Separation and identification of bacillus velezensis with antagonistic effect on nocardia and application of bacillus velezensis

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to separation and identification of bacillus velezensis with an antagonistic effect on nocardia and application of the bacillus velezensis. The bacillus velezensis JZGDFSBV241119 strain is separated from healthy micropterus salmoides culture water, has a good antagonistic effect on nocardia and various pathogenic bacteria, is safe to use, free of residues and hemolytic activity, nontoxic and harmless to fishes and high in high-temperature resistance, acid resistance and salt resistance, and can be applied to the field of fish breeding. And the compound is highly sensitive to 18 antibiotics such as penicillin, ampicillin, piperacillin and the like. The strain can enhance the immune function and enzyme activity of micropterus salmoides and other aquatic animals and reduce the case fatality rate, is beneficial to healthy aquaculture, and is suitable for improving intestinal flora of fishes due to the fact that the strain has the main function of antagonizing pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and specifically relates to the separation, identification and application of a Velezella veleci strain having antagonistic effect on Nocardia. Background Art

[0002] Aquaculture plays a vital role in the global food supply chain, but with the prevalence of intensive farming models, the aquatic environment is becoming increasingly deteriorating. According to statistics, in recent years, the economic losses caused by aquatic animal diseases in the entire aquaculture industry have reached 48 billion yuan each year, of which bacterial diseases account for a significant proportion and have become one of the key factors restricting the development of aquaculture. Under the threat of pathogens, farmers have low breeding efficiency, slow growth of aquatic animals, and high mortality rates.

[0003] At present, the mainstream solution strategy for bacterial diseases mainly relies on the use of antibiotics. Although this strategy can achieve significant therapeutic effects in a short period of time, the pathogen infection conditions of farmed animals are complex and diverse, and there are often multiple infections. Most breeders are often helpless in this regard, and even "try all kinds of medical treatments in a hurry", which is mainly manifested in the abuse of antibiotics, leading to a series of problems such as increased antibiotic resistance, large-scale deaths of farmed animals, low breeding efficiency, ecological damage, and unsafe quality of aquatic products, which seriously threaten human health. Although probiotics have been proven to prevent and treat diseases caused by specific pathogens to a certain extent, the exploration of probiotics that are effective against Nocardia is still relatively limited. Therefore, it is urgent to seek efficient, safe and green alternatives.

[0004] As a kind of aquatic feed additive, probiotics have been widely used in aquaculture. They have multiple beneficial effects such as anti-inflammatory and antioxidant effects, can effectively regulate the balance of intestinal flora, improve the immunity of farmed animals and the absorption efficiency of nutrients, inhibit the growth of pathogens, improve water quality, enhance the body's immunity, promote growth and improve survival rate, and have the advantages of antibiotic sensitivity, no pollution, no residue, safety and no toxic side effects, and are ideal substitutes. However, the relevant work on the prevention and control of bacterial diseases in aquatic animals with antagonistic bacteria is progressing relatively slowly, and the resources of antagonistic bacteria that can be used for the prevention and control of aquatic pathogens are scarce, which is far from meeting the current market demand.

[0005] Nocardiosis is a chronic infectious disease caused by Nocardia that is common to both humans and animals. It is extremely harmful to aquaculture. Nocardiosis is a conditional pathogen. Factors such as excessive stocking density, deterioration of water and bottom quality, lack of vitamin intake, low immunity, sudden weather changes, unsuitable water temperature, and spoilage of bait can all lead to the occurrence of the disease. The disease has a long epidemic season and a long incubation period. The disease is chronic and slow to come and go. There are no symptoms or no obvious symptoms in the early stage of the disease. The incubation period is long. It takes 15-20 days from fish infection to death. Nocardiosis is difficult to treat, and the focus is on prevention. Infected fish species include amberjack, pomfret, large yellow croaker, fortune fish, tilapia, snakehead, largemouth bass, grouper, four-finger threadfin, purple snapper, red snapper, and six-band trevally. With the increasing intensiveness of aquaculture, diseases in the aquaculture process are becoming more and more serious. The natural incidence of fish Nocardia can reach 15% to 30%, and in severe cases it can reach 60%. The mortality rate of artificial infection can be as high as 90% to 100%. Since Nocardia can damage the immune organs of sick fish and there is currently no specific medicine, the disease is difficult to treat and the focus is on prevention. However, the current prevention and treatment of fish Nocardia is still mainly based on chemical drugs, but there are problems such as damaging the water environment and endangering human health.

[0006] Bacillus velezensis is a newly defined spore-forming bacteria species that has the effects of promoting growth and inhibiting pathogens, and has broad-spectrum antibacterial properties. In the current situation where the demand for antibiotic substitutes is increasing, studies have shown that Bacillus velezensis can be used for plant epidemic prevention and control, and can improve feed digestibility, inhibit pathogenic bacteria, improve production performance and prevent and treat diseases in animal husbandry such as livestock and aquatic animal breeding. The application of Bacillus velezensis in aquaculture shows significant probiotic potential. It can not only effectively inhibit a variety of aquatic pathogens, but also enhance the immunity and antioxidant capacity of farmed animals, and has good biosafety.

[0007] In general, there is no specific drug for the prevention and treatment of Nocardia in aquatic products, so the disease is difficult to treat and the focus is on prevention. The existing technology has high investment and cost for the prevention and treatment of Nocardia, but the effect is minimal. Moreover, the use of a large number of drugs is harmful to the human body, and it is still necessary to consider food safety. It is easy to reduce the immunity of fish species against pathogens and affect their growth. At the same time, in the treatment of Nocardiosis, early studies have shown that feeding erythromycin can have a certain preventive effect, but in recent years, studies have shown that for erythromycin and spiramycin, the incidence of drug-resistant strains of Nocardia in yellowtail is as high as 69% and 65%, respectively. This shows that the existing antibiotic treatment regimen is facing serious drug resistance problems. As a preventive measure, early studies have shown that vaccination with formalin-inactivated vaccines or heat-inactivated vaccines can improve the serum agglutination antibody index of yellowtail, but recent studies have shown that after vaccination with these vaccines, no protection is shown after artificial attack. This shows that the protective effect of existing vaccines is limited. In addition, pathogenic Nocardia in aquatic animals grows slowly and the initial symptoms of infection are not obvious, but the disease has the characteristics of long duration, high morbidity and mortality, and it is difficult to diagnose and prevent the disease in the early stage of infection. Therefore, it is extremely important to establish rapid diagnostic technology and take effective prevention and control measures in time to control the spread and transmission of Nocardiosis.

[0008] In summary, the discovery of Bacillus Velezii with high antagonistic effect on Nocardia is of great significance for the prevention and control of Nocardiosis in fish. Summary of the invention

[0009] In order to overcome the deficiencies of the above-mentioned prior art, the present invention screens out a strain of Bacillus Velez JZGDFSBV241119 with a strong antagonistic effect on Nocardia from the aquaculture water of largemouth bass, and the strain is non-hemolytic and non-pathogenic to fish. Based on the unique therapeutic mechanism and environmental protection attributes of the strain, it can not only ensure the sustainable development of aquaculture, but also help reduce dependence on antibiotics, reduce environmental pollution risks, and improve breeding efficiency and product quality without destroying the ecological balance. In addition, the application of the strain also helps to protect public health and reduce health risks caused by antibiotic resistance. Therefore, the newly discovered Bacillus Velez JZGDFSBV241119 of the present invention can not only provide solutions to long-term problems in aquaculture, but also bring a new and sustainable treatment strategy to the field.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a Bacillus velezensis JZGDFSBV241119 strain, which was isolated from largemouth bass aquaculture water and deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on November 21, 2024, with the deposit number: CGMCC No.32730.

[0012] Preferably, the 16S rDNA sequence of the Bacillus velezinsis JZGDFSBV241119 strain is as shown in SEQ ID No: 1.

[0013] The second aspect of the present invention provides use of the Bacillus velezensis JZGDFSBV241119 strain described in the first aspect in the preparation of an inhibitor of aquatic pathogens, wherein the aquatic pathogens include Nocardia.

[0014] The present invention has found through research that the Velez Bacillus JZGDFSBV241119 strain can significantly inhibit the growth of Nocardia, and the prepared preparation can be used for preventing or treating bacterial diseases of aquatic animals.

[0015] Preferably, the aquatic pathogen inhibitor is a microbial preparation, which is used to enhance the immune function and enzyme activity of fish and improve the intestinal digestive function of fish.

[0016] Preferably, the aquatic pathogen inhibitor is a feed additive for enhancing the immune function and enzyme activity of fish and improving the intestinal digestive function of fish.

[0017] Preferably, the aquatic pathogen inhibitor is dry bacterial powder, fermentation broth or metabolite of JZGDFSBV241119 strain.

[0018] The third aspect of the present invention provides the use of the Bacillus velezensis JZGDFSBV241119 strain described in the first aspect in the preparation of a microecological preparation for improving the intestinal health of fish.

[0019] The present invention has found through research that after feeding the Bacillus Velezii JZGDFSBV241119 strain and common feed mixed, the damaged intestinal villus structure of fish infected with Nocardia was significantly restored, the villus length increased, and the crypt depth decreased, indicating that the intestinal absorption function was improved.

[0020] The fourth aspect of the present invention provides the use of the Bacillus velezensis JZGDFSBV241119 strain described in the first aspect in the preparation of a microecological preparation for enhancing the immune performance of fish.

[0021] The present invention has found through research that the application of the Bacillus velez JZGDFSBV241119 strain in fish farming can increase the expression of fish immune genes IL-1β, MHCI-α, and IL-8 in the spleen, and increase the ACP, AKP, SOD, and MDA enzyme activities in the serum, indicating that the strain can enhance the immune function and enzyme activity of fish, reduce the mortality rate, and is beneficial to the healthy farming of aquatic products.

[0022] The fifth aspect of the present invention provides an aquatic pathogen inhibitor, wherein the inhibitor uses the Bacillus velezensis JZGDFSBV241119 strain described in the first aspect as a main active ingredient.

[0023] Preferably, the inhibitor may also include auxiliary materials required for molding. The auxiliary materials include carriers and excipients. The excipients refer to diluents, adhesives, lubricants, disintegrants, solubilizers, stabilizers, etc. that can be used in the pharmaceutical field, as well as some pharmaceutical matrices. The carrier is a functional pharmaceutical excipient available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention isolates a strain of Bacillus velezensis JZGDFSBV241119 from a healthy largemouth bass breeding water body. The strain has a good antagonistic effect on Nocardia. At the same time, the strain is safe to use without residue, non-hemolytic, non-toxic and harmless to fish, has strong high temperature resistance, acid resistance and salt resistance, and is highly sensitive to 18 antibiotics such as penicillin, ampicillin, piperacillin, etc. The strain can enhance the immune function and enzyme activity of aquatic animals such as largemouth bass, reduce the mortality rate, and is beneficial to healthy aquaculture. Since the main function of the strain is to antagonize pathogens, it is suitable for improving the intestinal flora of fish. Specifically, the JZGDFSBV241119 strain of the present invention has the following advantages:

[0026] (1) The strain of Bacillus velezensis JZGDFSBV241119 has the ability to prevent and control a variety of aquatic animal diseases, which helps to reduce the use and residues of chemical drugs in aquaculture and reduce production costs.

[0027] (2) The Bacillus velezensis JZGDFSBV241119 strain can effectively inhibit the growth of the aquatic pathogen Nocardia spp. and is safe and has no toxic side effects. It reduces dependence on antibiotics and reduces the risk of environmental pollution. It can also improve aquaculture efficiency and product quality without destroying the ecological balance.

[0028] (3) In terms of the antagonistic effect against aquatic pathogens, the antagonistic bacteria obtained by the JZGDFSBV241119 strain from aquatic animals and their living environment are more effective than those from land. Antagonistic bacteria isolated from aquatic animals themselves and the environment can better adapt to the complex intestinal environment of fish and can adhere and colonize in the intestine. Therefore, it can be used to prevent the spread and transmission of Nocardia in the early stage of the disease.

[0029] (4) The Bacillus Velezii JZGDFSBV241119 strain can enhance the immune function of aquatic animals such as tilapia, reduce the occurrence of diseases, and is beneficial to healthy aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The morphology (left) and Gram staining (right) of Bacillus velezinoffe strain JZGDFSBV241119;

[0031] Figure 2 The phylogenetic tree of Bacillus Velezii JZGDFSBV241119 strain (in order to better aggregate the unknown strain sequence and the model strain and make the constructed phylogenetic tree more credible, the present invention chose to quote an exogenous bacterium of a different genus from Bacillus Velezii, i.e. Aeromonas hydrophila, codenamed ah (NCBI ID is MG428976.1) when constructing the tree);

[0032] Figure 3 This is a plate confrontation test between Bacillus Velezii JZGDFSBV241119 and Nocardia pathogen; Nocardia (left); JZGDFSBV241119 (right);

[0033] Figure 4 The effects of temperature, pH, and salinity on the growth ability of Bacillus velez JZGDFSBV241119;

[0034] Figure 5 The results of culturing blood plates with 1.5% Triton X-100, Bacillus velez JZGDFSBV241119 strain and PBS buffer for 24 hours;

[0035] Figure 6The effect of Bacillus Velezii JZGDFSBV241119 strain mixed with common feed and infected with Nocardia on the activities of malondialdehyde (MDA), total superoxide dismutase (T-SOD), alkaline phosphatase (ALP) and acid phosphatase (ACP) in largemouth bass was studied.

[0036] Figure 7 The effect of Bacillus Velezii JZGDFSBV241119 strain mixed with common feed and infected with Nocardia on the expression of immune genes IL-1β, MHCI-α, and IL-8 in largemouth bass.

[0037] Figure 8 These are intestinal sections of largemouth bass fed with Bacillus Velez-JZGDFSBV241119 and ordinary feed and infected with Nocardia; among them, ordinary group (left); Nocardia (middle); JZGDFSBV241119 (right). DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] The materials used in the following examples include:

[0041] (1) Test strains: Bacillus velez JZGDFSBV241119 and Nocardia seriolae strain 961113 isolated from healthy largemouth bass aquaculture water, donated by Huazhong Agricultural University. For details about the strains, see the literature: Hoang HH, Wang PC, Chen SC. Interleukin 34 Serves as a Novel Molecular Adjuvant against Nocardia Seriolae Infection in Largemouth Bass (Micropterus Salmoides). Vaccines (Basel). 2020 Mar 28; 8 (2): 151. doi: 10.3390 / vaccines8020151. PMID: 32231137; PMCID: PMC7349345.

[0042] (2) Experimental fish: Largemouth black bass fry (12.0±0.05 g) with a healthy physique were provided by Guangdong Heshi Aquatic Products Co., Ltd. They were temporarily raised for 2 weeks before the experiment, divided into groups according to experimental needs, and fed daily.

[0043] (3) The main instruments and equipment are shown in Table 1. The main reagents and solutions are shown in Table 2.

[0044] Table 1 Main instruments and equipment

[0045]

[0046] Table 2 Main reagents and sources

[0047]

[0048] Example 1: Isolation and Identification of Bacillus velez JZGDFSBV241119

[0049] 1. Screening of strains:

[0050] Isolation and purification of bacteria: Use sterile containers to take appropriate amounts of bottom mud, aquaculture sewage in the middle layer of the water body and aquaculture sewage in the upper layer of the water body from healthy largemouth bass aquaculture water (Foshan Agricultural Science Research Institute, Guangdong Province), and use sterile deionized water in a clean bench to perform 1, 10, 100 and 1000 times gradient dilution, and then fully oscillate and mix on a vortex shaker, and then take 100 μL each and evenly spread on LB, BHI, TSB and nutrient agar plates, seal with sealing film, and invert and culture at 28°C for 24h, pick single colonies with different morphologies, streak 4 times for purification and culture for 24h, and mix the cultured bacterial suspension with 50% glycerol 1:1 and store at -80°C for use. The strain numbered JZGDFSBV241119 was selected for subsequent research.

[0051] 2. Identification of strain JZGDFSBV241119

[0052] (1) Morphological observation

[0053] The bacterial solution in the seed tube was streaked and purified on the LB plate three times, and after 24 hours of recovery culture at 28°C, a single colony was picked and inoculated into 5 mL of LB liquid culture medium, and cultured at 28°C and 200 rpm for 24 hours. Subsequently, it was purified three times on the LB plate with an inoculation loop, and cultured at 28°C for 12 hours. The morphological characteristics of the colonies were observed and recorded. At the same time, the Gram staining kit (purchased from Beijing Solebow Technology Co., Ltd.) was used for staining. The staining process of the strain is as follows:

[0054] 1) Pipette 10 μL of fresh bacterial solution onto a glass slide and spread evenly;

[0055] 2) Pass over the outer flame of the alcohol lamp three times;

[0056] 3) After the bacterial solution is dried, add an appropriate amount of crystal violet staining solution for 1 minute, and then wash with an appropriate amount of water;

[0057] 4) Add appropriate amount of iodine solution to dye for 1 minute, and wash with appropriate amount of water;

[0058] 5) Add appropriate amount of decolorizing solution, decolorize for 30-40 seconds, wash with appropriate amount of water, and gently wipe the filter paper to dry the excess water on the surface of the biofilm;

[0059] 6) Add appropriate amount of safranin dye solution, dye for 1 minute, and rinse with water;

[0060] 7) Gently wipe the filter paper to dry the excess water on the surface of the biofilm or dry it at room temperature;

[0061] 8) Use an optical microscope to observe the Gram staining results and take photos.

[0062] like Figure 1 As shown, the colonies of the JZGDFSBV241119 strain are white with neat edges, round or irregular shapes, smooth colony surfaces, loose textures, and sometimes bifurcated or loose lines. Gram staining results show positive bacteria in the shape of rods.

[0063] (2) Physiological and biochemical identification

[0064] The detection methods of physiological and biochemical characteristics of candidate strains are as follows:

[0065] 1) Prepare a culture box and pour 5 mL of ddH2O into the bottom plate of the incubation box to fill all the holes and keep the gas environment moist at all times;

[0066] 2) After marking the edge of the incubation box bottom plate, use an inoculation loop to scrape 5 single colonies from the plate and transfer them to API50CHB medium to prepare 2McFarland bacterial suspension;

[0067] 3) Use a pipette to add the prepared bacterial suspension into each well of the corresponding reagent strip tube;

[0068] 4) Cover the culture lid and incubate at 28°C for 48 hours;

[0069] 5) After 48 hours, record the results, then enter the actual reaction results of the API reagent strips into the API WEB query system, and finally obtain the bacterial species identification results based on the reaction results.

[0070] The results are shown in Table 3. By matching with the data in the API database, it was confirmed that the physiological and biochemical characteristics of the strain were consistent with those of B. velezensis.

[0071] Table 3 Physiological and biochemical characteristics of JZGDFSBV241119 identified by API 50 CH bacterial identification system

[0072]

[0073] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0074] (3) Molecular biological identification

[0075] By observing the morphology, color and transparency of the colonies on the plate, different single colonies were picked and inoculated into 5 mL LB liquid culture medium. After culturing at 28°C and 200 rpm for 24 hours, a portion of the culture medium was taken out and mixed with 60% glycerol in equal proportions for seed preservation and stored at -80°C, and a portion was used to extract genomic DNA. The specific steps are as follows:

[0076] 1) Take 2 mL of bacterial culture medium, centrifuge at 10000 rpm for 1 min, and remove the supernatant;

[0077] 2) Add 200 μL of GA buffer to the bacterial pellet and vortex to mix;

[0078] 3) Add 20 μL of proteinase K and vortex to mix;

[0079] 4) Add 220 μL GB buffer, vortex mix for 15 seconds, place at 70°C for 10 minutes, and remove the fog droplets in the wall after the solution becomes clear;

[0080] 5) Add 220 μL of anhydrous ethanol, vortex mix for 15 seconds, and then centrifuge to remove the fog droplets on the tube wall;

[0081] 6) After mixing by pipetting, add the mixture to the adsorption column, centrifuge at 12000 rpm for 30 seconds, and discard the liquid;

[0082] 7) Add 500 μL of GD buffer to the column, centrifuge at 12000 rpm for 30 s, and discard the liquid;

[0083] 8) Add 500 μL PW rinse solution to the column, centrifuge at 12000 rpm for 30 s, and discard the solution;

[0084] 9) Repeat step 8;

[0085] 10) Return the column to the collection tube, spin at 12000 rpm for 2 min, discard the liquid, and leave at room temperature for 5 min;

[0086] 11) Place the column in an enzyme-free centrifuge tube, add 100 μL of TE elution buffer to the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution;

[0087] 12) The obtained DNA product was stored at -20°C for future use.

[0088] 13) PCR amplification: The universal primer for 16srRNA amplification and identification was 27F / 1492R, synthesized by Sangon Biotechnology Co., Ltd. The primer sequence is shown in Table 4. PCR amplification was performed according to the reaction system in Table 5 and the reaction program in Table 6.

[0089] Table 4 PCR identification primer sequences

[0090]

[0091] Table 5 PCR reaction system

[0092]

[0093] Table 6 PCR reaction program

[0094]

[0095]

[0096] (14) The PCR products were tested by 1-1.2% agarose gel electrophoresis to confirm the success of amplification and the size of the products. The strains with clear bands were sent for sequencing. The sequences with high homology in the search results were selected and downloaded on NCBI's BLAST, and then a phylogenetic tree was constructed in MEGA 11.

[0097] The build result is as follows Figure 2 As shown, PCR amplified bacterial genomic DNA to obtain a PCR product with a length of 1114. Using NCBI's BLAST homology search, it was found that the JZGDFSBV241119 strain had a small genetic distance and a close relationship with Bacillus velezensis (OM909106.1), with a homology of 98.20%. Therefore, the JZGDFSBV241119 strain was identified as a different strain of the same species of Bacillus velezensis.

[0098] Finally, the strain JZGDFSBV241119 was deposited, and the deposit information is as follows: deposit time: November 21, 2024; name of the deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit number: CGMCC No.32730; address of the deposit unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; classification name: Bacillus velezensis.

[0099] The 16S rDNA sequence of Bacillus velezensis JZGDFSBV241119 (SEQ ID No: 1) is:

[0100] GGAGCGTCTATCTGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTTTAATTCGAAGCAACGCGAAGAACCCTTACCAGGTCTTGA CATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTTCGGGGGCAGAGTGACAGGTGGTGCATGCTGTCGTCAGCTTCGTGTCGTGAGAATGTTGGTTTAGTCCCGCAACTAAGCGCCAACCCCTTGGATCTTAGGTTTGCCAAGCAATTTCA.

[0101] Example 2: Plate confrontation test between JZGDFSBV241119 strain and Nocardia pathogen

[0102] Add 10 μL of Noka to the center of BHI solid medium, BHI, and LB agar plates, and inoculate 5 μL of Bacillus velezensis JZGDFSBV241119 at 2 cm on three sides of the center of the plate. After culturing for 3-5 days, observe and record the growth status of each strain on the plate. Figure 3 The results showed that the growth rate of Bacillus Velezii JZGDFSBV241119 strain was relatively fast and could inhibit the growth of Nocardia.

[0103] Example 3: Study on the growth characteristics of JZGDFSBV241119 strain

[0104] 1. Effect of temperature on the growth ability of antagonistic strains

[0105] Use an inoculation loop to scrape the preserved strain, purify it three times on the LB plate, activate it at 28℃ for 24h, then pick a single colony in LB liquid culture medium, and culture it at 28℃ and 200rpm for 24h. Centrifuge at 5000rpm to obtain the bacteria, wash it with PBS three times, then take 100μL of the bacterial suspension and inoculate it into 5mL of LB liquid culture medium, and culture it at 10℃, 20℃, 30℃, 40℃, and 50℃ at 200rpm for 24h, and finally measure the OD of the cultured bacterial solution on an ELISA reader. 600 The results are as follows Figure 4 As shown, at 10°C, the growth activity of the strains was inhibited. At 20°C-40°C, the growth of the four strains was good.

[0106] 2. Effect of pH on the growth ability of antagonistic strains

[0107] Use an inoculation loop to scrape the preserved strain, purify it three times on an LB plate, activate it at 28°C for 24h, then pick a single colony in LB liquid culture medium, and culture it at 28°C and 200rpm for 24h. Centrifuge at 5000rpm to obtain the bacteria, wash it with PBS three times, then take 100μL of the bacterial suspension and inoculate it in 5mL of LB liquid culture medium with pH values ​​of 2, 4, 6, 8, 10 and 12, and culture it at 200rpm for 24h before measuring the OD of the bacterial solution on an ELISA reader. 600 The results are as follows Figure 4 As shown, at pH = 4, the OD of Bacillus velez JZGDFSBV241119 strain 600 The value was significantly higher than that of other groups (P<0.05); when pH = 4-6, the strain grew normally, and when pH = 6-12, the strain OD 600 The values ​​were significantly decreased (P<0.05).

[0108] 3. Effect of salinity on the growth ability of antagonistic strains

[0109] Use an inoculation loop to scrape the preserved strain, purify it three times on an LB plate, activate it at 28°C for 24 hours, then pick a single colony and place it in LB liquid culture medium. After shaking culture at 28°C and 200 rpm for 24 hours, centrifuge at 5000 rpm to obtain the bacteria. After washing with PBS three times, take 100 μL of the bacterial suspension and inoculate it in 5 mL of LB liquid culture medium with NaCl concentrations of 1.5%, 3.5%, 5.5%, 7.5%, 9.5%, 10.5% and 13.5%, respectively. After culture at 200 rpm for 24 hours, measure the OD of the bacterial solution on an ELISA reader. 600 The results are as follows Figure 4 As shown, when the salinity is 1.5%-7.5%, the bacterial growth ability gradually increases; when the salinity is 7.5%-13.5%, the growth ability of the strain gradually decreases.

[0110] Example 4: Antibiotic sensitivity test of JZGDFSBV241119 strain

[0111] The antagonistic strains were subjected to antibiotic sensitivity tests to evaluate their resistance to different antibiotics, and the antibiotics used are shown in Table 7. The diameter of the inhibition zone was determined to be extremely sensitive (E) when it was above 20 mm, highly sensitive (S) when it was 15-20 mm, moderately sensitive (I) when it was 10-15 mm, and insensitive (R) when it was less than 10 mm.

[0112] The specific steps are as follows:

[0113] (1) Use an inoculation loop to scrape the preserved strain, streak it on an LB plate for purification, and culture it at 28°C for 24 h;

[0114] (2) Pick a single colony from the plate and place it in LB liquid medium and culture it at 28°C and 200 rpm for 24 h;

[0115] (3) Take 1 mL of the cultured bacterial solution, centrifuge at 4000 rpm for 10 min to obtain the bacterial cells, wash them three times with PBS, and then take 100 μL of the bacterial solution and spread it evenly on the LB plate;

[0116] (4) Use sterile forceps to pick up 20 drug-sensitive tablets and place them on the surface of the plate;

[0117] (5) Seal with sealing film and place in an electric constant temperature incubator at 28°C for 24 h;

[0118] (5) Observe and accurately measure the diameter of each inhibition zone.

[0119] The results are shown in Table 8. The strain JZGDFSBV241119 was extremely sensitive to penicillin, ampicillin, piperacillin, cephalexin, cefazolin, cefuroxime sodium, ceftazidime, ceftriaxone, cefoperazone, gentamicin, kanamycin, tetracycline, minocycline, erythromycin, vancomycin, and doxycycline, highly sensitive to amikacin and streptomycin, moderately sensitive to polymyxin B, but insensitive to lincomycin.

[0120] Table 7 Antibiotics and their concentrations

[0121]

[0122]

[0123] Table 8 Inhibition zone diameter (cm) of JZGDFSBV241119 strain

[0124]

[0125]

[0126] Example 5: Biosafety Assessment of JZGDFSBV241119 Strain

[0127] 1. Hemolytic test

[0128] Place the enzyme-free filter paper on the surface of the blood plate and absorb 1 mL of fresh bacterial suspension. Centrifuge at 5000 rpm for 2 min, wash 3 times with PBS, and absorb 5 μL of the washed bacterial suspension on the filter paper. Incubate at 28°C for 12 h and observe the presence of hemolysis rings. Use 1.5% Triton X-100 as a positive control and PBS as a negative control. Figure 5As shown, the positive control (Triton X-100) formed an obvious transparent hemolytic ring on the blood plate, while the JZGDFSBV241119 strain did not produce a hemolytic ring, which was consistent with the result of the negative control (PBS).

[0129] 2. Strain toxicity detection

[0130] The selected strains are tested for toxicity to monitor their effects on fish health. The specific steps are as follows:

[0131] Healthy largemouth bass were randomly divided into two groups, 30 in each group, and placed in a fish canvas drum. The temperature in the drum was kept constant at 28±0.5℃ and aerated continuously. In one group, each fish was intraperitoneally injected with 200μL of fresh JZGDFSBV241119 bacterial suspension, and the concentration of bacterial suspension was adjusted to 1×10 8 CFU / mL. The negative control group was injected with 200μL PBS per fish, and the mortality of the two groups of fish was observed and recorded daily. The experiment lasted for 14 days. The results showed that the survival rate of the fish in the experimental group and the control group was 100% after intraperitoneal injection of JZGDFSBV241119 strain for 14 days.

[0132] Example 6: Determination of serum biochemical indicators of JZGDFSBV241119 strain

[0133] In the feeding experiment (feeding with Nocardia for 15 days, followed by feeding with Bacillus Velezii JZGDFSBV241119; the feed used was Zhengda California Bass No. 2 feed, in which the concentration of Nocardia was 1×10 6 CFU / mL, the concentration of Bacillus Velezii is 1×10 8 CFU / mL, fed twice a day) for 60 days, and the largemouth bass were fasted for 12 hours before sampling. Five largemouth bass were randomly selected from each experimental group, and the largemouth bass were anesthetized. The fish body surface was wiped with 75% alcohol, and blood was collected from the tail vein. 0.5-1mL of blood was collected from each tail, and the blood was allowed to stand at 37℃ for 1h. After standing at 4℃ overnight, the supernatant was collected by centrifugation at 4000r for 10min and stored at -80℃ for use in the determination of serum immunological indicators; Determination of immune-related enzyme activity in serum Five largemouth bass were randomly selected from each experimental group, and blood was collected from the tail with a sterile syringe. The enzyme activity kit was used for detection of the activities of malondialdehyde (MDA), total superoxide dismutase (T-SOD), alkaline phosphatase (ALP), acid phosphatase (ACP), etc. in the serum, and relevant bar graphs were drawn, such as Figure 6As shown in the figure, the activities of ACP, AKP, SOD and MDA enzymes in serum were all higher than those in the control group, among which ACP showed a highly significant difference between the control group NC (fed with ordinary feed after infection with Nocardia) and the BV group (fed with feed mixed with JZGDFSBV241119 strain after infection with Nocardia); the activity of T-SOD was also significantly different between the NC control group and the BV group.

[0134] Example 7: Determination of immune-related genes of JZGDFSBV241119 strain

[0135] In the feeding experiment (feeding with Nocardia for 15 days, followed by feeding with Bacillus Velezii JZGDFSBV241119; the feed used was Zhengda California Bass No. 2 feed, in which the concentration of Nocardia was 1×10 6 CFU / mL, the concentration of Bacillus Velezii is 1×10 8 CFU / mL, fed twice a day) and samples were collected 60 days after the start of sampling. The largemouth bass were fasted for 12 hours before sampling. The spleen and kidneys of 1-2 mm in size were cut aseptically and immersed in RNA preservation solution (RNAlater from Thermo Fisher Scientific) TM The samples were placed in an enzyme-free cryopreservation tube containing stabilizing solution and stored in a -80°C refrigerator for later use. Five samples were randomly selected from each test group for the determination of mRNA expression levels of relevant immune genes:

[0136] (1) After total RNA extraction, cDNA synthesis was performed according to Table 9;

[0137] (2) Real-time fluorescence quantitative PCR was performed according to Table 10: The cDNA obtained by reverse transcription was used as a template for real-time fluorescence quantitative RT-PCR reaction to detect the expression levels of IL-1β, MHCI-α, and IL-8 genes in fish liver, spleen, and head kidney tissues. Primers were designed using Primer3 with reference to the relevant gene sequences in GenBank. The primer sequences are shown in Table 11. Each sample was repeated 3 times, the internal reference gene was β-actin, and the relative expression of the gene was calculated using the 2-ΔΔct method.

[0138] like Figure 7 As shown, the expression levels of immune genes IL-1β, MHCI-α, and IL-8 in the spleen of the BV group (fed with JZGDFSBV241119 strain mixed feed after infection with Nocardia) were higher than those of the control group NC (fed with ordinary feed after infection with Nocardia).

[0139] Table 9 Reverse transcription reaction system

[0140]

[0141] Note: The reaction procedure is 37℃15min, 85℃5sec, 4℃∞.

[0142] Table 10 Real-Time PCR reaction system

[0143]

[0144] Note: The reaction program is 95℃30sec, 40 cycles (95℃5sec), 60℃30sec, 4℃∞.

[0145] Table 11 Primers for quantitative detection of immune-related genes

[0146]

[0147] Example 7: Preparation of intestinal tissue sections of JZGDFSBV241119 strain

[0148] In the feeding experiment (feeding with Nocardia for 15 days, followed by feeding with Bacillus Velezii JZGDFSBV241119; the feed used was Zhengda California Bass No. 2 feed, in which the concentration of Nocardia was 1×10 6 CFU / mL, the concentration of Bacillus Velezii is 1×10 8 CFU / mL, fed twice a day), 2 months after the start of the test, the test fish were dissected on ice under sterile conditions, and the midgut tissue with a width of 3-4 mm was cut with sterile scissors and forceps, and fixed in 4% paraformaldehyde after removing excess connective tissue, with 3-5 replicates in each group, for making tissue sections; the midgut tissue was fixed in 4% paraformaldehyde solution for more than 24 hours, dehydrated, clarified, and embedded in paraffin, and the embedded paraffin blocks were sliced, each slice was about 5 mm thick, and the slices were stained with hematoxylin-eosin (HE), and finally sealed with neutral gum, and the processed midgut tissue sections were observed under a microscope and photographed and analyzed.

[0149] The results are as follows Figure 8 As shown, the intestinal villi of the Nocardia group were damaged, atrophied and shed, with shed epithelial cells and a large amount of food residues in the intestinal cavity; in contrast, the damaged intestinal villus structure of the mice fed with the Velez-Bacillus JZGDFSBV241119 strain in the later stage was significantly restored, the villus length increased, and the crypt depth decreased, indicating that the intestinal absorption function was improved.

[0150] In summary, the present invention has isolated a new strain of Bacillus velezensis JZGDFSBV241119, and the strain has the following characteristics or effects:

[0151] (1) The JZGDFSBV241119 strain can significantly inhibit the growth of Nocardia, and the preparation can be used to prevent or treat bacterial diseases in aquatic animals; (2) The strain has high heat resistance, acid resistance and salt resistance; (3) The strain is non-hemolytic and non-toxic to fish; (4) The strain can significantly regulate the intestinal structure of fish, improve the growth performance of fish, improve the intestinal digestive function of fish, and make it better able to absorb nutrients in feed; (5) IL-1β and IL-8 are typical pro-inflammatory factors that can regulate inflammatory responses. Among them, immunoglobulins play a key role in the adaptive immune system and can help organisms fight pathogens. These indicators are important indicators that reflect the ability of fish to cope with pathogen infection and immune regulation, and significantly affect the nutrient absorption, immune response and disease resistance of fish. T-AOC is the sum of the body's antioxidant enzymes and various antioxidant substances, reflecting the body's overall antioxidant level. MDA is one of the lipid peroxidation products produced after the body's oxidative stress response. When the fish body resists the invasion of pathogenic microorganisms, innate immunity is the main defense tool. AKP is a typical regulatory enzyme that participates in the phosphorylation and dephosphorylation process of organisms, and plays a key role in the immune system as a lysosomal enzyme, and has the effect of improving the absorption of nutrients by fish. These indicators not only reveal the immune status of fish, but also reflect their nutritional and metabolic health status. The present invention has found through research that the application of JZGDFSBV241119 strain in fish farming can improve the above-mentioned indicators of fish, indicating that the strain can enhance the immune function and enzyme activity of fish, reduce the mortality rate, and is conducive to the healthy farming of aquatic products.

[0152] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus velezensis JZGDFSBV241119, characterized in that: The strain was isolated from the aquaculture water of largemouth bass and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 21, 2024, with the deposit number: CGMCC No.32730.

2. The Bacillus velezensis JZGDFSBV241119 strain according to claim 1, characterized in that: The 16S rDNA sequence of the Bacillus velezinsis JZGDFSBV241119 strain is shown in SEQ ID No:

1.

3. Use of the Bacillus velezensis JZGDFSBV241119 strain according to claim 1 or 2 in the preparation of aquatic pathogen inhibitors, characterized in that: The aquatic pathogens include Nocardia.

4. The use according to claim 3, characterized in that: The aquatic pathogen inhibitor is a microbial preparation used to enhance the immune function and enzyme activity of fish and improve the intestinal digestive function of fish.

5. The use according to claim 3, characterized in that: The aquatic pathogen inhibitor is a feed additive used to enhance the immune function and enzyme activity of fish and improve the intestinal digestive function of fish.

6. The use according to claim 3, characterized in that: The aquatic pathogen inhibitor is dry bacterial powder, fermentation liquid or metabolite of JZGDFSBV241119 strain.

7. Use of the Bacillus velezensis JZGDFSBV241119 strain according to claim 1 or 2 in the preparation of a microecological preparation for improving the intestinal health of fish.

8. Use of the Bacillus velezensis JZGDFSBV241119 strain according to claim 1 or 2 in the preparation of a microecological preparation for enhancing the immune performance of fish.

9. An aquatic pathogen inhibitor, characterized in that: The inhibitor uses the Bacillus velezensis JZGDFSBV241119 strain described in claim 1 or 2 as a main active ingredient.

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