Bacillus subtilis and its application in the prevention and treatment of diseases of Chinese mitten crab

Abstract

The invention relates to a bacillus subtilis, and in particular to a screened bacillus subtilis JK-2 having an antagonistic effect on pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 of the invention can be used to antagonize pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 of the invention can significantly improve the activity of lysozyme in the serum and hepatopancreas of Chinese mitten crabs, can enhance the ability of Chinese mitten crabs to eliminate pathogenic bacteria, and can be used for the prevention and treatment of diseases of Chinese mitten crabs.

Description

Technical Field

[0001] The invention relates to a Bacillus subtilis and application thereof in the prevention and treatment of diseases of Chinese mitten crab, in particular to Bacillus subtilis JK-2 and a microbial agent thereof, and application thereof in antagonizing pathogenic bacteria of aquatic animals and in the prevention and treatment of bacterial diseases of Chinese mitten crab, etc., belonging to the technical field of aquatic antimicrobial drugs. Background Art

[0002] In recent years, with the rapid development of intensive aquaculture, bacterial diseases have become the most common and most harmful diseases in Chinese mitten crab (Eriocheir sinensis) aquaculture, accounting for the largest proportion of disease outbreaks in each year.

[0003] For example, diseases such as gill rot, enteritis, tremor and hepatopancreatic necrosis caused by various pathogens such as Aeromonas punctata, Aeromonas hydrophila, Citrobacter freundii and Aeromonas vermiformis in aquaculture environments have caused huge economic losses to Chinese mitten crab farming in my country. In general, the problems of many types of pathogens, fast reproduction, strong pathogenicity and high incidence have seriously affected the healthy development of Chinese mitten crab farming in my country.

[0004] In the field of prevention and control of aquatic diseases, the hormone drugs, chemical drugs and antibiotics of the past have gradually been replaced by highly effective and zero-residue microbial agents due to their toxic side effects, drug resistance, and especially the destructiveness of drug residues to the aquaculture environment and the threat to the quality of aquatic products. Microbial agents are highly safe, can improve the aquaculture ecological environment, are not prone to drug resistance, can also improve the utilization efficiency of nutrients, enhance the immunity of aquatic animals, promote the body's metabolism and growth, and also have the advantages of low price and abundant sources.

[0005] How to use microbial agents to enhance the resistance of Chinese mitten crab to bacterial diseases has become an urgent problem to be solved in the aquaculture industry.

[0006] At present, enhancing the antioxidant capacity and immunity of the Chinese mitten crab is one of the most effective strategies for the prevention and control of bacterial diseases in the Chinese mitten crab. For example, some excellent strains of the genus Bacillus, such as Bacillus amylolyticus, Bacillus methylotrophicus, and Bacillus megaterium, which have been made public, have good antibacterial activity and have good effects in enhancing the antioxidant capacity and immunity of the body, greatly enriching the microbial resources for the development of Bacillus as a biological control product for bacterial diseases in the Chinese mitten crab.

[0007] Therefore, the art hopes to screen new strains that antagonize pathogenic bacteria in aquatic animals and develop effective microbial agents for preventing and controlling diseases of Chinese mitten crabs. Summary of the invention

[0008] In view of the above problems and / or other problems of the related art, the first aspect of the present invention provides a Bacillus subtilis JK-2, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: M20221568.

[0009] A second aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the above-mentioned Bacillus subtilis JK-2.

[0010] Preferably, the microbial agent comprises the fermentation broth of the above-mentioned Bacillus subtilis JK-2, or a bacterial liquid preparation made from the fermentation broth, or a bacterial powder preparation made by drying the fermentation broth.

[0011] The third aspect of the present invention provides the use of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent in antagonizing pathogenic bacteria of aquatic animals.

[0012] Specifically in the present application, aquatic animal pathogens refer to pathogens commonly found in freshwater aquaculture environments.

[0013] Preferably, the aquatic animal pathogenic bacteria are pathogenic bacteria of shrimps and crabs.

[0014] Specifically in the present application, pathogenic bacteria of shrimps and crabs refer to pathogenic bacteria that are commonly found in freshwater aquaculture environments and cause diseases in shrimps and crabs, for example, Aeromonas punctata, Aeromonas hydrophila, Citrobacter freundii, Aeromonas vermiformis, Providencia alcaligenes and other pathogens.

[0015] Preferably, the pathogenic bacteria of shrimps and crabs are selected from Aeromonas veronis, Aeromonas hydrophila, Aeromonas punctata, Providencia alcaligenes and Citrobacter freundii.

[0016] A fourth aspect of the present invention provides a product for antagonizing pathogenic bacteria of aquatic animals, wherein the product comprises the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent in improving the serum lysozyme activity and / or hepatopancreas lysozyme activity of Chinese mitten crab.

[0018] A sixth aspect of the present invention provides a Chinese mitten crab feed, which comprises a feed basic component and the above-mentioned Bacillus subtilis JK-2.

[0019] The seventh aspect of the present invention provides the use of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent or the above-mentioned Chinese mitten crab feed in the prevention and treatment of bacterial diseases of Chinese mitten crab.

[0020] The invention relates to a bacillus subtilis, in particular to a screened bacillus subtilis JK-2 having an antagonistic effect on pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 of the invention can be used to antagonize pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 of the invention can significantly improve the activity of lysozyme in the serum and hepatopancreas of Chinese mitten crabs, can enhance the ability of Chinese mitten crabs to eliminate pathogenic bacteria, and can be used for the prevention and treatment of diseases of Chinese mitten crabs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The antagonistic activity of the four strains (JK-1, JK-2, JK-3 and JK-4) isolated in the initial screening and the control strain against Aeromonas vernii HXH1;

[0022] Figure 2 The antagonistic activity of strain JK-2 against four other known aquatic pathogens;

[0023] Figure 3 This is a photo of the colony morphology of strain JK-2 formed on the nutrient agar plate;

[0024] Figure 4 This is a photo of strain JK-2 under a 100x optical microscope after Gram staining;

[0025] Figure 5 The effect of strain JK-2 on the serum lysozyme content of Chinese mitten crab;

[0026] Figure 6 The effect of strain JK-2 on the lysozyme content in the hepatopancreas of Chinese mitten crab;

[0027] Figure 7 Phylogenetic tree constructed based on the 16S rRNA sequence of strain JK2. DETAILED DESCRIPTION

[0028] The present invention is further described below through specific implementation modes, but the present invention is not limited to these specific implementation modes.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the following specific embodiments can be obtained from commercial sources. If no specific technology or conditions are specified, the technology or conditions described in the literature in the field or the product instructions are used.

[0030] 1. The following is an introduction to the operation process of the inventors of the present application to isolate and screen the new strain for preventing and treating bacterial diseases of Chinese mitten crabs, Bacillus subtilis JK-2 (Bacillus subtilis JK-2) CCTCC No: M20221568, from the natural environment.

[0031] 1) Initial screening

[0032] 1.0 g of aquaculture sludge (taken from a breeding pond in Weihai City, Shandong Province) was suspended in 9 mL of sterile saline, oscillated and mixed, and then placed in an 80°C water bath for 10 min. The strains were isolated and purified using the dilution coating plate method and the streak plate method on nutrient agar plates (nutrient agar purchased from Sinopharm Chemical Reagent Co., Ltd.).

[0033] The purified single colonies were inoculated into sterile nutrient broth (purchased from Sinopharm Chemical Reagent Co., Ltd.) and cultured in a shaker at 33°C and 180 r / min for 24 h. Then, 0.1 mL of the culture solution was aseptically pipetted and evenly spread on a nutrient agar plate. After constant temperature culture at 33°C for 24 h, the bacterial lawn was washed with 0.85% sterile saline to make a final concentration of 1.0 × 10 9 CFU / mL of bacterial suspension was stored for later use.

[0034] In the initial screening step, four strains (named: JK-1, JK-2, JK-3 and JK-4) were successfully isolated from the aquaculture sludge.

[0035] 2) Further screening

[0036] The pathogenic bacteria "Aeromonas vernix HXH1" (purchased from the National Aquatic Animal Pathogen Bank) was used as the screening indicator bacteria; 0.1 mL of Aeromonas vernix HXH1 bacterial suspension was aseptically pipetted and evenly spread on the nutrient agar plate, and then 10 μL of the bacterial suspension of the four strains obtained in the initial screening was aseptically pasted on the plate (concentration of 1.0×10 9 CFU / mL) were cultured at 33°C for 24 h and the ratio (H / C) of the inhibition zone diameter (H) to the colony diameter (C) was determined.

[0037] At the same time, the known Bacillus amyloliquefaciens G1 and Bacillus licheniformis PNB3 (purchased from the National Aquatic Animal Pathogen Bank) were used as positive control antagonistic strains to determine the ratio (H / C) of the inhibition zone diameter (H) and the colony diameter (C).

[0038] The experimental results can be found in Figure 1 .from Figure 1It can be seen that strain JK2 has the strongest antagonistic activity against the pathogenic bacterium Aeromonas veronii HXH1. Specifically, the H / C ratio of strain JK2 against Aeromonas veronii HXH1 was 2.66, which was 47.78% (P<0.05), 27.27% (P<0.05), and 97.04% (P<0.05) higher than the H / C of strains JK1, JK3, and JK4, respectively; and 10.83% (P<0.05) and 22.02% (P<0.05) higher than the H / C of the positive control antagonistic strains (Bacillus amyloliquefaciens G1 and Bacillus licheniformis PNB3) against Aeromonas veronii HXH1, respectively.

[0039] The experimental data in this article are expressed as mean ± standard deviation and statistically analyzed using SPSS 19.0 software. P < 0.05 indicates a significant difference (the same applies below).

[0040] To further verify the above antibacterial results, the candidate strains including strain JK-2 were further tested for the ratio (H / C) of the inhibition zone diameter (H) and the colony diameter (C) against four known aquatic animal pathogens, “Aeromonas hydrophila WS05”, “Aeromonas punctata DD5”, “Providencia alcaligenes P3” and “Citrobacter freundii C1” (all purchased from the National Aquatic Animal Pathogen Bank) according to the above operation.

[0041] The experimental results can be found in Figure 2 , it can be seen that strain JK2 also has good antagonistic activity against four known aquatic animal pathogens, “Aeromonas hydrophila WS05”, “Aeromonas punctata DD5”, “Providencia alcaligenes P3”, and “Citrobacter freundii C1”, with H / C values ​​greater than 1.80.

[0042] Therefore, strain JK-2 was selected as the superior strain.

[0043] Aeromonas vernix HXH1, Aeromonas hydrophila WS05, Aeromonas punctata DD5, Providencia alcaligenes P3, and Citrobacter freundii C1 are all relatively common pathogens of shrimps and crabs. Based on the above results, those skilled in the art can conceive of applying the screened strain JK-2 to antagonize pathogens of aquatic animals, especially to antagonize pathogens of shrimps and crabs; it can be conceived that the screened strain JK-2 is used in products for antagonizing pathogens of aquatic animals.

[0044] 2. The identification process of the strain JK-2 obtained by screening is as follows:

[0045] (1) Molecular biological identification

[0046] Based on the bacterial 16S rRNA sequence, phylogenetic analysis was used to perform molecular biological identification on the screened strain JK-2.

[0047] Specifically, the strain JK-2 obtained by screening was inoculated into 100 mL of sterile nutrient broth, cultured at 33°C and 180 r / min for 24 h, centrifuged at 4°C and 12000 r / min for 1 min, the supernatant was discarded, and then the genomic DNA of the strain JK-2 was extracted using the Ezup column genomic DNA extraction kit.

[0048] Then, the genomic DNA obtained was used as a template to perform PCR amplification of its 16S rRNA. The forward primer was 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, and the reverse primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′; the PCR amplification system (20 μL) was Premix Taq 10 μL, upstream and downstream primers 1 μL each, template 1 μL, ddH 2 O 7μL; PCR amplification conditions were: 94℃3min; 94℃1min, 60℃1min, 72℃1min, for a total of 35 cycles. The amplified 16S rRNA gene was sequenced by Shanghai Maipu Biotechnology Co., Ltd.

[0049] The DNA sequence corresponding to the 16S rRNA of strain JK-2 determined is as follows (SEQ ID NO.1):

[0050]

[0051] The 16S rRNA sequence of strain JK-2 was compared for homology using NCBI's BLAST. The comparison results showed that the 16S rRNA sequence of strain JK-2 naturally clustered with the 16S rRNA sequence of Bacillus subtilis in the GenBank database, with a homology of more than 99%.

[0052] The phylogenetic tree was constructed using the neighbor-joining method (repeat F1000) using MEGA 5.0 software, which further proved that strain JK-2 was most closely related to Bacillus subtilis A65.1 (GenBank accession number: ON366398); see Figure 7 .

[0053] (2) Morphological identification

[0054] The colony morphology of strain JK-2 on nutrient agar plates can be found in Figure 3 Its colony characteristics are white, irregular edges, smooth and easy to pick.

[0055] Strain JK-2 is a Gram-positive bacterium. The photo of the strain JK-2 under a 100x optical microscope after Gram staining is shown in Figure 4 , it can be seen that its shape is rod-shaped. It is similar to the common Bacillus subtilis in shape.

[0056] (3) Physiological and biochemical identification

[0057] Physiological and biochemical test traits were selected with reference to the relevant contents of identification of corresponding genera and species in the Manual of Systematic Identification of Common Bacteria (first edition) compiled by Dong Xiuzhu et al., and bacterial microbiochemical identification tubes were used to conduct physiological and biochemical identification of strain JK-2.

[0058] The identification results are shown in Table 1 below:

[0059]

[0060]

[0061] As can be seen from Table 1 above, the physiological and biochemical characteristics of strain JK-2 are completely consistent with those of Bacillus subtilis B25 (Literature: Lin Baoying, Analysis and Separation and Purification of Some Characteristics of Antifungal Active Substances from Bacillus subtilis B25, Hainan University, 2013).

[0062] In summary, combined with the results of 16S rRNA molecular identification, morphological observation and physiological and biochemical identification, it can be determined that the strain JK-2 obtained by the screening of the present invention is Bacillus subtilis.

[0063] III. Preservation of strains

[0064] The strain JK-2 of the present invention belongs to Bacillus subtilis, has been deposited in the China Center for Type Culture Collection, the address of the depository unit is Wuhan, China, the deposit date is October 14, 2022, and its deposit number is CCTCC No: M 20221568.

[0065] 4. Fermentation and culture method of strain JK-2 of the present invention

[0066] A single colony of the strain JK-2 of the present invention was inoculated into 20 mL of nutrient broth or other Bacillus fermentation medium, and cultured at 33°C and 180 rpm for 24 hours to obtain a bacterial solution concentration greater than 10 8 CFU / mL; then transfer it to 200mL nutrient broth or other Bacillus fermentation medium at a 10% inoculation amount, and culture it at 33°C and 180r / min shaking for 24 hours to obtain the fermentation broth of strain JK-2 of the present invention. After testing, the bacterial concentration of the fermentation broth is about 10 9 -10 10 CFU / mL.

[0067] The strain JK-2 of the present invention has a fast growth rate, and the operating conditions of the fermentation culture are relatively simple. Rapid high-density fermentation is carried out in an aerobic, sterile fermentation medium environment, energy is saved, and contamination by miscellaneous bacteria is reduced, and the quality of the obtained fermentation product is controllable. In summary, the strain JK-2 of the present invention is an excellent strain that can be applied to industrial fermentation production.

[0068] V. Safety test of Bacillus subtilis JK-2 of the present invention on Chinese mitten crab

[0069] The experiment set up 1 control group and 5 experimental groups, with 3 parallels in each group. Ten healthy Chinese mitten crabs were placed in a plastic aquarium (76cm×45cm×55cm) filled with 60L aerated tap water, and the water temperature was controlled at 28℃. The Chinese mitten crabs in the experimental group were injected with 0.2mL of a final concentration of 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 CFU / mL of Bacillus subtilis JK-2 suspension (bacterial suspension formed in sterile saline); the control group was injected with an equal amount of sterile saline. The experimental period was 7 days. The number of deaths of Chinese mitten crabs was recorded every day during the experiment, and the median lethal concentration (LC50) of Bacillus subtilis JK-2 on Chinese mitten crabs was calculated using the Bliss method. 50).

[0070] The safety test results of Bacillus subtilis JK-2 on Chinese mitten crab showed that the concentration of 1.0×10 5 ~1.0×10 9 CFU / mL of strain JK2 was injected into healthy Chinese mitten crabs, and the Chinese mitten crabs were all active, without limb weakness, fighting or death, and the control group also did not have any death or abnormal phenomenon, indicating that Bacillus subtilis JK-2 has a LC effect on Chinese mitten crabs. 50 >1.0×10 9 CFU / mL.

[0071] VI. Application effect of Bacillus subtilis JK-2 of the present invention on preventing and treating bacterial diseases of Chinese mitten crab

[0072] Healthy Chinese mitten crabs with no disease or injury, strong physique and uniform size were selected and placed in an aquarium. Tiles were placed in the box as cover and the Chinese mitten crabs were tamed with basic feed. The experiment was started after the crabs were eating normally without death and in a stable state.

[0073] The experiment consisted of 3 experimental groups and 1 control group. Each group had 3 parallel aquariums, and each aquarium contained 40 fish.

[0074] Experimental group: Bacillus subtilis JK-2 of the present invention was added to the feed; specifically, a bacterial suspension of Bacillus subtilis JK-2 (a bacterial suspension formed in sterile water) was added to the basic feed (Mudundao brand crab compound feed purchased from Jiangsu Hongxiang Feed Technology Co., Ltd.) so that the final content of Bacillus subtilis JK-2 in the feed was 1.0×10 5 , 1.0×10 6 , 1.0×10 7 CFU / g feed, used as the feed for the three experimental groups.

[0075] Control group: fed with basic feed (without adding microorganisms).

[0076] Feed once at 8:00 and 18:00 every day, and the feeding amount is 3% of the crab body weight. The test period is 20 days. During the test, the water temperature is maintained at 28℃, pH≤8.0, ammonia nitrogen≤0.20mg / L, nitrite≤0.01mg / L, dissolved oxygen≥5.5mg / L, and the water is changed once a day, and the water change amount is 1 / 3 of the water volume. A 40W fluorescent lamp is used as the light source, and the light and dark time are 12h respectively.

[0077] After the experiment, 5 crabs were randomly selected from each group to collect their hemolymph and hepatopancreas. The lysozyme content in serum and hepatopancreas was determined according to the instructions of the kit (lysozyme (LZM) test kit from Nanjing Jiancheng Bioengineering Institute). The results are shown in Figure 5 and Figure 6 .

[0078] Notes: Figure 5 and Figure 6 The T0 group represents the control group fed with basic feed (without microbial bacteria); the T1 group represents the control group fed with a final content of 1.0×10 5 CFU / g feed of Bacillus subtilis JK-2 in the experimental group 1; T2 group was fed with a final content of 1.0×10 6 CFU / g feed of Bacillus subtilis JK-2 in the experimental group 2; T3 group was fed with a final content of 1.0×10 7 CFU / g of feed containing Bacillus subtilis JK-2.

[0079] from Figure 5 The experimental results show that the final contents of T1-3 groups (using strain JK2) were 1.0×10 5 ~1.0×10 7 CFU / g of feed was continuously fed to Chinese mitten crab for 20 days, the serum lysozyme activity of Chinese mitten crab was significantly enhanced. Specifically, it was increased by 5.91% to 10.97% compared with the results of T0 group (fed with basic feed) (P<0.05).

[0080] from Figure 6 The experimental results show that the final contents of T1-3 groups (using strain JK2) were 1.0×10 5 ~1.0×10 7 CFU / g feed was continuously fed to Chinese mitten crab for 20 days, the hepatopancreatic lysozyme activity of Chinese mitten crab was significantly enhanced. Specifically, it was increased by 71.77% to 132.26% (P<0.05) compared with the results of T0 group (fed with basic feed); and the hepatopancreatic lysozyme activity was significantly increased with the increase of strain JK2 content.

[0081] This shows that adding the Bacillus subtilis JK-2 of the present invention to the basic feed can increase the activity of lysozyme in the serum and hepatopancreas of Chinese mitten crab, especially significantly increase the activity of lysozyme in the hepatopancreas, and can enhance the ability of Chinese mitten crab to eliminate pathogens.

[0082] Based on the above results, those skilled in the art can conceive of applying the Bacillus subtilis JK-2 of the present invention to improve the serum lysozyme activity and / or hepatopancreas lysozyme activity of Chinese mitten crab, to improve the ability of Chinese mitten crab to eliminate pathogens, and to prevent and treat bacterial diseases of Chinese mitten crab.

[0083] Based on the above results, those skilled in the art can conceive of adding the Bacillus subtilis JK-2 of the present invention to the feed of Chinese mitten crab. 50 >1.0×10 9 CFU / mL, as well as serum lysozyme and hepatopancreatic lysozyme activities with the increase of strain JK2 content (based on Figure 5 and Figure 6 Therefore, the concentration of Bacillus subtilis JK-2 in Chinese mitten crab feed can be 1.0×10 5 ~1.0×10 9 CFU / g feed, for example 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 or 1.0×10 9 CFU / g feed.

[0084] In order to further verify that "the Bacillus subtilis JK-2 of the present invention can enhance the ability of Chinese mitten crab to eliminate pathogens", 10 crabs were randomly selected from each group for artificial infection test, and the Chinese mitten crabs in the control group and the test group were fed with basal feed containing 1.0×10 7 CFU / g feed of Aeromonas vervetii HXH1. The pathogen feed was fed once at 8:00 and 18:00 every day, and the feeding amount was 3% of the crab body weight. After 7 consecutive days of feeding, the mortality of each group of Chinese mitten crabs was observed and recorded, and the mortality rate (%) was calculated; and the disease protection rate (%) was calculated according to the following formula (1).

[0085]

[0086] The statistical results are shown in Table 2 below:

[0087]

[0088] From the results in Table 2, it can be seen that the ability of the Chinese mitten crab in the test group (fed with feed containing Bacillus subtilis JK-2 of the present invention) to antagonize the infection of pathogenic bacteria (Aeromonas verticilli HXH1) is significantly enhanced. The mortality rate of the Chinese mitten crab in the T1-T3 test groups was reduced by 43.34% to 66.67% compared with the mortality rate in the control group T0 group (P<0.05).

[0089] This indicates that feeding the feed prepared by adding the Bacillus subtilis JK-2 of the present invention to the basic feed can significantly improve the anti-infection ability of Chinese mitten crab against pathogenic bacteria (Aeromonas veronii HXH1).

[0090] In summary, based on the above-mentioned antibacterial experimental results of Bacillus subtilis JK-2 of the present invention ( Figure 1 and Figure 2 According to the experimental results of the antagonistic pathogenic bacteria Aeromonas vernix HXH1, Aeromonas hydrophila WS05, Aeromonas punctata DD5, Providencia alcaligenes P3 and Citrobacter freundii C1), the physiological and biochemical identification results, and the safety test on Chinese mitten crab and the application experimental results of the fifth and sixth parts for the prevention and treatment of bacterial diseases of Chinese mitten crab, those skilled in the art can use the Bacillus subtilis JK-2 of the present invention as a biocontrol agent, and apply it to antagonize pathogenic bacteria of aquatic animals, especially antagonize pathogenic bacteria of shrimps and crabs. It can be added to the feed of Chinese mitten crab as a biocontrol agent to enhance the anti-infection ability of the Chinese mitten crab body (the ability to remove pathogenic bacteria), and be applied to the prevention and treatment of bacterial diseases of Chinese mitten crab.

[0091] Those skilled in the art can produce the Bacillus subtilis JK-2 of the present invention by fermentation using a conventional Bacillus subtilis fermentation method (e.g., the fourth part above), and can obtain the Bacillus subtilis JK-2 fermentation broth or a broth preparation prepared from the fermentation broth or a powder preparation prepared by drying the fermentation broth according to a conventional microbial industrial production method.

[0092] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0093] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus subtilis JK-2, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: M 20221568.

2. A microbial agent, characterized in that: The microbial agent comprises the Bacillus subtilis JK-2 as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent comprises the fermentation broth of Bacillus subtilis JK-2 as claimed in claim 1, or a broth preparation prepared from the fermentation broth, or a powder preparation prepared by drying the fermentation broth.

4. Use of the Bacillus subtilis JK-2 according to claim 1 or the microbial agent according to claim 2 in preparing products for antagonizing pathogenic bacteria of aquatic animals; characterized in that: The aquatic animal pathogenic bacteria are pathogenic bacteria of shrimps and crabs; the shrimps and crabs pathogenic bacteria are selected from Aeromonas vernix, Aeromonas hydrophila, Aeromonas punctata, Providencia alcaligenes and Citrobacter freundii.

5. A product for antagonizing pathogenic bacteria in aquatic animals, characterized by: The product comprises the Bacillus subtilis JK-2 as claimed in claim 1 or the microbial agent as claimed in claim 2.

6. A Chinese mitten crab feed, characterized in that: The Chinese mitten crab feed comprises a feed base component and the Bacillus subtilis JK-2 as claimed in claim 1.

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