Bacillus mojavensis NK-3, fungicide and application of bacillus mojavensis NK-3 in cultivation

Abstract

The invention belongs to the technical field of microbial agents. The invention provides bacillus mojavensis NK-3, a microbial agent and application of the bacillus mojavensis NK-3 in cultivation. The bacillus mojavensis NK-3 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.25408, and the preservation date is June 10, 2022. The invention further provides a bacillus mojavensis microbial inoculum, and the active ingredient of the bacillus mojavensis microbial inoculum is bacillus mojavensis NK-3. The bacillus mojavensis microbial agent disclosed by the invention is applied to culture and can inhibit staphylococcus aureus; the DNA / RNA content of pathogens is reduced, and the CT value of the mycoplasma synoviae of the laying hens is increased.

Description

Technical Field

[0001] This invention belongs to the field of microbial agent technology, specifically relating to Bacillus mojaverifolium NK-3, microbial agents, and their application in aquaculture. Background Technology

[0002] Staphylococcus aureus is a Gram-positive coccus, arranged in grape-like clusters, belonging to the Micrococcidae family. It is highly resistant to environmental factors, tolerating dryness, high temperatures (surviving 30 minutes at 80°C), and high salt (it can grow in media containing 7.5%-10% NaCl). Its marker is coagulase positivity (distinguishing it from coagulase-negative strains such as Staphylococcus epidermidis). Key virulence factors include coagulases: causing plasma coagulation, encapsulating bacteria to evade immune clearance; staphylococcal lysin: destroying erythrocytes; enterotoxin (SEA-E): heat-resistant, causing vomiting / diarrheal food poisoning; toxic shock syndrome toxin-1 (TSST-1): causing a systemic inflammatory response; and leukocidins: lysing neutrophils. Common infection types include post-traumatic skin and soft tissue infections, cross-contamination in livestock farms, and zoonotic infections.

[0003] Mycoplasma synoviae, a cell wall-less prokaryotic microorganism belonging to the class Mycoplasma, order Mycoplasma, is pleomorphic (spherical, filamentous), Gram-negative but difficult to stain. It primarily infects chickens and turkeys, with laying hens being particularly susceptible. The target organs are the synovial bursa (joints) and respiratory mucosa. Typical symptoms include: acute phase: unilateral or bilateral joint swelling, fever, tenderness, lameness or crouching posture, sternal cysts (occasionally); chronic phase: growth retardation, decreased egg production (reduction of 10%-30%), respiratory rales; secondary infections: Escherichia coli penetrating the joint cavity → suppurative arthritis; mixed viral infections exacerbate the condition. Transmission routes include horizontal transmission: inhalation of bacterial droplets / dust; vertical transmission: transmission from hatching eggs to chicks (late-stage infection); indirect transmission: contaminated feed, water, and equipment. Epidemiological characteristics: High incidence in young chickens aged 4-20 weeks, outbreaks are likely to occur during the peak egg production period; intensive feeding, high ammonia concentration, and stress factors (cold and heat stress) can induce infection; high rate of latent infection, and outwardly healthy chickens can carry the bacteria for a long time.

[0004] Since the beginning of the 21st century, food safety has become a new priority, and antibiotic contamination has received considerable attention. Antimicrobial lipopeptides produced by microbial metabolism have also garnered widespread attention due to their safe and highly effective antibacterial and antiviral effects. However, their low metabolic rate has become a bottleneck. These lipopeptides consist of a peptide ring composed of seven amino acids and a fatty acid side chain. The fatty acid side chain can directly penetrate the cell membrane of pathogens, thereby killing them. Current research on the production of antimicrobial lipopeptides using fermentation microorganisms mainly focuses on optimizing the culture medium and amino acids, primarily concentrating on the amino acid peptide ring. Research on the involvement of short-chain fatty acids is relatively limited. Regarding this aspect, the antimicrobial activity of antimicrobial lipopeptides produced by *Bacillus mojaveii* (a *Bacillus mojaveii* inoculant) needs further enhancement. Summary of the Invention

[0005] To address the problems existing in the background technology, this invention provides Bacillus mojaveii NK-3, a bacterial agent, and its application in aquaculture. During the fermentation process, the participation of short-chain fatty acid butyric acid can promote the formation of antimicrobial lipopeptide β-hydroxy fatty acid chains and promote bacterial metabolism, resulting in stronger antibacterial activity and enhanced and stable inhibitory effect against Staphylococcus aureus; it also reduces the DNA / RNA content of pathogens and increases the CT value of Mycoplasma synoviae in laying hens.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A species of Bacillus mojavensis, NK-3, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25408 and deposit date of June 10, 2022.

[0007] Preferably, the sieving method is as follows: Rhizosphere soil samples of Artemisia annua from the Ordos Desert in Inner Mongolia are brought back to the laboratory. 10g of the soil is weighed into a 90mL sterile Erlenmeyer flask containing physiological saline. Five sterile glass beads are placed in the flask. The flask is shaken at 180r / min for 30min and then allowed to stand for 10min to obtain a soil suspension. 1mL of the soil suspension is added to a sterile test tube containing 9mL of physiological saline to prepare 10... 2 Dilute the solution by 1:1 to obtain a diluted suspension; add 1 mL of the diluted suspension to another sterile test tube containing 9 mL of physiological saline, and repeat the process to prepare 10... 3 10 4 and 10 5 The serial dilution solution was prepared using a clean bench and the above procedures were performed in a laminar flow hood. The 10-fold serial dilution solution was then used. 3 10 4 and 10 5 The serially diluted solutions were inoculated onto LB solid agar medium using a sterile spreader. After inverting the plates in a 37°C incubator for 24 hours, the plates with large and sticky single colonies were selected. The single colonies were then purified on LB solid agar medium 2-3 times until no contaminants were found.

[0008] A Bacillus mojaverifolium agent, wherein the active ingredient of the Bacillus mojaverifolium agent is the aforementioned Bacillus mojaverifolium NK-3.

[0009] Preferably, the preparation method includes the following steps: S1. Inoculate a single colony of Bacillus mojaveii NK-3 into a seed culture medium and culture it to obtain a seed solution; S2. The seed culture is inoculated into a fermentation medium, and the pH is adjusted to 6.8-7.0 with butyric acid during the culture process to obtain the fermentation broth; S3. The fermentation broth is centrifuged, filtered, acid-precipitated, alcohol-oil extracted, adsorption-chromatographically concentrated and then dried to obtain the Bacillus mojavei inoculum.

[0010] Preferably, the seed culture medium comprises 10.00 g / L glucose, 10.00 g / L fish meal peptone, 5.00 g / L yeast extract, 5.00 g / L beef extract, and 5.00 g / L sodium chloride; the pH is adjusted to 7.2 with sodium hydroxide solution before sterilization, the liquid volume is 20%, and sterilization is carried out at 115°C for 30 min.

[0011] Preferably, the specific operation of the cultivation in S1 is as follows: cultivation for 20 hours in a constant temperature shaker at 35°C and 200 r / min.

[0012] Preferably, the fermentation medium comprises 35.00 g / L glucose, 8.00 g / L monosodium glutamate, 8.50 g / L enzymatically hydrolyzed soybean meal powder, 1.00 g / L yeast extract, 1.00 g / L beef extract, 0.50 g / L MgSO4, 0.50 g / L KCl, 1.00 g / L KH2PO4, 0.15 mg / L FeSO4, 5.00 mg / L MnSO4, and 0.20 mg / L CuSO4; the pH is adjusted to 7.5 with sodium hydroxide solution before sterilization, the fermentation tank is filled with 60% liquid, and sterilization is performed at 115°C for 30 min.

[0013] Preferably, in S2, the specific operation of the culture is as follows: fermentation temperature 35℃, rotation speed 180r / min, tank pressure 0.05, and ventilation ratio 1:1 for 20-22h; during this period, if the pH value is higher than 7.0, the pH value is adjusted with sterile butyric acid solution.

[0014] The application of a Bacillus mojaverii agent in aquaculture, wherein the active ingredient of the Bacillus mojaverii agent is the aforementioned Bacillus mojaverii NK-3, and the Bacillus mojaverii agent is used to inhibit Staphylococcus aureus; reduce the DNA / RNA content of pathogens; and increase the CT value of Mycoplasma synoviae in laying hens.

[0015] This application has the following beneficial effects: This invention provides a *Bacillus mojaverius* NK-3, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25408 and deposit date of June 10, 2022. This is the active ingredient in the *Bacillus mojaverius* inoculum prepared according to this invention. In the preparation of the *Bacillus mojaverius* inoculum of this invention, the participation of short-chain fatty acid butyric acid during fermentation promotes the formation of antimicrobial lipopeptide β-hydroxy fatty acid chains and promotes bacterial metabolism, resulting in stronger antibacterial activity and a strong and stable inhibitory effect against *Staphylococcus aureus*. Furthermore, it can reduce the DNA / RNA content of pathogens and increase the CT value of *Mycoplasma synoviae* in laying hens. Attached Figure Description

[0016] Figure 1 The inhibitory effect of the Bacillus mojavecifolius agent of this invention on Staphylococcus aureus is shown in the figure. Figure 2 Figure 1 shows the test report on the detection of the effect of the Bacillus mojavei bacterium agent on the CT value of Mycoplasma synoviae in laying hens. Figure 3 The experimental diagram showing the effect of the Bacillus mojaveii agent of this invention on Mycoplasma synovitis antibodies. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise specified, the raw materials used in the examples and comparative examples of the present application are all commercially available. Example

[0019] This embodiment provides a Mojavectomyces NK-3, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25408 and deposit date of June 10, 2022.

[0020] The screening method for obtaining Mojavec NK-3 is as follows: All Erlenmeyer flasks, test tubes, and glass beads used must be autoclaved (121°C, 20 min) to prevent contamination by other microorganisms. The sterilized glass beads are then used to physically break up soil aggregates, improving the efficiency of microbial release.

[0021] The rhizosphere soil sample of Artemisia annua from the Ordos Desert in Inner Mongolia was brought back to the laboratory. 10g of the sample was weighed into a 90mL sterile Erlenmeyer flask containing physiological saline. Five sterile glass beads were added to the flask. The mixture was shaken at 180 rpm for 30 minutes and then allowed to stand for 10 minutes to obtain a soil suspension. 1mL of this soil suspension was added to a sterile test tube containing 9mL of physiological saline to prepare 10... 2 Dilute the solution by 1:1 to obtain a diluted suspension; add 1 mL of the diluted suspension to another sterile test tube containing 9 mL of physiological saline, and repeat the process to prepare 10... 3 10 4 and 10 5 The serial dilution solution was prepared, and the above procedures were performed in a clean bench. The above 10 3 10 4 and 10 5 The serially diluted solutions were inoculated onto LB agar plates using a sterile spreader. After incubating upside down at 37°C for 24 hours, plates with large, sticky single colonies were selected and purified 2-3 times on LB agar plates until no contaminating bacteria were observed (microscopic examination confirmed uniform bacterial morphology). For streaking isolation, single colonies were streaked again onto fresh LB agar plates, and after incubation, only single colonies were observed. This yielded *Bacillus mojaverius* NK-3.

[0022] This embodiment also provides a Bacillus mojaveii inoculant, the active ingredient of which is the aforementioned Bacillus mojaveii NK-3.

[0023] The preparation method of this Bacillus mojavei inoculum includes the following steps: S1. Inoculate a single colony of Bacillus mojaveii NK-3 into a seed culture medium and culture it to obtain a seed solution.

[0024] Specifically, the seed culture medium includes 10.00 g / L glucose, 10.00 g / L fish meal peptone, 5.00 g / L yeast extract, 5.00 g / L beef extract, and 5.00 g / L sodium chloride. The weighing error of each component is ≤ ±0.1 g / L. After dissolving, the medium is brought to a final volume. Before sterilization, the pH is adjusted to 7.2 by slowly adding 1M sodium hydroxide solution dropwise (to avoid localized excessive alkalinity). The prepared culture medium is dispensed into Erlenmeyer flasks, with a filling volume of 20%, meaning each flask contains 20% of the flask's volume of culture medium. The flasks are then capped with cotton plugs or breathable membranes and autoclaved at 115°C for 30 minutes. After sterilization, the medium is cooled to room temperature before use. Sterility must be verified before use (a small amount of culture medium can be spread on an LB agar plate and incubated for 24 hours to observe for the presence of contaminants).

[0025] A single, typical colony was picked from a purified *Bacillus mojaveii* NK-3 single-colony agar plate using a sterile inoculation loop and inoculated into seed culture medium. The culture was carried out on a constant-temperature shaker at 35±0.5℃ and a rotation speed of 200±10 r / min for 20 hours to obtain the seed culture. The seed culture was uniformly turbid, odorless, and showed numerous free spores and vegetative cells under a microscope.

[0026] S2. Inoculate the seed culture into the fermentation medium, and use butyric acid to adjust the pH to 6.8-7.0 during the culture process to obtain the fermentation broth.

[0027] Specifically, the fermentation medium includes 35.00 g / L glucose, 8.00 g / L monosodium glutamate, 8.50 g / L enzymatically hydrolyzed soybean meal, 1.00 g / L yeast extract, 1.00 g / L beef extract, 0.50 g / L MgSO4, 0.50 g / L KCl, 1.00 g / L KH2PO4, 0.15 mg / L FeSO4, 5.00 mg / L MnSO4, and 0.20 mg / L CuSO4. Before sterilization, the pH is adjusted to 7.5 with 1M sodium hydroxide solution. The fermentation tank is filled with 60% liquid, meaning the fermentation liquid volume accounts for 60% of the working volume of the fermentation tank. Sterilization is performed at 115℃ for 30 min, and then cooled to 35℃ for later use.

[0028] Seed culture was inoculated at a volume ratio of 6%. Fermentation parameters were as follows: temperature 35±0.5℃, stirring speed 180±5 r / min, tank pressure maintained at 0.05 MPa (gauge pressure), aeration ratio of 1:1 (i.e., the volume of air introduced per minute equals the volume of fermentation liquid), and dissolved oxygen control maintained at approximately 30% saturation by adjusting stirring speed and aeration rate. pH control: Real-time monitoring was performed using an online pH electrode. When pH > 7.0, butyric acid solution (preset concentration 0.5%) was slowly added through a sterile microporous filter, with each adjustment decreasing by approximately 0.2 pH units to avoid drastic fluctuations and ensure pH control at 6.8-7.0. After 21 hours of co-fermentation, the termination time was determined by the following indicators: spore formation rate ≥ 80% observed under a microscope; OD 600 The fermentation broth reached a plateau; residual sugar concentration <1 g / L (detected by HPLC). Spore concentration in the fermentation broth ≥1.3×10⁻⁶. 10 CFU / mL.

[0029] S3. The fermentation broth is centrifuged, filtered, acid-precipitated, alcohol-oil extracted, adsorption-chromatographically concentrated and then dried to obtain Bacillus mojaveii inoculum.

[0030] Specifically, centrifuge at 8000-10000×g centrifugal force, 4℃ for 20-30 min, discard the supernatant and collect the bacterial sludge; then resuspend the bacterial sludge in physiological saline and repeat centrifugation twice to remove residual culture medium components.

[0031] Filtration: First, use double-layer gauze for coarse filtration to remove large particulate impurities, and then use a 0.22μm microporous membrane for fine filtration to obtain a clear filtrate.

[0032] Acid precipitation: Slowly add food-grade hydrochloric acid to the filtrate to adjust the pH to 4.5-5.0, and let it stand at low temperature (4℃) for 2 h to precipitate impurities such as proteins; then centrifuge (under the same conditions) to collect the supernatant and discard the precipitate.

[0033] Alcohol extraction: Add anhydrous ethanol and petroleum ether (boiling range 60-90℃) at a volume ratio of 1:1, mix thoroughly, and allow to stand for separation; the target product is enriched in the ethanol phase (upper layer), carefully remove it, and then remove the organic solvent by rotary evaporation.

[0034] Adsorption chromatography: Macroporous adsorption resin (such as XAD-16 type) is selected as the column material, and the column is packed by wet method; the concentrated solution is loaded onto the chromatography column and the flow rate is controlled at 1 BV / h (bed volume / hour); water, 30% ethanol and 60% ethanol are used for gradient elution, and the high-titer fraction is collected (the activity is determined by spore counting method).

[0035] Vacuum concentration and drying: Vacuum concentration at 45℃ to 1 / 10 of the original volume; then freeze drying technology (pre-freeze-sublimation drying) is used to protect spore activity; after drying, it is ground into powder, passed through an 80-mesh sieve, and packaged for storage in the dark.

[0036] Test Example 1 Experimental Methods: ① Culture Medium Preparation: Using existing aseptic techniques, a culture medium containing Staphylococcus aureus was precisely prepared. The pH of the medium was maintained between 6.5 and 7.5 to provide the optimal growth environment. The temperature was controlled at 37℃ to simulate human body temperature and promote bacterial growth. ② Hole Punching and Addition of Bacterial Agent: Using a sterile punch, holes were uniformly punched in the culture medium, with each hole having a diameter of 5 mm and a spacing of 30 mm. 100 μL of the prepared Bacillus mojaveii bacterial agent was precisely added to each hole. ③ Incubation and Observation: The prepared culture medium was placed in a 37℃ incubator and incubated for 36 hours, during which bacterial growth was observed. After incubation, the size of the inhibition zone around each hole was observed and recorded to evaluate the antibacterial effect of Bacillus mojaveii. Experimental Results: See [link to results]. Figure 1 .

[0037] Results Analysis: Figure 1 It can be seen that, Figure 1 The areas marked with numbers indicate the punching locations, where the Bacillus mojaverii agent formed distinct inhibition zones. This demonstrates that the Bacillus mojaverii agent of this invention has a significant inhibitory effect on Staphylococcus aureus.

[0038] Experimental Example 2 1. Experimental Methods: Laying hens were treated with the *Bacillus mojavemi* inoculum prepared in the previous example for 56 days, during which the CT values ​​of *Mycoplasma synoviae* in the hens were monitored regularly. The treatment was primarily achieved through feed additives, environmental optimization, and comprehensive health management, aiming to improve flock immunity and production performance. Regarding feed additives, normal feeding was maintained, with the *Bacillus mojavemi* inoculum prepared in the previous example continuously added to the feed at a mass ratio of 0.5%. Regarding the rearing environment, ventilation in the chicken house was improved, stocking density was reduced, and fresh air was maintained daily. Routine disinfection was carried out twice a week using a safe disinfectant (such as 0.1% Weidao brand disinfectant) to thoroughly spray the chicken house, waterers, and feed troughs. The temperature in the chicken house was controlled between 10℃ and 25℃, and the humidity between 55% and 65%, ensuring a comfortable and stable environment. Observe the chickens' feed intake, feces condition, feathers, and respiration daily, and promptly isolate sick or weak individuals to prevent disease transmission; avoid stressors such as changes in light or feed; maintain a fixed lighting time of 15 hours / day, and supplement drinking water with vitamins to alleviate stress. Experimental results: see [link to experiment]. Figure 2 .

[0039] Results Analysis: Figure 2 It can be seen that, Figure 2 The results of three experiments were presented, conducted on April 7, April 29, and June 2, 2025, respectively. Figure 2 The study meticulously recorded the fluorescence curves and CT values ​​for each experiment, including the CT values ​​and corresponding fluorescence intensities of different samples at different time points, providing strong data support for the experimental results. As the experimental time progressed, the CT value of *Mycoplasma synoviae* in laying hens showed a gradual increasing trend, indicating a gradual decrease in viral load, with some experimental results even reaching negative levels. This demonstrates that the *Bacillus mojavir* agent of this invention can reduce the pathogen's DNA / RNA content and increase the CT value of *Mycoplasma synoviae* in laying hens.

[0040] 2. Experimental Methods: The clinical production trial consisted of seven groups, with 21 laying hens in each group. Each group included hens with joint swelling. Before product use, blood was collected from under the wings of each group, and the average antibody level was measured using an ELISA kit. The average antibody level was measured again after 14 and 49 days of product use. Results: See [link to results]. Figure 3 .

[0041] Results analysis: such as Figure 3 As shown, after effective treatment, the pathogen lyses, and antigens are concentratedly exposed. Antibody levels begin to rise briefly at 2 weeks, then gradually the pathogen is cleared, antigen stimulation decreases, and antibody levels continue to decline. Overall, antibody levels show a trend of first rising and then falling.

[0042] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0043] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A type of Bacillus mojaverius NK-3, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25408 and deposit date of June 10, 2022.

2. The Mojavec bacillus NK-3 according to claim 1, characterized in that, The method for obtaining the sieve is as follows: Rhizosphere soil samples of Artemisia annua from the Ordos Desert in Inner Mongolia were brought back to the laboratory. 10g of the soil was weighed into a 90mL sterile Erlenmeyer flask containing physiological saline. Five sterile glass beads were placed in the flask. The mixture was shaken at 180r / min for 30min and then allowed to stand for 10min to obtain a soil suspension. 1mL of the soil suspension was added to a sterile test tube containing 9mL of physiological saline to prepare 10... 2 Dilute the solution by 1:1 to obtain a diluted suspension; add 1 mL of the diluted suspension to another sterile test tube containing 9 mL of physiological saline, and repeat the process to prepare 10... 3 10 4 and 10 5 10-fold serial dilution solution; the above 10 3 10 4 and 10 5 The serially diluted solutions were inoculated onto LB solid agar medium using a sterile spreader. After inverting the plates in a 37°C incubator for 24 hours, the plates with large and sticky single colonies were selected. The single colonies were then purified 2-3 times on LB solid agar medium to obtain the final product.

3. A Bacillus mojavecifolius inoculant, characterized in that, The active ingredient of the Mojave Bacillus agent is the Mojave Bacillus NK-3 as described in claim 1 or 2.

4. The Bacillus mojavenifolia inoculant according to claim 3, characterized in that, Its preparation method includes the following steps: S1. Inoculate a single colony of Bacillus mojaveii NK-3 into a seed culture medium and culture it to obtain a seed solution; S2. The seed culture is inoculated into a fermentation medium, and the pH is adjusted to 6.8-7.0 with butyric acid during the culture process to obtain the fermentation broth; S3. The fermentation broth is centrifuged, filtered, acid-precipitated, alcohol-oil extracted, adsorption-chromatographically concentrated and then dried to obtain the Bacillus mojavei inoculum.

5. The Bacillus mojavenifolia inoculant according to claim 4, characterized in that, The seed culture medium comprises 10.00 g / L glucose, 10.00 g / L fish meal peptone, 5.00 g / L yeast extract, 5.00 g / L beef extract, and 5.00 g / L sodium chloride; before sterilization, the pH is adjusted to 7.2 with sodium hydroxide solution, the liquid volume is 20%, and sterilization is carried out at 115℃ for 30 min.

6. The Bacillus mojavenifolia inoculum agent according to claim 4 or 5, characterized in that, In S1, the specific operation for cultivation is as follows: incubate for 20 hours at 35℃ and 200r / min in a constant temperature shaker.

7. The Bacillus mojavecifolius agent according to claim 4, characterized in that, The fermentation medium comprises 35.00 g / L glucose, 8.00 g / L monosodium glutamate, 8.50 g / L enzymatically hydrolyzed soybean meal powder, 1.00 g / L yeast extract, 1.00 g / L beef extract, 0.50 g / L MgSO4, 0.50 g / L KCl, 1.00 g / L KH2PO4, 0.15 mg / L FeSO4, 5.00 mg / L MnSO4, and 0.20 mg / L CuSO4. Before sterilization, the pH is adjusted to 7.5 with sodium hydroxide solution. The fermentation tank is filled with 60% liquid and sterilized at 115°C for 30 minutes.

8. The Bacillus mojavenifolia inoculum agent according to claim 4 or 7, characterized in that, In S2, the specific operation for cultivation is as follows: fermentation temperature 35℃, rotation speed 180r / min, tank pressure 0.05, and ventilation ratio 1:1 for 20-22h; during this period, if the pH value is higher than 7.0, the pH value is adjusted with sterile butyric acid solution.

9. The application of a Bacillus mojaverii in aquaculture, characterized in that, The active ingredient of the Bacillus mojaveii agent is Bacillus mojaveii NK-3 as described in claim 1 or 2. The Bacillus mojaveii agent is used to inhibit Staphylococcus aureus, reduce the DNA / RNA content of pathogens, and increase the CT value of Mycoplasma synoviae in laying hens.

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