Embedded microspheres for in-situ culture of Vibrio harveyi and culture method

Abstract

The invention discloses embedded microspheres for in-situ culture of Vibrio harveyi and a culture method, and relates to the technical field of microbial carriers. The preparation method of the embedded microspheres comprises the steps of preparing a blending solution and preparing the embedded microspheres. The method for in-situ culture of the vibrio harveyi comprises the steps of preparing a blended solution, preparing a suspended bacterial solution and preparing the vibrio harveyi embedded microspheres. The prepared embedded microspheres are high in mechanical strength, excellent in permeability, high in vibrio harveyi embedding rate and good in culture effect.

Description

Technical Field

[0001] This invention relates to the field of microbial carrier technology, specifically to an embedded microsphere for in situ culture of Vibrio harveyi and a culture method thereof. Background Technology

[0002] Sodium alginate is a natural polysaccharide extracted from brown seaweed. It is formed by the combination of the carboxyl group at the 6-position of alginate and sodium ions. Its molecular backbone is a linear copolymer of α-L-mannuronic acid and β-D-guluronic acid linked by 1,4-glycosidic bonds. Sodium alginate can be cross-linked with divalent ions such as Ca²⁺, Ba²⁺, and Co²⁺ to form gels. Among these, calcium ions are preferred as cross-linking agents in the food and biotechnology fields due to their non-toxicity. Calcium alginate microspheres prepared by cross-linking sodium alginate with Ca²⁺ exhibit biocompatibility, biodegradability, swelling behavior, good diffusion characteristics, and surface characteristics. However, the pore size of this microsphere network is too large, and the mechanical strength of the gel beads is poor, resulting in low reusability. Further optimization and improvement through technical means are needed.

[0003] Polyvinyl alcohol (PVA) is a synthetic hydrophilic polymer with excellent elasticity, swelling properties, non-toxicity, high tolerance, and chemical stability, making it widely used in pharmaceutical and biological fields. However, forming it into microspheres is challenging. Existing technology, CN118813596A, discloses a PVA spherical microbial carrier, its preparation method, and its application. This technology mixes PVA with sodium alginate and uses boric acid as a crosslinking agent to prepare PVA microspheres. While this improves the mechanical strength of the microspheres, it also reduces swelling and permeability. Furthermore, boric acid is harmful to microorganisms such as Vibrio harveyi, making it unsuitable for encapsulating and culturing Vibrio harveyi. In summary, while current technologies have improved certain properties of calcium alginate microspheres to some extent, they still suffer from the following technical problems: insufficient mechanical strength, poor permeability, low encapsulation rate for Vibrio harveyi, and poor culturing effect. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides an in-situ culture method for embedding microspheres of Vibrio harveyi, achieving the following objectives: to prepare embedding microspheres with high mechanical strength, excellent permeability, high embedding rate of Vibrio harveyi, and good culture effect.

[0005] To achieve the above objectives, the following technical solution is adopted: A method for preparing embedded microspheres of Vibrio harveyi in situ culture, comprising the following steps: Step 1: Preparation of the blend solution Polyvinyl alcohol (PVA) was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 20-25°C for 20-30 minutes at a stirring speed of 100-200 rpm, then placed at 80-85°C and stirred for another 40-50 minutes. Sodium alginate was then added, and stirring continued for 30-40 minutes. Nano-hydroxyapatite was then added, and the stirring speed was increased to 150-250 rpm, continuing to stir for 20-30 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fractions of PVA, sodium alginate, and nano-hydroxyapatite were 1-2% and 0.5-1.5% respectively. The ultrasonic treatment was performed at a power of 300-500 W for 20-30 minutes.

[0006] Step 2: Prepare embedded microspheres Dissolve calcium chloride in deionized water to prepare a calcium chloride solution with a mass fraction of 4-6%.

[0007] Encapsulated microspheres were prepared using a microencapsulation generator. The blended solution was loaded into the syringe of the microencapsulation generator, which was connected to a syringe pump. The injection rate of the syringe pump was adjusted to 10-25 mL / h, and the gas pressure of the microencapsulation generator was adjusted to 100-300 mbar. A beaker containing calcium chloride solution was placed at the nozzle of the microencapsulation generator. The blended solution was sprayed through the nozzle to form encapsulated microspheres, which were collected with calcium chloride solution. The microspheres were then centrifuged, and the precipitate was collected. The precipitate was washed 2-3 times with sterile seawater, followed by centrifugation to obtain the encapsulated microspheres.

[0008] The centrifugation process involves centrifuging at 3000-4000 rpm for 15-20 minutes.

[0009] This invention also provides a method for in situ culture of Vibrio harveyi, comprising the following steps: Step 1: Preparation of the blend solution A measured amount of polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 20-25℃ for 20-30 minutes at a stirring speed of 100-200 rpm, then placed at 80-85℃ and stirred for another 40-50 minutes. Sodium alginate was then added, and stirring continued for 30-40 minutes. Nano-hydroxyapatite was then added, and the stirring speed was increased to 150-250 rpm, continuing to stir for 20-30 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fraction of polyvinyl alcohol was 1-2%, the mass fraction of sodium alginate was 1-2%, and the mass fraction of nano-hydroxyapatite was 0.5-1.5%. The ultrasonic treatment was performed at a power of 300-500 W for 20-30 minutes.

[0010] Step 2: Preparation of bacterial suspension Casein peptone, yeast extract, and ferric phosphate were dissolved in seawater. The pH of the system was adjusted to 7.6-7.8 with NaOH solution, and then seawater was added to a final volume of 1L. The mixture was then autoclaved at 120-122℃ and 0.1-0.105 MPa for 20-30 minutes to obtain a liquid culture medium. Vibrio harveyi was inoculated into the liquid culture medium and placed in a constant temperature incubator at 36-38℃ for 16-18 hours. After incubation, the bacterial cells were collected by centrifugation at 3000-3500 rpm for 15-20 minutes, and then sterilized seawater was added to obtain a bacterial suspension.

[0011] Step 3: Preparation of Vibrio harveyi-embedded microspheres Add the suspended bacterial solution to the sterilized blending solution and stir until homogeneous to obtain the bacterial mixture.

[0012] Dissolve calcium chloride in deionized water to prepare a calcium chloride solution with a mass fraction of 4-6%.

[0013] Assemble the microencapsulation generator, load the bacterial mixture into a syringe, connect the syringe pump, and adjust the injection rate to 10-25 mL / h. Adjust the generator pressure to 100-300 mbar. Place a beaker containing calcium chloride solution at the generator nozzle and collect the microspheres using the calcium chloride solution. Then, centrifuge the microspheres, collect the precipitate, and wash the precipitate 2-3 times with sterile seawater. After centrifugation, obtain Vibrio harveyi-embedded microspheres. Add the Vibrio harveyi-embedded microspheres to sterile seawater to obtain a sterile seawater suspension. Add this suspension to liquid culture medium and incubate at 36-38℃. Take samples periodically for analysis.

[0014] The centrifugation process involves centrifuging at 3000-4000 rpm for 15-20 minutes.

[0015] The beneficial effects of this invention are as follows: (1) The microspheres of Vibrio harveyi cultured in situ of the present invention have better permeability to small molecules such as riboflavin and water-soluble molecules such as bovine serum albumin. The cumulative release rate of riboflavin of the prepared microspheres is 66.38% after 5 hours; the cumulative release rate of bovine serum albumin of the prepared microspheres is 16.81% after 300 minutes.

[0016] (2) The microspheres for in situ culture of Vibrio harveyi of the present invention have a good encapsulation effect on Vibrio harveyi. The encapsulation rate of the prepared Vibrio harveyi microspheres for Vibrio harveyi is 91.2746%±1.2213%. (3) The method for in situ culture of Vibrio harveyi of the present invention has a good culture effect on Vibrio harveyi. After 6 hours of culture, the OD value of Vibrio harveyi is 0.074; after 18 hours of culture, the OD value of Vibrio harveyi is 0.592. Attached Figure Description

[0017] Appendix Figure 1 This is an optical microscope image of the embedded microspheres prepared in Example 2.

[0018] Appendix Figure 2 This is a comparison chart showing the sustained release results of riboflavin by the encapsulated microspheres prepared in Example 2 and Comparative Example 1.

[0019] Appendix Figure 3 This is a comparison chart showing the sustained release results of bovine serum albumin (BSA) by the encapsulated microspheres prepared in Example 2 and Comparative Example 1.

[0020] Appendix Figure 4 This is a comparison diagram of the growth of Vibrio harveyi in Example 4 and Comparative Example 2. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0022] Example 1: A method for preparing embedded microspheres of Vibrio harveyi in situ culture A method for preparing embedded microspheres of Vibrio harveyi in situ culture, comprising the following steps: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 20°C for 30 minutes at a stirring speed of 100 rpm, then placed at 80°C and stirred for another 50 minutes. Sodium alginate was then added, and stirring continued for 40 minutes. Nano-hydroxyapatite was added, and the stirring speed was increased to 150 rpm, with stirring continuing for another 30 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fraction of polyvinyl alcohol was 1%, the mass fraction of sodium alginate was 2%, and the mass fraction of nano-hydroxyapatite was 0.5%. The ultrasonic treatment was performed at a power of 300 W for 30 minutes.

[0023] Step 2: Prepare embedded microspheres Dissolve calcium chloride in deionized water to prepare a 4% calcium chloride solution.

[0024] Encapsulated microspheres were prepared using a microencapsulation generator. The blended solution was loaded into the syringe of the microencapsulation generator, which was connected to a syringe pump. The injection rate of the syringe pump was adjusted to 10 mL / h, and the gas pressure of the microencapsulation generator was adjusted to 100 mbar. A beaker containing calcium chloride solution was placed at the nozzle of the microencapsulation generator. The blended solution was sprayed through the nozzle to form encapsulated microspheres, which were collected with calcium chloride solution. The microspheres were then centrifuged, and the precipitate was collected. The precipitate was washed twice with sterile seawater, and then centrifuged again to obtain the encapsulated microspheres.

[0025] The centrifugation process was carried out at a speed of 3000 rpm for 20 minutes.

[0026] Example 2: A method for preparing embedded microspheres of Vibrio harveyi in situ culture A method for preparing embedded microspheres of Vibrio harveyi in situ culture, comprising the following steps: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 25°C for 25 minutes at a stirring speed of 150 rpm, then placed at 85°C and stirred for another 45 minutes. Sodium alginate was then added, and stirring continued for 35 minutes. Nano-hydroxyapatite was added, and the stirring speed was increased to 200 rpm, with stirring continuing for 25 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fractions of polyvinyl alcohol, sodium alginate, and nano-hydroxyapatite were 1%. The ultrasonic treatment was performed at a power of 400 W for 25 minutes.

[0027] Step 2: Prepare embedded microspheres Dissolve calcium chloride in deionized water to prepare a 5% calcium chloride solution.

[0028] Encapsulated microspheres were prepared using a microencapsulation generator. The blended solution was loaded into the syringe of the microencapsulation generator, which was connected to a syringe pump. The injection rate of the syringe pump was adjusted to 13 mL / h, and the gas pressure of the microencapsulation generator was adjusted to 250 mbar. A beaker containing calcium chloride solution was placed at the nozzle of the microencapsulation generator. The blended solution was sprayed through the nozzle to form encapsulated microspheres, which were collected with calcium chloride solution. The microspheres were then centrifuged, and the precipitate was collected. The precipitate was washed three times with sterile seawater, followed by centrifugation to obtain the encapsulated microspheres.

[0029] The centrifugation process was carried out at a speed of 3500 rpm for 20 minutes.

[0030] Example 3: A method for preparing embedded microspheres of Vibrio harveyi in situ culture A method for preparing embedded microspheres of Vibrio harveyi in situ culture, comprising the following steps: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 25°C for 20 minutes at a stirring speed of 200 rpm, then placed at 85°C and stirred for another 40 minutes. Sodium alginate was then added, and stirring continued for 30 minutes. Nano-hydroxyapatite was added, and the stirring speed was increased to 250 rpm, with stirring continuing for 20 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fraction of polyvinyl alcohol was 2%, the mass fraction of sodium alginate was 1%, and the mass fraction of nano-hydroxyapatite was 1.5%. The ultrasonic treatment was performed at a power of 500 W for 20 minutes.

[0031] Step 2: Prepare embedded microspheres Dissolve calcium chloride in deionized water to prepare a 6% calcium chloride solution.

[0032] Encapsulated microspheres were prepared using a microencapsulation generator. The blended solution was loaded into the syringe of the microencapsulation generator, which was connected to a syringe pump. The injection rate of the syringe pump was adjusted to 25 mL / h, and the gas pressure of the microencapsulation generator was adjusted to 300 mbar. A beaker containing calcium chloride solution was placed at the nozzle of the microencapsulation generator. The blended solution was sprayed through the nozzle to form encapsulated microspheres, which were collected with calcium chloride solution. The microspheres were then centrifuged, and the precipitate was collected. The precipitate was washed three times with sterile seawater, followed by centrifugation to obtain the encapsulated microspheres.

[0033] The centrifugation process was carried out at a speed of 4000 rpm for 15 minutes.

[0034] Comparative Example 1 A method for preparing embedded microspheres of Vibrio harveyi in situ culture, comprising the following steps: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 25°C for 25 minutes at a stirring speed of 150 rpm, then placed at 85°C and stirred for another 45 minutes. Sodium alginate was then added, and the mixture was stirred for another 35 minutes. Finally, the mixture was ultrasonically treated and passed through a 200-mesh sieve to obtain a blended solution. The mass fraction of polyvinyl alcohol and sodium alginate in the blended solution was 1.5%. The ultrasonic treatment was performed at a power of 400 W for 25 minutes.

[0035] Step 2: Prepare embedded microspheres This step is the same as the "preparation of embedded microspheres" step in Example 2.

[0036] Performance Test 1 (a) The particle size distribution of the embedded microspheres prepared in Examples 1-3 and Comparative Example 1 was determined using a Winner2000ZD laser particle size analyzer. The specific test results are shown in Table 1.

[0037] Table 1 Example 1 Example 2 Example 3 D50 (μm) 45.3 47.8 46.6 Average particle size (μm) 51.36 55.24 53.67 PDI 1.12 1.03 1.23 As shown in Table 1, the D50 of the embedded microspheres prepared in Examples 1-3 is 45.3-47.8 μm, the average particle size is 51.36-55.24 μm, and the PDI is 1.03-1.23. It can be seen that Example 2 has the largest average particle size and the smallest dispersion coefficient, indicating that the embedded microspheres prepared in Example 2 have better uniformity. Therefore, Example 2 is selected as the optimal example.

[0038] Example 4: A method for in situ culture of Vibrio harveyi In situ culture of Vibrio harveyi was performed using the method described in Example 2, as follows: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 25°C for 25 minutes at a stirring speed of 150 rpm, then placed at 85°C and stirred for another 45 minutes. Sodium alginate was then added, and stirring continued for 35 minutes. Nano-hydroxyapatite was added, and the stirring speed was increased to 200 rpm, with stirring continuing for 25 minutes. The mixture was then subjected to ultrasonic treatment and passed through a 200-mesh sieve to obtain a blended solution. In the blended solution, the mass fractions of polyvinyl alcohol, sodium alginate, and nano-hydroxyapatite were 1%. The ultrasonic treatment was performed at a power of 400 W for 25 minutes.

[0039] Step 2: Preparation of bacterial suspension Dissolve 5g casein peptone, 1g yeast extract, and 0.1g ferric phosphate in seawater. Adjust the pH of the system to 7.7 with NaOH solution, then add seawater to a final volume of 1L. Autoclave at 121℃ and 0.105MPa for 20 minutes to obtain a liquid culture medium. Inoculate 20ml of Vibrio harveyi into the liquid culture medium and incubate at 37℃ for 16 hours. After incubation, collect the bacterial cells by centrifugation at 3000rpm for 15 minutes, and add 5ml of sterilized seawater to obtain a bacterial suspension.

[0040] Step 3: Preparation of Vibrio harveyi-embedded microspheres Add the suspended bacterial solution to 30 ml of sterile blending solution and stir well to obtain the bacterial mixture.

[0041] Dissolve calcium chloride in deionized water to prepare a 5% calcium chloride solution.

[0042] Encapsulated microspheres were prepared using a microencapsulation generator. The blended solution was loaded into the syringe of the microencapsulation generator, which was connected to a syringe pump. The injection rate of the syringe pump was adjusted to 13 mL / h, and the gas pressure of the microencapsulation generator was adjusted to 250 mbar. A beaker containing calcium chloride solution was placed at the nozzle of the microencapsulation generator. The blended solution was sprayed through the nozzle to form encapsulated microspheres, which were collected with calcium chloride solution. The microspheres were then centrifuged, and the precipitate was collected. The precipitate was washed 2-3 times with sterile seawater, followed by centrifugation to obtain Vibrio harveyi encapsulated microspheres. The Vibrio harveyi encapsulated microspheres were added to 5 ml of sterile seawater to obtain a sterile seawater suspension. This suspension was then added to 30 ml of liquid culture medium and incubated at 37°C. 3 ml samples were periodically taken for analysis.

[0043] The centrifugation process was carried out at a speed of 3500 rpm for 20 minutes.

[0044] Comparative Example 2 A method for in situ culture of Vibrio harveyi is as follows: Step 1: Preparation of the blend solution Polyvinyl alcohol was placed in an Erlenmeyer flask, and distilled water was added. The mixture was first stirred at a constant temperature of 25°C for 25 minutes at a stirring speed of 150 rpm, then placed at 85°C and stirred for another 45 minutes. Sodium alginate was then added, and the mixture was stirred for another 35 minutes. Finally, the mixture was ultrasonically treated and passed through a 200-mesh sieve to obtain a blended solution. The mass fraction of polyvinyl alcohol and sodium alginate in the blended solution was 1.5%. The ultrasonic treatment was performed at a power of 400 W for 25 minutes.

[0045] Step 2: Preparation of bacterial suspension This step is the same as the "Preparation of Suspended Bacterial Solution" step in Example 4.

[0046] Step 3: Preparation of Vibrio harveyi-embedded microspheres This step is the same as the step in "Preparing Vibrio harveyi-embedded microspheres" in Example 4. Performance Test 2 (I) Using riboflavin as a sustained-release model, the sustained-release effect of the encapsulated microspheres prepared in Example 2 and Comparative Example 1 on riboflavin was tested. The specific test results are attached. Figure 2 As shown.

[0047] From the appendix Figure 2It can be seen that the sustained-release trend of riboflavin in the encapsulated microspheres prepared in Example 2 and Comparative Example 1 is consistent, both consisting of two stages: a burst release stage from 0 to 2 hours, a gradual release stage from 2 to 5 hours, and a sustained-release equilibrium after 5 hours. The cumulative release rate of the encapsulated microspheres prepared in Example 2 was 66.38% at 5 hours, while that of the encapsulated microspheres prepared in Comparative Example 1 was 47.23% at 5 hours. This indicates that the encapsulated microspheres prepared in this invention have better permeability to small molecules.

[0048] (II) Using bovine serum albumin (BSA) as a sustained-release model, the sustained-release effect of the encapsulated microspheres prepared in Example 2 and Comparative Example 1 on BSA was tested. Specific test results are attached. Figure 3 As shown.

[0049] From the appendix Figure 3 It can be seen that the sustained-release trend of BSA in the encapsulated microspheres prepared in Example 2 and Comparative Example 1 is consistent. There is a significant burst release phenomenon in the first 0-20 min, a gradual release phase in the first 20-240 min, and a sustained-release equilibrium is basically reached after 300 min. The cumulative release rate of the encapsulated microspheres prepared in Example 2 is 16.81% at 300 min, while the cumulative release rate of the encapsulated microspheres prepared in Comparative Example 1 is 13.58% at 5 h. This indicates that the encapsulated microspheres prepared in this invention have better permeability to water-soluble molecules.

[0050] (III) Add 4 ml of sodium citrate to the sterile seawater suspensions prepared in Example 4 and Comparative Example 2 respectively, centrifuge, discard the supernatant, rinse with sterile seawater, centrifuge again, add 5 ml of sterile seawater to each and mix well. Using sterile seawater as a blank, measure the absorbance value at a wavelength of 600 nm using an ultraviolet spectrophotometer. The encapsulation rate is calculated using the following formula: . The specific test results are shown in Table 2.

[0051] Table 2 Example 4 Comparative Example 2 Encapsulation rate (%) 91.2746%±1.2213 89.3515%±0.5118 As shown in Table 2, the encapsulation rate of Vibrio harveyi microspheres prepared in Example 4 was 91.2746% ± 1.2213%, proving that the encapsulation effect of the microspheres prepared in this invention on Vibrio harveyi was better.

[0052] (iv) The growth of Vibrio harveyi in the embedded microspheres prepared in Example 4 and Comparative Example 2 was detected using a UV spectrophotometer. The specific test results are attached. Figure 4 As shown.

[0053] From the appendix Figure 4As can be seen, in the microspheres prepared in Example 4 and Comparative Example 2, the detected OD values ​​of Vibrio harveyi gradually increased with the extension of culture time, indicating a gradual increase in bacterial density. Bacterial growth was very slow from 0-6 hours. At 6 hours, the OD value of Vibrio harveyi in the microspheres prepared in Example 4 was 0.074, while the OD value of Vibrio harveyi in the microspheres prepared in Comparative Example 2 was 0.021. After 6 hours, bacterial growth accelerated, because the newly encapsulated bacteria needed an adaptation period. The figure shows that the bacterial growth activity in the encapsulated microspheres prepared in Example 4 was consistently higher than that in the encapsulated microspheres prepared in Comparative Example 2. At 18 hours, the OD value of Vibrio harveyi in the microspheres prepared in Example 4 was 0.592, while the OD value of Vibrio harveyi in the microspheres prepared in Comparative Example 2 was 0.126. This indicates that the encapsulated microspheres prepared in this invention have higher permeability to both macromolecules and small molecules, making it easier for nutrient molecules to enter.

[0054] The specific parameters of the raw materials used in this invention are as follows: The Vibrio harveyi strain was obtained from a commercially available source, and the isolation source was Vibrio harveyi strain BB170, numbered Bio-67732.

[0055] The casein peptone and yeast extract were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0056] The seawater has a pH value of 7.6-7.8.

[0057] The sodium alginate was purchased from Qingdao Haizhilin Biotechnology Development Co., Ltd.

[0058] The calcium chloride was purchased from BASF Chemical Co., Ltd. in Tianjin.

[0059] The polyvinyl alcohol has an average degree of polymerization of 1799±50 and a molecular weight of 1750±50.

[0060] Obviously, there are many other possible implementation methods under the concept of this invention. It should be stated here that any changes made under the inventive concept of this invention will fall within the protection scope of this invention.

Claims

1. A method for preparing embedded microspheres of Vibrio harveyi in situ culture, characterized in that: The method for preparing the embedded microspheres includes the steps of preparing a blended solution and obtaining the embedded microspheres. Preparation of the blend solution: Polyvinyl alcohol is placed in an Erlenmeyer flask, distilled water is added, and the mixture is first stirred at a constant temperature of 20-25℃, then stirred at 80-85℃; sodium alginate is then added and stirred; nano-hydroxyapatite is added and stirred; then ultrasonic treatment is performed, and the mixture is sieved to obtain the blend solution. The process for obtaining the embedded microspheres involves: loading the blended solution into the syringe of the micro-embedding generator, connecting the syringe to an injection pump, placing a beaker containing calcium chloride solution at the nozzle of the micro-embedding generator, collecting the microspheres, then centrifuging them, rinsing the microsphere precipitate with sterile seawater, and centrifuging again to obtain the embedded microspheres.

2. The method for preparing embedded microspheres of Vibrio harveyi in situ culture according to claim 1, characterized in that: In the blended solution, the mass fraction of polyvinyl alcohol is 1-2%, the mass fraction of sodium alginate is 1-2%, and the mass fraction of nano-hydroxyapatite is 0.5-1.5%.

3. The method for preparing embedded microspheres of Vibrio harveyi in situ culture according to claim 1, characterized in that: The ultrasonic treatment involves using an ultrasonic power of 300-500W for 20-30 minutes.

4. The method for preparing embedded microspheres of Vibrio harveyi in situ culture according to claim 1, characterized in that: The calcium chloride solution has a mass fraction of 4-6%.

5. The method for preparing embedded microspheres of Vibrio harveyi in situ culture according to claim 1, characterized in that: The centrifugation process is carried out at a speed of 3000-4000 rpm for 15-20 min. The injection pump has an injection rate of 10-25 mL / h and a gas pressure of 100-300 mbar for the micro-embedding generator.

6. A method for in situ culture of Vibrio harveyi, characterized in that: The method for in situ culture of Vibrio harveyi includes the following steps: preparing a blended solution, preparing a suspension of bacteria, and obtaining Vibrio harveyi-encapsulated microspheres. The preparation of the suspended bacterial solution involves dissolving casein peptone, yeast extract, and ferric phosphate in seawater, adjusting the pH of the system, adding seawater, and autoclaving to obtain a liquid culture medium. Vibrio harveyi is inoculated into the liquid culture medium and placed in a constant temperature incubator for cultivation. After cultivation, the bacterial cells are collected by centrifugation, and sterilized seawater is added to obtain the suspended bacterial solution.

7. The method for in situ culture of Vibrio harveyi according to claim 6, characterized in that: The high-pressure sterilization is carried out at a temperature of 120-122℃, a pressure of 0.1-0.105MPa, and a sterilization time of 20-30min. The pH value of the system is adjusted to 7.6-7.8 using NaOH solution. The temperature of the constant temperature incubator is 36-38℃, and the incubation time is 16-18h.

8. The method for in situ culture of Vibrio harveyi according to claim 6, characterized in that: The preparation of Vibrio harveyi-embedded microspheres involves: adding the bacterial suspension to a sterile blending solution and stirring until homogeneous to obtain a bacterial mixture; assembling a micro-embedding generator, loading the bacterial mixture into a syringe, connecting it to an injection pump, placing a beaker containing calcium chloride solution at the generator nozzle, and collecting the microspheres; then centrifuging the microspheres, rinsing the precipitate with sterile seawater, and centrifuging again to obtain Vibrio harveyi-embedded microspheres; adding the Vibrio harveyi-embedded microspheres to sterile seawater to obtain a sterile seawater suspension, and then culturing it in a liquid culture medium.

9. The method for in situ culture of Vibrio harveyi according to claim 8, characterized in that: The centrifugation process is carried out at a speed of 3000-4000 rpm for 15-20 min; the calcium chloride solution has a mass fraction of 4-6%; the injection pump has an injection rate of 10-25 mL / h and the micro-embedding generator has a pressure of 100-300 mbar.

10. The method for in situ culture of Vibrio harveyi according to claim 8, characterized in that: The culture process is carried out at a temperature of 36-38℃.

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