Bacillus safensis and application thereof in aquaculture

Abstract

The invention discloses a bacillus safensis strain and application thereof in aquaculture, and relates to the technical field of aquaculture disease prevention and control, the obtained bacillus safensis strain ZY-1 forms spores under a certain fermentation condition, can be prepared into powder for preservation, has a long shelf life, has the bacteriostatic activity retention rate of more than or equal to 85% after being stored in a dark place for 12 months at 4-25 DEG C, and has the antibacterial activity of more than or equal to 7.2%. The production, transportation and storage cost is saved, main bacteriostatic effect factors of the bacillus safensis ZY-1 strain exist in extracellular metabolites, the bacillus safensis ZY-1 strain is stable in performance and resistant to high temperature, acid and alkali, ultraviolet rays, protease and the like, and fermentation supernatant of the bacillus safensis ZY-1 strain can be directly applied or prepared into spray-dried powder and is suitable for various aquaculture application scenes such as pond splashing and feed mixing and feeding. The metabolite spray-dried powder has a remarkable killing effect on high-pathogenicity vibrios VpAHPND and VpTPD, the minimum inhibitory concentrations of metabolite spray-dried powder respectively reach 23.44-46.88 mu g / ml and 46.88-93.75 mu g / ml, and the metabolite spray-dried powder can be used for preventing and controlling prawn infectious hepatopancreas necrosis and prawn transparent larva diseases induced by the metabolite spray-dried powder and also has a certain killing function on pathogens of other aquatic animals.

Description

Technical Field

[0001] This invention relates to the field of disease prevention and control technology in aquaculture, and in particular to a strain of Bacillus sabolicii and its application in aquaculture. Background Technology

[0002] Aquaculture plays a vital role in the global food supply; however, disease problems have consistently hampered its development. Pathogenic bacteria, common and highly damaging pathogens in aquaculture, have dealt a heavy blow to the industry. Data from 2024 shows that with the development of high-density intensive aquaculture, the ecological balance of aquaculture waters is easily disrupted, and some pathogens have undergone new mutations in their virulence genes, significantly reducing the effectiveness of traditional chemical drugs and antibiotics. Diseases caused by various Vibrio species, such as Vibrio parahaemolyticus and Vibrio alginolyticus, occur frequently and exhibit multidrug resistance, becoming a key factor seriously affecting the profitability of aquaculture. Entering 2025, the threat of bacterial diseases continues to intensify. Researchers predict that El Niño will cause abnormal water temperatures, increasing the reproduction rate of aquatic pathogens by 30%, and the traditional "high-temperature period control" model faces the risk of failure. Simultaneously, cross-infection is becoming commonplace, and cases of pathogens co-infecting viruses and parasites are increasing. Under these severe circumstances, finding new and effective methods for pathogen control is urgently needed.

[0003] Vibrio species are numerous, and diseases caused by them are among the major bacterial diseases in marine aquaculture animals, posing serious threats to fish, shrimp, crabs, and shellfish. In recent years, some Vibrio strains have evolved highly pathogenic virulence genes and carry plasmids containing genes for resistance to multiple antibiotics. This study focuses on a highly pathogenic Vibrio parahaemolyticus strain. Vibrio parahaemolyticus ( Vp Acute hepatopancreatic necrosis syndrome (AHPND) in shrimp is caused by *Vibrio parahaemolyticus* (AHPND). Shrimp larvae infected with AHPND can experience a mortality rate of up to 100% within 10-35 days after stocking. Another strain of *Vibrio parahaemolyticus* (...) Vp TPD (Transparent Proliferative Diarrhea) can also cause transparent larval disease (TPD) in shrimp. Infected shrimp become completely transparent, lose vitality, stop feeding, and experience extremely high mortality rates in a short period, severely impacting shrimp larval production. In fish, Vibrio infection can lead to diseases such as body surface ulceration. For example, approximately 66.7% of diseases in grouper are vibrio infections, occurring at all stages of their growth, with mortality rates reaching as high as 50%. In marine aquaculture, common species such as turbot, sea bass, and large yellow croaker are also frequently affected by Vibrio. Crustaceans such as the swimming crab (Portunus trituberculatus) are also susceptible to Vibrio infection, experiencing slow growth, weakened immunity, and even mass mortality after infection.

[0004] For a long time, chemical drugs and antibiotics have been widely used in the prevention and control of diseases in aquaculture. However, with the large-scale use of antibiotics, their drawbacks have become increasingly prominent. On the one hand, the resulting selective pressure leads to the emergence, persistence, and spread of drug-resistant bacteria, making the effectiveness of drug control for vibriosis increasingly poor. On the other hand, the overuse of antibiotics poses a huge threat to the environment and food safety. Vaccines, as another prevention and control measure, can enhance the immunity of aquatic animals to some extent, but due to the heterogeneity, adaptability, and ubiquitous presence of Vibrio in the marine environment, the use of vaccines has certain limitations. It is difficult to provide effective immune protection against all strains of Vibrio, and since crustaceans in aquaculture are lower invertebrates, they lack specific immune functions, making vaccines unsuitable for these organisms.

[0005] In recent years, facing the problem of increasingly strong Vibrio resistance, aquaculture farmers have fallen into a vicious cycle of "the more drugs used, the harder it is to treat." Therefore, developing safe, efficient, and environmentally friendly new prevention and control products has become an urgent issue for the aquaculture industry. Probiotics, as a class of microorganisms with broad ecological adaptability and stress resistance, have received much attention in aquaculture in recent years. However, the core functions of currently used microecological preparations in aquaculture are concentrated on regulating water quality and promoting growth. Their targeted control of Vibrio diseases is still insufficient, making it difficult to directly solve the prevention and control problems caused by the current emergence of Vibrio resistance. Specifically, probiotics secrete various enzymes, such as proteases and amylases, to decompose organic matter in water, reduce the concentration of toxic substances such as ammonia nitrogen and nitrite, improve the aquatic environment, and provide good ecological conditions for the growth of aquatic animals. Secondly, the presence of a large number of probiotics can compete with pathogenic microorganisms for living space and nutrients, thereby inhibiting the growth and reproduction of pathogenic microorganisms to a certain extent. In addition, some microorganisms can promote the growth of aquatic animals, improve feed utilization and conversion rate by regulating the balance of intestinal flora, and activate the immune system of aquatic animals, thereby enhancing their immunity and disease resistance to a certain extent.

[0006] However, according to market research, current aquatic probiotic agents are not effective against highly pathogenic Vibrio parahaemolyticus. Vp AHPND、 Vp TPD has insufficient antagonistic effect and cannot effectively control acute hepatopancreatic necrosis syndrome and transparent larval disease in shrimp. Most probiotic metabolites are easily inactivated under high temperature (>60℃), extreme pH (pH<4 or pH>10) or ultraviolet radiation, making it difficult to adapt to complex scenarios such as open ponds, processing and transportation. Some strains have broad-spectrum antibiotic resistance, and long-term use can easily lead to the accumulation of drug-resistant genes in the aquaculture environment, threatening food safety and ecological balance. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of existing aquatic probiotic agents being ineffective against highly pathogenic Vibrio parahaemolyticus. Vp AHPND、 Vp TPD has problems such as insufficient antagonistic effect, poor environmental adaptability, and potential safety hazards.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A strain of Bacillus safranin, named Bacillus safranin ZY-1, has the accession number CGMCC No. 35025 and the GenBank genome sequence number CM129639.1.

[0009] This invention also provides a microbial powder, the active ingredient of which is *Bacillus salsa* ZY-1 and its metabolites, wherein the viable bacterial content of *Bacillus salsa* ZY-1 in the powder is [missing information]. It is a preparation made by fermenting and culturing Bacillus salsa ZY-1 and then drying it.

[0010] This invention also provides a method for preparing microbial powder, comprising the following steps: (1) Seed culture: Bacillus sabolicii ZY-1 was inoculated into 2216E liquid medium and cultured at 30℃ and 220r / min for 24h to obtain seed culture; (2) Fermentation culture: The seed culture was inoculated into the fermentation tank at an inoculation rate of 5 wt% (for 50-500L fermentation tanks). The fermentation culture medium consisted of 5 g / L peptone, 3 g / L yeast extract, 20 g / L sodium chloride, and 0.5 g / L magnesium sulfate (or standard 2216E liquid medium was used). There was no significant difference between the two culture media in the growth of Bacillus sabolicii ZY-1 and the spore formation rate (the spore formation rate was ≥90%). The culture was carried out at 28℃ and oxygen supply of 6 L / min for 36 h until the spore formation rate was ≥90%. (3) Preparation of preparation: Collect the fermentation liquid, concentrate it and remove some water, spray dry it under the conditions of air inlet temperature of 150-160℃ and air outlet temperature of 80-90℃, pulverize it and pass it through an 80-mesh sieve to obtain microbial powder.

[0011] Preferably, the *Bacillus sarcodactylis* ZY-1 has the following physiological and biochemical characteristics: it can ferment rhamnose, sorbitol, and lactose to produce acid, but cannot ferment inositol, calendula alcohol, D-xylose, L-arabinose, and D-mannitol; it is negative for arginine dihydrolase test, positive for gelatin liquefaction test, positive for VP test, negative for nitrate reduction test, negative for starch hydrolysis test, positive for citrate utilization test, and positive for propionate utilization test; it has the ability to form spores, is Gram-positive, and has a short rod-shaped morphology with flagella.

[0012] Preferably, the moisture content of the bacterial powder is ≤5%, and the particle size is 125-180µm. After being stored at 4-25℃ in the dark for 12 months, the antibacterial activity retention rate is ≥85%.

[0013] Preferably, the metabolites contained in the bacterial powder are effective against highly pathogenic Vibrio parahaemolyticus. Vp The minimum inhibitory concentration (MIC) of AHPND is 23.44-46.88 μg / mL, which is effective against highly pathogenic Vibrio parahaemolyticus. Vp The minimum inhibitory concentration (MIC) of TPD is 46.88-93.75 μg / mL.

[0014] Preferably, the metabolites contained in the bacterial powder also have inhibitory effects on Vibrio parahaemolyticus TX1, Vibrio anguillarum, Vibrio alginolyticus, Vibrio harveyi, Edwardsiella tarda, and Flavobacterium columnare, wherein the MIC for Vibrio parahaemolyticus TX1 and Vibrio anguillarum is 23.44-46.88 μg / mL, the MIC for Vibrio alginolyticus and Vibrio harveyi is 46.88-93.75 μg / mL, and the MIC for Edwardsiella tarda and Flavobacterium columnare is 187.5-375 μg / mL.

[0015] Preferably, in step (2), 0.03 wt% of Dow fermentation defoamer DF103 is added during the fermentation process to prevent foam overflow. After fermentation, the spore formation rate in the fermentation liquid needs to be tested. Only when the spore formation rate is ≥90% can the subsequent preparation steps be carried out.

[0016] Preferably, the bacterial powder is applied in aquaculture by pond sprinkling or mixing with feed. When used for pond sprinkling, the dosage is 0.5-2 g / m³ of water, and when used for feed mixing, the dosage is 2-20 μg / g of feed.

[0017] The beneficial effects of this invention are: 1. The Bacillus sarfusae ZY-1 obtained in this application forms spores under certain fermentation conditions, which can be made into powder for preservation. It has a long shelf life. After being stored at 4-25℃ in the dark for 12 months, the spore reactivation rate is ≥85%, saving production, transportation and storage costs.

[0018] 2. The main antagonistic effect factor of the Bacillus salsa ZY-1 strain of this application is found in extracellular metabolites. It is stable and resistant to high temperature, acid and alkali, ultraviolet light, protease, etc. When it is made into spray-dried powder together with spores, it is suitable for various aquaculture applications such as pond sprinkling and feed mixing.

[0019] 3. The metabolites of Bacillus sabovellae ZY-1 in this application are effective against highly pathogenic Vibrio. Vp AHPND and VpTPD has a significant bactericidal effect. The minimum inhibitory concentrations of the metabolites contained in Bacillus sabovellatus ZY-1 spray dry powder reach 23.44-46.88μg / ml and 46.88-93.75μg / ml, respectively. It can be used to control infectious hepatopancreatic necrosis and transparent larval disease induced by it in shrimp, and also has a certain bactericidal function against other aquatic animal pathogens.

[0020] 4. The *Bacillus salsa* ZY-1 strain described in this application shows resistance to only 7 antibiotics (gentamicin, ceftazidime, ceftriaxone, amikacin, chloramphenicol, cefuroxime, and polymyxin B), intermediate resistance to 14 antibiotics (intermediate concentrations are all higher than twice the commonly used therapeutic dose in aquaculture, with no clinical drug use risk), and sensitivity to 9 antibiotics. It meets the 'broad-spectrum drug resistance judgment standard (≤10 types of resistant drugs)' in the "Safety Evaluation Specification for Aquatic Microecological Preparations". It does not exhibit broad-spectrum drug resistance and will not exacerbate the accumulation of drug-resistant genes in the aquaculture environment during use, thus having higher safety. Attached Figure Description

[0021] Figure 1 This invention relates to *Bacillus safranin* against highly pathogenic *Vibrio parahaemolyticus*. Vp AHPND and Vp Morphology of inhibition zones of pathogens such as TPD (inhibition zone diameter is approximately 16mm-32mm).

[0022] Figure 2 This invention relates to the morphological observation of Bacillus salsa ZY-1. (A is a natural morphological image of the colony, B is a Gram-stained morphological image of Bacillus salsa ZY-1, C is a morphological image of Bacillus salsa ZY-1 observed under a transmission electron microscope, and D is a morphological image of Bacillus salsa ZY-1 observed under a scanning electron microscope).

[0023] Figure 3 This is a phylogenetic tree of the 16S rRNA gene of Bacillus safensis of the present invention (showing that this strain is most closely related to Bacillus safensis strain MTR-NT01 in terms of evolution).

[0024] Figure 4 The graph shows the results of the thermostability test of the metabolites of Bacillus sabolicus in this invention (showing that the change rate of the diameter of the inhibition zone after treatment at 40-100℃ is ≤15%).

[0025] Figure 5 The graph shows the results of the acid-base tolerance test of the metabolites of Bacillus salsa ZY-1 in this invention (showing that the change rate of the inhibition zone diameter after pH 1-11 treatment is ≤20%).

[0026] Figure 6The image shows the UV light tolerance test results of the metabolites of Bacillus salsa ZY-1 of this invention (showing that the change rate of the inhibition zone diameter is ≤18% within 6 hours of UV irradiation).

[0027] Figure 7 The figure shows the effect of the elastase of the present invention on the antibacterial activity of Bacillus sabolicii ZY-1 metabolites (showing no significant difference in the diameter of the inhibition zone after treatment with different concentrations of elastase).

[0028] Figure 8 The figure shows the effect of the chymotrypsin of the present invention on the antibacterial activity of Bacillus sabolicii ZY-1 metabolites (showing no significant difference in the diameter of the inhibition zone after treatment with different concentrations of chymotrypsin).

[0029] Figure 9 This is a comparison chart of the MIC values ​​of the Bacillus salsa ZY-1 metabolite powder of the present invention against eight pathogens (showing the MIC values ​​against...). Vp AHPND, Vibrio parahaemolyticus TX1, and Vibrio anguillarum had the lowest MICs, ranging from 23.44 to 46.88 μg / mL.

[0030] Figure 10 The images show pathological sections of the intestines and hepatopancreas of Litopenaeus vannamei after feeding with the Bacillus salsa ZY-1 powder of this invention (showing intact intestinal villi and neatly arranged hepatopancreas cells in both the treatment and control groups, with no pathological damage).

[0031] Figure 11 The images show pathological sections of the heart, liver, spleen, and kidney tissues of turbot after feeding with the Bacillus salsa powder ZY-1 of this invention (showing that the myocardial cells, hepatocytes, spleen cells, and nephrons in the treatment group and the control group have normal structures and no pathological features). Detailed Implementation

[0032] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are only preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0033] 2216E liquid culture medium: 5.0g peptone, 1.0g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.034g strontium chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g ammonium nitrate, 0.008g disodium hydrogen phosphate, 1000mL artificial seawater. Adjust pH to 7.6-7.8 and sterilize at 121℃ for 20 minutes. 2216E solid medium: Add 1.5%-2.0% agar to 2216E liquid medium and sterilize at 121℃ for 20 minutes; LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 20 minutes; MH liquid culture medium: 2g beef extract powder, 17.5g acid-hydrolyzed casein, 1.5g starch, 1000mL distilled water, pH 7.3±0.1, sterilized at 121℃ for 15 minutes; MHA agar medium: 2g beef extract powder, 17.5g acid-hydrolyzed casein, 1.5g starch, 17g agar, 1000mL distilled water, pH 7.3±0.1, sterilized at 121℃ for 15 minutes.

[0034] Example 1: Isolation and Identification of Bacillus salsa ZY-1 1. Strains Isolation Sediment samples were collected from the seabed of Jiaozhou Bay, Qingdao (depth 50m, latitude 36°10′N, longitude 120°13′E). 10g of sample was weighed and added to 90mL of sterile physiological saline. The mixture was shaken for 30 minutes and then allowed to stand for 10 minutes. The supernatant was then diluted to a final concentration of 1000 ml. 0.1 mL of the diluted solution was spread onto 2216E solid medium and incubated at 28°C for 48 h. Single colonies with different morphologies were picked and purified, resulting in a total of 312 bacterial strains. The core purpose of this isolation was to screen strains with special antagonistic activities from the marine environment to provide candidate strains for the subsequent control of aquatic vibriosis AHPND and TPD.

[0035] 2. Screening of antagonistic strains Screening by punching Vp AHPND and Vp Strains exhibiting antagonistic activity against TPD were inoculated into 2216E liquid medium and cultured at 30°C and 220 rpm for 24 h. The fermentation supernatant was then collected, and simultaneously... Vp AHPND、 VpTPD was inoculated into MH liquid medium and incubated at 30℃ until OD600 = 0.5. 1 mL of the bacterial culture was mixed thoroughly with 20 mL of LB solid medium (45-50℃) and poured into petri dishes to prepare pathogen agar plates. Wells (6 mm) were punched in the plates using a sterile punch, and 100 μL of the fermentation supernatant of the strain to be screened was added to each well. A control was prepared using the fermentation supernatant of non-resistant Bacillus subtilis. After drying at room temperature for 10 minutes, the plates were incubated at 30℃ for 24 hours, and the formation of inhibition zones was observed. One strain resistant to TPD was screened. Vp AHPND and Vp The strain that could form inhibition zones ≥16mm in diameter under TPD was named ZY-1 (later identified as Bacillus safranin, see [link]). Figure 1 Bacillus sarfusae ZY-1 Vp The inhibition zone morphology of AHPND (approximately 32 mm in diameter) was observed. The screening process strictly controlled the culture conditions and control settings to ensure the accuracy and reliability of the screening results.

[0036] 3. Morphological identification ZY-1 strain was inoculated onto 2216E solid medium and cultured at 28°C for 48 hours. Single colony morphology was observed; the colonies were moist, opaque, convex in the center, viscous, with irregular edges, and easily picked up. Single colonies were Gram-stained; the staining result was observed to be purple under an optical microscope, indicating Gram-positive bacteria. Logarithmic growth phase bacterial suspension was fixed with 2.5% glutaraldehyde, subjected to gradient dehydration, and gold sputtering. Scanning electron microscopy and transmission electron microscopy revealed that the bacterial cells were short rod-shaped and possessed flagella (see...). Figure 2 These morphological characteristics provide a basis for the preliminary classification and identification of the strains.

[0037] 4.16S rRNA gene identification ZY-1 cells were inoculated into 2216E liquid medium and cultured at 30℃ and 220 r / min for 24 hours. The cells were collected by centrifugation at 4℃ and 8000×g for 5 minutes. Genomic DNA was extracted using the Ezup column-based bacterial genomic DNA extraction kit (purchased from Shanghai Sangon Biotech Co., Ltd.). Using the genomic DNA as a template, PCR amplification was performed using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3') for the 16S rRNA gene. The expected amplified fragment length was approximately 1500 bp. The PCR reaction system (20 μL) consisted of: 14 μL sterile double-distilled water, 2 μL 10×Buffer, 1.6 μL dNTPs, 0.6 μL 10 μM primer 27F, 0.6 μL 10 μM primer 1492R, 1 μL genomic template, and 0.2 μL PrimerSTAR DNA Polymerase (Takara). The PCR program was: 95℃ pre-denaturation for 5 minutes, 30 cycles (95℃ 45 seconds, 55℃ 45 seconds, 72℃ 1.5 minutes), and extension at 72℃ for 10 minutes. The PCR products were detected by 1.0% agarose gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the 16S rRNA gene sequence of ZY-1 was 1546 bp in length. This sequence was BLAST-aligned in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA11 software (see [link to PCR results]). Figure 3 The results showed that this strain was most closely related to Bacillus safensis strain MTR-NT01 and Bacillus sp. (in: firmicutes)strain XIXJ297 in terms of evolution. Based on morphological characteristics, the strain was identified as Bacillus safensis and named Bacillus safensis strain ZY-1, with accession number CGMCC No. 35025. The depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is June 27, 2025. The whole genome sequencing of this strain has been completed, and the relevant data has been submitted to the GenBank database with sequence number CM129639.1. The gene identification process strictly followed the standard molecular biology experimental procedures to ensure the scientific validity of the sequencing results and phylogenetic analysis.

[0038] 5. Physiological and biochemical identification Commercial kits from Guangdong Huankai Microbial Technology Co., Ltd. and HBI Bacillus biochemical identification strips (HBIG14) from Qingdao Haibo Biotechnology Co., Ltd. were used to detect the fermentation capacity of ZY-1 index for carbon sources and its physiological and biochemical indicators, following the instructions of the kits. The detection items included rhamnose, sorbitol, inositol, calendula alcohol, lactose fermentation, arginine dihydrolase, gelatin liquefaction, nitrate reduction, starch hydrolysis, VP test, citrate utilization, propionate utilization, D-xylose utilization, L-arabinose utilization, D-mannitol utilization, and spore formation. The results are shown in Table 1.

[0039] Table 1: Physiological and biochemical characteristics of Bacillus salsa ZY-1 Reagent strip corresponding tube / substrate Test results Reagent strip corresponding tube / substrate Test results Rhamnose + Nitrate reduction - Sorbitol + Starch hydrolysis - Inositol - VP test + Calendula alcohol - citrate + lactose fermentation + propionate + Arginine dihydrolase - D-xylose - Gelatin liquefaction + L-arabinose - Spore formation + D-Mannitol - Rhamnose + Nitrate reduction - As shown in Table 1, ZY-1 tested positive for both the acetylmethane methanol test (VP test) and the gelatin liquefaction test, but negative for the nitrate reduction test. It can utilize sorbitol, rhamnose, lactose, citrate, and propionate as carbon sources, but cannot utilize inositol, calendula alcohol, D-xylose, L-arabinose, D-mannitol, or starch. Furthermore, it can form spores. These physiological and biochemical characteristics further validate the strain's taxonomic position, indicating that its morphological, physiological, and biochemical characteristics are similar to those of Bacillus species, and providing crucial information for understanding its metabolic capacity and environmental adaptability.

[0040] 6. Antibiotic susceptibility testing The Kirby-Bauer disk diffusion method was used to assess the susceptibility of strain ZY-1 to commonly used antibiotics. ZY-1 strain was inoculated into 2216E liquid medium and incubated overnight at 30°C and 220 rpm, followed by centrifugation at 4°C and 8000×g for 5 minutes. The bacterial cells were then resuspended in sterile physiological saline and the concentration was adjusted to 10. 8CFU / mL, 0.1 mL of bacterial suspension was evenly spread onto Mueller-Hinton agar (MHA) plates (manufactured by Beijing Luqiao Technology Co., Ltd.). Antibiotic susceptibility testing discs (purchased from Hangzhou Microbial Reagent Co., Ltd.) were affixed to the plate surface. After aerobic incubation at 30℃ for 24 hours, the diameter of the inhibition zone was measured to determine the susceptibility of the strain to antibiotics (susceptible S, intermediate I, resistant R). The test was repeated 3 times. The antibiotic susceptibility testing discs and concentrations used were as follows: midemycin (30 μg), cefoperazone (75 μg), piperacillin (100 μg), furazolidone (300 μg), ofloxacin (5 μg), erythromycin (15 μg), kanamycin (30 μg), gentamicin (10 μg), cephalosporin (10 μg), ciprofloxacin (10 μg), ciprofloxacin (10 μg), cephalosporin ... The following drugs were administered: Peptoxin (30 μg), Cefazolin (30 μg), Ceftazidime (30 μg), Ceftriaxone (30 μg), Tetracycline (30 μg), Vancomycin (30 μg), Amikacin (30 μg), Chloramphenicol (30 μg), Neomycin (30 μg), Clindamycin (2 μg), Oxyxacillin (1 μg), Ampicillin (10 μg), Carbenicillin (100 μg), Minocycline (30 μg), Ciprofloxacin (5 μg), Doxycycline (30 μg), Cefadroxil (30 μg), Cefuroxime (30 μg), Polymyxin B (300 μg), Compound Sulfamethoxazole (19 μg), Penicillin (10 μg), Norfloxacin (10 μg). The results are shown in Table 2.

[0041] Table 2: Antibiotic susceptibility of ZY-1. Note: Intermediate status is determined according to the CLSI Aquatic Animal Pathogen Susceptibility Testing Standards. When the intermediate concentration (MIC) is greater than twice the clinically recommended concentration, it is considered as having no risk of drug use.

[0042] The data in Table 2 show that strain ZY-1 was sensitive to nine antibiotics, including midecamycin, piperacillin, furazolidone, ofloxacin, cefazolin, vancomycin, doxycycline, trimethoprim-sulfamethoxazole, and norfloxacin; it showed intermediate sensitivity to 14 antibiotics, including erythromycin, kanamycin, cephalexin, tetracycline, neomycin, clindamycin, oxacillin, ampicillin, carbenicillin, minocycline, ciprofloxacin, cefadroxil, and penicillin; and it showed resistance to seven antibiotics, including gentamicin, ceftazidime, ceftriaxone, amikacin, chloramphenicol, cefuroxime, and polymyxin B. These results indicate that strain ZY-1 does not exhibit broad-spectrum resistance and will not exacerbate antibiotic resistance problems in aquaculture environments during use, demonstrating high safety.

[0043] Example 2: Antibacterial spectrum and physicochemical properties of Bacillus salsa metabolites 1. Antibacterial activity spectrum analysis Fermentation culture of Bacillus sabovella ZY-1 was performed by inoculating the strain into 2216E liquid medium and incubating with shaking at 30℃ and 220 r / min until the bacterial concentration reached the target value. The concentration of CFU / mL was used to obtain a fermentation broth containing antibacterial active substances. The broth was then filtered through a 0.22 μm membrane to remove bacterial cells, and the sterile fermentation supernatant was collected for later use. Twenty aquatic and common pathogenic bacteria preserved in the laboratory were selected as indicator bacteria, specifically including: highly pathogenic Vibrio parahaemolyticus. Vp AHPND, highly pathogenic Vibrio parahaemolyticus Vp TPD, Vibrio parahaemolyticus TX1, Vibrio fischeri, Staphylococcus aureus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio parsini, Proteus, Aeromonas salmonidae, Vibrio rotiferus, Vibrio harveyi, Vibrio splenti, Citrobacter fischeri, Oligotrophozoites, Enterococcus pyogenes, Pseudomonas aeruginosa, Flavobacterium columnare, Edwardsiella tarda, and Streptococcus dolphinus.

[0044] The above-mentioned indicator bacteria were inoculated into MH liquid medium and cultured with shaking at 30℃ and 220r / min until the OD600 value of the bacterial solution reached 0.5 to obtain the pathogenic bacterial solution. 1 mL of the pathogenic bacterial solution was mixed thoroughly with 20 mL of MHA solid medium at 45-50℃ and poured into a sterile petri dish. After the medium solidified, pathogenic bacterial plates were prepared. The antibacterial activity of ZY-1 fermentation supernatant was determined using the perforation method. Sterile perforators were used to punch holes (6 mm in diameter) in the pathogenic bacteria agar plates. 100 μL of sterile fermentation supernatant of *Bacillus saboidis* ZY-1 was added to each well, with *Bacillus subtilis* culture supernatant used as a blank control. After standing at room temperature for 10 minutes, the plates were incubated at 30°C for 24 hours, and the formation of transparent inhibition zones was observed. The results are as follows Figure 1 As shown, the fermentation supernatant of the selected Bacillus saboidis ZY-1 was effective against eight pathogens (highly pathogenic Vibrio parahaemolyticus). Vp AHPND, highly pathogenic Vibrio parahaemolyticus Vp TPD showed good antibacterial effects against Vibrio parahaemolyticus TX1, Vibrio anguillarum, Vibrio alginolyticus, Flavobacterium columnare, Edwardsiella tarda, and Vibrio harveyi, all forming clear, transparent inhibition zones. No obvious inhibition zones were observed against the remaining 12 pathogens (Vibrio fischeri, Staphylococcus aureus, Vibrio parcinii, Proteus, Aeromonas salmonidae, Vibrio rotiferus, Vibrio splenium, Citrobacter fischeri, Oligotrophozoites, Enterococcus pyogenes, Pseudomonas aeruginosa, and Streptococcus dolphinus), indicating no significant antibacterial activity.

[0045] 2. Physicochemical property analysis of antibacterial metabolites Highly pathogenic Vibrio parahaemolyticus VpAHPND was used as a representative pathogenic strain. The thermostability, acid-base tolerance, UV resistance, and protease tolerance of ZY-1 strain metabolites were tested. Each treatment was performed in triplicate, with untreated sterile fermentation supernatant serving as a control. The experimental apparatus and procedures are as follows: (1) Thermal stability test Experimental instruments: constant temperature water bath (model: HH-S, Jintan Dadi), vernier calipers.

[0046] Experimental Procedure: The sterile supernatant from *Bacillus sabolicii* ZY-1 fermentation for 24 hours was collected and treated in constant temperature water baths at 40℃, 60℃, 80℃, and 100℃ for 30 minutes each. After treatment, the supernatant was allowed to cool naturally to room temperature, and its effects on [unclear - possibly related to a specific organism or process] were detected using a perforation method. Vp The antibacterial activity of AHPND was assessed by measuring the diameter of the inhibition zone and calculating the rate of change. The test results are as follows: Figure 4 As shown.

[0047] The results showed that the diameter of the inhibition zone changed by ≤15% after treatment at 40-100℃. This indicates that the metabolites have good thermal stability and can withstand temperature fluctuations in aquatic processing and aquaculture environments.

[0048] (2) Acid-base tolerance test Experimental instruments: pH meter (model: PHS-3C, Leici), constant temperature water bath, vernier caliper. Experimental Procedure: Take sterile supernatant and adjust its pH to 1, 3, 5, 7, 9, and 11 respectively using 1 mol / L HCl solution or 1 mol / L NaOH solution. Let it stand at room temperature for 1 hour. Then, adjust the pH of the supernatant back to its original pH (8.3) using the above acid / base solutions. The effect of the supernatant on pH is then tested using the perforation method. Vp The antibacterial activity of AHPND was recorded, the diameter of the inhibition zone was recorded, and the test results were as follows: Figure 5 As shown. The results showed that the change rate of the inhibition zone diameter after treatment with pH 1-11 was ≤20%. This indicates that the metabolites maintain antibacterial activity over a wide acid-base range and can adapt to different pH environments in aquaculture water (such as seawater pH 7.5-8.2 and freshwater pH 6.5-7.5). (3) Ultraviolet light tolerance test Experimental instruments: UV lamp (model: ZW30S19W, Philips), sterile petri dishes, vernier calipers.

[0049] Experimental Procedure: Pour sterile supernatant into sterile petri dishes, controlling the liquid layer thickness to 2 mm. Place the petri dishes under a UV lamp (30W power, irradiation distance 30cm) for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours respectively. After irradiation, cool to room temperature in the dark. Use the perforation method to detect the effect of the supernatant on the [unclear - possibly related to UV radiation]. Vp The antibacterial activity of AHPND was analyzed, and the effect of UV irradiation duration on the antibacterial effect was examined. The test results are as follows: Figure 6 As shown. The results showed that the diameter of the inhibition zone changed by ≤18% within 6 hours of UV irradiation. This indicates that the metabolites have strong UV resistance and are not easily deactivated by UV radiation when used in open-air pond aquaculture.

[0050] (4) Protease tolerance test Experimental instruments: constant temperature water bath, microplate reader (model: InfiniteM200, Tecan), vernier calipers.

[0051] Experimental Procedure: Considering the common concentrations of elastase and chymotrypsin in host or bacterial cultures, both proteases were added to sterile supernatants. Elastase concentrations were set at four gradients: 0.2 μg / mL, 2 μg / mL, 20 μg / mL, and 200 μg / mL. Chymotrypsin concentrations were set at four gradients: 0.12 μg / mL, 0.6 μg / mL, 1.2 μg / mL, and 12 μg / mL. The supernatants containing the proteases were placed in a 37°C water bath for 1 hour, followed by boiling for 10 minutes to inactivate the proteases. After cooling, the effects on the bacteria were detected using a perforation method. Vp The antibacterial activity of AHPND was compared with the antibacterial effect after treatment with different protease concentrations. The test results are as follows: Figure 7 and Figure 8 As shown. The results showed that there was no significant difference in the diameter of the inhibition zone after treatment with different concentrations of protease (P>0.05). This indicates that the metabolites are not easily degraded by proteases and can stably exert antibacterial effects in the intestines of aquatic animals.

[0052] Example 3: Preparation of Bacillus salsa powder and metabolite powder 1. Experimental Materials and Instruments Experimental materials: Bacillus sabolicus ZY-1 strain, 2216E liquid culture medium, DF103 Dow fermentation defoamer (purchased from Dow Chemical), and sterile physiological saline. Experimental instruments: Fermenter (Model: BLBIO-500SJ, Bailun), Spray dryer (Model: Szsgz 2, Zhousheng) 2. Preparation steps (1) Inoculation and activation: Bacillus sabolicii ZY-1 strain was inoculated onto 2216E solid medium and cultured at 28℃ for 48 hours. Single colonies were picked and inoculated into 200 mL of 2216E liquid medium and cultured at 30℃ and 220 r / min for 24 hours to obtain the fermentation seed liquid (bacterial concentration ≥ (CFU / mL) indicates that the seed solution has been activated.

[0053] (2) First expansion culture: The activated seed liquid in step (1) was inoculated into 2216E liquid medium in a 5L fermenter for the first expansion culture. The culture conditions were: oxygen supply of 6L / min, temperature of 28℃, culture for 24 hours. During the fermentation process, 0.03% DF103 Dow fermentation defoamer was added to prevent foam overflow from affecting cell growth.

[0054] (3) Second expansion culture: The bacterial culture from the first expansion culture in step (2) is inoculated into a 500L fermenter. The fermentation medium can be the simplified medium of claim 3 (5g / L peptone, 3g / L yeast extract, 20g / L sodium chloride, 0.5g / L magnesium sulfate, 2g / L calcium chloride and 0.5g / L manganese sulfate) or the standard 2216E medium of this embodiment (the two media are equivalent). The culture conditions (oxygenation, temperature, time and amount of defoamer added) are kept consistent with the first expansion culture. The second expansion culture is carried out. After 36 hours, the fermentation is terminated and a large amount of fermentation product is obtained.

[0055] 3. Powder quality testing Tests showed that the microbial powder had a moisture content of ≤5%, uniform particle size (125-180µm), and after being stored at 4-25℃ in the dark for 12 months, the antibacterial activity retention rate of the metabolite powder was ≥85%, and the survival rate of Bacillus spores was ≥85%, which meets the industry standards for aquatic microecological preparations.

[0056] Example 4: MIC determination of metabolites in Bacillus salsa powder and its application in aquaculture. 1. Determination of the minimum inhibitory concentration (MIC) of the powder against aquatic pathogens (1) Experimental materials and instruments Experimental materials: metabolites of Bacillus sabovella ZY-1, 8 kinds of aquatic pathogens ( Vp AHPND、 Vp TPD, Vibrio parahaemolyticus TX1, Vibrio alginolyticus, Edwardsiella tarda, Vibrio anguillarum, Flavobacterium columnare, Vibrio harveyi, MH liquid medium, sterile PBS buffer (pH 7.4). Experimental instruments: 96-well cell culture plate (Corning), microplate reader (model: TecanInfinite200Pro), centrifuge, incubator. (2) Experimental steps (I) Preparation of pathogens: Eight pathogens were inoculated into antibiotic-free LB liquid medium and cultured at 37°C (220 r / min) until the logarithmic growth phase. The cultures were then centrifuged at 3000 × g for 5 minutes at room temperature, the supernatant was discarded, and the bacterial suspension was resuspended in sterile PBS buffer and washed three times to remove residual culture medium. Finally, the bacterial cells were resuspended in MH liquid medium, and the bacterial concentration was adjusted to [specific values ​​to be filled in]. CFU / mL. (II) Gradual dilution of metabolites: The fermentation supernatant from the second expansion culture was separately prepared into spray-dried powder to form a spray-dried powder of metabolites. The metabolite powder was resuspended in MH liquid medium (pH 7.4) and diluted to 3000 μg / mL. Subsequently, it was serially diluted twice in MH liquid medium (pH 7.4) to obtain powder solutions with a total concentration gradient of 8: 1500 μg / mL, 750 μg / mL, 375 μg / mL, 187.5 μg / mL, 93.75 μg / mL, 46.88 μg / mL, 23.44 μg / mL and 11.72 μg / mL. (III) MIC determination: In a sterile 96-well cell culture plate, 100 μL of metabolite solutions of different concentrations were added to each well, followed by 100 μL of diluted pathogenic bacterial solution to each well. (CFU / mL) to make the final volume of each well 200 μL. Blank control wells containing only MH medium and positive control wells containing only pathogenic bacterial suspension were set up. After incubating the 96-well plate in a 28°C incubator for 24 hours, the OD600 absorbance of each well was measured using a microplate reader. (iv) MIC determination criteria: The minimum inhibitory concentration (MIC) is defined as the lowest metabolite concentration that can completely inhibit bacterial growth (OD600 value ≤ 0.05, with no significant difference from the blank control well). If two adjacent concentrations can inhibit growth, the range between the two concentrations is recorded. (3) Measurement results The MIC values ​​of the powder against eight pathogens are shown in Table 3.

[0057] Table 3: Minimum Inhibitory Concentrations (MICs) of Powders against Aquatic Pathogens Types of pathogens Minimum inhibitory concentration (μg / mL) of ZY-1 metabolite powder Vibrio parahaemolyticus AHPND 23.44-46.88 Vibrio parahaemolyticus TPD 46.88-93.75 Vibrio parahaemolyticus TX1 23.44-46.88 Vibrio alginolyticus 46.88-93.75 Edwardsiella tarda 187.5-375 Vibrio anguillarum 23.44-46.88 Flavobacterium columnare 187.5-375 Vibrio harveyi 46.88-93.75 Table 3 shows the effects of metabolites on... Vp The inhibitory effects on AHPND, Vibrio parahaemolyticus TX1, and Vibrio anguillarum were the strongest, with MIC values ​​of 23.44-46.88 μg / mL; [the text abruptly ends here, likely due to an incomplete sentence or missing information.] VpThe inhibitory effects on TPD, Vibrio alginolyticus, and Vibrio harveyi were moderate, with MIC values ​​of 46.88-93.75 μg / mL; the inhibitory effects on Edwardsiella tarda and Flavobacterium columnare were relatively weak, with MIC values ​​of 187.5-375 μg / mL. (See also: [link to relevant documentation]). Figure 9 .

[0058] 2. Demonstration effect of aquaculture application (1) Safety and farming effect of Litopenaeus vannamei (I) Experimental Design: Healthy Litopenaeus vannamei shrimp (initial body length 4.2±0.4cm, initial weight 0.42±0.1g) were selected and divided into 3 groups. Group B1: ZY-1 powder was added to the feed, and the amount of spores added was... CFU / g; Group B2: ZY-1 powder was added to the feed, and the amount of spores added was [missing information]. CFU / g; Group B0 (control group): No powder was added to the feed, which was the basal feed. Each group had 6 replicates, with 30 shrimp per replicate, housed in 50L glass aquariums with continuous oxygenation (dissolved oxygen ≥5mg / L), water temperature controlled at 28±1℃, and salinity 25‰. The average daily feed amount was 2%-5% of the shrimp's body weight, adjusted according to shrimp growth and water temperature changes. Feeding was done three times daily (8:00, 14:00, 20:00), with 1 / 3 of the water changed daily to maintain stable water quality. After 30 days of rearing, [the following was added to the water...] Vp AHPND was subjected to immersion infection to achieve a final concentration of CFU / mL. Shrimp larvae mortality was observed and recorded every 6 hours, and the cumulative mortality rate was calculated.

[0059] (II) Detection Indicators and Results: After 30 days of culture, 18 shrimp were randomly selected from each group, and their body length and weight were measured. Intestinal and hepatopancreatic tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm thick), stained with hematoxylin and eosin (HE), and observed under a light microscope for pathological changes. Tables 4 and 5 show that the body length and weight of shrimp in group B2 were significantly higher than those in group B0 (P < 0.05). At 30 days, the body length reached 5.97 ± 0.32 cm, and the weight reached 1.25 ± 0.15 g. Pathological section observation (scale bar: 100 μm) showed that the intestinal villi of shrimp in all three groups were intact, and the hepatopancreatic cells were neatly arranged, with no pathological features observed. The structure was as follows: Figure 10 As shown, there were no deaths due to feed safety issues during the breeding period, indicating that the addition of Bacillus salsa ZY-1 powder to the feed is safe for Litopenaeus vannamei and has a significant growth-promoting effect.

[0060] Table 4: Shrimp body length (cm) growth after 30 days of feeding

[0061] Table 5: Weight gain (g) of shrimp after 30 days of feeding

[0062] (III) Results of the challenge experiment After 30 days of culture with added Bacillus subtilis powder, farmed shrimp were subjected to immersion infection. Vp The cumulative mortality rate of AHPND B0 group over 7 days was 83.3%, the cumulative mortality rate of B1 experimental group was 4.2%, and mortality occurred in B2 group. Bacillus salsa ZY-1 showed a good protective effect.

[0063] (2) Safety test on turbot (I) Evaluation of the safety of Bacillus salsa ZY-1 in turbot by intraperitoneal injection Experimental materials: Healthy turbot (weight 50±5g), Bacillus sabolicii ZY-1 bacterial suspension (cultured at 30℃ for 24 hours, concentration 10). 9 CFU / mL), sterile PBS buffer. Experimental design: Turbot were divided into 4 groups, with 3 replicates in each group and 15 fish in each replicate, and were raised in a 100L recirculating aquaculture system (water temperature 25±1℃, dissolved oxygen ≥6mg / L).

[0064] The overnight cultured Bacillus salsa ZY-1 fermentation broth was centrifuged (3000g, 10min), the supernatant was removed, and the bacterial cells were resuspended in sterile PBS. The concentration was adjusted using a turbidimeter. CFU / mL CFU / mL The experiment was divided into 4 groups, with CFU / mL as the concentration. Group A1: 100 μL of CFU / mL was injected intraperitoneally into each fish. ZY-1 bacterial suspension at CFU / mL; Group A2: 100 μL of ZY-1 bacterial suspension was injected intraperitoneally into each fish at a concentration of CFU / mL. ZY-1 bacterial suspension at CFU / mL; Group A3: 100 μL of ZY-1 bacterial suspension was injected intraperitoneally into each fish. ZY-1 bacterial suspension at CFU / mL; Group A0: 100 μL sterile PBS injected intraperitoneally into each fish. Feeding, swimming behavior, and mortality of the turbot were observed daily. Survival rate was recorded after 15 days, and any abnormal symptoms observed in each group were also recorded.

[0065] (II) Test Results After a 15-day rearing period, the survival and status data of the turbot in each group are shown in Table 6. Table 6: Safety test of intraperitoneal injection of Bacillus salsa ZY-1 in turbot Group mortality rate(%) Feeding status Behavior A1 0 normal normal A2 0 normal normal A3 0 normal normal A0 0 normal normal Table 6 shows that the mortality rate was 0 in the control group and groups A1, A2, and A3, with no individual deaths. All groups of turbot exhibited normal feeding behavior (daily intake of approximately 3%-4% of body weight), active swimming, and no abnormal symptoms such as surface congestion, abdominal swelling, or sluggishness were observed. These results indicate that even with intraperitoneal injection of high-concentration bacterial solution, *Bacillus saffron* ZY-1 remains safe for turbot and does not cause harm.

[0066] (3) Safety of oral administration of Bacillus salsa ZY-1 to turbot Experimental materials: Healthy turbot (weight 50±5g), Bacillus sabolicii ZY-1 bacterial suspension (cultured at 30℃ for 24 hours, concentration 10). 9 CFU / mL), sterile PBS buffer. Experimental Design: Turbot were divided into 3 groups, with 3 replicates per group and 15 fish per replicate. They were housed in a 100L recirculating aquaculture system (water temperature 25±1℃, dissolved oxygen ≥6mg / L). Group C1: ZY-1 powder was added to the feed, with a spore addition amount of [missing information]. CFU / g; Group C2: ZY-1 powder was added to the feed, and the amount of spores added was [missing information]. CFU / g; C0 group (control group): No powder was added to the feed, which was the basal feed. The average daily feed amount was 2%-5% of the turbot's body weight, adjusted according to the turbot's growth and water temperature changes. Feeding was carried out 3 times a day (8:00, 14:00, 20:00), and 1 / 3 of the water was changed daily to maintain stable water quality. After 30 days of feeding, 6 turbot were selected from each group, and four tissues were collected: heart, liver, spleen, and kidney. All tissue samples were fixed with 4% paraformaldehyde for 24 hours, dehydrated in a gradient, embedded in paraffin, and then prepared into 5μm thick tissue sections. Hematoxylin and eosin (HE) staining was performed, and the morphology of the tissue cells was observed under an optical microscope. Observation results: In the group treated with Bacillus salsa ZY-1, the cardiomyocytes in the heart tissue of turbot were neatly arranged without breakage or lysis; the liver tissue showed clear hepatocyte outlines without fatty degeneration or necrosis; the spleen tissue showed intact lymphoid follicle structure without inflammatory cell infiltration; and the kidney tissue showed no swelling of renal tubular epithelial cells and normal glomerular structure. Figure 11 As shown in the figure (scale bar 20 μm), the morphology of the tissue cells in all treatment groups was completely consistent with that in the control group, and the growth status was good. None of them showed any pathological characteristics, indicating that Bacillus sabolicii ZY-1 does not cause damage to the important organs and tissues of turbot, further verifying its safety in turbot farming.

[0067] In summary, the present invention isolated and screened *Bacillus sabolicus* ZY-1 (accession number CGMCC No. 35025) from the sediment of Jiaozhou Bay. Its taxonomic position was confirmed through morphological, molecular biological, and physiological-biochemical identification. Its metabolites exhibit broad-spectrum antibacterial activity against eight common aquatic pathogens (especially highly pathogenic *Vibrio parahaemolyticus*). Vp AHPND、 Vp TPD (ThinPrep) exhibits significant inhibitory effects, and its metabolites possess excellent thermal stability, acid-base tolerance, UV resistance, and protease tolerance, allowing it to maintain stable efficacy in the complex environment of aquaculture. The powder, prepared through a two-step expansion culture and spray drying process, meets industry standards, exhibits a low minimum inhibitory concentration (MIC) (23.44-375 μg / mL), and high antibacterial efficiency. Aquaculture application experiments show that this powder significantly promotes the growth of Litopenaeus vannamei (30-day weight gain increased by 13.6% compared to the control), and is non-toxic to both Litopenaeus vannamei and turbot, causing no damage to the intestines, hepatopancreas, heart, liver, or other tissues. Furthermore, ZY-1 does not exhibit broad-spectrum antibiotic resistance and will not exacerbate antibiotic resistance issues in the aquaculture environment during use. It is a safe, efficient, and environmentally friendly aquatic microecological preparation that can be widely used for disease control and healthy aquaculture in shrimp, turbot, and other aquatic animals, possessing significant industrial application value.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Bacillus safranin, characterized in that, The name is Bacillus sarfusca ZY-1, its accession number is CGMCCNo.35025, and its genome sequence number in GenBank is CM129639.

1.

2. A microbial powder, characterized in that, The active ingredient is *Bacillus salsa* ZY-1 and its metabolites as described in claim 1, wherein the viable bacterial content of *Bacillus salsa* ZY-1 in the bacterial powder is [missing information]. It is a preparation made by fermenting and culturing Bacillus salsa ZY-1 and then drying it.

3. The method for preparing the microbial powder according to claim 2, characterized in that, Includes the following steps: (1) Seed culture: Bacillus sabolicii ZY-1 was inoculated into 2216E liquid medium and cultured at 30℃ and 220r / min for 24h to obtain seed culture; (2) Fermentation culture: The seed culture was inoculated into the fermentation tank at an inoculation rate of 5 wt% (for 50-500L fermentation tanks). The fermentation culture medium consisted of: 5 g / L peptone, 3 g / L yeast extract, 20 g / L sodium chloride, 0.5 g / L magnesium sulfate, 2 g / L calcium chloride and 0.5 g / L manganese sulfate (or standard 2216E liquid medium was used). There was no significant difference between the two culture media in terms of the growth and spore formation rate of Bacillus sabolicii ZY-1. The culture was carried out at 28℃ and 6 L / min oxygenation for 36 h until the spore formation rate was ≥90%. (3) Preparation of preparation: Collect the fermentation liquid, concentrate it and remove some water, spray dry it under the conditions of air inlet temperature of 150-160℃ and air outlet temperature of 80-90℃, and pass it through an 80-mesh sieve to obtain microbial powder.

4. The *Bacillus safortifolius* according to claim 1, characterized in that, The *Bacillus sarcodactylis* ZY-1 has the following physiological and biochemical characteristics: it can ferment rhamnose, sorbitol, and lactose to produce acid, but cannot ferment inositol, calendula alcohol, D-xylose, L-arabinose, and D-mannitol; It is negative for arginine dihydrolase test, positive for gelatin liquefaction test, positive for VP test, negative for nitrate reduction test, negative for starch hydrolysis test, positive for citrate utilization test, and positive for propionate utilization test; it has the ability to form spores, is Gram-positive, and has a short rod-shaped morphology with flagella.

5. The microbial powder according to claim 2, characterized in that, The bacterial powder has a moisture content of ≤5% and a particle size of 125-180µm. After being stored at 4-25℃ in the dark for 12 months, the antibacterial activity retention rate is ≥85%.

6. The microbial powder according to claim 2, characterized in that, The bacterial powder contains metabolites that are effective against highly pathogenic Vibrio parahaemolyticus. Vp The minimum inhibitory concentration (MIC) of AHPND is 23.44-46.88 μg / mL, which is effective against highly pathogenic Vibrio parahaemolyticus. Vp The minimum inhibitory concentration (MIC) of TPD is 46.88-93.75 μg / mL.

7. The microbial powder according to claim 6, characterized in that, The metabolites contained in the bacterial powder also have inhibitory effects on Vibrio parahaemolyticus TX1, Vibrio anguillarum, Vibrio alginolyticus, Vibrio harveyi, Edwardsiella tarda, and Flavobacterium columnare. The MICs against Vibrio parahaemolyticus TX1 and Vibrio anguillarum are 23.44-46.88 μg / mL, against Vibrio alginolyticus and Vibrio harveyi are 46.88-93.75 μg / mL, and against Edwardsiella tarda and Flavobacterium columnare are 187.5-375 μg / mL.

8. The method for preparing the microbial powder according to claim 3, characterized in that, In step (2), 0.03 wt% of Dow fermentation defoamer DF103 is added during the fermentation process to prevent foam overflow. After fermentation, the spore formation rate in the fermentation broth needs to be tested. Only when the spore formation rate is ≥90% can the subsequent preparation steps be carried out.

9. The microbial powder according to claim 2, characterized in that, The bacterial powder can be applied in aquaculture by pond spraying or mixing with feed. When used for pond spraying, the dosage is 0.5-2 g / m³ of water. When used for mixing with feed, the dosage is 2-20 μg / g of feed.

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