Bacillus velezensis, culture and application of bacillus velezensis in prevention or treatment of aquatic animal diseases

Abstract

The invention discloses bacillus velezensis, a culture and application of the bacillus velezensis to prevention or treatment of aquatic animal diseases, and relates to the cross technical field of biotechnology and aquatic disease prevention and control. The preservation number of the bacillus velezensis is CCTCC (China Center For Type Culture Collection) NO: M 20252822, and the bacillus velezensis is classified and named as bacillus velezensis LB7. The bacillus velezensis has broad-spectrum antibacterial activity and can destroy biofilms of pathogenic bacteria. Through culture medium optimization, the bactericidal activity of bacillus velezensis can be further improved. The culture supernatant of the bacillus velezensis LB7 is mixed with feed to feed grass carps infected with flavobacterium clostridium, so that the survival rate of the grass carps can be remarkably increased, and a treatment effect is achieved. The fermentation product provided by the invention has remarkable sterilization and bacteriostasis effects, and can be used as a synergist of antibiotics florfenicol and enrofloxacin, so that the investment of the two antibiotics in aquaculture is reduced.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biotechnology and aquatic disease control, specifically to a Bacillus belye, its culture, and its application in the prevention or treatment of aquatic animal diseases. Background Technology

[0002] grass carp( Ctenopharyngodon idella Grass carp, as one of the most important aquaculture species in China, accounts for 21.9% of the total freshwater fishery output annually (Qi et al., 2024; Wu et al., 2024). However, the continuous increase in stocking density has led to frequent outbreaks of diseases (Liu et al., 2022; Luo et al., 2024). The most common bacterial diseases in grass carp farming include those caused by Flavobacterium columnare (…). Flavobacterium columnare Columnar disease caused by Aeromonas versicolor and by Aeromonas villiformis ( Aeromonas veronii Aeromonas sepsis caused by (Guo et al., 2024; Ma et al., 2024).

[0003] Currently, antibiotics such as enrofloxacin and florfenicol are widely used for the prevention and treatment of diseases caused by Flavobacterium columnare and Aeromonas vesiculosus (Schrader et al., 2013; Ferreira et al., 2022; Chen et al., 2024). However, repeated use of antibiotics can lead to increased antibiotic resistance in pathogens (Gieseker et al., 2022) and can also disrupt the mucosal flora of fish. Biocontrol is an eco-friendly method of disease control and has been recognized as a sustainable disease management strategy. Studies have shown that probiotics can enhance host immunity and promote growth by regulating the microbial community structure and synthesizing antimicrobial compounds (Hemarajata & Versalovic, 2013; Woldemariamyohannes et al., 2020; Qiu et al., 2025). Bacillus can produce a variety of antimicrobial substances, including lipopeptides, polyketides, and nonribosomal peptides, which have significant inhibitory effects on a variety of aquatic pathogens.

[0004] In the production of probiotics and metabiotics, changes in culture conditions significantly affect their probiotic capacity (Marco & Tachon, 2013; Oana et al., 2023). Therefore, optimizing culture conditions is crucial for improving probiotic yield and activity. Kamoun et al. (2022) successfully synthesized Bacillus amyloliquefaciens (BAM) by optimizing the culture medium composition. Bacillus amyloliquefaciensThe spore yield increased by approximately three times. Currently, Response Surface Methodology (RSM) is widely used as a classic method in culture medium optimization (Orhan et al., 2023). However, this method usually requires screening culture medium components through single-factor experiments, making it difficult to fully consider the optimal combination of components (Yao & Asayama, 2007; Combe & Sokolenko, 2021). Furthermore, RSM has limitations in handling nonlinear complex systems and is no longer sufficient to meet the current needs of culture medium optimization (Parampalli et al., 2007; Singh et al., 2009). In recent years, machine learning (ML) models based on Artificial Intelligence (AI), such as Extreme Gradient Boosting (XGBoost), Random Forest (RF), and Artificial Neural Network (ANN), have proven to be effective strategies for predicting complex culture medium composition and optimizing culture medium parameters (Rabiya and Sen, 2022). Furthermore, the Shapley Additive exPlanations (SHAP) method can be used to interpret predictive models and determine key parameters, and combined with genetic algorithms, it can further optimize culture medium models (Yan et al., 2024).

[0005] The demand for ecological control of aquatic diseases is increasing, and Bacillus is gradually being recognized as a biocontrol probiotic. Developing Bacillus strains with broad-spectrum and highly effective antagonistic effects against aquatic pathogens is of great significance.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a Bacillus belye, its culture, and its application in the prevention or treatment of diseases in aquatic animals to solve the above-mentioned technical problems.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a Bacillus belyesense, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252822, and is classified as follows: Bacillus velezensis LB7, deposited on December 10, 2025, at Wuhan University, Wuhan, China, is identified as viable.

[0009] Secondly, the present invention also provides a culture of Bacillus belesiensis, wherein the culture refers to the fermentation broth, cell-free supernatant, bacterial sludge, extract of fermentation broth, or concentrate, gelatinized product, dried product, diluted product, and broken product of Bacillus belesiensis.

[0010] Thirdly, this aquatic animal invention also provides the application of Bacillus belye or the aforementioned cultures in the prevention or treatment of livestock and poultry diseases.

[0011] Fourthly, the present invention also provides a microbial preparation or feed comprising the above-mentioned Bacillus belye or the above-mentioned culture.

[0012] Fifthly, the present invention also provides a fermentation medium for culturing the above-mentioned Bacillus belye, comprising the following raw materials in the following mass concentrations: 15.5-17.5 g / L sucrose, 9.0-11.0 g / L glucose, 6.5-8.5 g / L tryptone, 0.50-0.70 g / L casein peptone, 0.20-0.40 g / L yeast extract, 9.5-11.5 g / L urea, 0.3-0.5 g / L potassium nitrate, and 0.5-2.5 g / L magnesium chloride.

[0013] In a sixth aspect, the present invention also provides a method for preventing or treating diseases in aquatic animals or livestock, comprising the following steps: feeding the aquatic animals or livestock with the aforementioned microbial preparation or feed.

[0014] The present invention has the following beneficial effects: This invention isolated a potential probiotic strain with broad-spectrum antibacterial activity—Bacillus belyss LB7—from water samples of Poyang Lake. The culture supernatant of this bacterium can antagonize Flavobacterium columnare G4 (… Flavobacterium cloumnare G4), Aeromonas verrucosa ASW ( Aeromonas veronii Multiple aquatic bacterial pathogens, including ASW, were identified. Whole-genome identification analysis revealed that LB7, a potential probiotic with antibacterial activity, is *Bacillus belyssae* (ASW). Bacillus velezensis LB7 contains a genome containing 12 gene clusters of secondary metabolites, including polyketides and lipopeptides.

[0015] Through culture medium optimization, it was found that fermenting *Bacillus vesicularis* in a specific fermentation medium can improve its antibacterial activity to a certain extent. The effective antibacterial components of the cell-free supernatant of *Bacillus vesicularis* possess characteristics such as heat resistance and protease resistance. Freeze-drying the cell-free supernatant of *Bacillus vesicularis* and mixing it into feed can significantly improve the survival rate of infected grass carp. The fermentation product of this invention has significant bactericidal and bacteriostatic effects and can be used as a synergist for the antibiotics florfenicol and enrofloxacin, reducing the input of these two antibiotics in aquaculture. Therefore, the *Bacillus vesicularis* and its culture provided by this invention have good application prospects for the prevention or treatment of diseases in aquatic animals or livestock. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The graphs show the antibacterial effect of Bacillus blazei LB7 (A is the Oxford cup test graph of the antibacterial effect of the potential probiotic bacterial solution and cell-free supernatant, B is the bar graph of the antibacterial effect of the potential probiotic cell-free supernatant, and C is the bactericidal effect of the potential probiotic LB7 cell-free supernatant on Flavobacterium columnare G4 (left) and the antibacterial effect on Aeromonas verrucosa ASW (right). Figure 2 This is a graph showing the average nucleic acid similarity analysis of LB7 based on the whole genome sequence. Figure 3 The graph shows the effect of cell-free supernatant of Bacillus belyss LB7 on the destruction or inhibition of biofilm development of Aeromonas versicolor ASW (A) and Flavobacterium columnare G4 (B) at different stages (6 h represents biofilm still developing, 12 h represents mature biofilm). Figure 4 The following figures illustrate the results of optimizing the culture medium to improve the antibacterial effect of Bacillus belyss LB7 cell-free supernatant against Flavobacterium columnare G4: (A. Prediction accuracy analysis based on the extreme gradient boosting model; B. Iterative calculation results based on the genetic algorithm; C. Antibacterial effect of cell-free supernatant based on the genetic algorithm results; D. Visualization of the importance ranking of each feature obtained from SHAP analysis based on the extreme gradient boosting model; E. Antibacterial effect of cell-free supernatant obtained after simplifying the culture medium composition based on SHAP analysis). Figure 5 To investigate the antibacterial effect of cell-free supernatant of Bacillus belyss LB7 under different treatment conditions on Flavobacterium columnare G4; Figure 6 Figure A shows the experimental results of the therapeutic and protective effect of Bacillus vesiculosus LB7 cell-free supernatant on grass carp infected with Flavobacterium columnare G4. A. Schematic diagram of the experimental procedure; After 14 days of temporary rearing, 2.5 × 10⁻⁶ cells were used... 6 Grass carp were soaked in CFU / mL Flavobacterium columnare G4 for 3 h, and then each group was fed with feed equivalent to 1.5% of their body weight twice a day (9:00 and 18:00) for 10 consecutive days; B. The therapeutic effect of adding Bacillus berleis LB7 cell-free supernatant to the feed on grass carp infected with Flavobacterium columnare G4 was statistically analyzed between each treatment group and the control group (G4 infection + ordinary feed). Detailed Implementation

[0018] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0019] In a first aspect, the present invention provides a Bacillus belyesense, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252822, and is classified as follows: Bacillus velezensis LB7, deposited on December 10, 2025, at Wuhan University, Wuhan, China, is identified as viable.

[0020] Bacillus belyssus isolated from water samples of Poyang Lake, and after whole-genome identification and analysis, the potential probiotic LB7 with antibacterial activity was identified as Bacillus belyssus (LB7). Bacillus velezensis LB7, whose genome contains gene clusters of 12 secondary metabolites, including polyketides and lipopeptides. The culture supernatant of *Bacillus belyssiensis* LB7 exhibits a bactericidal effect against *Flavobacterium columnare* G4 and an inhibitory effect against *Aeromonas vesiculosus* ASW. The culture supernatant of *Bacillus belyssiensis* LB7 can inhibit the development and maturation of biofilms of *Flavobacterium columnare* G4 and *Aeromonas vesiculosus* ASW. Therefore, *Bacillus belyssiensis* is a potential probiotic with broad-spectrum antibacterial activity. Secondly, this invention also provides a culture of *Bacillus belyssiensis*, which refers to the fermentation broth, cell-free supernatant, bacterial sludge, extract of the fermentation broth, or concentrate, gelatinized product, dried product, diluted product, and broken product of *Bacillus belyssiensis*.

[0021] Culture protection is not limited to solid-state or liquid fermentation.

[0022] The dried materials include, but are not limited to, spray-dried materials, freeze-dried materials, vacuum-dried materials, drum-dried materials, etc.

[0023] In a preferred embodiment of the present invention, the cell-free supernatant is obtained by filtration of Bacillus belye bacterial culture.

[0024] Thirdly, the present invention also provides the use of Bacillus belye or the above-mentioned cultures in the prevention or treatment of diseases in aquatic animals or livestock.

[0025] In a preferred embodiment of the present invention, the diseases of aquatic animals or livestock are caused by at least one of the following pathogens: Flavobacterium columnare, Aeromonas verrucosa, Edwardsiella tinctoria, Streptococcus agalactiae, and Vibrio parahaemolyticus.

[0026] In a preferred embodiment of the present invention, the aquatic animal is selected from any one or more of the following: grass carp, mandarin fish, largemouth bass, yellow catfish, catfish, crucian carp, common carp, silver carp, bighead carp, black carp, yellow eel, snakehead, eel, tilapia, redfin pufferfish, yellow pufferfish, and zebrafish.

[0027] Livestock and poultry include, but are not limited to: pigs, cattle, horses, sheep, donkeys, camels, rabbits, deer, dogs, chickens, ducks, geese, turkeys, pigeons, quails, minks, foxes, raccoon dogs, ostriches, and emus.

[0028] In a preferred embodiment of the present invention, the application includes at least one of the following application methods: (1) destroying the biofilm of pathogens; (2) improving the survival rate of aquatic animals.

[0029] Fourthly, the present invention also provides a microbial preparation or feed comprising the above-mentioned Bacillus belye or the above-mentioned culture.

[0030] In a preferred embodiment of the present invention, each 100g of feed contains 10-500ml of dried product of Bacillus belysin liquid or liquid culture; the base feed is commercially available aquatic feed or poultry feed.

[0031] In a preferred embodiment of the present invention, each 100g of feed contains 10-500 ml of dried product of cell-free supernatant of Bacillus belysinus.

[0032] Fifthly, the present invention also provides a fermentation medium for culturing the above-mentioned Bacillus belye, comprising the following raw materials in the following mass concentrations: 15.5-17.5 g / L sucrose, 9.0-11.0 g / L glucose, 6.5-8.5 g / L tryptone, 0.50-0.70 g / L casein peptone, 0.20-0.40 g / L yeast extract, 9.5-11.5 g / L urea, 0.3-0.5 g / L potassium nitrate, and 0.5-2.5 g / L magnesium chloride.

[0033] In one embodiment, the fermentation medium comprises the following raw materials in mass concentrations: 15.5, 15.8, 16, 16.5, 17 or 17.5 g / L of sucrose, 9.0, 9.5, 10, 10.5 or 11.0 g / L of glucose, 6.5, 7, 8 or 8.5 g / L of tryptone, 0.50, 0.60 or 0.70 g / L of casein peptone, 0.20, 0.30 or 0.40 g / L of yeast extract, 9.5-10 or 11-11.5 g / L of urea, 0.3, 0.4 or 0.5 g / L of potassium nitrate, and 0.5-2.5 g / L of magnesium chloride.

[0034] Fermentation of Bacillus belyssus under this culture medium formulation can enhance its antibacterial activity to a certain extent. This culture medium formulation has the technical advantages of low cost and significant antibacterial effect.

[0035] Sixthly, the present invention also provides a method for preventing or treating diseases in aquatic animals or livestock, comprising the following steps: Feeding aquatic organisms or livestock with the above-mentioned microbial preparations or feeds; In a preferred embodiment of the present invention, the aquatic animals or livestock are fed a diet containing Bacillus vesiculosus culture supernatant at a rate of 1.5% to 2% of their body weight, twice a day, once in the morning and once in the evening. For example, the diet containing Bacillus vesiculosus culture supernatant is fed at a rate of 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% of the aquatic animals or livestock's body weight.

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 This embodiment provides an experiment on the isolation and antibacterial effect detection of candidate probiotic strain LB7.

[0039] Water samples were collected from the Piaotou area of ​​Poyang Lake, Yongxiu County, Jiangxi Province in November 2022. After incubation at 80℃ for 20 min, the samples were diluted and spread onto Brain Heart Infusion Broth (BHI) agar plates, and incubated at 28℃ for 24 h. Different single colonies were streaked, purified, and numbered, and then stored at -80℃ with 50% glycerol added. The antibacterial effect of the potential probiotics was detected using the Oxford cup method. Flavobacterium columnare G4 (OD) was used as an example. 600nm =0.3) and Aeromonas vesiculosus ASW (OD 600nm =0.5) was transferred at a 1:10 ratio to Shieh's semi-solid medium and LB semi-solid medium to prepare semi-solid plates with Oxford cups. 150 μL of cell-free supernatant containing potential probiotics (obtained by filtering the probiotic supernatant obtained from culture through a 0.22 μm filter membrane) was added to each well of the Oxford cup, and the plates were incubated at 28°C for 24 h. The size of the inhibition zone was observed for probiotic screening. Strains exhibiting the best antibacterial effects against Flavobacterium columnare G4 and Aeromonas velutipes ASW were selected.

[0040] Results reference Figure 1 As shown in A and B, it can be seen that the bacterial solution and cell-free supernatant of the potential probiotic LB7 exhibit significant antibacterial activity against both aquatic pathogens. Figure 1 SSB5, YAB4, and SAB3 are all strain numbers.

[0041] Subsequently, *Flavobacterium columnare* G4 and *Aeromonas vesiculosus* ASW were inoculated into Shieh's medium and LB medium, respectively, and cultured overnight at 28°C. After transfer to fresh medium, the bacterial culture was transferred to 24-well plates, and each well was treated with different proportions of *Bacillus belyssiensis* LB7 cell-free supernatant, and incubated at 28°C. The absorbance (OD) of each well was measured using a microplate reader at different time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, and 28 h). 600nm ).

[0042] Results reference Figure 1 As shown in C, the cell-free supernatant of Bacillus belyss LB7 can exert a significant bactericidal effect on Flavobacterium columnare G4, and this effect is time-dependent and concentration-dependent. At the same time, it mainly exerts a significant inhibitory effect on Aeromonas verrucosa ASW.

[0043] Example 2 This embodiment provides a molecular identification test for the candidate probiotic strain LB7.

[0044] The genome of LB7 was isolated from the fresh LB7 bacterial culture in Example 1 using a DNA extraction kit (Tiangen Biotech, DP302-02). The whole genome of LB7 was further sequenced and analyzed, and the average nucleotide identity (ANI) analysis based on the whole genome sequencing results was performed.

[0045] Results reference Figure 2 As shown, LB7 has the highest similarity to Bacillus belyssus SQR9, therefore LB7 is classified as Bacillus belyssus. Online analysis using AntiSMASH v7.1.0 revealed that the Bacillus belyssus LB7 genome contains 12 secondary metabolite-related biosynthetic gene clusters, involving surfactantin, bacilysin, bacillibactin, difficidin, fengycin, bacillaene, macrolactin H, and butirosin (Table 1).

[0046] Table 1: Secondary metabolite-related biosynthetic gene clusters in the genome of Bacillus belyss LB7

[0047] Example 3 This embodiment provides an assay to inhibit biofilm formation of *Bacillus belyssae* LB7 cell-free supernatant against *Flavobacterium columnare* G4 and *Aeromonas vesiculosus* ASW. The cell-free supernatant of *Bacillus belyssae* LB7 was obtained by filtering the LB7 culture supernatant through a 0.22 μm filter membrane.

[0048] Overnight cultured Flavobacterium columnare G4 and Aeromonas vera were transferred to fresh Shieh's and LB media at a 1:10 ratio, respectively. Then, 900 µL of the bacterial culture was transferred to 24-well plates containing round glass slides. After static incubation at 28°C for 6 h and 12 h, 100 µL of different concentrations of Bacillus belyssus LB7 cell-free supernatant were added to prepare Bacillus belyssus LB7 cell-free supernatant to total solution volumes of 10%, 5%, 2.5%, 1.25%, and 0.625%. After co-incubation for 4 hours, the bacterial culture was aspirated, and the coverslips were gently rinsed three times with sterile PBS to remove surface airborne bacteria. Then, 400 µL of 4% paraformaldehyde was added to each well, and the plates were fixed overnight at 4 °C. The 4% paraformaldehyde fixative was discarded, and the plates were air-dried. Each well was stained with 400 µL of 0.1% crystal violet for 30 min. The staining solution was discarded, and the plates were washed three times with sterile PBS, air-dried, and photographed. 200 µL of 1% SDS was added to each well to dissolve the crystal violet, and the cell culture plates were placed on a shaker in the dark for 10 min until the crystal violet was completely dissolved. The OD was measured using a microplate reader.630nm Absorbance value.

[0049] Results reference Figure 3 As shown, treatment with cell-free supernatant of Bacillus belye LB7 can inhibit the formation of early-stage (immature) biofilms of Aeromonas versicolor in a concentration-dependent manner, but does not disrupt mature biofilms. Figure 3 (Figure A in the figure); it showed a disruptive effect on both the early and mature biofilm formation of Flavobacterium columnare G4, and the effect was concentration-dependent. Figure 3 (Figure B in the diagram).

[0050] Example 4 This embodiment provides an experiment to improve the antibacterial effect of Bacillus leucis LB7 cell-free supernatant by optimizing the culture medium.

[0051] First, in this embodiment, 70 culture media were prepared according to the following scheme. Each culture medium contains seven common carbon sources (sucrose, glucose, sorbitol, mannitol, arabinose, fructose, and lactose), six common nitrogen sources (tryptone, casein, yeast extract, urea, ammonium chloride, and beef extract), and four common inorganic salts (sodium chloride, potassium nitrate, calcium chloride, and magnesium chloride). Furthermore, the concentrations of the same type of component vary within each medium. There are ten options for carbon and nitrogen source concentrations (0.1 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 20 g / L), and ten options for inorganic salt concentrations (0.01 g / L, 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L). 0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, 2 g / L).

[0052] According to this specification, 70 culture media were randomly generated using R language random functions. Then, using *Flavobacterium columnare* G4 as the pathogen indicator, cell-free supernatants obtained from culturing *Bacillus belyssiensis* LB7 in each culture medium were used for antimicrobial testing using the Oxford cup method. An extreme gradient boosting model was constructed using the concentration of each component of the culture medium as the explanatory variable and the size of the inhibition zone as the response variable. Subsequently, a genetic algorithm (using the "GA" package in R software v4.3.0) was used to optimize the optimal composition of the culture medium. The optimal composition of the culture medium optimized using the genetic algorithm was: sucrose, glucose, sorbitol, mannitol, arabinose, fructose, lactose, tryptone, casein peptone, yeast extract, urea, ammonium chloride, beef extract, sodium chloride, potassium nitrate, calcium chloride, and magnesium chloride. The diameter of the inhibition zone in the culture supernatant obtained from culturing LB7 in the optimized medium was 21.78 mm (8 mm excluding the Oxford cup diameter).

[0053] Based on this, the culture conditions were optimized to be adequately oxygenated. Then, the optimal culture medium components were reintroduced into the model, and the Shapley algorithm (using the "iml" package in R software v4.3.0) was used to analyze the contribution of each component in the optimal culture medium. The components with the lowest contribution were gradually reduced to further simplify the culture medium composition while maintaining antibacterial activity.

[0054] After relieving oxygen restriction using a breathable membrane, the inhibition zone size was increased to 30.17 mm ± 0.23 mm (8 mm diameter excluding the Oxford cup). During LB culture, the inhibition zone diameter was 7.33 ± 0.47 mm (8 mm diameter excluding the Oxford cup). Furthermore, SHAP analysis was used to gradually reduce the least contributing culture medium components while maintaining antimicrobial activity, further simplifying the culture medium composition. Finally, the culture medium composition was determined to be: sucrose, glucose, tryptone, casein peptone, yeast extract, urea, potassium nitrate, and magnesium chloride, with the inhibition zone size maintained at 25.83 ± 0.23 mm (8 mm diameter excluding the Oxford cup). During LB culture, the inhibition zone diameter was 7.50 ± 0.41 mm (8 mm diameter excluding the Oxford cup).

[0055] Results reference Figure 4 As shown, the coefficient of determination (R²) of the established extreme gradient boosting model is... 2 The mean squared error (MSE) was 0.002524, indicating that the model can make accurate predictions. After optimization using a genetic algorithm with this model, the antibacterial effect of cell-free supernatant against Flavobacterium columnare G4 was improved to 21.78 mm (8 mm diameter excluding Oxford cups). Further improvement was achieved by using a breathable membrane to relieve oxygen limitation, increasing the antibacterial effect to 30.17 ± 0.23 mm (8 mm diameter excluding Oxford cups). Finally, shaply analysis was used to further simplify the culture medium composition, ultimately stabilizing the antibacterial activity of the cell-free supernatant at 25.83 ± 0.23 mm (8 mm diameter excluding Oxford cups).

[0056] Example 5 This embodiment provides a stability test for cell-free supernatant of Bacillus leucis LB7.

[0057] The cell-free supernatant of Bacillus belyss LB7 obtained using optimized culture medium was treated using the following steps: (1) in water baths at 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 30 min; (2) treated with 1.0 mg / mL proteinase K or trypsin for 30 min; (3) treated with different pH values ​​(1.82, 4.88, 9.50, and 12.80) adjusted with HCl (1.0 mol / L) and NaOH (1.0 mol / L) for 30 min; and (4) stored at 4℃ for 14 days. The inhibitory activity of the cell-free supernatant of Bacillus belyss LB7 under different treatments against Flavobacterium columnare G4 was then determined using the Oxford cup method.

[0058] Experimental results refer to Figure 5 As shown, when the pH was adjusted to 1.82 and 12.80, and after heating at 90℃ and 100℃ for 30 min and storing at 4℃ for 14 days, the antibacterial activity of Bacillus belyss LB7 cell-free supernatant was significantly reduced. Furthermore, proteinase K and trypsin had no significant effect on the antibacterial effect of LB7 cell-free supernatant. Therefore, the effective antibacterial components of Bacillus belyss LB7 cell-free supernatant possess characteristics such as heat resistance and proteinase resistance.

[0059] Example 6 This embodiment demonstrates the therapeutic effect of Bacillus bellis LB7 cell-free supernatant mixed with feed on grass carp infected with Flavobacterium columnare G4. Grass carp with a body length of 7.0 ± 1.0 cm were used as experimental subjects. They were fed commercial feed (Tongwei, product number: 240708001) at 1.5% - 2% of their body weight daily, twice a day, morning and evening, and temporarily housed in aquariums with a water temperature of 26.0 ± 2.0℃ for two weeks.

[0060] Furthermore, cell-free supernatants of *Bacillus belyssus* obtained from fermentation on LB medium and optimized medium were prepared separately, and dehydrated using a freeze dryer to obtain dried supernatant products. Then, different volumes of the dried supernatant products were dissolved in 5 mL of sterile water and evenly sprayed onto 100 g of feed. The specific preparation volumes are as follows: florfenicol feed (50 mg florfenicol / 100 g feed), LB medium fermented feed (CFS-LB, 500 mL LB medium fermentation broth supernatant dried product / 100 g feed), high-concentration optimized medium fermented feed (CFS-OP-H, 500 mL optimized medium fermentation broth supernatant dried product / 100 g feed), medium-concentration optimized medium fermented feed (CFS-OP-M, 100 mL optimized medium fermentation broth supernatant dried product / 100 g feed), and low-concentration optimized medium fermented feed (CFS-OP-L, 10 mL optimized medium fermentation broth supernatant dried product / 100 g feed).

[0061] Fifty healthy grass carp were randomly divided into 5 groups of 10 each. Three to five monoclonal strains of Flavobacterium columnare G4 were pre-selected and incubated overnight in a Shieh's liquid medium at 28°C. The culture was then transferred to fresh Shieh's liquid medium at a 1:10 ratio and incubated until the final OD concentration was reached. 600 =Approximately 0.3. After centrifugation, resuspend in fresh Shieh's medium, and obtain a final concentration of 1.29 × 10⁻⁶ by serial dilution of Flavobacterium columnare G4. 3 CFU / mL, 1.29 × 10 4 CFU / mL, 1.29 × 10 5 CFU / mL, 1.29×10 6 The bacterial suspension was prepared at CFU / mL. Group 5 was supplemented with an equal volume of fresh Shieh's medium solution as a negative control. After immersion in the infection solution for 1 hour, the grass carp were transferred to the culture water and observed for one week. Disease and mortality rates were recorded, and the median lethal concentration (LD50) was calculated to be 1.45 × 10⁻⁶. 5 CFU / mL.

[0062] As shown in the diagram Figure 6 Two hundred and ten healthy grass carp were randomly divided into seven groups of 30 fish each. Single clones of Flavobacterium columnare G4 were pre-selected and cultured overnight in a Shieh liquid medium at 28°C. The culture was then transferred to fresh Shieh liquid medium at a 1:10 ratio and cultured until the final OD concentration was reached. 600 =Approximately 0.3. After centrifugation, resuspend in fresh Shieh medium and dilute to a final concentration of 2.5 × 10⁻⁶. 6 The grass carp were divided into 6 groups and infected with CFU / mL feed. They were then immersed in the infected feed for 3 hours before being transferred to aquaculture water. Each group was then fed twice daily, at 1.5% of their body weight: regular feed, florfenicol feed, LB medium fermentation supernatant feed (CFS-LB), high-concentration optimized medium fermentation supernatant feed (CFS-OP-H), medium-concentration optimized medium fermentation supernatant feed (CFS-OP-M), and low-concentration optimized medium fermentation supernatant feed (CFS-OP-L), twice a day (morning and evening). A seventh group, with an equal volume of fresh Shieh's medium solution added to simulate infection and fed a regular feed, served as a negative control. Pathological mortality was recorded promptly, and dead animals were removed immediately. The group was observed for 10 consecutive days, and the experiment was terminated only after three consecutive days without grass carp mortality.

[0063] Results reference Figure 6As shown, after infection with Flavobacterium columnare G4, the survival rate was 10.00% in the normal feed group, 73.33% (relative protection rate of 70.37%) in the group fed with high-concentration optimized culture medium fermentation supernatant, 63.33% (relative protection rate of 59.30%) in the group fed with medium-concentration optimized culture medium fermentation supernatant, 46.67% (relative protection rate of 40.74%) in the group fed with low-concentration optimized culture medium fermentation supernatant, 55.67% (relative protection rate of 51.85%) in the group fed with LB culture medium fermentation supernatant, and 63.33% (relative protection rate of 59.25%) in the group fed with florfenicol.

[0064] In summary, the cell-free supernatant obtained by culturing Bacillus bellis LB7 in an optimized culture medium, when mixed with ordinary feed, can significantly improve the protection rate of grass carp.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0066] References Combe M, Sokolenko S. 2021. Quantifying the impact of cell culturemedia on CHO cell growth and protein production. Biotechnology Advance 50,107761. Chen Q, Xing YF, Lei Y, Tong GX, Lin XB, He PC, Tang SL, Zheng FF, Zeng H, Wei XX, Zhang YA, Zhou Y. 2024. Genetic diversity, antibioticresistance, and pathogenicity of Aeromonas veronii isolated from farmedlargemouth bass ( Micropterus salmoides ) in the main aquaculture regions of China. Aquaculture 592, 741150. Ferreira DDAR, Assane IM, Vaneci-Silva D, Oliveira ADV, Tamashiro GD,Hashimoto DT, Pilarshi F.2022.Morpho-molecular identification, pathogenicityfor piaractus mesopotamicus , and antimicrobial susceptibility of avirulent flavobacterium columnare isolated from nile tilapia cultured inbrazil. Aquaculture . 560, 738486. Gieseker CM, Gaunt PS, Hawke JP, Crosby TC, Hasbrouck NR, Gao DX,Declercq AM.2022. Epidemiological Cutoff Values for Standard BrothMicrodilution Susceptibility Testing of ​ Isolated fromFishes. ​ . 28, 9. Guo MX, Peng R, Jin KL, Zhang X, Mo HL, Li X, Qu FF, Tang JZ, Cao YH,He ZM, Mao ZW, Fan JD, Li JZ, Liu Z.2024. Effects of ​ infection on theimmune system, physical barriers and microflora structure in the intestine ofjuvenile grass carp ( ​ ). ​ .153,109790. Hemarajata P, Versalovic J.2013. Effects of probiotics on gutmicrobiota: mechanisms of intestinal immunomodulation andneuromodulation. ​. 6(1), 39. Kamoun F, Weekers F, Ayed RB, Mechri S, Jabeur F, Thonart P, JaouadiB.2022. Multiple linear regression models to simulate spore yields of ​ ​ BS13 through optimization of mediumcomposition. ​ . 69(6), 2686-2697. Liu WZ, Xue MY, Yang T, Li YQ, Jiang N, Fan YD, Meng Y, Luo XW, ZhouY, Zeng LB.2022. Characterization of a Novel RNA Virus Causing MassiveMortality in Yellow Catfish, ​ , as an Emerging Genusin ​ ( ​ ). ​ . 10(4), e00624-22. Luo WS, Xu ZH, He QY, Peng J, Wang F, Li J, Luo SW.2024. Comparativeanalyses of immune gene profiles and antioxidant capabilities in the midgutand liver of three species of grass carps ( ​ ) followinggut infection with ​ . ​ . Marco ML, Tachon S.2013. Environmental factors influencing theefficacy of probiotic bacteria. ​ . 24(2), 207-213. Ma YB, Zhou XQ, Jiang WD, Wu P, Liu Y, Li SW, Tang L, Zhang L, Mi HF,Feng L.2024. Tea polyphenols protect against ​ -inducedgill injury via suppression of oxidative stress, inflammation, and apoptosisin grass carp. ​ . 254,127050. Oana K, Shimizu K, Takada T, Makino H, Yamazaki M, Katto M, Ando M,Kurakawa T, Oishi K.2023. Manipulating the growth environment through co-culture to enhance stress tolerance and viability of probiotic strains in thegastrointestinal tract. ​ . 89,12. Orhan F, Ceyran E, Akincioglu A.2023. Optimization of ectoineproduction from ​ and one-step ectoinepurification. ​ . 371, 128646. Parampalli A, Eskridge K, Smith L, Meagher MM, Mowry MC, SubramanianA.2007. Developement of serum-free media in CHO-DG44 cells using a centralcomposite statistical design. ​ . 54(1), 57–68. Qi XZ, Xue MY, Shi K, Wang GX, Ling F.2024. Evaluating ​ ​JK-1 as an in-feed probiotic: Enhancing growth, immune-antioxidant,disease resistance and modulating gut microflora composition in grass carp( ​ ). ​ . 585, 740715. Qiu CW, Shi YH, Huang XX, Chen ZZ.2025. Impact of Probiotics onEnzyme Activities, Intestinal Microbial Remodeling, and Metabolic Pathways inAmerican Shad ( ​ ) at High Temperatures. Marine Biotechnology .27, 58. Rabiya R, Sen R.2022. Artificial intelligence driven advancedoptimization strategy vis-`a-vis response surface optimization of productionmedium: Bacterial exopolysaccharide production as a case-study. Biochemical Engineering Journal . 178, 108271. Singh V, Khan M, Khan S, Tripathi CK.2009. Optimization ofactinomycin V production by Streptomyces triostinicus using artificial neuralnetwork and genetic algorithm. Applied Microbiology Biotechnology . 82(2), 379–385. Schrader KK, Harries MD, Darwish AM.2013. In vitro comparisons of theinhibitory activity of florfenicol, copper sulphate and potassiumpermanganate towards aeromonas hydrophila and flavobacterium columnare . Aquaculture Research . 44(2), 212-219. Woldemariamyohannes K, Wan Z, Yu Q, Li H, Sun B.2020. Prebiotic,probiotic, antimicrobial, and functional food applications of bacillus amyloliquefaciens . Journal of Agricultural and Food Chemistry . 68(50), 14709-14727. Wu HL, Yin JY, Li SM, Wang H, Jiang P, Li PF, Ding ZY, Yan H, Chen B,Wang LC, Wang Q.2024. Oral immunization with recombinant L. lactis expressingGCRV-II VP4 produces protection against grass carp reovirus infection. Fish& Shellfish Immunology . 147, 109439. Yao T, Asayama Y.2017. Animal-cell culture media: history,characteristics, and current issues. Reproductive Medicine and Biology . 16(2),99–117. Yan TT, Xing XQ, Xia TB, Wang D.2024. Relation between faultcharacteristic frequencies and local interpretability shapley additiveexplanations for continuous machine health monitoring. Engineering Applications of Artificial Intelligence . 136, 109046。

Claims

1. A type of Bacillus belesii, characterized in that, It is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20252822, and its classification name is... Bacillus velezensis LB7, deposited on December 10, 2025, at Wuhan University, Wuhan, China, is identified as viable.

2. A culture of Bacillus belesiensis as described in claim 1, characterized in that, The culture refers to the fermentation broth, cell-free supernatant, bacterial sludge, extract of fermentation broth, or concentrate, gelatinized product, dried product, diluted product, and broken product of Bacillus vesiculosus.

3. The culture according to claim 2, characterized in that, The cell-free supernatant was obtained by filtering the bacterial culture of the Bacillus belye.

4. The use of Bacillus belye as described in claim 1 or the culture as described in any one of claims 2-3 in the prevention or treatment of diseases in aquatic animals or livestock.

5. The application according to claim 4, characterized in that, The diseases of aquatic animals or livestock are caused by at least one of the following pathogens: Flavobacterium columnare, Aeromonas verrucosa, Edwardsiella tinctoria, Streptococcus agalactiae, and Vibrio parahaemolyticus.

6. The application according to claim 5, characterized in that, The aquatic animals are selected from any one or more of the following: grass carp, mandarin fish, largemouth bass, yellow catfish, catfish, crucian carp, common carp, silver carp, bighead carp, black carp, yellow eel, snakehead, eel, tilapia, redfin pufferfish, yellow pufferfish, and zebrafish.

7. The application according to claim 5, characterized in that, The application includes at least one of the following methods: (1) destroying the biofilm of pathogens; (2) improving the survival rate of aquatic organisms.

8. A microbial preparation or feed, characterized in that, It includes the Bacillus belyssus as described in claim 1 or the culture as described in any one of claims 2-3; Preferably, each 100g of the feed contains 10-500ml of the dried product of the Bacillus vesiculosus liquid or liquid culture; Preferably, each 100g of the feed contains 10-500ml of the dried product of the cell-free supernatant of Bacillus belyssum.

9. A fermentation medium for cultivating Bacillus belye as described in claim 1, characterized in that, It includes the following raw materials in a mass concentration meter: 15.5-17.5 g / L sucrose, 9.0-11.0 g / L glucose, 6.5-8.5 g / L tryptone, 0.50-0.70 g / L casein, 0.20-0.40 g / L yeast extract, 9.5-11.5 g / L urea, 0.3-0.5 g / L potassium nitrate, and 0.5-2.5 g / L magnesium chloride.

10. A method for preventing or treating diseases in aquatic animals or livestock, characterized in that, It includes the following steps: Feeding aquatic animals or livestock with the microbial preparation or feed described in claim 8; Preferably, the aquatic animals or livestock are fed a diet containing Bacillus vesiculosus culture supernatant at a rate of 1.5% to 2% of their body weight, twice a day, once in the morning and once in the evening.

Images