Bacillus velezensis microcapsule slow-release complex microbial inoculant and application thereof

Abstract

The invention provides a bacillus velezensis microcapsule slow-release composite microbial agent. The bacillus velezensis microcapsule slow-release composite microbial agent comprises an effective amount of composite bacillus velezensis viable bacteria and a microcapsule slow-release structure for loading the bacillus velezensis viable bacteria. The bacillus velezensis microcapsule slow-release complex microbial inoculant provided by the invention can inhibit broad-spectrum bacterial pathogens in marine aquaculture, and the four strains generate a synergistic bacteriostatic effect; the microcapsule slow-release structure not only obviously prolongs the survival and release time of thalli in a water body, but also improves the tolerance of the bacterial strain to environmental factors such as salinity, pH, temperature and the like. In addition, the complex microbial inoculant disclosed by the invention can be in various forms such as particles, powder and sustained-release tablets, and can meet the requirements of different breeding scenes.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparations and aquaculture disease prevention and control technology, specifically relating to a Bacillus vesiculosus microcapsule sustained-release compound bacterial agent and its application. Background Technology

[0002] Existing microbial agents used in marine aquaculture have several problems: low survival rate, with common agents easily inactivated in water and having short survival times, making it difficult to maintain effective antibacterial concentrations; inconsistent release of live bacteria, with rapid release and short duration of action, requiring frequent application to effectively inhibit harmful microorganisms; limited antibacterial spectrum, with most existing agents only effective against single or a few pathogens; limited application methods, relying mainly on sprinkling or mixing with feed, making precise dosing difficult; and poor stress resistance, with existing agents easily inactivated when salinity, pH, and temperature change significantly.

[0003] Bacillus belye is a biocontrol strain widely used in agriculture and aquaculture, exhibiting good antagonistic, growth-promoting, and environmental adaptability. In recent years, with the increasing problem of antibiotic resistance due to overuse, microbial preparations have gradually gained attention as a green alternative.

[0004] Therefore, considering the aforementioned advantages of Bacillus belyssus, it is necessary to study the preparation of microbial agents for marine aquaculture based on Bacillus belyssus strains in order to overcome the shortcomings of existing technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this application is to provide a Bacillus berghei microcapsule sustained-release compound microbial agent, wherein the Bacillus berghei microcapsule sustained-release compound microbial agent comprises an effective amount of at least one of the following: Bacillus berghei with accession number CCTCC No: M2025378, Bacillus berghei with accession number CCTCC No: M 2025379, Bacillus berghei with accession number CCTCC No: M 2025380, and Bacillus berghei with accession number CCTCC No: M 2025381, and a microcapsule sustained-release structure for loading the Bacillus berghei live bacteria.

[0006] This application solves the first problem by using a compound bacterial agent of four live Bacillus belyssus bacteria in combination with multiple antagonistic strains to broaden the antibacterial spectrum and enable them to exert a synergistic effect, thereby more effectively inhibiting harmful microorganisms on the surface and inside the body of marine aquatic organisms.

[0007] As some embodiments of this application, the *Bacillus beryl* microcapsule sustained-release compound bacterial agent contains four live bacteria: *Bacillus beryl* with accession number CCTCC No: M 2025378, *Bacillus beryl* with accession number CCTCC No: M 2025379, *Bacillus beryl* with accession number CCTCC No: M 2025380, and *Bacillus beryl* with accession number CCTCC No: M 2025381; the live bacteria content of *Bacillus beryl* with accession number CCTCC No: M2025378 is ≥1×10⁻⁶. 8 The viable count of *Bacillus belyssus* with accession number CCTCC No: M2025379 was ≥1×10⁻⁶ CFU / mL. 8 The viable count of *Bacillus belyssus* with accession number CCTCC No: M2025380 was ≥1×10⁻⁶ CFU / mL. 8 The viable count of *Bacillus belyssus* with accession number CCTCC No: M2025381 was ≥1×10⁻⁶ CFU / mL. 8 CFU / mL

[0008] The viable count of *Bacillus belyssus* microcapsule sustained-release compound bacterial agent with accession number CCTCC No: M 2025378 in the aforementioned agent can be 1×10⁻⁶. 8 CFU / mL, 2×10 8 CFU / mL, 3×10 8 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL, 6×10 8 CFU / mL, 7×10 8 CFU / mL, 8×10 8 CFU / mL, 9×10 8 CFU / mL, 1×10 9 CFU / mL, 2×10 9 CFU / mL, 3×10 9 CFU / mL, 4×10 9 CFU / mL, 5×10 9 CFU / mL, 6×10 9 CFU / mL, 7×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL, 1×10 10 CFU / mL, 2×10 10 CFU / mL, 3×10 10CFU / mL, 4×10 10 CFU / mL, 5×10 10 CFU / mL, 6×10 10 CFU / mL, 7×10 10 CFU / mL, 8×10 10 CFU / mL, 9×10 10 CFU / mL, 1×10 11 CFU / mL, 2×10 11 CFU / mL, 3×10 11 CFU / mL, 4×10 11 CFU / mL, 5×10 11 CFU / mL, 6×10 11 CFU / mL, 7×10 11 CFU / mL, 8×10 11 CFU / mL, 9×10 11 CFU / mL, 1×10 12 CFU / mL, 2×10 12 CFU / mL, 3×10 12 CFU / mL, 4×10 12 CFU / mL, 5×10 12 CFU / mL, 6×10 12 CFU / mL, 7×10 12 CFU / mL, 8×10 12 CFU / mL, 9×10 12 CFU / mL, 1×10 13 CFU / mL, 2×10 13 CFU / mL, 3×10 13 CFU / mL, 4×10 13 CFU / mL, 5×10 13 CFU / mL, 6×10 13 CFU / mL, 7×10 13 CFU / mL, 8×10 13 CFU / mL, 9×10 13 CFU / mL, 1×10 14 CFU / mL, 2×10 14 CFU / mL, 3×10 14 CFU / mL, 4×10 14 CFU / mL, 5×10 14 CFU / mL, 6×10 14 CFU / mL, 7×10 14 CFU / mL, 8×10 14 CFU / mL, 9×1014 CFU / mL, 1×10 15 CFU / mL

[0009] As some embodiments of this application, the viable content of Bacillus belyceae with accession number CCTCC No: M 2025378 is 1 × 10⁻⁶ per milliliter. 8 ~1×10 10 The viable count of *Bacillus belyssus* CFU with accession number CCTCC No: M 2025379 was 1 × 10⁻⁶ cells / mL. 8 ~1×10 10 The viable count of *Bacillus belyssus* CFU with accession number CCTCC No: M2025380 was 1 × 10⁻⁶ cells / mL. 8 ~1×10 10 The viable count of *Bacillus belyssus* CFU with accession number CCTCC No: M2025381 was 1 × 10⁻⁶ cells / mL. 8 ~1×10 10 CFU.

[0010] As some embodiments of this application, the microcapsule sustained-release structure is made by encapsulating sodium alginate and chitosan.

[0011] The microcapsule structure of this application can effectively shield against adverse external environments and prolong survival time. Simultaneously, the sustained-release design enables continuous release of the bacteria, extending the action period and thus solving the second problem. Furthermore, microcapsule encapsulation technology improves the strain's adaptability to the environment.

[0012] As some embodiments of this application, the Bacillus belyss microcapsule sustained-release compound bacterial agent is prepared by encapsulation through the following steps: Step (1) encapsulating the bacterial suspension of Bacillus belyss with accession number CCTCC No:M 2025378, the bacterial suspension of Bacillus belyss with accession number CCTCC No:M 2025379, the bacterial suspension of Bacillus belyss with accession number CCTCC No:M2025380, and the bacterial suspension of Bacillus belyss with accession number CCTCC No:M After mixing the bacterial suspension of Bacillus beryl of 2025381, a Bacillus beryl complex bacterial suspension was obtained; Step (2) The Bacillus beryl complex bacterial suspension was mixed with sodium alginate at a volume ratio of 1 to 5:1, wherein the sodium alginate solution concentration was 1.0 to 10.0 w / v%; Step (3) CaCl2 solution was added dropwise to the mixture of sodium alginate and Bacillus beryl complex bacterial suspension to form microspheres, wherein the CaCl2 solution concentration was 0.5 to 5.0 w / v%, and the diameter of the formed microspheres was 0.5 to 5 mm; Step (4) The microspheres were immersed in chitosan solution to form a coating, wherein the chitosan solution concentration was 0.1 to 5 w / v%; Step (5) The coated Bacillus beryl bacterial agent was dried to obtain the Bacillus beryl microcapsule sustained-release complex bacterial agent.

[0013] As some embodiments of this application, in step (1) of the preparation of the Bacillus vesiculosus microcapsule sustained-release compound bacterial agent, the bacterial suspensions of Bacillus vesiculosus with accession number CCTCC No: M 2025378, Bacillus vesiculosus with accession number CCTCC No: M 2025379, Bacillus vesiculosus with accession number CCTCC No: M 2025380, and Bacillus vesiculosus with accession number CCTCC No: M2025381 are mixed in a ratio of 2-5:0.1-3:0.5-4:0.5-3 to obtain the Bacillus vesiculosus compound bacterial suspension.

[0014] The *Bacillus vesiculosus* complex bacterial suspension and sodium alginate were selected by volume ratios of 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2... .8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1.

[0015] The sodium alginate solution concentrations were selected from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, and 5.4. ,5.5,5.6,5.7,5.8,5.9,6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7. 8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0w / v%.

[0016] The CaCl2 solution concentration is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0 w / v%.

[0017] The diameters of the formed microspheres are selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0 mm.

[0018] The chitosan solution concentration was selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 w / v%.

[0019] As some embodiments of this application, the concentration of sodium alginate solution in step (2) is 3.0 w / v, and the concentration of CaCl2 solution in step (3) is 1.5 w / v.

[0020] As some embodiments of this application, the composite bacterial agent microspheres forming a chitosan coating are further reinforced with a sodium alginate solution, wherein the concentration of the sodium alginate solution used for reinforcement is 0.01–0.5 w / v.

[0021] The concentration of the reinforced sodium alginate solution was selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0 .38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5w / v%.

[0022] As some embodiments of this application, the dosage forms of the Bacillus vesiculosus microcapsule sustained-release compound microbial agent include powder, granules and sustained-release tablets.

[0023] The Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application can be made into granules, tablets or powder, and is suitable for various administration / feeding methods.

[0024] This application also provides the application of the aforementioned Bacillus vesiculosus microcapsule sustained-release compound microbial agent in the preparation of microbial agents, wherein the microbial agent has any of the following functions: a. inhibiting harmful microorganisms; b. reducing the incidence of diseases in marine aquaculture; c. prolonging survival time and prolonging the antibacterial time; d. adapting to salinity changes; e. adapting to pH changes; f. adapting to temperature changes.

[0025] As some embodiments of this application, the harmful microorganisms include at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Flavobacterium columnare, and Streptococcus.

[0026] This application also provides a method for improving the marine aquaculture environment using the aforementioned Bacillus vesiculosus microcapsule slow-release compound bacterial agent, the method comprising applying the aforementioned Bacillus vesiculosus microcapsule slow-release compound bacterial agent to the marine aquaculture water.

[0027] As described above, the Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application and its application have the following beneficial effects:

[0028] 1. The Bacillus velezensis microcapsule sustained-release compound bacterial agent of this application is prepared using multiple strains of Bacillus velezensis. It can be used to inhibit common bacterial pathogens in marine aquaculture (such as Vibrio, Streptococcus, etc.) and has the advantages of broad-spectrum antibacterial, environmental friendliness and strong sustainability.

[0029] 2. The compound bacterial agent composed of four strains of Bacillus belye in this application has a synergistic inhibitory effect on pathogens and has a synergistic inhibitory effect on a variety of pathogens.

[0030] 3. The Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application adopts a microcapsule sustained-release structure design, namely calcium alginate-chitosan composite encapsulation technology, which significantly prolongs the survival and release time of the bacteria in water.

[0031] 4. The Bacillus belyss microcapsule sustained-release compound bacterial agent of this application adopts an environmental adaptability enhancement design: through microcapsule material screening and process optimization, the strain's tolerance to environmental factors such as salinity, pH, temperature, and resistance to ocean waves is improved.

[0032] 5. The Bacillus vesiculosus microcapsule sustained-release compound microbial agent of this application has multiple application forms: granules, powders, sustained-release tablets and other forms are developed to meet the needs of different breeding scenarios. Detailed Implementation

[0033] To make the technical means, creative features, achieved objectives, and effects of this application readily understandable, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, and test strains used in the following examples are commercially available.

[0035] Example 1: Preparation of Bacillus belyss microcapsule sustained-release compound bacterial agent according to this application

[0036] 1.1 Isolation and Identification of Bacillus belyssus strains

[0037] Water samples were collected from the aquaculture area. The water samples were serially diluted 10-fold six times with sterile physiological saline. Using a pipette, 100 μL of each dilution was transferred to an LB agar plate, spread evenly with a spreader, numbered, and three replicates were performed. After even spreading, the plates were placed in a laminar flow hood for 5–10 minutes to allow for full absorption of the bacterial culture on the surface. Finally, the plates were inverted and incubated at 30°C for 24 hours. Colonies of different morphologies were selected and inoculated onto LB agar plates for isolation and purification.

[0038] The antibacterial properties of isolated and purified bacteria were determined using the Oxford cup plate inhibition method. After extensive isolation and screening, four strains were obtained that showed good inhibitory effects against pathogenic bacteria such as Vibrio parahaemolyticus, Vibrio alginolyticus, Flavobacterium columnare, and Streptococcus. The four microorganisms were all Gram-positive, with short rod-shaped, singly arranged, and non-motile colonies; colonies were 3–4 mm in size, round, raised, milky white, relatively viscous, with neat edges and a raised, wrinkled surface.

[0039] Pure cultures of the four microbial strains were inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm for 24 h. The bacterial cells were then collected by centrifugation. Total DNA was extracted from each of the four microbial strains using a centrifuge column-type bacterial genomic DNA extraction kit. The 16S rRNA gene of the strains was amplified using universal primers. Homology analysis of the 16S rRNA sequence confirmed that all four microbial strains were *Bacillus velezensis*. *Bacillus velezensis* is a Gram-positive, strictly aerobic bacterium. After culturing in LB medium at 30°C for 48 h, the colonies formed were milky white, translucent, with neat edges and a raised, wrinkled surface.

[0040] The four microbial strains in this application are named Bacillus belyssus LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4, respectively, and are submitted for deposit.

[0041] The four Bacillus velezensis strains collected in this application are classified and named as Bacillus velezensis LTS-AF1, Bacillus velezensis LTS-AF2, Bacillus velezensis LTS-AF3, and Bacillus velezensis LTS-AF4, respectively. They are all deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC No:M 2025378, CCTCC No:M 2025379, CCTCC No:M 2025380, and CCTCC No:M 2025381, respectively, and the deposit date is March 5, 2025.

[0042] 1.2 Preparation of Bacillus belyssus complex bacterial solution

[0043] Bacillus belyssus strains with accession numbers CCTCC No:M 2025378, CCTCC No:M 2025379, CCTCC No:M2025380, and CCTCC No:M 2025381 were inoculated onto LB slant agar and activated at 37°C for 24 hours. The bacterial growth was scraped off and inoculated into sterile water, then shaken to prepare a bacterial suspension (approximately 10 μL). 8 CFU / ml); inoculate 2% (V / V) into a 500ml Erlenmeyer flask containing 100ml LB medium, and culture at 37℃ and 180r / min for 48h with shaking, resulting in a spore rate of over 95%.

[0044] The Bacillus beryl bacterial suspensions described in CCTCC No:M 2025378, CCTCC No:M 2025379, CCTCC No:M2025380, and CCTCC No:M 2025381 were mixed in a ratio of 2:1:1:1 to obtain the Bacillus beryl composite bacterial suspension.

[0045] 1.3 Determination of the antibacterial function of Bacillus belyssus

[0046] Four strains of Bacillus berleis with antibacterial function and pathogenic bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas vesiculosus, Vibrio harveyi, and Fusarium moniliforme were inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm for 24 h. The cultures were then resuspended in sterile physiological saline for counting. The final concentrations of the four Bacillus berleis bacterial suspensions, the Bacillus berleisis compound suspension (LTS-AF1:LTS-AF2:LTS-AF3:LTS-AF4 in a ratio of 2:1:1:1), and the pathogenic bacterial suspensions were all 1×10⁻⁶.6 CFU / ml; 100 μl of pathogenic bacterial suspension was spread onto different LB agar plates and incubated for 30 min in a sterile environment to allow for full absorption of the bacterial suspension from the surface of the culture medium. Two sterile Oxford cups (6 mm inner diameter) were placed on the pathogenic bacterial plates. One Oxford cup contained 50 μl of Bacillus belysse bacterial suspension, and the other contained 50 μl of PBS as a blank control. After 24 h of incubation, the diameter of the inhibition zone was measured. The inhibition zones of the four Bacillus belysse strains are shown in Table 1, while no inhibition zone was observed on the PBS blank control.

[0047] Table 1

[0048]

[0049] As shown in Table 1, Bacillus belyss LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 all exhibited good inhibitory effects against Escherichia coli, Aeromonas hydrophila, Aeromonas vesiculosus, Vibrio harveyi, and Fusarium oxysporum. The Bacillus belysss composite bacterial solution showed even better inhibitory effects against various pathogenic microorganisms when the viable count was comparable to that of a single-species solution, indicating that the composite bacterial solution produced a synergistic inhibitory effect against pathogenic microorganisms.

[0050] 1.4 Microcapsule Encapsulation Process

[0051] (1) Pretreatment of embedding materials

[0052] Sodium alginate solution: 3.0 wt% (i.e., 30 g / L), sterilized at 70°C for 15 minutes, cooled to 25°C for later use; Chitosan solution: 0.8–1.0 wt% (dissolved in 1% acetic acid, pH adjusted to 5.8 with NaOH); Calcium chloride solution: 1.5 wt% (15 g / L), sterilized at 121°C for 20 minutes;

[0053] (2) Microcapsule preparation process

[0054] The composite bacterial suspension was mixed with 3.0% sodium alginate solution at a volume ratio of 2.5:1 (bacterial suspension: sodium alginate), and magnetically stirred for 20 minutes until homogeneous. The mixture was then dropped into 1.5% calcium chloride solution from a height of 20 cm using a syringe (needle diameter 0.8 mm), and crosslinked at room temperature for 20 minutes to form gel microspheres with a diameter of 1.5–2.0 mm. The microspheres were removed, washed three times with sterile water, and then immersed in 0.9% chitosan solution and shaken for 10 minutes.

[0055] Secondary curing: Immerse in 0.1% sodium alginate solution for 5 minutes (to enhance the density of the outer layer);

[0056] (3) Drying and Formulation

[0057] Freeze-drying: The microspheres were pre-frozen at -80℃ for 4 hours and then freeze-dried for 24 hours (cold trap temperature -50℃, vacuum degree 0.1mbar) to obtain loose and porous microcapsule particles.

[0058] Spray drying: inlet temperature 120℃, outlet temperature 55℃, feed rate 5ml / min, to obtain microspheres with a particle size of 80~150μm;

[0059] Final product form:

[0060] Granules: Microcapsule granules are mixed with sodium carboxymethyl cellulose (2%) and diatomaceous earth (5%) and granulated.

[0061] Sustained-release tablets: Microcapsule powder + microcrystalline cellulose (30%) + hydroxypropyl methylcellulose (3%) compressed into tablets.

[0062] Example 2: Determination of the antibacterial effect of the Bacillus vesiculosus compound bacterial solution of this application.

[0063] Four pathogenic bacteria—Vibrio parahaemolyticus, Vibrio alginolyticus, Flavobacterium columnare, and Streptococcus—were inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm for 24 h. The cultures were then resuspended in sterile physiological saline for counting. The Bacillus bellis complex culture was prepared according to the method described in 1.2 of Example 1. The final concentration of both the four pathogenic bacteria and the Bacillus bellis complex culture was 1 × 10⁻⁶. 8 CFU / ml; 100 μl of pathogenic bacterial suspension was spread onto different LB agar plates and incubated for 30 min in a sterile environment to allow the bacterial suspension on the surface of the culture medium to be fully absorbed. Two sterile Oxford cups (6 mm inner diameter) were placed on the pathogenic bacterial plates. 50 μl of Bacillus belysse bacterial suspension was added to one Oxford cup, and 50 μl of PBS was added to the other as a control. After 24 h of incubation, the diameter of the inhibition zone was measured.

[0064] Experimental results showed that the Bacillus vesiculosus compound bacterial solution used in this application exhibited an inhibition zone diameter of 32 mm against Vibrio parahaemolyticus, 30 mm against Vibrio alginolyticus, 28 mm against Flavobacterium columnare, and 33 mm against Streptococcus; no inhibition zone was observed in the blank control group. Therefore, the Bacillus vesiculosus compound bacterial solution used in this application has a significant inhibitory effect on common pathogens in seawater aquaculture.

[0065] Example 3: Comparison of the antibacterial effects of the Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application with traditional bacterial agents.

[0066] Take equal volumes of aquaculture water containing equal amounts of Vibrio parahaemolyticus (concentration 1.5 × 10⁻⁶). 5CFU / ml), the same concentration of the bacterial agent of this application and the traditional bacterial agent were added respectively, and a blank control group (with an equal volume of sterile water added) was set up; each group was placed under the same culture conditions, and after culturing for 24 hours, the number of Vibrio parahaemolyticus in the water of each group was determined by plate counting method, and the inhibition rate was calculated (inhibition rate = (number of pathogens in the control group - number of pathogens in the treatment group) / number of pathogens in the control group × 100%).

[0067] The experimental results showed that the number of Vibrio parahaemolyticus in the blank control group was 1.2 × 10⁻⁶. 5 CFU / ml, the traditional bacterial agent treatment group was 4.8 × 10⁻⁶. 4 CFU / ml, with an inhibition rate of 68%; the bacterial agent treatment group in this application is 2.0×10⁻⁶. 4 The bacterial agent has a concentration of CFU / ml and an inhibition rate of 86.7%. The antibacterial efficiency of this application is 18.7% higher than that of traditional bacterial agents.

[0068] Example 4: Survival time and release cycle of the Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application.

[0069] The bacterial agent of this application and the traditional bacterial agent were respectively inoculated into artificially prepared seawater (salinity 3.0%), so that the initial concentration of both was 1×10⁻⁶. 7 The inoculum was incubated at 25°C for CFU / ml. Samples were taken at 1, 3, 5, 7, 10, 15, and 20 days after incubation, and the viable count was determined using the plate count method. The results showed that the viable count of the traditional inoculum decreased to 10 after 3-5 days of incubation. 3 CFU / ml below; the bacterial agent of this application still had a survival rate of 5×10⁻⁶ after 15 days of incubation. 5 CFU / ml, 2×10⁻⁶ after 20 days of culture. 4 With a concentration of CFU / ml, the survival time can reach 15-20 days, which is 3-4 times longer than that of traditional bacterial agents.

[0070] The compound bacterial agent of this application was placed in a container containing 1000 ml of seawater, with an initial concentration of 5 × 10⁻⁶. 7 CFU / ml, incubated at 25℃. Daily samples were taken to determine the number of released bacteria in the water, and the antibacterial effect against *Vibrio parahaemolyticus* was measured using the agar diffusion method described above, until the inhibition zone diameter was less than 10 mm. From day 1 to 14, the daily bacterial release was approximately 1×10⁻⁶. 6 -1×10 7 Between CFU / ml, with 8×10 on day 1. 6 CFU / ml, 5×10 on day 7. 6 CFU / ml, 3×10 on day 14. 6CFU / ml; the diameter of the inhibition zone against Vibrio parahaemolyticus was greater than 10 mm from day 1 to day 14, and the diameter of the inhibition zone was 8 mm on day 15, indicating that the inhibition could be sustained for 10-14 days.

[0071] Example 5: Environmental adaptability evaluation of the Bacillus vesiculosus microcapsule sustained-release compound bacterial agent of this application.

[0072] Salinity adaptability

[0073] Seawater solutions with salinities of 0.5%, 1.5%, 2.5%, and 3.5% were prepared, and the compound bacterial agent of this application was inoculated into them at an initial concentration of 1×10⁻⁶. 7 CFU / ml, three replicates per group, incubated at 25℃ for 24 h. The number of viable bacteria was determined by plate counting after incubation. Results showed that the number of viable bacteria was consistently above 1×10⁻⁶ at all salinity levels. 6 CFU / ml or higher, with a salinity of 0.5% being 7 × 10⁻⁶. 6 CFU / ml, 5×10 at a salinity of 3.5%. 6 CFU / ml indicates that it can survive normally in a salinity range of 0.5%-3.5%.

[0074] pH adaptability

[0075] The seawater pH was adjusted to 6.0, 7.0, 8.0, and 9.0, respectively, and then inoculated with the bacterial agent of this application at an initial concentration of 1×10⁻⁶. 7 CFU / ml, three replicates per group, incubated at 25℃ for 24 h. The number of surviving cells was counted after incubation. Results showed that the number of surviving cells exceeded 1×10⁻⁶ under all pH conditions. 6 CFU / ml, 6×10 at pH 6.0 6 CFU / ml, 4×10 at pH 9.0 6 CFU / ml indicates that it can survive normally within the pH range of 6.0-9.0.

[0076] Temperature resistance

[0077] High-temperature test: The bacterial agent of this application was placed in a 50℃ water bath for 30 minutes, while the control group was treated at 25℃. Each group was repeated three times. After treatment, the survival rate was measured (survival rate = number of surviving cells in the treatment group / number of surviving cells in the control group × 100%). The results showed that after treatment at 50℃, the survival rate of the bacterial agent was 88%, which is greater than 85%.

[0078] Low-temperature test: The bacterial agent of this application was placed in an environment of 10℃ and cultured for 7 days. The bacterial activity was observed regularly, and dormancy was determined by measuring the content of organic acids produced by bacterial metabolism (the production of organic acids is extremely low in the dormant state). The results showed that the production of organic acids remained stable at a certain level within 7 days, indicating that the bacteria did not become dormant at a low temperature of 10℃.

[0079] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any way. Although this application has disclosed the preferred embodiment as above, it is not intended to limit this application. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above to create equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of this application without departing from the content of the technical solution of this application shall still fall within the scope of the technical solution of this application.

Claims

1. A Bacillus velezensis microcapsule slow-release complex microbial inoculant, characterized in that, The Bacillus velezensis microcapsule sustained-release composite microbial agent comprises at least one of the live Bacillus velezensis with a preservation number of CCTCC No: M 2025378, the live Bacillus velezensis with a preservation number of CCTCC No: M 2025379, the live Bacillus velezensis with a preservation number of CCTCC No: M 2025380, and the live Bacillus velezensis with a preservation number of CCTCC No: M 2025381, and a microcapsule sustained-release structure for loading the live Bacillus velezensis.

2. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 1, characterized in that, The Bacillus velezensis microcapsule sustained-release composite microbial agent comprises the four live Bacillus velezensis with a preservation number of CCTCC No: M 2025378, a preservation number of CCTCC No: M 2025379, a preservation number of CCTCC No: M 2025380, and a preservation number of CCTCC No: M 2025381. The viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025378 is ≥ 1 x 10 8 CFU / mL, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025379 is ≥ 1 x 10 8 CFU / mL, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025380 is ≥ 1 x 10 8 CFU / mL, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025381 is ≥ 1 x 10 8 CFU / mL.

3. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 2, characterized in that, The viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025378 is 1 x 10 8 ~1 x 10 10 CFU per milliliter, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025379 is 1 x 10 8 ~1 x 10 10 CFU per milliliter, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025380 is 1 x 10 8 ~1 x 10 10 CFU per milliliter, the viable cell content of Bacillus velezensis with the preservation number of CCTCC No: M 2025381 is 1 x 10 8 ~1 x 10 10 CFU per milliliter.

4. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 1, characterized in that, The microcapsule sustained-release structure is made of sodium alginate and chitosan complex embedding.

5. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 4, characterized in that, The Bacillus velezensis microcapsule sustained-release composite microbial agent is made by embedding the following steps: Step (1) mixing the bacterial liquid of the Bacillus velezensis with a preservation number of CCTCC No: M 2025378, the bacterial liquid of the Bacillus velezensis with a preservation number of CCTCC No: M 2025379, the bacterial liquid of the Bacillus velezensis with a preservation number of CCTCC No: M 2025380, and the bacterial liquid of the Bacillus velezensis with a preservation number of CCTCC No: M 2025381 to obtain a Bacillus velezensis composite bacterial suspension; Step (2) mixing the Bacillus velezensis composite bacterial suspension with sodium alginate at a volume ratio of 1-5:1, wherein the concentration of the sodium alginate solution is 1.0-10.0 w / v%; Step (3) dropping CaCl2 solution into the mixture of the sodium alginate and the Bacillus velezensis bacterial suspension to form microspheres, wherein the concentration of the CaCl2 solution is 0.5-5.0 w / v%, and the diameter of the formed microspheres is 0.5-5 mm; Step (4) soaking the microspheres in a chitosan solution to form a coating, wherein the concentration of the chitosan solution is 0.1-5 w / v%; Step (5) drying the coated Bacillus velezensis microbial agent to obtain the Bacillus velezensis microcapsule sustained-release composite microbial agent.

6. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 5, characterized in that, In step (2), the concentration of the sodium alginate solution is 3.0 w / v%, and in step (3), the concentration of the CaCl2 solution is 1.5 w / v%; The chitosan-coated composite microbial agent microspheres are reinforced again using a sodium alginate solution, and the concentration of the reinforced sodium alginate solution is 0.01-0.5 w / v%.

7. The Bacillus velezensis microcapsule slow-release compound microbial inoculant according to claim 1, characterized in that, The dosage form of the Bacillus velezensis microcapsule sustained-release composite microbial agent includes powder, granules, and sustained-release tablets.

8. Use of the Bacillus velezensis microcapsule slow-release compound microbial inoculant according to any one of claims 1 to 7 in the preparation of a microbial inoculant, wherein, The microbial agent has any of the following functions: a. inhibiting a broad-spectrum of harmful microorganisms in seawater; b. reducing the incidence of diseases in seawater aquaculture organisms; c. a function of prolonging survival time and prolonging bacteriostatic time; d. a function of salinity change adaptability; e. a function of pH change adaptability; f. a function of temperature change adaptability.

9. Use according to claim 8, characterized in that, The harmful microorganism includes at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Flavobacterium columnare and Streptococcus.

10. A method for improving the environment of seawater aquaculture by using the Bacillus velezensis microcapsule slow-release compound microbial inoculant according to any one of claims 1-7, characterized in that, The method comprises applying the Bacillus velezensis microcapsule slow-release complex microbial agent to a seawater aquaculture water body.

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