Bacillus velezensis and application thereof

Abstract

The invention discloses bacillus velezensis Bve1910, which is preserved in the China Center for Type Culture Collection (CCTCC) and has the preservation number of CCTCC NO. M 20221965. The strain and metabolites thereof have strong antagonistic effects on more than ten types of marine culture animal pathogenic bacteria such as vibrio harveyi, vibrio erwiniae, vibrio canbayi, vibrio parahaemolyticus, vibrio alginolyticus, vibrio anguillarum, vibrio cholerae, vibrio shewanii, vibrio rotifer, pseudoalteromonas and photobacterium damarae, are strong in antibacterial activity and wide in antibacterial spectrum, and can be used for preparing marine culture animal pathogenic bacteria. According to the preparation method, the traditional Chinese medicine composition is simple in preparation method and rich and stable in metabolite, an ammonium sulfate precipitation extract, a hydrochloric acid precipitation methanol extraction extract, an ethyl acetate extraction extract and a chloroform extraction extract all have a broad-spectrum bacteriostatic effect, functional substances playing the broad-spectrum antagonistic bacteriostatic effect are compounded and diversified, and the directional selection effect on bacteria in the environment and ecology is not generated; and pathogenic bacteria are not easy to generate tolerance, so that the method has good application safety and ecological safety properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus velezensis and its application. Background Art

[0002] With the rapid development of the aquaculture industry, the problem of diseases has become increasingly serious. Frequent deaths of farmed animals caused by bacteria, viruses, and parasites as the main pathogens have led to huge economic losses, severely restricting the development speed of China's aquaculture industry. In some cases, the aquaculture of certain important aquatic animals has even stagnated or declined, causing a serious blow to the aquaculture industry. The prevention and control of aquaculture diseases has always been a very active research field worldwide. At present, antibiotics and chemical drugs are still the main means for preventing and controlling aquaculture diseases. However, with the aggravation of diseases, the large-scale and blind use of chemical drugs such as antibiotics has led to changes in the genetic and metabolic characteristics of aquaculture-related bacteria, making them no longer effective in treating bacterial diseases and even leading to the spread of the risk of bacterial drug resistance. The resulting problem of bacterial drug resistance has a large potential ecological safety hazard. In addition, food safety problems caused by drug residues due to the abuse of drugs are common, such as drug residue incidents in shrimp, turbot, mandarin fish, hairy crabs, eels, etc., which seriously threaten people's food health and safety. Reducing and replacing antibiotics is an urgent need to ensure the quality and safety of aquatic products and the green development of the industry, and it is also an inevitable trend of industrial development. Only green aquaculture can increase production and efficiency for the aquaculture industry. Therefore, it is urgent to develop green and safe antibiotic alternatives for application in aquaculture.

[0003] Microecological preparations are a general term for a class of beneficial microorganisms isolated from the living environment of microorganisms, which can achieve the effect of controlling bacteria with bacteria under natural conditions and are a hot spot for developing safe and green aquaculture prevention and control technologies. Microecological preparations play an important role in the aquaculture industry through various forms such as improving the balance of the microecological system and the aquaculture environment, enhancing the stress resistance and disease resistance of farmed aquatic animals, regulating body metabolism, and providing nutrients, and have the characteristics of being green, environmentally friendly, and safe. The currently developed microecological preparation products include different types of strains such as photosynthetic bacteria, Bacillus, Lactobacillus, yeast, Bifidobacterium, Nitrobacter, antibacterial peptides, and active polysaccharides. Among them, Bacillus is one of the earliest discovered bacteria by humans, which can improve the water environment, promote growth, enhance immunity, and inhibit pathogenic bacteria, becoming a suitable alternative for the prevention and control of animal diseases and having broad application prospects. However, at present, the quality of Bacillus strains is uneven, and some Bacillus may even cause diseases in aquatic animals. In addition, as a potential carrier of virulence and drug resistance genes in the environment, probiotics have the risk of becoming a transmission channel for virulence and drug resistance genes. Therefore, obtaining a probiotic strain with broad-spectrum high efficiency and ecological safety is a common problem faced by researchers.

[0004] At present, many Bacillus strains have been proven to have inhibitory effects on various pathogenic bacteria, including Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus velezensis, etc. However, there is currently no report that these Bacillus strains can simultaneously have strong inhibitory effects on more than a dozen pathogenic bacteria of marine aquaculture animals, such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio campbellii, Photobacterium damselae, Vibrio cholerae, Shewanella spp., Edwardsiella spp., Vibrio rotiferianus, and Pseudoalteromonas spp. In addition, there are also reports in China on Bacillus velezensis that has significant inhibitory effects on Aeromonas hydrophila, Vibrio harveyi, Vibrio parahaemolyticus, etc. It has been proven by artificial infection that it is not lethal to specific cultured organisms, and then its safety has been evaluated. However, there is a lack of a perfect evaluation standard for the direct or indirect harm risks it poses to aquatic animals and the environment, and there is no involvement in the multiple joint evaluation of the biological characteristics and ecological safety of Bacillus velezensis. Therefore, as a microorganism released into the environment, Bacillus not only has to have good pathogenic antagonistic effects but also must have no potential ecological risks. Summary of the Invention

[0005] The purpose of the present invention is to provide a Bacillus velezensis and its application to solve the problems raised in the above background technology.

[0006] To achieve the above purpose, the present invention provides the following technical solution: A Bacillus velezensis, the Bacillus velezensis is Bve1910, which was isolated from seawater, aquaculture animal samples, and sediment at the bottom of the water treatment pool collected from the seedling cultivation area and the tail water treatment area. It was deposited with the China Center for Type Culture Collection on December 13, 2022. The classification name is Bacillus velezensis Bve1910, and the deposit number is CCTCC NO.M 20221965.

[0007] Preferably, the 16S rDNA sequence of the Bacillus velezensis Bve1910 is as shown in SEQ NO.1.

[0008] Preferably in any of the above solutions, the colonies formed by the Bacillus velezensis Bve1910 after culturing on TSB medium for 36 h are milky white, semi-transparent, with smooth edges and convex and wrinkled surfaces.

[0009] A bacterial agent, the bacterial agent includes Bacillus velezensis Bve1910.

[0010] A probiotic preparation, the probiotic preparation contains Bacillus velezensis Bve1910 and / or a pure culture, fermentation broth, ammonium sulfate precipitation extract of the fermentation broth, hydrochloric acid precipitation methanol extraction extract, ethyl acetate extraction extract, and chloroform extraction extract of the bacterial agent.

[0011] Application of Bacillus velezensis Bve1910 in preparing medicament for preventing and treating bacterial diseases of marine cultured animals caused by marine cultured animal pathogenic bacteria.

[0012] Preferably, in any of the above solutions, the marine cultured animal pathogenic bacteria include Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio owensii, Vibrio campbellii, Photobacterium damselae, Vibrio cholerae, Shewanella sp., Edwardsiella sp., Vibrio rotiferianus, and Pseudoalteromonas sp.

[0013] Application of Bacillus velezensis Bve1910, bacterial agent, and probiotic preparation as partial components in feed in aquaculture.

[0014] Application of Bacillus velezensis Bve1910, bacterial agent, and probiotic preparation in preparing aquaculture feed.

[0015] Technical effects and advantages of the present invention: 1. The Bacillus velezensis provided by the present invention is Bve1910, which is isolated from seawater, cultured animal samples, and sediment at the bottom of the water treatment pool collected from the seedling cultivation area and the tail water treatment area. This strain and its metabolites have strong antagonistic effects against more than ten kinds of pathogenic bacteria of marine cultured animals such as Vibrio harveyi, Vibrio owensii, Vibrio campbellii, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio anguillarum, Vibrio cholerae, Shewanella sp., Vibrio rotiferianus, Pseudoalteromonas sp., and Photobacterium damselae, with strong bacteriostasis and a wide bacteriostatic spectrum.

[0016] 2. The metabolites of Bacillus velezensis Bve1910 provided by the present invention are rich and stable. Its ammonium sulfate precipitation extract, hydrochloric acid precipitation methanol extraction extract, ethyl acetate extraction extract, and chloroform extraction extract all have broad-spectrum bacteriostatic effects. The functional substances that play a broad-spectrum antagonistic bacteriostatic role are complex and diversified, and will not produce directional selection effects on bacteria in the environmental ecosystem, nor are they likely to make pathogenic bacteria develop tolerance, having good application safety and ecological safety properties.

[0017] 3. Bacillus velezensis Bve1910 provided by the present invention is safe and non-toxic. The strain and its metabolites have no hemolytic property and are safe and non-toxic to marine cultured animals such as Litopenaeus vannamei, Epinephelus sp., and Scophthalmus maximus. Feed additives and environmental probiotic preparations prepared with the pure culture of the strain as the active ingredient can significantly reduce the number of pathogenic bacteria in the environment and animals and the infection mortality rate, having a broad application prospect in the prevention and treatment of bacterial diseases of marine cultured animals.

[0018] 4. Bacillus velezensis Bve1910 can be used as a feed additive and a probiotic preparation to reduce the mortality rate of aquatic animals caused by multi-pathway infection of pathogenic bacteria. Description of the Drawings

[0019] Figure 1 Inhibitory activity diagram of Bacillus velezensis Bve1910 against pathogenic bacteria of different species and genera;

[0020] Figure 2 Colony morphology diagram of Bacillus velezensis Bve1910 on TSB plate;

[0021] Figure 3 Morphology diagram of single colony of Bacillus velezensis Bve1910;

[0022] Figure 4 Hemolysis effect test diagram of Bacillus velezensis Bve1910 on blood agar plate;

[0023] Figure 5 Phylogenetic tree of Bacillus velezensis Bve1910 based on 16S rDNA sequence;

[0024] Figure 6 Protease and amylase activity diagrams of Bacillus velezensis Bve1910;

[0025] Figure 7 Histopathological change diagram of grouper against bacterial disease infection after feeding with Bacillus velezensis Bve1910. Detailed implementation manners

[0026] The following further explains the detailed implementation manners of the present invention with reference to the accompanying drawings. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Example 1: Isolation and screening of Bacillus velezensis strain Bve1910

[0028] 1. Isolation of strains

[0029] Taking a seawater aquaculture animal seedling farm in Haiyang City, Shandong Province as the investigation point, this seedling farm breeds various economic seawater aquaculture animal seedlings such as shrimp seedlings, grouper seedlings, turbot seedlings, and half-smooth tongue sole seedlings. Samples of seawater, aquaculture animals, and sediment at the bottom of the water treatment pool were collected from different seedling cultivation areas and the tail water treatment area, and were packed and taken back to the laboratory at low temperature for microbial cultivation.

[0030] Use a pipette to aspirate seawater samples and sediment diluents and spread them on TSB solid medium (3 replicates). (The formula is: tryptone 17.0 g / L, peptone 3.0 g / L, sodium chloride 20 g / L, agar 15 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3 ± 0.2). After standing and drying for 15 min, incubate in an inverted position at 28 °C for 36 h. Pick colonies with the ability to produce endospores and inoculate them on a new medium for isolation and purification.

[0031] Weigh 0.5 g of healthy shrimp larvae (about 300 tails), rinse them repeatedly with sterile 1.5% NaCl solution, and then perform low-temperature tissue homogenization. Subsequently, centrifuge at 2000 rpm for 1 min and take the supernatant tissue suspension. Gradient dilute the suspension 100 times and 1000 times. Use a pipette to aspirate the diluent and spread it on TSB solid medium (3 replicates). After standing and drying for 15 min, incubate in an inverted position at 28 °C for 36 h. Pick colonies with the ability to produce endospores and inoculate them on a new medium for isolation and purification.

[0032] Select 10 healthy fish fry, cut out the visceral mass with sterile dissection tools, mix them well, weigh 1 g, add sterile 1.5% NaCl solution and perform low-temperature tissue homogenization. Subsequently, centrifuge at 2000 rpm for 1 min and take the supernatant tissue suspension. Gradient dilute the suspension 100 times and 1000 times. Use a pipette to aspirate the diluent and spread it on TSB solid medium (3 replicates). After standing and drying for 15 min, incubate in an inverted position at 28 °C for 36 h. Pick colonies with the ability to produce endospores and inoculate them on a new medium for isolation and purification.

[0033] 2. Screening of strains

[0034] Inoculate the isolated Bacillus strains on sheep blood agar medium. Add 5% sterile defibrinated sheep blood to the TSB solid medium to prepare a blood agar plate. Observe whether there is hemolysis after culturing in a constant temperature incubator at 28 °C for 36 h. Using pathogenic Vibrio parahaemolyticus, Vibrio harveyi, Photobacterium damselae, and Vibrio anguillarum in seawater fish and shrimp as indicator bacteria, use the Oxford cup plate antibacterial method to test the antibacterial ability of the isolated and purified Bacillus strains, measure the size of the antibacterial zone of different Bacillus strains, and screen out the antagonistic strain with no hemolytic activity and the largest antibacterial zone, and name it Bve1910.

[0035] Example 2: Analysis of the broad-spectrum antibacterial activity of Bacillus Bve1910

[0036] The Oxford cup plate antibacterial method was used to determine the antibacterial activity of the isolated and purified Bacillus Bve1910: Pick the purified Bacillus colony, perform shaking culture using liquid TSB medium, and then resuspend the centrifuged bacterial sludge with sterile 1.5% NaCl solution to prepare a bacterial suspension with a concentration of 4×10 8 CFU / mL (OD≈0.8). In addition to the 4 pathogenic indicator bacteria used in the screening process, the pathogenic bacteria also include Vibrio alginolyticus, Vibrio owensii, Vibrio campbellii, Vibrio cholerae, Shewanella sp., Vibrio rotiferianus, Edwardsiella tarda, and Pseudoalteromonas sp., a total of 8 pathogenic bacteria. After culturing the pathogenic bacteria in liquid TSB medium (formula: tryptone 17.0 g / L, peptone 3.0 g / L, sodium chloride 20 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3±0.2) until the logarithmic phase, resuspend with 1.5% NaCl solution to prepare a bacterial suspension with a concentration of 2×10 6 CFU / mL (OD≈0.4). Take 100 μL of the pathogenic bacteria suspension and spread it on the surface of the TSB medium. Then add 100 μL of the Bacillus Bve1910 bacterial suspension into the Oxford cup wells, use sterile 1.5% NaCl solution as a control, let it stand for 10 min, and then culture it at 28 °C for 36 h. Observe the inhibitory effect of Bacillus Bve1910 on 5 pathogenic bacteria and measure the diameter of the antibacterial circle.

[0037] As Figure 1 shown, the results show that Bacillus Bve1910 has inhibitory effects on all 12 pathogenic bacteria. The diameters of the antibacterial circles are as follows: Vibrio parahaemolyticus 20.1 mm, Vibrio alginolyticus 18.8 mm, Vibrio harveyi 27.6 mm, Vibrio owensii 30.9 mm, Vibrio campbellii 25.5 mm, Vibrio rotiferianus 19.8 mm, Photobacterium damselae subsp. piscicida 23.6 mm, Vibrio anguillarum 20.8 mm, Vibrio cholerae 19.6 mm, Shewanella sp. 22.1 mm, Edwardsiella tarda 24.7 mm, Pseudoalteromonas sp. 28.7 mm. The antibacterial effect is significant and has certain broad-spectrum antibacterial characteristics.

[0038] Example 3 Safety of Bacillus Bve1910 Strain and Its Extracellular Products to Aquaculture Animals

[0039] The safety of Bacillus Bve1910 for cultured animals was detected by intramuscular injection (for shrimp) and intraperitoneal injection (for fish). The specific operation was as follows: Litopenaeus vannamei (5.2 ± 1.2 g / tail), Epinephelus coioides (8.3 ± 1.4 g / tail), and Scophthalmus maximus (12.6 ± 2.1 g / tail) were temporarily cultured for 7 days and then grouped. 20 tails were randomly selected from each group. The experiment set up 2 experimental groups with different bacterial liquid concentrations, 1 experimental group of fermentation broth, and 1 blank control group. The experimental groups of bacterial liquid were injected with 100 μL of Bacillus Bve1910 bacterial suspension with concentrations of 4×10 9 CFU / mL and 4×10 8 CFU / mL, the fermentation broth group was injected with 100 μL of the fermentation broth of the metabolite of Bacillus Bve1910, and the negative control group was injected with 100 μL of sterile 1.5% NaCl solution. The experimental observation period was 7 days, and the symptoms and deaths of the experimental animals were recorded every day.

[0040] The experimental results showed that during the experimental period, no deaths or disease symptoms occurred in the experimental group infected with Bacillus Bve1910 strain and the experimental group infected with fermentation broth, indicating that the strain Bve1910 is safe and non-toxic to seawater cultured animals such as Litopenaeus vannamei, Epinephelus coioides, and Scophthalmus maximus, and has high biosafety.

[0041] Example 4: Identification and genetic metabolic characteristic analysis of Bacillus Bve1910

[0042] (1) Identification of Bacillus Bve1910

[0043] The purified strain Bve1910 was streaked and inoculated on TSB solid medium and cultured at 28 °C for 36 h to observe the colony morphology, as Figure 2 shown. The genomic DNA of strain Bve1910 was extracted according to the instructions of the DNA extraction kit of Takara Company. The target gene was amplified using the universal primers for 16S rDNA gene. The sequences of the universal primers for 16S rDNA gene were: 27F: 5′-AGAGTTTGATCATGGCTCAG-3′, 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′. The positive PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The measured gene sequences were subjected to Nucleotide BLAST alignment analysis in the NCBI database, the gene sequences were aligned by Clustal W in Mega 7.0, and then the neighbor-joining method was used with Kimura 2-pammeter as the nucleotide substitution model and Bootstrap (1000 repetitions) as the test method to construct a phylogenetic tree.

[0044] As Figure 2and Figure 3 As shown in Figure 3 , the results showed that the colonies formed by Bacillus Bve1910 after culturing on TSB medium for 36 h were milky white, translucent, with smooth edges, convex and wrinkled surfaces, and had typical Bacillus apparent properties. The gene sequencing results showed that the full length of the 16S rDNA gene of Bacillus Bve1910 was about 1500 bp, and its 16S rDNA sequence was as follows,

[0045]

[0046] As Figure 5 shown, the results of phylogenetic analysis showed that the strain Bve1910 had the highest homology with the genus Bacillus, with a similarity of 100%. The gene sequence was used for homologous sequence retrieval analysis in the NCBI database, and the sequence with the highest homology was used to construct a phylogenetic tree. By comparison, it was found that the strain Bve1910 had the highest similarity with Bacillus velezensis CR 502. Combining the morphological and genetic characteristics of bacteria, the strain Bve1910 was identified as Bacillus velezensis.

[0047] (2) Extracellular enzyme activity test of Bacillus velezensis Bve1910

[0048] 18.5 g of soluble starch agar and 21.0 g of casein agar were added to 500 mL of deionized water respectively, and after sterilization, a starch plate for measuring the amylase decomposition ability of the strain and a casein plate for measuring the protease decomposition ability were prepared. The purified Bacillus velezensis Bve1910 was resuspended in sterile 1.5% sodium chloride solution to prepare a bacterial suspension with a concentration of 10 8 CFU / mL. Subsequently, 5 μL of the bacterial suspension was pipetted and inoculated onto the starch plate and the casein plate. After incubation at 28 °C for 48 h, the hydrolysis zones around the bacterial lawns were observed and measured, and the amylase and protease decomposition abilities of the strain were recorded. As Figure 6 shown, Bacillus velezensis Bve1910 could decompose both protease and amylase, and the diameters of the hydrolysis zones were 21.6 mm and 18.5 mm respectively.

[0049] (3) Detection of the risk of carrying drug-resistant genes in Bacillus Bve1910

[0050] The risk of carrying drug-resistant genes in Bacillus velezensis Bve1910 was detected by PCR. A total of 18 genes, including tetA, tetB, tetE, blaTEM, ampC, blaZ, ant(3)-Ia (aadA), aph(6’)-Ib (strB), sul1, sul2, sul3, ermA, ermX, floR, cfr, qnrA, qnrB, and qnrS, were detected. The PCR reaction system was (25 μL): 12.5 μL of 2×Rapid Taq Master Mix, 0.5 μL each of the upstream and downstream primers of the drug-resistant gene (10 μmol / L), 1 μL of template DNA, and 10.5 μL of double-distilled water. The PCR amplification conditions were: 94 °C for 5 min; 94 °C for 30 s, 52.5 °C for 30 s, 72 °C for 1.5 min, for 30 cycles; 72 °C for 10 min, and stored at 4 °C. The amplification products were detected by 1.0% agarose gel electrophoresis.

[0051] The results showed that Bacillus velezensis Bve1910 only carried the tetracycline resistance gene tetB, and no other genes were detected, indicating that the risk of Bacillus Bve1910 spreading drug resistance genes in the environment was extremely low.

[0052] (4) Detection of the risk of carrying virulence genes by Bacillus Bve1910

[0053] The PCR method was used to detect the presence of virulence genes in Bacillus amyloliquefaciens Bve1910. A total of 10 virulence genes including hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM, bceT, and ces were detected. The PCR reaction system was (25 μL): 2×Rapid Taq Master Mix 12.5 μL, upstream and downstream primers of virulence genes (10 μmol / L) 0.5 μL each, template DNA 1 μL, and double-distilled water 10.5 μL. The PCR amplification conditions were: 94°C for 5 min; 94°C for 30 s, 50°C for 30 s, 72°C for 1.5 min, for 30 cycles; 72°C for 10 min, and stored at 4°C. The amplified products were detected by 1.0% agarose gel electrophoresis.

[0054] The results showed that no virulence genes were detected in Bacillus velezensis Bve1910, indicating that the pathogenic risk of Bacillus Bve1910 to cultured organisms and the risk of spreading virulence genes in the environment were extremely low, and it had high biological and ecological safety.

[0055] Example 5: Analysis of the characteristics of the antibacterial active components of Bacillus Bve1910

[0056] (1) Preparation of the cell lysate of Bacillus Bve1910

[0057] A single colony of Bacillus Bve1910 was picked and inoculated into 100 mL of TSB liquid medium, and cultured with shaking at 28°C and 180 r / min for 16 h to prepare a seed solution. Subsequently, 10 mL of the seed solution was pipetted and inoculated into 200 mL of liquid TSB medium, and cultured with shaking at 28°C and 180 r / min for 36 h to make the viable cell concentration in the medium reach 2×10 11 CFU / mL (OD value ≈ 1.8). The culture solution was dispensed into 50 mL sterile centrifuge tubes, and then placed in ice water for ultrasonic disruption. The ultrasonic frequency was 50 Hz, and the operation-stop cycle was 3 s:5 s, and ultrasonic disruption was carried out for 30 min. The disrupted cell solution was filtered again with a microporous membrane with a diameter of 0.22 μm to ensure no viable bacteria remained, and 100 μL of the disrupted solution was pipetted and spread on TSB medium to test for the presence of viable bacteria. After determining that no colonies grew on the medium, it was a qualified Bve1910 cell lysate.

[0058] (2) Effects of digestive enzymes on the antibacterial activity of the cell lysate of Bacillus sp. Bve1910

[0059] The cell lysate of Bve1910 was divided into six portions, each with a volume of 20 mL. Trypsin, papain, proteinase K, pepsin, and lipase with a final concentration of 1 mg / mL were used for enzymatic digestion, respectively. The enzyme-untreated solution supplemented with an equal amount of sterile PBS was used as the control. The mixtures were placed in a water bath at 37 °C for 1 h. Subsequently, the Oxford cup plate antibacterial method was used to determine the antibacterial activity of the metabolites of strain Bve1910 against four test pathogenic bacteria, namely Vibrio alginolyticus, Vibrio harveyi, Photobacterium damselae subsp. piscicida, and Vibrio parahaemolyticus.

[0060] The results showed that there were slight differences in the antibacterial effects of the cell lysate of strain Bve1910 against the four pathogenic indicator bacteria after treatment with the five enzymes compared with the control group: the antibacterial activities against Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi decreased, while there was no significant change in the inhibitory activity against Photobacterium damselae subsp. piscicida. This indicated that the antibacterial substances in strain Bve1910 were somewhat sensitive to the five enzymes, and the antibacterial substances in the extracellular products contained corresponding protein components but were not the main effective substances. At the same time, the differences in antibacterial activities among different pathogenic bacteria also showed that the material basis mediating the broad-spectrum antibacterial activity of strain Bve1910 should be the synergistic action of various substances of different categories.

[0061] (3) Effects of temperature on the antibacterial activity of the cell lysate of Bacillus sp. Bve1910

[0062] The original cell lysate of Bve1910 was divided into six portions, each with a volume of 50 mL. They were incubated at -20 °C, 4 °C, 30 °C, 60 °C, 80 °C, and 100 °C for 30 min, respectively. Then, they were taken out and placed in a laminar flow hood to cool to room temperature, and filtered again using a 0.22-μm microporous membrane to prevent contamination by miscellaneous bacteria during the operation. The untreated cell lysate was used as the control. The Oxford cup plate antibacterial method was used to determine the antibacterial activity of the cell lysate after different temperature treatments against four test pathogenic bacteria, namely Vibrio alginolyticus, Vibrio harveyi, Photobacterium damselae subsp. piscicida, and Vibrio parahaemolyticus.

[0063] The results showed that the antibacterial activities of the extracellular products of Bacillus sp. Bve1910 treated at -20 °C, 4 °C, and 30 °C were not significantly different from those of the control group at room temperature. After treatment at temperatures higher than 60 °C to 100 °C, the antibacterial activity of the ECP showed a downward trend, and the product treated at 100 °C almost completely lost its antibacterial activity, indicating that the main antibacterial substances in strain Bve1910 should include temperature-sensitive substances such as antibacterial peptides.

[0064] (4) Effects of pH on the antibacterial activity of the cell lysate of Bacillus sp. Bve1910

[0065] The original solution of the Bve1910 cell lysate was divided into six portions, and the pH values were adjusted to 1, 3, 5, 7, 9, and 11 with 1 mol / L HCl and 1 mol / L NaOH respectively. After standing at room temperature for 2 h, the pH values were adjusted back to neutral, and then filtered through a 0.22-μm microporous membrane to prevent contamination by miscellaneous bacteria during the operation process. The untreated cell lysate was used as a control. The Oxford cup plate antibacterial method was used to determine the antibacterial activities of the cell lysates treated at different pH values against four test pathogenic bacteria, namely Vibrio harveyi, Vibrio alginolyticus, Photobacterium damselae subsp. piscicida, and Vibrio parahaemolyticus.

[0066] The results showed that there were no significant differences in the antibacterial activities of the Bve1910 cell lysates treated at pH values of 1, 3, 5, and 7 against the four indicator bacteria compared with the control group. As the pH increased to 9 - 11, the antibacterial activity of the Bve1910 cell lysate decreased significantly, indicating that the antibacterial active components of Bacillus Bve1910 contained substances with strong acid tolerance and alkali sensitivity.

[0067] Example 6: Identification of the antibacterial active components of Bacillus Bve1910

[0068] Ammonium sulfate precipitation was used to extract protein substances from the Bve1910 cell lysate of Bacillus, and their antibacterial activities against four test pathogenic bacteria, namely Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, and Photobacterium damselae subsp. piscicida, were determined. The specific operation was as follows: 200 mL of the prepared original solution of the Bve1910 cell lysate of Bacillus was taken, and ammonium sulfate powder was slowly added until the saturation was 70%. Then it was placed at 4 °C overnight for precipitation, and then centrifuged at 8000 r / min for 10 min. The precipitate was collected and dissolved and fixed to 10 mL with PBS (pH = 7.0). The solution obtained at this time was the crude extract solution of protein substances in the Bve1910 cell lysate of Bacillus. 100 μL of the suspensions of the four test pathogenic bacteria with a concentration of 2×10 6 CFU / mL were respectively spread on the surface of the TSB medium. Then, 200 μL of the crude extract solution of protein substances in the Bve1910 cell lysate of Bacillus extracted was added into the Oxford cup wells. The precipitate dissolution solution obtained by performing the same treatment with the blank culture solution with an ammonium sulfate saturation of 70% was used as a control, and each treatment was repeated 3 times. After standing for 10 min, it was cultured at 28 °C for 24 h, and the antibacterial effect was observed. The results showed that the diameters of the antibacterial zones of the protein products in Bacillus Bve1910 against the four test strains were: 15.7 mm for Vibrio harveyi, 16.4 mm for Vibrio parahaemolyticus, 21.3 mm for Vibrio alginolyticus, and 18.4 mm for Photobacterium damselae subsp. piscicida, all showing good antibacterial activities, indicating that the antagonistic substances produced by Bacillus Bve1910 included protein substances.

[0069] Compare with the above inhibition zones to see if the rules of the inhibition zone sizes for different strains are consistent, and if they are the same as those of proteinous substances, there should be some differences

[0070] The antibacterial activity of lipopeptides in the cell lysate of Bacillus Bve1910 against 4 test pathogenic bacteria was determined by the method of hydrochloric acid precipitation and methanol extraction. The specific operation was as follows: Take 200 mL of the prepared original cell lysate of Bacillus Bve1910, adjust the pH to 2.0 with 6 mol / L HCl, then place it at 4 °C overnight for precipitation, and then centrifuge at 4 °C and 8000 r / min for 10 min to collect the precipitate. Extract the precipitate with methanol repeatedly 3 times. After shaking for 10 min, centrifuge at 4 °C and 8000 r / min for 10 min to collect the supernatant. Rotate and evaporate the supernatant to concentrate it to 10 mL at 40 °C and 60 r / min, dissolve it in 10 mL of methanol solution after freeze-drying, adjust the pH to 7.0 with 1 mol / L NaOH, and filter it with a 0.22 μm organic filter membrane. The solution obtained at this time is the crude extract solution of lipopeptide active substances in the cell lysate of Bacillus Bve1910. Respectively, pipette 100 μL of the suspensions of 4 test pathogenic bacteria, Vibrio campbellii, Vibrio harveyi, Photobacterium damselae subsp. piscicida, and Vibrio parahaemolyticus, with a concentration of 2×10 6 CFU / mL onto the surface of TSB medium. Then add 200 μL of the prepared lipopeptide active substance solution of Bacillus Bve1910 into the Oxford cup wells, and use the blank culture solution extracted in the same way as the control. Each treatment was repeated 3 times. After standing for 10 min, culture it at 28 °C for 24 h to observe the antibacterial effect. The results showed that the diameters of the inhibition zones of the lipopeptide products produced by Bacillus Bve1910 against the 4 test strains were: Vibrio harveyi 16.2 mm, Vibrio parahaemolyticus 14.7 mm, Vibrio alginolyticus 15.5 mm, and Photobacterium damselae subsp. piscicida 13.4 mm, indicating that the broad-spectrum antagonistic substances produced by Bacillus Bve1910 contain lipopeptide active substances.

[0071] The ethyl acetate and chloroform extraction methods were used to determine the antibacterial activities of organic small molecules, alkaloids and other substances in the cell lysate of Bacillus Bve1910 against four test pathogenic bacteria, namely Vibrio campbellii, Vibrio harveyi, Photobacterium damselae and Vibrio parahaemolyticus. The specific operation was as follows: Take 2 portions of 200 mL of the prepared original cell lysate of Bacillus Bve1910. Add an equal volume of ethyl acetate to one portion, shake well for 3 - 5 min and then let it stand. After the separation of the aqueous phase and the organic phase, collect the upper organic phase, and concentrate it to 10 mL by rotary evaporation at 40 °C and 60 r / min, and then dissolve it in 10 mL of methanol after freeze-drying; Add an equal volume of chloroform to the other portion, shake for 3 - 5 min and then let it stand. After the separation of the aqueous phase and the organic phase, collect the upper organic phase, concentrate it to 10 mL by rotary evaporation at 40 °C and 60 r / min, and then dissolve it in 10 mL of methanol after freeze-drying. The solution obtained at this time is the extract of organic small molecules and other organic substances in the cell lysate of Bacillus Bve1910. Respectively absorb 100 μL of the suspensions of 4 test pathogenic bacteria, namely Vibrio campbellii, Vibrio harveyi, Photobacterium damselae and Vibrio parahaemolyticus, with a concentration of 2×10 6 CFU / mL and spread them on the surface of the TSB medium. Then add 200 μL of the ethyl acetate and chloroform extraction solutions of Bacillus Bve1910 prepared into the Oxford cup holes respectively, and use the blank culture solution extracted in the same way as the control. Each treatment was repeated 3 times. After standing for 10 min, culture it at 28 °C for 24 h and observe the antibacterial effect. The results showed that the diameters of the antibacterial zones of the ethyl acetate extract and chloroform extract of the metabolites of Bacillus Bve1910 against Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus and Photobacterium damselae were all > 15 mm, indicating that the antagonistic substances produced by Bacillus Bve1910 contain a variety of polar active substances.

[0072] The results of this example showed that the ammonium sulfate precipitation extract, hydrochloric acid precipitation methanol extraction extract, ethyl acetate extraction extract and chloroform extraction extract of Bacillus Bve1910 all had antibacterial effects against 4 test pathogenic bacteria, namely Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus and Photobacterium damselae, indicating that the functional substances of Bacillus Bve1910 that play a broad-spectrum antagonistic antibacterial role are complex and diversified, do not produce directional selection effects on bacteria in the environmental ecosystem, and are not likely to make pathogenic bacteria develop tolerance, and have good application safety and ecological safety attributes.

[0073] Example 7: Evaluation of the prevention and control effect of Bacillus Bve1910 on the anti-vibriosis infection of Litopenaeus vannamei

[0074] Pick a single colony of Bacillus Bve1910 and inoculate it into TSB liquid medium. After culturing with shaking at 28 °C and 180 r / min for 48 h, centrifuge to collect the bacterial cells, resuspend them with sterile PBS solution, and then add the bacterial suspension to the pellet expanded feed to prepare a probiotic bait with a bacterial content of 2×10 8 CFU / g. The experimental shrimp have a body length of 7.8 ± 1.3 cm. Each group has a culture water volume of 300 L, a culture density of 100 shrimp per barrel, and a total of 6 barrels are cultured (3 barrels in the experimental group and 3 barrels in the control group). During the experiment, the water temperature is 28 - 30 °C, keep appropriate aeration, feed twice a day, and the feeding amount is 2% of the shrimp body weight. The control group is fed with the same amount of untreated pellet expanded feed. After 21 days of the experiment, an artificial infection test is carried out.

[0075] The pathogenic bacterium used in the artificial infection experiment is Vibrio parahaemolyticus pathogenic to shrimp preserved in the laboratory (patent application number 202410713083.2). Inoculate the pathogenic bacterium into liquid TSB medium and culture it overnight, centrifuge to collect the bacterial cells and resuspend them with sterile PBS solution to prepare a bacterial suspension. In the oral infection experimental group, add the bacterial suspension to the pellet expanded feed to prepare a pathogenic bacterium bait with 3×10 6 CFU / g for feeding the infection test. Feed the pathogenic bacterium bait once a day and a normal bait once a day, and the feeding amount is 1% of the shrimp body weight. The bait containing the pathogenic bacterium is prepared for each meal. After the experimental group is continuously fed for 3 days, it is changed to ordinary feed. The control group is fed with the same amount of untreated pellet expanded feed. Record the death and disease occurrence of shrimp in each group every day and continuously observe for 7 days. In the immersion infection experiment, directly sprinkle the pathogenic bacterium into the water body, and the final concentration of the pathogenic bacterium is 3×10 6 CFU / mL. Change the water twice a day, each time changing 1 / 2 of the volume and equally supplementing the pathogenic bacterium to the original concentration. After 3 days of infection, no more bacterial suspension is added. Feed ordinary pellet expanded feed twice a day, and the feeding amount is 1% of the shrimp body weight. Record the death and disease occurrence of shrimp in each group every day and continuously observe for 7 days.

[0076] The results show that in the feeding infection experiment, the cumulative mortality rate of the control group is 76.1%, and the cumulative mortality rate of the shrimp in the experimental group fed with probiotics is 43.8%; in the immersion infection experiment, the cumulative mortality rate of the control group is 52.5%, and the cumulative mortality rate of the shrimp in the experimental group fed with probiotics is 30.6%. It shows that adding Bacillus Bve1910 to the feed can significantly improve the resistance of shrimp to oral and surface infections by pathogenic bacteria.

[0077] Example 8: Evaluation of the prevention and control effect of Bacillus Bve1910 on grouper against Vibrio infection

[0078] The experiment was conducted in a farm in Rizhao City. The length of grouper was 17.8±2.4cm, the weight was 76.2±4.5g, the aquaculture water volume of each group was 300L, the aquaculture density was 50 fish / barrel, the water temperature was 24-28℃ during the experiment, and the aeration was maintained continuously. A single colony of Bacillus Bve1910 was selected and inoculated into TSB liquid culture medium. After 48h of shaking culture at 28℃ and 180r / min, the bacteria were collected by centrifugation, resuspended in sterile PBS solution, and added to the pelleted extruded feed to prepare 2×10 8 CFU / g of probiotic feed. Feed twice a day, the feeding amount is 2% of the body weight of the flatfish, and the control group is fed with the same amount of untreated pelleted feed. After feeding for 21 days, artificial infection test was carried out.

[0079] The pathogen used in the artificial infection experiment was the pathogenic Vibrio harveyi preserved in the laboratory (Xu Yue, 2018). Vibrio harveyi was cultured in liquid TSB and then centrifuged and resuspended in sterile PBS solution to a concentration of 3×10 6 CFU / mL of bacterial suspension was used for artificial injection infection test, and the injection infection volume was 100μL / tail. The death and morbidity were recorded every day and observed continuously for 7 days. At the same time, 3 groupers were randomly collected from each group 48h after infection, and the liver, spleen, kidney, intestine and other tissues were removed and cut into appropriate sizes. They were fixed with Davidson's fixative for 24h and then transferred to 75% ethanol solution for preservation. The fixed tissues were then dehydrated, waxed, embedded, sliced, dewaxed, eluted, stained with hematoxylin-eosin and sealed with neutral resin to prepare tissue sections. The pathological changes of different tissues were then observed under a microscope, such as Figure 7 shown.

[0080] The results showed that after the injection of pathogens, the cumulative mortality of grouper in the experimental group fed with Bacillus was 18.2%, and the cumulative mortality of the control group was 45.7%, indicating that the addition of Bacillus Bve1910 in feed can improve the disease resistance of marine cultured fish. Histopathological observation showed that after infection with Vibrio harveyi, the liver, spleen, kidney and intestinal tissues of the two groups of grouper showed lesions to varying degrees, especially the difference in intestinal tissue structure. The intestinal structure of grouper in the experimental group with Bacillus Bve1910 was relatively intact, and the hepatocytes were relatively compact. In the experimental group without probiotics, the villi in the midgut were obviously enlarged and necrotic, the chorion fell off, the hepatocytes were obviously vacuolated, the renal tubules were narrowed, and the glomeruli were obviously atrophied. It shows that long-term feeding of functional feed containing Bacillus Bve1910 has the effect of enhancing the resistance of fish tissues, and then plays a barrier role and secretes antibacterial substances in the process of pathogen infection, achieving the effect of preventing and treating pathogen infection to a certain extent.

[0081] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Bacillus velezensis, characterized in that: The Bacillus velezensis is Bve1910, and its preservation unit is the China Center for Type Culture Collection, with the preservation number: CCTCC NO.M 20221965.

2. A Bacillus velezensis according to claim 1, characterized in that: The 16S rDNA sequence of the Bacillus velezensis Bve1910 is as shown in SEQ NO.

1.

3. A Bacillus velezensis according to claim 1, characterized in that: The colonies formed after culturing the Bacillus velezensis Bve1910 on TSB medium for 36 hours are milky white, translucent, with smooth edges, and convex and wrinkled surfaces.

4. A bacterial agent, characterized in that: The microbial agent includes the Bacillus velezensis Bve1910 described in claim 1.

5. A probiotic preparation, characterized in that: The probiotic preparation contains one of the pure culture, fermentation broth, ammonium sulfate precipitation extract of the fermentation broth, hydrochloric acid precipitation methanol extraction extract, ethyl acetate extraction extract, and chloroform extraction extract of the Bacillus velezensis Bve1910 described in claim 1 and / or the microbial agent described in claim 4.

6. Application of the Bacillus velezensis Bve1910 described in claim 1 in the preparation of a drug for preventing and treating bacterial diseases of marine aquaculture animals caused by marine aquaculture animal pathogenic bacteria.

7. The application according to claim 6, wherein: The marine aquaculture animal pathogenic bacteria include Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio owensii, Vibrio campbellii, Photobacterium damselae subsp. piscicida, Vibrio cholerae, Shewanella spp., Edwardsiella spp., Vibrio rotiferianus, and Pseudoalteromonas spp.

8. Application of the Bacillus velezensis Bve1910 described in any one of claims 1-3, the microbial agent described in claim 4, and the probiotic preparation described in claim 5 as partial components in feed in aquaculture.

9. Application of the Bacillus velezensis Bve1910 described in any one of claims 1-3, the microbial agent described in claim 4, and the probiotic preparation described in claim 5 in the preparation of aquaculture feed.

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