure a purity >99%. The concentration of each strain was adjusted to 1×10⁻⁶ with sterile physiological saline. 8CFU / mL was mixed at a ratio of 1:1:1 (v / v / v) to prepare a multi-host bacterial suspension, which was then refrigerated at 4°C for later use.

[0018] S120. Collect mid-layer water samples, bottom sediment, and intestinal contents of healthy turtles from the turtle farming pond. Prepare suspensions of the bottom sediment and intestinal contents separately at a 1:10 (w / v) ratio using sterile physiological saline. Place the suspensions on a shaker at 25℃ and 150 rpm for 30 minutes. Take the supernatant and mix it with the mid-layer water sample at a 1:1 volume ratio to obtain a mixed sample. Add the multi-host bacterial suspension prepared in S110 to the mixed sample to achieve a final host bacterial concentration of 1×10⁻⁶. 7 Incubate at CFU / mL at 37℃ for 6-8 hours with gentle shaking every 2 hours to promote specific binding of phages to host bacteria. After induction, add penicillin to a final concentration of 100 μg / mL to inhibit host bacterial proliferation, and continue culturing for 2 hours to improve phage enrichment efficiency.

[0019] S130. Take the induced sample suspension and centrifuge at 8000×g for 10 min at 4℃. Take the supernatant and filter it through a 0.22 μm polyethersulfone membrane to remove bacteria and impurities. In this example, the double-layer plate method is used for screening. Specifically, mix 100 μL of filtrate with 100 μL of multi-host bacterial suspension, incubate at 37℃ for 15 min, add 5 mL of 0.7% semi-solid LB medium (45℃, to avoid high-temperature inactivation of bacteriophages), mix quickly, and spread on 1.5% solid LB medium plates. Incubate at 30-32℃ for 24 h and observe plaque formation. Phage plaques with a diameter >2 mm, clear edges, and transparency were selected. The core of the plaque was picked using a sterile toothpick and inoculated into 5 mL of single-host bacterial suspension (corresponding to Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila, respectively). The suspension was incubated at 37℃ and 150 rpm for 6 h with shaking until the bacterial solution changed from turbid to clear. The supernatant was filtered through a 0.22 μm filter to obtain pure cultures of single phages. The lysis profiles of each phage were determined: using the spot method, 10 common pathogenic bacteria of soft-shelled turtles (including 3 strains of Bacillus cereus, 3 strains of Vibrio parahaemolyticus, and 4 strains of Aeromonas hydrophila) were plated separately on LB agar plates. Pure cultures of each phage (titer 1×10⁻⁶) were then incubated. 9 PFU / mL was dropped onto the surface of a plate and incubated at 37°C for 24 h. The diameter of the inhibition zone was measured. Strains with a lysis rate of >95% against the target pathogen and an inhibition zone diameter of >15 mm were screened. Finally, one strain each of phage PH-Bc against Bacillus cereus, phage PH-Vp against Vibrio parahaemolyticus, and phage PH-Ah against Aeromonas hydrophila were selected to obtain the target phages.

[0020] S140. Mix the phage cocktail mixture in equal proportions and inoculate it into 500 mL of multi-host bacterial suspension (concentration 1×10⁻⁶). 8(CFU / mL), incubate at 37℃ with shaking at 150 rpm for 8-10 h, and measure the OD of the bacterial culture every 1 h. 600 Value, when OD 600 When the value drops from 0.8 to below 0.1, stop the culture. Centrifuge the culture at 4℃ and 8000×g for 15 min, and collect the supernatant as the phage proliferation solution. The titer can be determined using the double-layer plate method, reaching 1×10¹. 0 PFU / mL or higher.

[0021] S2. After purifying the phage proliferation broth to remove endotoxins, a high-purity phage solution was obtained; Specifically: S210: A polyethersulfone (PES) membrane with a molecular weight cutoff of 300 kDa was used. The membrane was pre-washed with 0.1 mol / L NaOH solution for 30 min, followed by rinsing with sterile water until neutral. The phage proliferation solution was passed through a tangential flow filtration system at an operating pressure of 0.12 MPa, a temperature of 25 °C, and a flow rate of 15 mL / min for cross-flow filtration concentration to 5% of the original volume, removing small molecule impurities such as proteins, salts, and metabolites. The concentrate was washed three times with SM buffer (100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris-HCl, pH 7.5, pre-autoclaved and cooled to 4 °C), concentrating to 5% of the original volume after each wash for preliminary phage purification and removal of residual impurities.

[0022] S220, using an anion exchange membrane column (Q-agarose packing material, column volume 10 mL), equilibrate the column with SM buffer (pH 7.5) until the pH of the eluent matches that of the equilibration buffer. Load the concentrated phage solution at a flow rate of 5 mL / min to ensure thorough binding of the phage to the column packing material. Rinse the column with SM buffer containing 0.1 M NaCl to remove unbound impurities, then elute the phage with SM buffer containing 0.3 M NaCl and collect the eluent. Detect the endotoxin content in the eluent using dynamic turbidimetry. Mix 100 μL of the eluent with Limulus Amebocyte Lysate (LAL) reagent, incubate at 37 °C for 60 min, and measure the absorbance. If the endotoxin content is >0.5 EU / mL, pass the eluent through an endotoxin removal column (affinity chromatography packing material) and repeat the detection until the endotoxin content is <0.5 EU / mL. Obtain a high-purity phage solution. Determine the titer using the double-layer plate method, maintaining a concentration of 5 × 10⁻⁶. 9 Store at 4°C for use if PFU / mL or higher.

[0023] S3. High-purity bacteriophage fluid and embedding material are prepared into microcapsule oral formulations using an electrostatic spraying device.

[0024] Specifically: 310. Weigh 2g of sodium alginate, add 100mL of sterile water, and stir in a 60℃ constant temperature water bath for 1h until completely dissolved and clear. Then add 0.1g of nano silica (particle size 20-50nm, purity >99.5%), place in an ultrasonic cell disruptor, and sonicate at 200W for 10min to prepare an embedding solution. Refrigerate at 4℃ for later use. Mix the purified phage solution and the embedding solution at a ratio of 1:4 (v / v), add 0.5g of glycine-EDTA composite synergist (glycine and EDTA mixed at a mass ratio of 3:1), and stir at 25℃ and 100r / min for 15min to ensure uniform dispersion of the phage. A coaxial electrostatic spraying device with an inner phase nozzle diameter of 0.2 mm was used. A high-voltage power supply of 18 kV was set, and the distance between the nozzle and the receiving liquid was 15 cm. The receiving liquid was a 0.2 M CaCl2 solution (containing 0.1% Tween-80 to reduce surface tension). The device was placed on a magnetic stirrer and slowly stirred at 50 rpm. The flow rate was adjusted to 0.3 mL / h, and the device was started to spray the mixture into the receiving liquid. The mixture was allowed to stand at 30°C for 20 min to allow the microcapsules to fully cross-link and solidify. The microcapsules were rinsed three times with sterile water, centrifuged at 5000 × g for 5 min after each rinse to remove excess CaCl2 and uncured embedding material from the surface.

[0025] S320. Spread the solidified microcapsules evenly on a freeze-drying tray and pre-freeze them in a -40℃ freezer for 6 hours to ensure complete freezing. Place the tray in a vacuum freeze dryer, set the vacuum degree to 10Pa, the temperature to -50℃, and dry for 12 hours until the moisture content of the microcapsules is <3%. After drying, pass the microcapsules through an 80-mesh standard sieve to obtain the phage composite agent.

[0026] The phage compound preparation prepared in this embodiment is used during the seedling cultivation period of soft-shelled turtles, the early stage of adult turtle farming, and one week before the peak period of disease incidence. The dosage is 0.2% of the turtle's body weight. The phage compound agent is evenly mixed into the turtle's formulated feed, and a small amount of sterile water is added to make the agent adhere to the surface of the feed. After air-drying for 30 minutes, it is fed. Continuous use for 7 days can control the incidence of bacterial diseases in soft-shelled turtles to below 8%.

[0027] The specific embodiments described above do not constitute a limitation on the scope of protection of this disclosure. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. A method for preparing a phage complex preparation of Chinese soft-shelled turtle, characterized in that, The method includes the following steps: S1. Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease, respectively. They were mixed to prepare a multi-host bacterial suspension. The multi-host bacterial suspension was mixed with environmental bacteriophage samples for enrichment and induction. After screening out the target bacteriophages, the bacteriophages were purified and cultured to obtain the bacteriophage proliferation solution. S2. After purifying the phage proliferation broth to remove endotoxins, a high-purity phage solution was obtained; S3. High-purity bacteriophage fluid and embedding material are prepared into microcapsule oral formulations using an electrostatic spraying device.

2. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 1, characterized in that, Step S1 includes the following steps: S110. Isolation and Purification of Multi-Host Bacteria: Bacillus cereus, Vibrio parahaemolyticus, and Aeromonas hydrophila were isolated from turtles suffering from head-shaking disease, shell rot, and hemorrhagic disease, respectively. After culture and purification, the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, mixed at a volume ratio of 1:1:1 to prepare a multi-host bacterial suspension; S120. Phage sample collection and induction enrichment: Collect water samples, bottom mud and intestinal contents of healthy turtles from turtle farming ponds, prepare mixed samples, add multi-host bacterial suspension to induce enrichment for 6-8 hours, and add penicillin to inhibit host bacterial proliferation. S130. Phage screening and purification: The double-layer plate method was used to screen phage plaques. Clear phage plaques with a diameter >2mm were selected, and after proliferation, filtration and purification, target phages with a lysis rate >95% against pathogenic bacteria were screened. S140. Phage amplification culture: Inoculate the target phage into a multi-host bacterial suspension, incubate at 37°C with shaking for 8-10 hours, centrifuge and collect the supernatant to obtain a titer ≥1×10¹. 0 Phage proliferation medium with PFU / mL.

3. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 1, characterized in that, Step S2 includes the following steps: S210, Tangential flow filtration concentration: Use a polyethersulfone membrane with a molecular weight cutoff of 300-500kDa to perform tangential flow filtration on the phage proliferation solution to concentrate it to 5%-10% of the original volume, and wash with SM buffer. S220. Membrane chromatography purification and endotoxin removal: Purification was performed using an anion exchange membrane chromatography column, followed by elution with SM buffer containing 0.3M NaCl, and then treatment with an endotoxin removal column to achieve an endotoxin content of <0.5 EU / mL, resulting in a high-purity phage solution.

4. The method for preparing the phage complex preparation of the Chinese soft-shelled turtle according to claim 1, characterized in that, Step S3 includes the following steps: S310. Electrostatic spraying preparation of microcapsules: 1-3% w / v sodium alginate solution and high-purity bacteriophage solution are mixed at a ratio of 4-8:

1. After stirring evenly to obtain a mixture, 0.5-1% glycine-EDTA composite synergist is added to the total mass of the mixture. Microcapsules are prepared using a coaxial electrostatic spraying device. The receiving liquid is 0.2M CaCl2 solution. The sodium alginate solution contains 0.1% w / v nano silica. S320. Microcapsule drying and shaping: The microcapsules are pre-frozen and then freeze-dried under vacuum to obtain oral phage microcapsule formulations.

5. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 2, characterized in that, In step S110, Bacillus cereus is cultured on BHI medium, Vibrio parahaemolyticus on TCBS medium, and Aeromonas hydrophila on LB medium. The culture temperature is 30-37℃ and the culture time is 18-24h.

6. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 3, characterized in that, In step S210, the tangential flow filtration operates at a pressure of 0.1-0.2 MPa, a temperature of 25-30℃, and a flow rate of 15-20 mL / min; the SM buffer solution consists of 100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris-HCl, and a pH of 7.

5.

7. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 4, characterized in that, In step S310, the electrostatic spray device has a nozzle diameter of 0.2 mm, a voltage of 18 kV, a receiving distance of 15 cm, and a flow rate of 0.3 mL / h.

8. The method for preparing the bacteriophage complex preparation of the Chinese soft-shelled turtle according to claim 4, characterized in that: In step S320, the vacuum freeze-drying conditions are a vacuum degree of 10-100 Pa, a freeze-drying temperature of -40 to -50°C, and a freeze-drying time of 12-24 h.