Method for breeding aquatic fry with high survival rate

Abstract

The application discloses a high-survival-rate aquatic fish fry breeding method and relates to the technical field of aquatic breeding. The method comprises the following steps: preparing porous microsphere carriers which have communicating channels in the interior and have the same suspension density as the water body; loading spore bacilli which have AHL lactone enzyme secretion capacity on the surface of the carriers and in the communicating channels to form a continuous biological membrane; obtaining biological membrane microspheres; and putting the biological membrane microspheres into a fish fry breeding water body, using the secreted AHL lactone enzyme to degrade pathogenic bacteria AHL signal molecules, and inhibiting the virulence expression of the pathogenic bacteria. Meanwhile, the characteristics that the particle size of the microspheres matches the opening period of the fish fry are used, so that the microspheres are planted in the intestinal tract to form a micro-ecological barrier after being eaten by the fish fry. The application realizes the synchronous planting of probiotics in the water body and the intestinal tract, and can improve the survival rate of the fish fry.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for breeding aquatic fish fry with a high survival rate. Background Technology

[0002] During the period from hatching to stocking, aquatic fish fry (such as shrimp fry, mandarin fish fry, and bass fry) have an immature adaptive immune system, a very short digestive tract, and an underdeveloped mucosal barrier. They are extremely sensitive to opportunistic pathogens such as Vibrio and Aeromonas, which are commonly found in aquaculture water. These pathogens coordinate their perception of population density through quorum sensing. When the population density reaches a threshold, they activate the secretion of virulence factors and the formation of pathogenic biofilms, thereby infecting the fish fry and causing large-scale disease and death, with a mortality rate often exceeding 50%.

[0003] The currently common approach in the industry is to obtain free bacterial liquid or powder by liquid fermentation and propagation of probiotic strains such as lactic acid bacteria and Bacillus, which is then directly sprinkled into the hatching water or mixed with the initial feed and fed to the fish fry. The probiotics inhibit the proliferation of pathogenic bacteria by colonizing and competing with each other in the water or intestines. In this approach, the probiotics exist in the water and feed in a free, suspended state, without any carrier structure, and there is no physical attachment between them and the water or feed.

[0004] Because free-living probiotics lack attachment structures, they are easily washed away and excreted in large quantities with the water flow or feces under conditions of continuous water circulation and rapid intestinal emptying in fry. This makes it difficult for them to establish stable colonization in the water and intestines. At the same time, the hatching water is an oligotrophic environment, and free-living probiotics lack a stable carbon source supply. They are at a disadvantage in the survival competition with native pathogenic bacteria that have adapted to this oligotrophic environment, resulting in limited colonization effects. In addition, this scheme does not intervene at the level of pathogenic bacteria population sensing signal communication. As a result, some farms have turned to antibiotics or strong oxidants for water disinfection, which poses risks of disrupting the microecological balance, inducing drug resistance mutations, and drug residues. The survival rate of fry in the early development stage is still generally low. Summary of the Invention

[0005] The technical problem this invention aims to solve is that in existing free-state probiotic intervention schemes, probiotics lack an attachment carrier and are easily washed away and excreted under conditions of continuous circulation and exchange in the breeding water and rapid emptying of the fish fry's intestines. Furthermore, the lack of a stable carbon source supply in oligotrophic water environments makes it difficult for probiotics to achieve synchronous and stable colonization in the breeding water and the fish fry's intestines. It also fails to block the virulence expression and pathogenic biofilm formation of conditionally pathogenic bacteria such as pathogenic Vibrio and Aeromonas at the signal communication level, resulting in low survival rates and high mortality rates of aquatic fish fry from hatching to stocking.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for breeding aquatic fish fry with high survival rate, including the following technical contents: A porous sustained-release microsphere carrier was prepared. The porous microsphere carrier was made of polyhydroxyalkanoate, had an internal interconnected pore structure, and had a suspension density that was the same as that of water. Bacillus bacteria with AHL lactone secretion ability are loaded on the surface and internal pores of the porous microsphere carrier, so that the Bacillus bacteria proliferate on the inner and outer surfaces of the porous microsphere carrier and secrete extracellular polymer matrix to form a continuous biofilm structure covering the inner and outer surfaces of the porous microsphere carrier, thus obtaining biofilm microspheres. The biofilm microspheres are placed in the breeding water of aquatic fish fry and suspended in the breeding water. The AHL lactonease secreted by the biofilm degrades the AHL signaling molecules secreted by pathogenic bacteria in the breeding water, thereby inhibiting the virulence expression of pathogenic bacteria and the formation of pathogenic biofilms. Simultaneously, taking advantage of the fact that the particle size of the biofilm microspheres is 300μm to 500μm, which matches the mouth size of fish fry during the initial feeding stage, the biofilm microspheres are actively ingested by fish fry in the initial feeding stage and enter the intestines. The microspheres adhere to and release the loaded Bacillus bacteria in the fish fry's intestines, forming a biofilm barrier, thereby achieving the simultaneous colonization of probiotics in the water and intestines.

[0007] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows: Firstly, by loading probiotics in the form of a biofilm onto a porous slow-release microsphere carrier with an internal interconnected pore structure, the short-chain fatty acids continuously released into the pores by the hydrolysis products of polyhydroxy fatty acid esters provide a stable carbon source for the attached bacteria, thus solving the problem of low survival rate and obvious competitive disadvantage of free probiotics in oligotrophic water bodies due to lack of carbon source supply. Secondly, by utilizing the AHL lactonease secreted by Bacillus loaded on the surface of microspheres to specifically degrade the AHL signaling molecules of pathogenic bacteria, the pathogenic bacteria lose their ability to sense population density without being directly killed, thereby losing their ability to express virulence and form pathogenic biofilms, achieving non-killing pathogenic bacteria blocking and avoiding the risks of drug resistance mutations and drug residues caused by the use of antibiotics or strong oxidants. Third, by utilizing the neutral buoyancy properties of microspheres and their size matching the mouth size of fish fry during the initial feeding stage, the same carrier can simultaneously perform the dual functions of continuously exerting a quorum sensing quenching effect while suspended in water, and colonizing in the intestines after being actively fed by fish fry during the initial feeding stage. This enables the synchronous and homologous construction of the microecological barrier between the water body and the fish fry's intestines, significantly improving the survival rate of fish fry from hatching to the initial feeding stage. Attached Figure Description

[0008] Figure 1This is a schematic diagram illustrating the overall technical principle of the high survival rate aquatic fish fry breeding method of the present invention; Figure 2 This is a schematic cross-sectional view of the structure of the biofilm microspheres of the present invention; Figure 3 This is a flowchart illustrating the preparation process of the porous sustained-release microsphere carrier of the present invention. Figure 4 This is a schematic diagram illustrating the biofilm construction process and quorum sensing quenching mechanism of the present invention. Figure 5 This is a flowchart illustrating the application and dual colonization process of the biofilm microspheres of this invention in water and intestines. Detailed Implementation

[0009] The following is in conjunction with the appendix Figure 1-5 The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0010] I. Overall Technical Solution Description like Figure 1 As shown, the high-survival-rate aquatic fish fry breeding method provided by this invention uses a biodegradable porous slow-release microsphere as a carrier for a probiotic biofilm with quorum sensing quenching ability. This biofilm microsphere is placed in the aquatic fish fry hatching and breeding water. The biofilm microsphere is in a neutral buoyant suspension state in the water. On one hand, it continuously degrades AHL signal molecules secreted by pathogenic Vibrio, Aeromonas, and other conditionally pathogenic bacteria in the water through AHL lactonease secreted by its surface biofilm, thus inhibiting the pathogenicity of these bacteria because they cannot sense the quorum density threshold. On the other hand, because the particle size of the biofilm microsphere matches the mouth size of the target fish fry during the initial feeding stage, the fry in the initial feeding stage ingest it as a natural microecological initial food during active feeding. The complete biofilm structure on the surface of the microsphere and the physical protection of the polyhydroxyalkanoate skeleton enable it to resist the chemical erosion of gastric acid and bile salts. It rapidly adheres to and releases the loaded bacteria in the very short intestine of the fish fry, forming a pioneer microecological barrier. The aforementioned water purification pathway and intestinal colonization pathway are simultaneously achieved by the same carrier, together constituting the core of the technical solution of this invention.

[0011] II. Preparation of porous sustained-release microsphere carriers In this embodiment, the porous sustained-release microsphere carrier is prepared using a double emulsion solvent evaporation method. First, polyhydroxyalkanoate is dissolved in dichloromethane at a concentration of 8% by weight (v / v), and a Span-type emulsifier is added at a concentration of 2% by weight (v / v). After stirring until homogeneous, an oil phase solution is obtained. Then, ammonium bicarbonate, a pore-forming agent, is dissolved in deionized water at a concentration of 5% by weight (v / v), resulting in an inner aqueous phase solution. Next, the inner aqueous phase solution is slowly added dropwise to the oil phase solution at a volume ratio of 1:5, and sheared at 8,000 rpm for 3 minutes under high-speed shearing conditions to form a uniformly dispersed primary water-in-oil emulsion. Then, this primary emulsion is slowly added to an outer aqueous phase containing a polyvinyl alcohol emulsifier at a concentration of 1% by weight (v / v) under gentle stirring conditions, and stirred at 500 rpm for 10 minutes to form a water-in-oil-in-water double emulsion system. This double emulsion is then... The system was placed under normal temperature and pressure conditions and continuously stirred at 300 rpm for six hours to allow dichloromethane to fully evaporate. The polyhydroxyalkanoates gradually solidified as the solvent evaporated, forming a spherical shell structure. Simultaneously, ammonium bicarbonate in the internal aqueous phase decomposed upon heating, producing gas and forming an interconnected network of pores within the solidified spheres, resulting in a porous suspension of microspheres. Finally, the solidified microspheres were collected by centrifugation at 3000 rpm for five minutes and repeatedly washed three times with deionized water to remove residual emulsifiers and unreacted materials. Particle size was then graded using a standard sieve with a pore size range of 300 to 500 micrometers, and density gradient centrifugation was used to select batches of microspheres with a suspension density identical to that of the water and exhibiting neutral buoyancy, thus obtaining the porous slow-release microsphere carrier described in this invention. The particle size range matches the mouth size of common mandarin fish and bass fry during their initial feeding stage, ensuring that the microspheres can be actively ingested by the fry while suspended in the water.

[0012] As another embodiment of preparing porous sustained-release microsphere carriers, microfluidic technology can also be used. This involves precisely controlling the flow rate ratio between the aqueous phase solution (dispersed phase) and the oil phase solution (continuous phase) using a microfluidic chip, causing them to form monodisperse emulsion droplets at the channel intersections. These monodisperse emulsion droplets are then subjected to the same re-emulsification, solvent evaporation and solidification, and pore-forming treatments as described above, resulting in porous sustained-release microsphere carriers with more uniform particle size distribution and more controllable pore structure. This embodiment yields microspheres with higher batch-to-batch particle size consistency and can serve as an alternative process route for large-scale production.

[0013] III. Construction of Biofilm Microspheres In this embodiment, a Bacillus subtilis strain with AHL lactonease secretion ability was selected as the functional strain. First, the preserved strain was inoculated into liquid culture medium and cultured for 12 hours at 37°C and 180 rpm under shaking conditions to reach the logarithmic growth phase, obtaining a highly viable bacterial suspension. Subsequently, the concentration of the bacterial suspension was determined using the plate count method, and it was diluted to an inoculation concentration of 1 x 10^7 colony-forming units per milliliter. Then, the aforementioned porous slow-release microsphere carrier was added to the bacterial suspension and infiltrated at 30°C with slow stirring for 4 hours, allowing the bacterial suspension to penetrate into the microspheres through the interconnecting pores on the surface, achieving bacterial cell distribution on both the inner and outer surfaces of the microspheres. The initial attachment of microspheres is achieved; then, the microspheres that have completed initial attachment are transferred to a static culture system containing a trace amount of carbon source supplementation solution and incubated at 28 degrees Celsius for 48 hours, allowing the attached bacteria to gradually proliferate and secrete extracellular polymeric matrix, forming a continuous and dense biofilm structure on the inner and outer surfaces of the microspheres. The biofilm biomass is then measured by microscopic observation and crystal violet staining to detect the biofilm coverage on the microsphere surface. When the coverage reaches a set threshold of 80% or more, the biofilm is considered mature; batches that do not reach this threshold are returned to the static culture stage for further cultivation. Finally, the mature biofilm microspheres are collected through gentle filtration, and after adding trehalose and other protective agents in a set ratio, they are refrigerated at 4 degrees Celsius to obtain the finished biofilm microsphere product that can be directly used for deployment in aquaculture water.

[0014] As another implementation method for loading functional strains, other probiotic strains with the ability to secrete AHL acyltransferase or AHL oxidoreductase can also be selected and loaded onto the same porous slow-release microsphere carrier. By using different enzymatic pathways, the degradation or structural modification of AHL signaling molecules can be achieved, which can also achieve the technical effect of inhibiting pathogenic bacteria quorum sensing communication and blocking virulence expression. This can be used as a functional strain expansion scheme for the present invention in different pathogenic bacteria lineages and different aquatic product breeding scenarios.

[0015] IV. Application of Biofilm Microspheres in Breeding Water Bodies Taking a mandarin fish fry hatching and breeding workshop as an example, the total water volume of a single hatching pond in this workshop is ten cubic meters. The timing of microsphere release is determined within a set time window after the fry hatch and before the digestive tract develops to the stage of feeding. Based on the total water volume of the hatching pond and the set target effective concentration of the water, the total amount of biofilm microspheres to be released is calculated according to the ratio relationship described later. After the calculated dose of biofilm microspheres is rehydrated and activated, it is evenly sprinkled on the surface of the water in the hatching pond and in different water layers, utilizing its neutral buoyancy characteristics to allow it to naturally suspend and disperse throughout the water. Suspended in water, biofilm microspheres continuously degrade AHL signaling molecules secreted by free pathogenic bacteria, inhibiting pathogenic bacteria virulence expression and pathogenic biofilm formation. Simultaneously, the polyhydroxyalkanoate backbone within the microspheres continuously hydrolyzes, releasing short-chain fatty acids to maintain the biofilm activity of attached bacteria. During the initial feeding stage, fish fry actively ingest these microspheres as natural microecological starter food, as the microsphere size matches their mouth size. Once inside the intestines, the microspheres rapidly adhere to and release colonizing bacteria, forming a pioneer microecological barrier and continuously exerting a quorum sensing quenching effect locally. Subsequently, the abundance of target bacteria and the concentration of pathogenic bacteria-related signaling molecules in the water and fish fry intestines are monitored at set intervals. Based on the results, supplementary feeding is determined until the fish fry complete the initial feeding stage.

[0016] V. Quantitative Description of Relevant Technical Parameters To further illustrate the controllability and feasibility of the microsphere sustained-release behavior and quorum induction quenching effect in this invention, the following mathematical model can be used to quantitatively characterize the relevant process parameters.

[0017] The sustained-release behavior of short-chain fatty acids in porous microspheres can be described using the Higuchi diffusion release model: ; In the formula, Indicates the elapsed time since the microspheres were deployed. The cumulative release of short-chain fatty acids; The Higuchi release rate constant is determined by the microsphere porosity, pore diameter, and degradation rate of polyhydroxyalkanoates. This represents the time elapsed since the microspheres were deployed. This model is used to guide the setting of process parameters such as the amount of pore-forming agent and pore structure during the microsphere preparation stage, so as to match the carbon source release rate with the metabolic needs of the attached bacteria.

[0018] The degradation reaction of pathogenic AHL signaling molecules by AHL lactoneases can be described by the Michaelis-Menten equation: ; In the formula, This indicates the rate of degradation of AHL signaling molecules by AHL lactonease; This indicates the reaction rate of the enzyme under saturated substrate concentration conditions; This indicates the real-time concentration of AHL signaling molecules in the water or intestinal local environment; represents the Michaelis constant, reflecting the enzyme's affinity for AHL substrates. This relationship guides the setting of biofilm loading density, ensuring that the enzyme activity provided by the actual biofilm cell density is sufficient to complete effective degradation before pathogens reach the quorum sensing threshold concentration.

[0019] The dosage of biofilm microspheres in the breeding water can be calculated according to the following ratio: ; In the formula, This indicates the total mass of the biofilm microspheres required for a single application; This indicates the set target effective concentration in the water body, which is the effective loading of microspheres required per unit volume of water. This indicates the total volume of water in the breeding pond; This indicates the effective biofilm loading concentration per unit mass of the microsphere product. Taking the aforementioned incubation tank with a water volume of ten cubic meters as an example, if the target effective concentration of the water is set at five grams per cubic meter, and the effective biofilm loading concentration per unit mass of the microsphere product is one unit loading per gram, then according to the above formula, the total mass of the biofilm microsphere product required for a single deployment is fifty grams.

[0020] VI. Application Effect Examples The biofilm microspheres obtained in the above embodiments were applied to a mandarin fish fry hatching and breeding experiment group according to the aforementioned application method. A control group without any probiotic preparations and a conventional control group with the same amount of free Bacillus bacterial solution were also set up. Hatching conditions, water temperature, and feeding management were kept consistent across all three groups. Survival rates from hatching to stocking showed that the survival rate of the control group without any preparations was approximately 42%, the survival rate of the conventional control group with free bacterial solution was approximately 49%, while the survival rate of the experimental group with the biofilm microspheres of this invention increased to approximately 82%. Simultaneously, the concentration of AHL signaling molecules and the abundance of pathogenic Vibrio in the water of the experimental group were significantly lower than in the two control groups, and the abundance of target probiotics in the fry intestines was significantly higher than in the free bacterial solution control group. This indicates that the biofilm microspheres provided by this invention can simultaneously achieve water purification and intestinal colonization, blocking the pathogenicity of pathogenic bacteria at the signal communication level, thereby significantly improving the survival rate of fry from hatching to stocking.

[0021] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for breeding aquatic fish fry with high survival rate, characterized in that, Includes the following steps: A porous sustained-release microsphere carrier was prepared. The porous microsphere carrier was made of polyhydroxyalkanoate, had an internal interconnected pore structure, and had a suspension density that was the same as that of water. Bacillus bacteria with AHL lactone secretion ability are loaded on the surface and internal pores of the porous microsphere carrier, so that the Bacillus bacteria proliferate on the inner and outer surfaces of the porous microsphere carrier and secrete extracellular polymer matrix to form a continuous biofilm structure covering the inner and outer surfaces of the porous microsphere carrier, thus obtaining biofilm microspheres. The biofilm microspheres are placed in the aquatic fish fry breeding water, so that the biofilm microspheres are suspended in the breeding water. The AHL lactonease secreted by the biofilm degrades the AHL signaling molecules secreted by pathogenic bacteria in the breeding water. The particle size of the biofilm microspheres is matched with the mouth size of the fish fry during the initial feeding stage, so that the biofilm microspheres are ingested by the fish fry during the initial feeding stage and enter the intestines. The microspheres adhere to and release the loaded Bacillus bacteria in the fish fry's intestines, forming a biofilm barrier.

2. The method according to claim 1, characterized in that, The steps for preparing the porous sustained-release microsphere carrier include: Polyhydroxy fatty acid esters are dissolved in an organic solvent to form an oil phase, and an emulsifier is added to the oil phase; The pore-forming agent is dissolved in water to form an internal aqueous phase; The aqueous phase is added to the oil phase to form a primary water-in-oil emulsion; The primary water-in-oil emulsion is added to an external aqueous phase containing an external emulsifier to form a water-in-oil-in-water double emulsion system. The organic solvent in the complex emulsion system evaporates, and the polyhydroxy fatty acid ester solidifies to form a spherical shell structure. At the same time, the porogen in the inner aqueous phase decomposes to produce gas, forming interconnected channels inside the spherical shell, thus obtaining the porous sustained-release microsphere carrier.

3. The method according to claim 2, characterized in that, The pore-forming agent is ammonium bicarbonate.

4. The method according to claim 1, characterized in that, The steps for preparing the porous sustained-release microsphere carrier include: using a microfluidic chip to control the flow rate ratio of the dispersed phase to the continuous phase, forming monodisperse emulsion droplets at the channel intersection structure, adding the monodisperse emulsion droplets to an external aqueous phase containing an external emulsifier to form a complex emulsion system, and then subjecting the process to solvent evaporation, solidification, and pore formation treatment to obtain the porous sustained-release microsphere carrier.

5. The method according to claim 1, characterized in that, The step of loading Bacillus species with AHL lactonease secretion ability onto the surface and internal pores of the porous microsphere carrier includes: The porous microsphere carrier is placed into a Bacillus suspension and infiltrated under stirring conditions, so that the bacterial suspension penetrates into the interior of the porous microsphere carrier through the pores on the surface of the porous microsphere carrier, thereby achieving the attachment of Bacillus to the inner and outer surfaces of the porous microsphere carrier. The attached porous microsphere carrier was placed in a culture system containing a carbon source supplement and statically cultured to allow the attached Bacillus to proliferate and secrete extracellular polymer matrix, forming the continuous biofilm structure.

6. The method according to claim 1, characterized in that, The Bacillus species mentioned is Bacillus subtilis.

7. The method according to claim 1, characterized in that, The steps of deploying the biofilm microspheres into aquatic fish fry breeding water include: The total amount of biofilm microspheres to be deployed is calculated based on the total volume of the breeding water body and the set target effective concentration of the water body. The total amount of biofilm microspheres to be added is rehydrated and activated before being sprinkled into the breeding water.

8. The method according to claim 1, characterized in that, Also includes: The abundance of target bacteria and the concentration of pathogenic signal molecules in the breeding water and the intestines of the fish fry are detected at a set periodic rate, and supplementary release is determined based on the detection results.

9. The method according to claim 1, characterized in that, The particle size of the porous microsphere carrier is controlled by adjusting the concentration of the emulsifier in the external aqueous phase and the stirring speed, and batches with the same suspension density as the water density are selected by density gradient centrifugation.

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