Saline-alkali-tolerant vibrio-inhibiting aquatic probiotics and preparation process thereof
Through gradient acclimation culture and double-layer microencapsulation technology, combined with specific strains and functional synergists, the stability and antibacterial efficacy of aquatic probiotics in the saline-alkali environment are solved, long-term antibacterial and microecological restoration are achieved, and environmental adaptability and product quality of aquaculture are improved.
Patent Information
- Application Number
- CN202510376525.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-01
AI Technical Summary
Traditional aquatic probiotic preparations have poor adaptability in saline-alkali environments, insufficient targeted antibacterial efficacy, and prone to inactivation of active ingredients, which cannot achieve long-term antibacterial and microecological repair.
The combination of Bacillus halophilus, Lactobacillus alkali-resistant Lactobacillus and Viaphage phage lyase engineering bacteria is adopted, combining functional synergists such as oleosaccharides, nanosilica-loaded γ-polyglutamic acid, and ecological regulation auxiliary materials such as modified zeolite powder. Through gradient domestication culture and double-layer microencapsulation encapsulation technology, a physical-biochemical dual antibacterial barrier and dynamic microecological regulation network are formed.
The stable colonization and long-term antibacterials of probiotics in a high saline-alkali environment are achieved, the tolerance and functional stability of active ingredients are improved, and the three-dimensional protection system is formed, ensuring the continuous antibacterial effect in aquaculture water.
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Figure CN120227402A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of probiotics, and specifically relates to an aquatic probiotic resistant to salinity and alkali and inhibiting vibrio and its preparation process. Background Art
[0002] Aquatic probiotics are a class of beneficial microorganisms applied in the field of aquaculture, mainly including lactic acid bacteria, bacillus, yeast, etc., and have various functions such as regulating water quality, promoting the growth of aquatic animals, and improving immunity. These probiotics can improve the water environment, reduce the content of harmful substances such as ammonia nitrogen and nitrite, and maintain the ecological balance of the water body by secreting beneficial metabolites such as enzymes, vitamins, and antibiotics. At the same time, probiotics can also adhere to the intestinal mucosa of aquatic animals, enhance the intestinal barrier function, inhibit the growth of harmful bacteria, improve feed utilization rate, and promote the healthy growth of aquatic animals. Reasonable use of probiotics in aquaculture helps to achieve a green, efficient, and sustainable aquaculture model, improve the quality of aquatic products, and ensure food safety.
[0003] However, traditional aquatic probiotic preparations generally have defects such as poor adaptability to saline-alkali environments, insufficient targeted antibacterial efficacy, and easy inactivation of active ingredients. Conventional strains are difficult to stably colonize in high-salinity and high-alkalinity water bodies without directional domestication, and antibacterial relying on single metabolites is likely to cause drug resistance of vibrio; the protection ability of ordinary embedding technology against gastric acid and osmotic pressure is limited, resulting in low viable cell survival rate and short action time. At the same time, there is a lack of systematic regulation of toxin clearance in the aquaculture environment and the ecological balance of the flora, and the synergistic effect of long-term antibacterial and microecological repair cannot be achieved. Summary of the Invention
[0004] The purpose of the present invention is to provide an aquatic probiotic resistant to salinity and alkali and inhibiting vibrio and its preparation process to solve the above-mentioned problems.
[0005] The technical solution adopted by the present invention is as follows: An aquatic probiotic resistant to salinity and alkali and inhibiting vibrio, the aquatic probiotic includes:
[0006] Core combined probiotics: 120 parts of halophilic bacillus, 80 parts of marine rhodotorula, 100 parts of alkali-tolerant lactobacillus, and 60 parts of vibriophage lysozyme engineering bacteria;
[0007] Functional metabolism synergist: 150 parts of fucoidan oligosaccharide, 80 parts of nano-silica loaded γ-polyglutamic acid, 70 parts of Dunaliella salina extract, and 40 parts of Fe(III)-EDTA chelate;
[0008] Ecological regulation adjuvant: 180 parts of modified zeolite powder, 90 parts of freeze-dried powder of kelp fermentation supernatant, and 30 parts of tetrahymena cyst powder.
[0009] In a preferred embodiment, the preparation process includes the following steps:
[0010] S1: Halophilic Bacillus, alkali-resistant Lactobacillus and marine red yeast were cultured in gradually increasing salinity and pH to screen for salt- and alkali-resistant dominant strains.
[0011] S2: Inoculate the Vibrio phage lytic enzyme engineered bacteria into a specific culture medium to induce them to express VP4 lytic enzyme, inactivate them and collect the bacteria for later use.
[0012] S3: Brown algae oligosaccharides were prepared by enzymatic hydrolysis of kelp extract, nano-silica loaded with γ-polyglutamic acid was synthesized by sol-gel method, and chelates were generated by reacting FeCl3 with EDTA.
[0013] S4: The zeolite powder is acid-washed and modified and loaded with antibacterial ingredients, the kelp fermentation liquid is freeze-dried and powdered, and the Tetrahymena cysts are sterilized and dried.
[0014] S5: Thoroughly mix the domesticated core probiotics, engineered bacteria and functional enhancers in a low-temperature inert gas environment.
[0015] S6: Add modified zeolite powder, kelp freeze-dried powder and Tetrahymena cyst powder into the mixed system, and use fluidized bed coating technology to achieve uniform coating.
[0016] S7: Sodium alginate-chitosan was used to embed the bacteria as the inner layer, and the outer layer was covered with a composite membrane of poly-γ-glutamic acid and montmorillonite to control the particle size of the microcapsule.
[0017] S8: The finished product is obtained by freeze drying and low-temperature spray drying, and the number of viable bacteria, lytic enzyme activity and tolerance to saline-alkali environment are tested.
[0018] In a preferred embodiment, in step S1, halophilic Bacillus and alkali-resistant Lactobacillus are inoculated in a liquid culture medium with an initial salinity of 0% and a pH of 8.0, respectively, and cultured at 37°C with shaking at 150 rpm for 24 hours; then the salinity is gradually increased to 8%, 12%, and 15%, and the pH is simultaneously increased to 8.5, 9.0, and 9.5, and each stage is cultured for 48 hours to screen out stable strains resistant to 15% salinity and pH 9.5. Marine red yeast uses a seawater yeast culture medium with a salinity of 20‰, and is statically cultured at 25°C for 72 hours to finally obtain high-density biomass.
[0019] In a preferred embodiment, in step S2, the Vibrio phage lytic enzyme engineered bacteria are inoculated into LB liquid culture medium containing 0.1% glycine, the initial pH is adjusted to 7.2, and fermented at 30°C and 200rpm for 18 hours; 0.5mM IPTG inducer is added, and fermentation is continued for 6 hours to activate VP4 lytic enzyme expression. After the fermentation is completed, the bacteria are collected by centrifugation at 8000rpm for 10 minutes, and inactivated in a water bath at 80°C for 30 minutes to retain the lytic enzyme activity.
[0020] In a preferred embodiment, in step S3, the fucoidan oligosaccharide is prepared by hydrolyzing kelp extract with fucoidan lyase. The reaction conditions are 50 °C and pH 6.8 for 6 hours, and the oligosaccharide component with a molecular weight of 500 - 1000 Da is obtained by ultrafiltration membrane separation. The nano-silica loaded γ-polyglutamic acid is synthesized by the sol-gel method. A 10% concentration of γ-polyglutamic acid solution is mixed with mesoporous silica at a mass ratio of 1:5, and loading is achieved by stirring at 40 °C for 12 hours. The Fe(III)-EDTA chelate is prepared by reacting FeCl3 with disodium EDTA at a molar ratio of 1:1.2 in a 60 °C water bath for 2 hours, and after adjusting the pH to 6.5, it is cooled and crystallized.
[0021] In a preferred embodiment, in step S4, the modified zeolite powder is soaked in a 0.5 M hydrochloric acid solution for 2 hours, washed with water until neutral, dried, and then mixed and adsorbed with a subtilisin solution at a concentration of 2.5 mg / g for 12 hours. The kelp fermentation broth is prepared by inoculating Lactobacillus plantarum into kelp slurry and anaerobically fermenting at 35 °C for 72 hours. After centrifuging to obtain the supernatant, it is freeze-dried into powder at -50 °C. The Tetrahymena cysts are induced to form by 4 °C low-temperature stress and carbon source starvation treatment for 48 hours, and then sterilized by ultraviolet irradiation for 20 minutes after hot air drying at 60 °C.
[0022] In a preferred embodiment, in step S5, the halophilic Bacillus sp., alkali-tolerant Lactobacillus sp., and Rhodotorula marina cells after gradient acclimation are mixed with the inactivated engineered bacteria according to the formulated ratio, and fucoidan oligosaccharide, nano-silica loaded γ-polyglutamic acid, Dunaliella salina extract, and Fe(III)-EDTA chelate are added. The mixture is stirred at a low speed of 50 rpm in a nitrogen protection environment at 4 °C for 30 minutes to ensure uniform dispersion of the materials and no anaerobic damage to the live bacteria.
[0023] In a preferred embodiment, in step S6, the modified zeolite powder, freeze-dried kelp powder, and Tetrahymena cyst powder are added to the mixed system in three batches. Using a fluidized bed coating equipment, with a 2% hydroxypropyl methylcellulose solution as the binder, controlling the inlet air temperature at 38 °C and the atomization pressure at 0.3 MPa, the coating weight gain rate is controlled within 8% - 10%, and the coating uniformity error is less than 5%.
[0024] In a preferred embodiment, in step S7, the inner layer microencapsulation uses the high-voltage electrostatic forming technology. The mixed bacterial population is mixed with a 2% sodium alginate - 1% chitosan mixture to form droplets through an 8 kV voltage and then dropped into a 2% calcium chloride solution for curing for 10 minutes. The outer layer uses a suspension of 1% poly-γ-glutamic acid and 3% montmorillonite as the coating material and is secondarily coated at 12 kV voltage and cured in a 0.1 M sodium bicarbonate solution. Finally, the microcapsule particle size is controlled within the range of 150 - 200 μm.
[0025] In a preferred embodiment, in step S8, the microcapsule product is first vacuum freeze-dried at -45°C for 24 hours to remove moisture, and then secondarily dried in a spray drying tower with an inlet air temperature of 60°C until the water content is ≤5%. Quality inspection includes detecting the viable count by the plate counting method (required to be ≥1×10 11 CFU / g), measuring the VP4 lyase activity by the colorimetric method (≥800 U / g), and testing the survival rate under a simulated saline-alkali environment (15% NaCl, pH 9.5) (≥85%).
[0026] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:
[0027] 1. In the present invention, the gradient domestication culture of the core flora and the directed expression technology of engineering bacteria achieve the active adaptation ability of probiotics to the high saline-alkali environment. After the stepwise salinity and pH adaptive evolution of Halobacillus halophilus and Lactobacillus alkalitolerans, they can stably colonize in the aquaculture water body and continuously secrete antibacterial metabolites; while the phage lyase expressed by the engineering bacteria can precisely destroy the cell wall structure of Vibrio, forming a physical-biochemical double antibacterial barrier. At the same time, the collaborative design of the functional synergist and the ecological adjuvant not only optimizes the metabolic activity of the flora through components such as alginate oligosaccharides and nanocarriers, but also constructs a dynamic microecological regulation network with materials such as modified zeolite and Tetrahymena cysts, effectively blocking the expression of Vibrio virulence genes and removing environmental toxins, forming a three-dimensional protection system from strains to ecosystems.
[0028] 2. In the present invention, the integrated application of the double-layer microencapsulation and the low-temperature drying process overcomes the technical bottleneck of the easy inactivation of active ingredients. The sodium alginate-chitosan inner embedding technology ensures the survival rate of viable bacteria during processing, storage, and in the digestive tract, and the outer poly-γ-glutamic acid composite membrane realizes the slow release control of functional components, enabling the probiotics to continuously release antibacterial factors in the saline-alkali water body for more than 72 hours. This process not only greatly improves the tolerance of the product to environmental stress, but also ensures the uniformity of the flora distribution and the functional stability through precise control technologies such as fluidized bed coating and electrostatic microcapsules, providing a reliable guarantee for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 It is the process schematic diagram of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0030] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0031] Example:
[0032] Refer to Figure 1 ,
[0033] An aquatic probiotic for resisting salinity and alkalinity and inhibiting Vibrio, the aquatic probiotic comprising:
[0034] Core combined probiotics: 120 parts of Bacillus halodurans, 80 parts of Rhodotorula marina, 100 parts of Lactobacillus alkalitolerans, and 60 parts of Vibrio phage lysozyme engineering bacteria;
[0035] Functional metabolic synergist: 150 parts of fucoidan oligosaccharide, 80 parts of nano-silica loaded γ-polyglutamic acid, 70 parts of Dunaliella salina extract, and 40 parts of Fe(III)-EDTA chelate;
[0036] Ecological regulation adjuvant: 180 parts of modified zeolite powder, 90 parts of freeze-dried powder of kelp fermentation supernatant, and 30 parts of Tetrahymena cyst powder.
[0037] The preparation process comprises the following steps:
[0038] S1: Gradually increasing the salinity and pH adaptability culture of Bacillus halodurans, Lactobacillus alkalitolerans, and Rhodotorula marina, and screening to obtain salinity- and alkalinity-resistant dominant strains.
[0039] S2: Inoculating the Vibrio phage lysozyme engineering bacteria into a specific medium, inducing the expression of VP4 lysozyme, inactivating, and collecting the bacterial cells for standby.
[0040] S3: Preparing fucoidan oligosaccharide by enzymatically hydrolyzing kelp extract, synthesizing nano-silica loaded γ-polyglutamic acid by the sol-gel method, and generating a chelate by reacting FeCl3 with EDTA.
[0041] S4: Acid-washing and modifying the zeolite powder and loading antibacterial components, freeze-drying the kelp fermentation broth to make powder, and sterilizing and drying the Tetrahymena cyst.
[0042] S5: Fully mixing the domesticated core probiotics, engineering bacteria, and functional synergist in a low-temperature inert gas environment.
[0043] S6: Adding the modified zeolite powder, freeze-dried kelp powder, and Tetrahymena cyst powder to the mixing system, and realizing uniform coating by the fluidized bed coating technology.
[0044] S7: Using sodium alginate-chitosan to embed the bacterial cells as the inner layer, and covering the outer layer with a composite membrane of poly-γ-glutamic acid and montmorillonite to control the microcapsule particle size.
[0045] S8: Obtaining the finished product by freeze-drying and low-temperature spray drying, and detecting the viable bacteria count, lysozyme activity, and salinity and alkalinity environment tolerance.
[0046] In step S1, Halobacillus and Lactobacillus alkalitolerans were respectively inoculated into a liquid medium with an initial salinity of 0% and a pH of 8.0, and cultured with shaking at 150 rpm at 37°C for 24 hours; subsequently, the salinity was gradually increased to 8%, 12%, and 15%, and the pH was synchronously increased to 8.5, 9.0, and 9.5, and cultured for 48 hours at each stage to screen out stable strains resistant to 15% salinity and pH 9.5. Rhodotorula marina was cultured statically in a seawater yeast medium with a salinity of 20‰ at 25°C for 72 hours to finally obtain a high-density biomass.
[0047] In step S2, the engineered Vibrio phage lyase bacteria were inoculated into an LB liquid medium containing 0.1% glycine, the initial pH was adjusted to 7.2, and fermentation culture was carried out at 30°C and 200 rpm for 18 hours; 0.5 mM IPTG inducer was added and fermentation continued for 6 hours to activate the expression of VP4 lyase. After fermentation, the cells were collected by centrifugation at 8000 rpm for 10 minutes, inactivated in a water bath at 80°C for 30 minutes, and the lyase activity was retained.
[0048] In step S3, fucoidan oligosaccharides were prepared by hydrolyzing kelp extracts with alginate lyase, and the reaction conditions were 50°C and pH 6.8 for 6 hours. The oligosaccharide fraction with a molecular weight of 500 - 1000 Da was obtained by ultrafiltration membrane separation. Nano-silica-supported γ-polyglutamic acid was synthesized by the sol-gel method. A 10% concentration of γ-polyglutamic acid solution was mixed with mesoporous silica at a mass ratio of 1:5 and stirred at 40°C for 12 hours for loading. The Fe(III)-EDTA chelate was prepared by reacting FeCl3 with disodium EDTA at a molar ratio of 1:1.2 in a water bath at 60°C for 2 hours, and the pH was adjusted to 6.5 and then cooled and crystallized.
[0049] In step S4, the modified zeolite powder was soaked in 0.5 M hydrochloric acid solution for 2 hours, washed with water until neutral and then dried, and mixed with a subtilin solution with a concentration of 2.5 mg / g for adsorption for 12 hours. The kelp fermentation broth was obtained by inoculating Lactobacillus plantarum into kelp slurry and anaerobically fermenting at 35°C for 72 hours. After centrifugation, the supernatant was freeze-dried into powder at -50°C. Tetrahymena cysts were induced to form by 4°C low-temperature stress and carbon source starvation treatment for 48 hours, and sterilized by ultraviolet irradiation for 20 minutes after hot air drying at 60°C.
[0050] In step S5, the gradient-domesticated Halobacillus, Lactobacillus alkalitolerans, and Rhodotorula marina cells were mixed with the inactivated engineered bacteria according to the formulated ratio, and fucoidan oligosaccharides, nano-silica-supported γ-polyglutamic acid, Dunaliella salina extract, and Fe(III)-EDTA chelate were added. Stirring was carried out at a low speed of 50 rpm in a nitrogen protection environment at 4°C for 30 minutes to ensure uniform dispersion of the materials and no anaerobic damage to the live bacteria.
[0051] In step S6, the modified zeolite powder, the freeze-dried powder of seaweed, and the cysts powder of Tetrahymena are added to the mixed system in three times. Using a fluidized bed coating equipment, with a 2% hydroxypropyl methylcellulose solution as the binder, controlling the inlet air temperature at 38°C and the atomization pressure at 0.3 MPa, the coating weight gain rate is controlled at 8%-10%, and the coating uniformity error is less than 5%.
[0052] In step S7, the inner layer microencapsulation adopts the high-voltage electrostatic forming technology. The mixed flora and the 2% sodium alginate - 1% chitosan mixed solution form droplets through an 8 kV voltage and fall into the 2% calcium chloride solution for curing for 10 minutes. The outer layer uses a suspension of 1% poly-γ-glutamic acid and 3% montmorillonite as the coating material and is secondarily coated at a voltage of 12 kV and cured in a 0.1 M sodium bicarbonate solution. Finally, the microcapsule particle size is controlled within the range of 150 - 200 μm.
[0053] In step S8, the microcapsule product is first vacuum freeze-dried at -45°C for 24 hours to remove moisture, and then secondarily dried in a spray drying tower with an inlet air temperature of 60°C until the water content ≤ 5%. Quality inspection includes detecting the viable bacteria count by the plate counting method (required to be ≥ 1×10 11 CFU / g), measuring the VP4 lyase activity by the colorimetric method (≥ 800 U / g), and testing the survival rate under a simulated saline-alkali environment (15% NaCl, pH 9.5) (≥ 85%).
[0054] It can be known from the above that:
[0055] In the present invention, the gradient domestication culture of the core flora and the engineering bacteria directed expression technology achieve the active adaptation ability of probiotics to the high saline-alkali environment. After the step-by-step salinity and pH adaptive evolution of Halophilic Bacillus and Alkaliphilic Lactobacillus, they can stably colonize in the aquaculture water body and continuously secrete antibacterial metabolites; while the phage lyase expressed by the engineering bacteria can precisely destroy the cell wall structure of Vibrio, forming a physical-biochemical double antibacterial barrier. At the same time, the collaborative design of the functional synergist and the ecological adjuvant not only optimizes the metabolic activity of the flora through components such as fucoidan and nano-carriers, but also constructs a dynamic microecological regulation network with materials such as modified zeolite and Tetrahymena cysts, effectively blocking the expression of Vibrio virulence genes and removing environmental toxins, forming a three-dimensional protection system from strains to ecosystems.
[0056] In the present invention, the integrated application of the double-layer microencapsulation packaging and the low-temperature drying process overcomes the technical bottleneck of the easy inactivation of active ingredients. The sodium alginate-chitosan inner layer embedding technology ensures the survival rate of viable bacteria during processing, storage, and in the digestive tract. The outer layer of poly-γ-glutamic acid composite membrane realizes the slow release control of functional components, enabling the probiotics to continuously release antibacterial factors in the saline-alkali water body for more than 72 hours. This process not only greatly improves the tolerance of the product to environmental stress, but also ensures the uniformity of the flora distribution and the functional stability through precise control technologies such as fluidized bed coating and electrostatic microcapsules, providing a reliable guarantee for large-scale production.
[0057] It should be noted that in this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the said element.
[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements 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 salt-alkali resistant aquatic probiotic that inhibits Vibrio, characterized by: The aquatic probiotics include: Core combination probiotics: 120 parts of halophilic Bacillus, 80 parts of marine red yeast, 100 parts of alkali-resistant Lactobacillus and 60 parts of Vibrio phage lytic enzyme engineered bacteria; Functional metabolic enhancer: 150 parts of brown algae oligosaccharide, 80 parts of nano-silica-loaded γ-polyglutamic acid, 70 parts of Dunaliella salina extract and 40 parts of Fe(III)-EDTA chelate; Ecological regulation auxiliary materials: 180 parts of modified zeolite powder, 90 parts of kelp fermentation supernatant freeze-dried powder and 30 parts of Tetrahymena cyst powder.
2. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: The preparation process comprises the following steps: S1: Halophilic Bacillus, alkali-tolerant Lactobacillus and marine red yeast were cultured in gradually increasing salinity and pH to screen for salt-alkali-tolerant dominant strains; S2: Inoculate the Vibrio phage lytic enzyme engineered bacteria into a specific culture medium to induce them to express VP4 lytic enzyme, inactivate them and collect the bacteria for later use; S3: Preparation of brown algae oligosaccharides by enzymatic hydrolysis of kelp extract, synthesis of nano-silica loaded with γ-polyglutamic acid by sol-gel method, and formation of chelate by reaction of FeCl3 and EDTA; S4: acid-washing and modifying the zeolite powder and loading the antibacterial components, freeze-drying the kelp fermentation liquid to make powder, and sterilizing and drying the Tetrahymena cysts; S5: fully mix the domesticated core probiotics, engineered bacteria and functional enhancers under a low-temperature inert gas environment; S6: adding modified zeolite powder, kelp freeze-dried powder and Tetrahymena cyst powder to the mixed system, and achieving uniform coating using fluidized bed coating technology; S7: Sodium alginate-chitosan was used to embed the bacteria as the inner layer, and the outer layer was covered with a composite membrane of poly-γ-glutamic acid and montmorillonite to control the particle size of the microcapsule; S8: The finished product is obtained by freeze drying and low-temperature spray drying, and the number of viable bacteria, lytic enzyme activity and tolerance to saline-alkali environment are tested.
3. The preparation process of a salt-alkali tolerant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In the step S1, halophilic bacillus and alkali-resistant lactobacillus are inoculated into a liquid culture medium with an initial salinity of 0% and a pH of 8.0, respectively, and cultured at 37°C with shaking at 150 rpm for 24 hours; then the salinity is gradually increased to 8%, 12%, and 15%, and the pH is simultaneously increased to 8.5, 9.0, and 9.5, and each stage is cultured for 48 hours to screen out a stable strain resistant to 15% salinity and pH 9.5; and marine red yeast uses a seawater yeast culture medium with a salinity of 20‰ and is statically cultured at 25°C for 72 hours.
4. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In the step S2, the Vibrio phage lytic enzyme engineered bacteria are inoculated into an LB liquid culture medium containing 0.1% glycine, the initial pH is adjusted to 7.2, and fermentation is cultured at 30°C and 200rpm for 18 hours; 0.5mM IPTG inducer is added, and the fermentation is continued for 6 hours to activate the expression of VP4 lytic enzyme; after the fermentation is completed, the bacteria are collected by centrifugation at 8000rpm for 10 minutes, and inactivated in an 80°C water bath for 30 minutes to retain the lytic enzyme activity.
5. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In the step S3, brown algae oligosaccharides are prepared by hydrolyzing kelp extract with alginate lyase, the reaction conditions are 50°C and pH 6.8 for 6 hours, and oligosaccharide components with a molecular weight of 500-1000Da are obtained by ultrafiltration membrane separation; nano-silica-loaded γ-polyglutamic acid is synthesized by a sol-gel method, a 10% concentration of γ-polyglutamic acid solution is mixed with mesoporous silica in a mass ratio of 1:5, and the loading is achieved by stirring at 40°C for 12 hours; Fe(III)-EDTA chelate is reacted with FeCl3 and disodium EDTA in a molar ratio of 1:1.2 in a 60°C water bath for 2 hours, and the pH is adjusted to 6.5 and then cooled for crystallization.
6. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In the step S4, the modified zeolite powder is soaked in a 0.5M hydrochloric acid solution for 2 hours, washed with water until neutral and then dried, and mixed with a 2.5mg / g subtilisin solution for adsorption for 12 hours; the kelp fermentation liquid is prepared by inoculating kelp slurry with plant lactobacillus, anaerobically fermenting at 35°C for 72 hours, centrifuging the supernatant and freeze-drying it at -50°C into powder; the Tetrahymena cysts are induced to form by low temperature stress at 4°C and carbon source starvation for 48 hours, and then hot air dried at 60°C and sterilized by ultraviolet irradiation for 20 minutes.
7. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In the step S5, the gradient-acclimated halophilic Bacillus, alkali-resistant Lactobacillus, and marine red yeast cells are mixed with the inactivated engineering bacteria according to the formula ratio, and brown algae oligosaccharides, nano-silica-loaded γ-polyglutamic acid, salina algae extract, and Fe(III)-EDTA chelate are added, and stirred at a low speed of 50 rpm for 30 minutes in a nitrogen protection environment at 4°C to ensure that the materials are evenly dispersed and the live bacteria are not damaged by oxygen.
8. The process for preparing a salt-alkali tolerant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In step S6, modified zeolite powder, kelp freeze-dried powder and Tetrahymena cyst powder are added to the mixed system three times, and a fluidized bed coating device is used, with 2% hydroxypropyl methylcellulose solution as a binder, the inlet air temperature is controlled at 38° C., the atomization pressure is controlled at 0.3 MPa, the coating weight gain rate is controlled at 8%-10%, and the coating uniformity error is less than 5%.
9. The preparation process of a salt-alkali resistant aquatic probiotic that inhibits Vibrio as claimed in claim 1, characterized in that: In step S7, the inner layer microencapsulation adopts high-voltage electrostatic forming technology, and the mixed bacterial community and 2% sodium alginate-1% chitosan mixture are formed into droplets through 8kV voltage, and fall into 2% calcium chloride solution for curing for 10 minutes; the outer layer is coated with 1% poly-gamma-glutamic acid and 3% montmorillonite suspension as the packaging material, and is secondary coated at 12kV voltage and cured in 0.1M sodium bicarbonate solution. The final microcapsule particle size is controlled in the range of 150-200μm.
10. The process for preparing a salt-alkali resistant aquatic probiotics that inhibits Vibrio according to claim 1, characterized in that: In step S8, the microcapsule product is first subjected to vacuum freeze drying at -45°C for 24 hours to remove moisture, and then secondary drying in a spray drying tower with an inlet air temperature of 60°C to a moisture content of ≤5%; quality inspection includes plate count method to detect the number of viable bacteria, colorimetric method to determine VP4 lyase activity, and survival rate test in a simulated saline-alkali environment.