Brevibacillus laterosporus for inhibiting aquatic pathogenic vibrio and application of brevibacillus laterosporus

Abstract

The invention belongs to the technical field of microorganisms, and provides brevibacillus laterosporus for inhibiting aquatic pathogenic vibrio and application of the brevibacillus laterosporus. The brevibacillus laterosporus strain provided by the invention is obtained by screening from intestinal epithelium of healthy penaeus vannamei boone, and is preserved in Guangdong Microbial Culture Collection Center on May 22, 2024, and the preservation number is GDMCC NO: 64668. The brevibacillus laterosporus and the metabolite thereof have a good inhibition effect on common halophilic aquatic pathogenic vibrios such as vibrio parahaemolyticus and vibrio alginolyticus.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Brevibacillus laterosporus capable of inhibiting aquatic pathogenic Vibrio and application thereof. Background Art

[0002] In recent years, although the aquaculture industry has made significant progress, it has been accompanied by the rise of intensive and high-density farming models and the improper use of drugs such as antibiotics. These changes have had a serious impact on the ecological environment of aquatic animals. This series of problems has not only led to a sharp deterioration in the farming environment, but also triggered a chain reaction of increased drug resistance of aquatic pathogens, prominent antibiotic residue problems, and decreased immunity of aquatic animals. In particular, the Vibrio problem has become the top priority of farmers, which has seriously hindered the sustainable and healthy development of the aquaculture industry.

[0003] Vibrio, as a conditional pathogen widely present in water and shrimp, occupies a dominant position in the seawater environment with its strong adaptability and resistance to adversity, and often causes outbreaks of diseases. In shrimp farming, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cholerae are particularly common pathogenic Vibrio. Once Vibrio invades the shrimp, they will quickly colonize the digestive tract, release toxins, cause tissue damage, and disrupt the hepatopancreatic function and digestive system. Initial infection may manifest as symptoms such as red body, yellow gills, and broken whiskers. In severe cases, it will cause hepatopancreatic necrosis, causing enteritis, white stools, empty jejunum and stomach, and even stealing death. Therefore, in the process of farming white shrimp, it is particularly important to control the number of Vibrio in the pond within a safe range. However, because many farmers ignore the regulation of bacterial phase, the pond bacteria are single and the bacterial phase environment is fragile. Once the environmental conditions change, the bacterial phase is easy to collapse, and the pathogenic strains multiply in large numbers, thereby inducing vibriosis. This highlights the extreme importance of biocontrol of Vibrio in shrimp farming.

[0004] In recent years, probiotics have attracted much attention due to their advantages such as non-toxicity, pollution-free, safety and reliability. Probiotics, including live bacteria, inactive microbial cells or crude cell extracts, as feed additives can significantly improve the survival rate and growth performance of aquatic animals, enhance disease resistance, and optimize the intestinal flora structure. More importantly, the use of probiotics can also effectively reduce the number of Vibrio in water and shrimp.

[0005] Regarding the technical solution of applying Bacillus laterosporus to inhibit freshwater aquaculture pathogens, there are documents that disclose that the screened Bacillus laterosporus has a good inhibitory effect on Vibrio parahaemolyticus and water-type Pseudomonas punctata in freshwater aquaculture. However, Vibrio is halophilic, and its reproduction rate is fastest and its species are more numerous in water bodies with higher salinity. It is unknown whether the Bacillus laterosporus in the above documents also has a good inhibitory effect on Vibrio in seawater. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a strain of Brevibacillus laterosporus for inhibiting aquatic pathogenic Vibrio and its application. The Brevibacillus laterosporus provided by the present invention has a good inhibitory effect on halophilic Vibrio, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 22, 2024. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC NO: 64668 and a classification name of Brevibacillus laterosporus.

[0007] The active ingredients of the bacterial agent prepared by using Brevibacillus laterosporus include Brevibacillus laterosporus and its metabolites.

[0008] The application of the above-mentioned Brevibacillus laterosporus and the bacterial agent prepared therefrom in inhibiting halophilic pathogenic Vibrio in aquatic products is also the key protection content of the present invention.

[0009] The preparation method of the bacterial agent mainly comprises the following steps:

[0010] (1) After the CBD230301 Brevibacillus laterosporus is cultured, fermented in a seed tank, and fermented in a secondary fermentation tank, the obtained fermentation liquid is spray-dried to obtain bacterial powder;

[0011] (2) The bacterial powder and maltodextrin are fully mixed to obtain the bacterial agent, namely the "Hu Yidao" product.

[0012] Preferably, in (1), 3.0% magnesium sulfate is added to the fermentation broth before spray drying.

[0013] Preferably, the amount of maltodextrin in (2) is 5.0 wt%.

[0014] Compared with the prior art, the present invention has the following advantages and effects:

[0015] (1) The Brevibacillus laterosporus provided by the present invention has a significant inhibitory effect on halophilic aquatic pathogenic Vibrio. The strain is screened from the intestinal epithelium of healthy white shrimp. Through the preparation process, a bacterial agent containing Brevibacillus laterosporus and its metabolites can be obtained. This bacterial agent performs well in inhibiting common aquatic pathogenic Vibrio such as Vibrio parahaemolyticus and Vibrio alginolyticus, and can effectively reduce the number of Vibrio in water and shrimp, thereby preventing and controlling the outbreak of vibriosis and improving the survival rate and production performance of aquaculture;

[0016] (2) The Brevibacillus laterosporus and its bacterial agent provided by the present invention are a microecological preparation, which has the advantages of being non-toxic, pollution-free, safe and reliable. Its use can effectively improve the breeding environment, optimize the flora structure, promote ecological balance, reduce the use of antibiotics, and reduce the drug resistance of pathogens, thereby promoting the green and healthy development of aquaculture;

[0017] (3) The Brevibacillus laterosporus and its bacterial agent provided by the present invention have achieved remarkable results in practical applications. Through the verification of a series of application cases, the bacterial agent can significantly reduce the number of Vibrio in aquaculture water and shrimp, improve the growth of shrimp, and increase the survival rate. This not only improves the economic benefits of aquaculture, but also provides consumers with a safer and healthier food source. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Evaluation of the antibacterial effect of different batches of Brevibacillus laterosporus fermentation broth in Example 1 on Vibrio parahaemolyticus and Vibrio alginolyticus;

[0019] Figure 2 The antibacterial effect evaluation of the parallel treatment of different batches of Brevibacillus laterosporus fermentation broth in Example 1 on Vibrio parahaemolyticus and Vibrio alginolyticus;

[0020] Figure 3 This is the change of Vibrio plate in the water and shrimp bodies of Mr. Zhang’s breeding pond in Application Case 1 after using Vibrio Yidao (or Brevibacillus laterosporus);

[0021] Figure 4 This is the change in the number of Vibrio in the water and shrimp bodies of Mr. Zhang’s breeding pond in Application Case 1 after Vibrio was removed using Vibrio Yidao (or Brevibacillus laterosporus);

[0022] Figure 5 For application case 2, the changes in the Vibrio plate after the use of Vibrio Yidao (or Brevibacillus laterosporus) in the water and shrimp bodies of the Chinese and Korean masters’ breeding ponds;

[0023] Figure 6 This is the change of Vibrio plate in the water and shrimp bodies of Mr. Zhang’s breeding pond in application case 3 after using Vibrio Yidao (or Brevibacillus laterosporus);

[0024] Figure 7 This is the change in the number of Vibrio in the water and shrimp bodies of Mr. Zhang’s breeding pond in Application Case 3 after Vibrio was removed using Vibrio Yidao (or Brevibacillus laterosporus);

[0025] Figure 8 This is the change of Vibrio plate in the water and shrimp bodies of Mr. Li’s breeding pond in Application Case 4 after using Vibrio Yidao (or Brevibacillus laterosporus);

[0026] Fig. 9 This is the change in the number of Vibrio in the water and shrimp bodies of Mr. Li’s breeding pond in Application Case 4 after using Vibrio Yidao (or Brevibacillus laterosporus);

[0027] Fig.10 It is a comparative evaluation of the antibacterial effect of Brevibacillus laterosporus and commercially available Vibrio inhibitory competitors in the comparative example;

[0028] Fig.11 The single bacteria separation and purification of Vibrio F (left) and Vibrio R (right) after screening on the Weifang diseased material plate in Example 1. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0030] Example 1: Analysis of the antibacterial effect of Brevibacillus laterosporus on Vibrio

[0031] 1.1 Culture medium and consumables

[0032] (1) Liquid LB medium: 1% sodium chloride, 0.5% yeast extract powder, 1% peptone; pH 7.2±0.2; 121°C, 0.1 MPa, 30 min sterilization, used for Vibrio proliferation culture.

[0033] (2) Complete culture medium: 1% glucose, 1% beef extract powder, 1% peptone, 0.5% yeast extract powder, 0.5% sodium chloride; pH 7.2±0.2; sterilization at 121°C, 0.1 MPa, 30 min; 1% agar powder, for inoculation and culture of Vibrio.

[0034] (3) Plain agar medium: 1.5% agar powder, dissolved in pure water, sterilized at 121°C, 0.1 MPa, 30 min, used for fixing the Oxford cup.

[0035] (4) Physiological saline: 0.85% sodium chloride, sterilized at 121°C, 0.1 MPa, 30 min, used to dilute samples.

[0036] (5) Consumables: Petri dishes, Oxford cups, tweezers, 200 μL pipette tips, 5 mL pipette tips, test tubes, 5 mL centrifuge tubes, 50 mL centrifuge tubes, vials, 10 mL syringes, 0.22 μm filter membranes, filters, and 250 mL conical flasks with glass beads. Sterilize at 121°C, 0.1 MPa, for 30 min before use.

[0037] 1.2 Test content and analysis results

[0038] The Brevibacillus laterosporus provided by the invention is screened from the intestinal epithelium of healthy white shrimp, and the production batch numbers thereof are CBD230202, CBD230301, CBD230302-LD and CBD230304 respectively.

[0039] There are two types of Vibrio species used in the present invention, namely, Vibrio species F (Vibrio parahaemolyticus, also known as Vibrio chlororaphis) and Vibrio species R (Vibrio alginolyticus, also known as Vibrio flavus). Fig.11 As shown, the left picture is the separation and purification of Vibrio species F, and the right picture is the separation and purification of Vibrio species R. Both were screened from water sample plates of farmers in Hanting District, Weifang City. The water samples are seawater aquaculture water samples, so the screened Vibrio parahaemolyticus and Vibrio alginolyticus are both halophilic, which is completely different from the Vibrio in fresh water.

[0040] The test mainly includes the following steps:

[0041] (1) Inoculation of Vibrio activation solution: Use an inoculation loop to pick a loop from the Vibrio plate and inoculate it into liquid LB medium. Incubate overnight at 37°C in a constant temperature shaker for 16 h.

[0042] (2) Pour the agar plates in advance, about 10 mL per plate, and cool them for later use (only for fixing the Oxford cup).

[0043] (3) Weigh 36 g of physiological saline into a 250 mL conical flask with glass beads, weigh 4 g of Brevibacillus laterosporus sample using weighing paper, pour it into the conical flask, and shake it manually.

[0044] (4) The dissolved Brevibacillus laterosporus sample was placed at room temperature and shaken at 200 rpm / min for 30 min.

[0045] (5) Pour the shaken Bacillus laterosporus sample into a 50 mL centrifuge tube (about 20 mL), balance it, and centrifuge it at 4°C, 10,000 rpm, for 10 min to obtain the supernatant.

[0046] (6) The supernatant of the sample to be tested is filtered using a 10 mL syringe, a 0.22 μm filter membrane, and a filter. The filtered sample is placed in a 5 mL centrifuge tube for later use.

[0047] (7) Mark Vibrio F and R on the plain agar plate, then place the Oxford cup on the plain agar plate and place it upright in the clean bench.

[0048] (8) Take out 50 μL of the cultured Vibrio F and R and add it to a test tube. Then add 12 mL of complete culture medium to the test tube. Cover the test tube with a stopper and mix it up and down (4 to 5 times). After mixing, pour it into a plain agar plate with an Oxford cup. After solidification, remove the Oxford cup.

[0049] (9) Inject 100 μL of Brevibacillus laterosporus sample into the holes formed in the Oxford cup and incubate at 37°C for 16 h. Observe and measure the diameter of the transparent circle with a vernier caliper.

[0050] (10) Analyze the antibacterial data of the samples to be tested.

[0051] The test results are analyzed as follows:

[0052] Table 1 Evaluation of the antibacterial effect of Brevibacillus laterosporus

[0053] Pathogens in treatment groups CBD230202 CBD230301 CBD230302-LD CBD230304 Vibrio F / mm 18.15 19.20 18.15 19.15 Vibrio R / mm 15.12 16.17 14.05 15.05

[0054] From the above Table 1, Figure 1 , Figure 2 It can be seen that different production batches of Brevibacillus laterosporus have a certain inhibitory effect on the two Vibrio species, among which the Brevibacillus laterosporus strain with production batch number CBD230301 has the most obvious inhibitory effect on Vibrio species F, and its inhibition zone can reach 19.20mm.

[0055] The Brevibacillus laterosporus strain with production batch number CBD230301 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 22, 2024, with the deposit number: GDMCC NO:64668.

[0056] Example 2 Preparation of the bacterial agent "Hu Yi Dao"

[0057] The bacterial agent "Hu Yi Dao" was prepared using the Brevibacillus laterosporus strain with production batch number CBD230301 screened out in Example 1. The preparation was mainly carried out by the following steps:

[0058] (1) CBD230301 Brevibacillus laterosporus in a glycerol tube was inoculated into a plate culture medium (composed of 0.5% sodium chloride, 1.0% yeast extract powder, 1% peptone, and 2% agar). After 16 h of culture, a single colony was transferred to a slant culture medium (with the same composition as the plate culture medium). After 16 h of subculturing, a sample was retained for fermentation.

[0059] (2) The cultured slant culture medium was transferred to a seed tank (volume 1.5t, culture medium composition: 0.5% sodium chloride, 1.2% yeast extract powder, 1.5% peptone, 1.0% glucose). After fermentation in the seed tank for 12 hours, it was transferred to a secondary fermentation tank (volume 15t, culture medium composition: 4% soybean meal, 1.5% glucose, 0.8% magnesium sulfate, 0.8% ammonium sulfate). After fermentation in the secondary fermentation tank for 18 hours, it was transferred to post-extraction.

[0060] (3) After the fermentation is completed, 3.0% magnesium sulfate is added to the fermentation liquid and the liquid is transferred to a spray tower for spray drying. After spray drying, CBD230301 bacterial powder is obtained.

[0061] (4) The spray-dried bacterial powder was thoroughly mixed with 5.0% by mass of maltodextrin and then packaged. The preparation of the bacterial agent "Hu Yidao" was completed.

[0062] In summary, the present invention provides a bacterial agent whose active ingredients are Brevibacillus laterosporus and its metabolites, which can effectively inhibit halophilic Vibrio in seawater.

[0063] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

[0064] Comparative Example

[0065] In this comparative experiment, the culture medium formula was upgraded

[0066] 1. Purpose of the test

[0067] Verify the antibacterial effect of the "Hu Yidao" provided by the present invention on Vibrio compared with competing products.

[0068] 2. Test subjects

[0069] "Hu Yi Dao": active ingredient Brevibacillus laterosporus (CBD230301) and its metabolites;

[0070] Competitor A: The active ingredients are Bacillus subtilis and its metabolites;

[0071] Competitor B: The active ingredient is lipopeptide substances;

[0072] Competitor C: The active ingredient is Bdellovibrio;

[0073] Competitor D: The active ingredient is Bacillus subtilis;

[0074] Competitor E: The active ingredient is Bacillus laterosporus;

[0075] Competitor F: The active ingredient is antimicrobial lipopeptides;

[0076] Competitor G: Specific ingredients are not indicated.

[0077] Note: Competitors A~G are all commercially available Vibrio inhibitors, and the above active ingredients are labeled on the product packaging.

[0078] The Vibrio species were Vibrio species F (Vibrio parahaemolyticus) and Vibrio species R (Vibrio alginolyticus).

[0079] 3. Experimental content and results analysis

[0080] 3.1 Culture medium and consumables

[0081] (1) 3% sodium chloride alkaline peptone water: 3% sodium chloride, 1% peptone; pH 8.5; 121°C, 0.1 MPa, 30 min sterilization, used for Vibrio proliferation.

[0082] (2) 3% sodium chloride peptone medium: 3% sodium chloride, 1% peptone; pH 7.2; 121°C, 0.1 MPa, 30 min sterilization; 1.0% agar powder, used for Vibrio inoculation and culture.

[0083] (3) Plain agar medium: 1.5% agar powder, dissolved in pure water, sterilized at 121°C, 0.1 MPa, 30 min, used for fixing the Oxford cup.

[0084] (4) Physiological saline: 0.85% sodium chloride, sterilized at 121°C, 0.1 MPa, 30 min, used for dilution.

[0085] (5) Consumables: Petri dishes, Oxford cups, tweezers, 200 μL pipette tips, 5 mL pipette tips, test tubes, 5 mL centrifuge tubes, 50 mL centrifuge tubes, vials, 10 mL syringes, 0.22 μm filter membranes, filters, and 250 mL conical flasks with glass beads. Sterilize at 121°C, 0.1 MPa, for 30 min before use.

[0086] 3.2 Operation steps

[0087] (1) One day in advance, inoculate pathogens (Vibrio parahaemolyticus, Vibrio alginolyticus, etc.) from a slant into a conical flask containing 3% sodium chloride alkaline peptone water and culture at 37°C and 200 rpm / min for 16-18 hours.

[0088] (2) Pour about 5 mL of agar into each plate in advance and cool it for later use (only for fixing the Oxford cup).

[0089] (3) Weigh 36 g of normal saline into a 250 mL conical flask with glass beads, weigh 4 g of the sample to be tested using weighing paper, pour it into the conical flask, and shake it manually.

[0090] (4) Place the dissolved sample at room temperature and shake at 200 rpm / min for 30 min.

[0091] (5) Pour the shaken sample into a 50 mL centrifuge tube (about 20 mL), balance it, and centrifuge it at 4°C, 10,000 rpm, for 10 min to obtain the supernatant.

[0092] (6) The supernatant of the sample to be tested is filtered using a 10 mL syringe, a 0.22 μm filter membrane, and a filter. The filtered sample is placed in a 5 mL centrifuge tube for later use.

[0093] (7) Mark the pathogens on the plain agar plate, then place the Oxford cup on the plain agar plate and place it in the clean bench.

[0094] (8) Take out 50 μL of the cultured pathogen (Vibrio) and add it to a test tube. Then add 12 mL of 3% sodium chloride peptone medium to the test tube. Cover the test tube with a stopper and mix up and down (4-5 times). After mixing, pour it into a plain agar plate with an Oxford cup. After solidification, remove the Oxford cup.

[0095] (9) Inject 100 μL of the sample to be tested into the holes formed in the Oxford cup, incubate at 37°C for 16 h, and observe and measure the diameter of the transparent circle with a vernier caliper.

[0096] 3.3 Test results and analysis

[0097] Table 2 Comparison of antibacterial effects of Brevibacillus laterosporus CBD230301 and competing products

[0098]

[0099] Note: “ / ” represents no inhibition zone, i.e. no antibacterial effect

[0100] By optimizing the Vibrio enrichment medium and inoculation medium, the antibacterial effect of Brevibacillus laterosporus on Vibrio is more obvious. At the same time, compared with other Vibrio inhibition products on the market, the relevant data can be found in Fig.10 , combined with Table 2, it can be seen that the inhibition zone of Brevibacillus laterosporus provided by the present invention is larger, and has a significant inhibitory effect on Vibrio parahaemolyticus and Vibrio alginolyticus.

[0101] Actual case application and effect analysis

[0102] A product with significant inhibitory effect on aquatic pathogenic Vibrio, whose core active ingredient is Brevibacillus laterosporus and its metabolites, is now named "Hu Yi Dao". In order to further verify its actual application effect, a series of actual application tests have been planned for the "Hu Yi Dao" product.

[0103] Application Case 1

[0104] (1) Breeding species and cycle: Penaeus vannamei (hereinafter referred to as shrimp), 10 days after stocking, stocking specification 400 heads / catties, 40,000 stockings;

[0105] (2) Case details:

[0106] On May 18, we observed the feed table and found that there was a serious amount of leftover feed, and 5 to 6 dead shrimps were found by the pond;

[0107] On May 19, we reduced the feed input, but the leftover feed was still serious and dead shrimps were still found by the pond. We preliminarily diagnosed the disease as shrimp stealth syndrome, and on the same day we used bacteriophage, Bdellovibrio, lipopeptide and other Vibrio disinfection products. However, the situation did not improve until May 21, the shrimps still refused to eat and the shrimps continued to die by the pond. Master Zhang had already made plans to re-stock the shrimps.

[0108] (3) Treatment plan:

[0109] Considering that Mr. Zhang's shrimps have not eaten the feed, he first used the product "Hu Yidao" provided by the present invention to disinfect Vibrio in the water. On May 22, he began to use "Hu Yidao" for spraying, with a spraying amount of 200g / time, and the spraying frequency was once every three days. A small amount of feed was added to the feed, and the addition ratio was 3% of the weight of the feed.

[0110] (4) Application effect:

[0111] Table 3 Effect of Master Zhang’s “Arc Knife”

[0112] date May 22 May 23 May 24 May 25 May 27 Vibrio count in water (CFU / mL) 3325 525 50 25 <10 Vibrio count in shrimp (CFU / mL) 3000 750 570 204 16

[0113] pass Figure 3 , Figure 4 According to the data analysis in Table 3, we observed that the number of Vibrio in the pond water and shrimp bodies changed significantly during the continuous monitoring from May 22 to May 27. Specifically, starting from May 22, before the use of "Arc-1 Knife", the number of Vibrio in the aquaculture water was as high as 3325 CFU / mL, and the number of Vibrio in the shrimp bodies was also seriously exceeded. However, after the application of "Arc-1 Knife", by May 27, the number of Vibrio in the aquaculture water was effectively controlled, and the number of Vibrio in the shrimp bodies was also significantly reduced and controlled below 16 CFU / mL. The number of shrimps on the feed table gradually recovered from 1 shrimp on May 24 to more than 10 shrimps on May 28. This series of data fully proves the significant effect of "Arc-1 Knife" in inhibiting Vibrio.

[0114] Application Case 2

[0115] (1) Breeding species and cycle: Whiteleg shrimp, 27 days of stocking, stocking specification 1,400 heads / catties, 93,000 shrimps stocked;

[0116] (2) Case situation: On May 25, Mr. Han observed through plate coating that the vibrio chlororaphis in the shrimp was seriously exceeded and the shrimp was in a serious stress state;

[0117] (3) Treatment plan: In view of the excessive Vibrio in Mr. Han’s shrimp, he first used “Hu Yidao” to mix the feed and add it at a ratio of 2% of the feed weight. At the same time, the breeding water was sprayed and disinfected, with a spraying amount of 200g / time;

[0118] (4) Application effect:

[0119] Table 4 Effect of Master Han’s “Arc Knife”

[0120] date May 25 May 27 May 28 May 29 Vibrio count in water (CFU / mL) / 825 275 ＜10 Vibrio count in shrimp (CFU / mL) 2000+ 124 72 ＜10

[0121] Note: “ / ” indicates that the number of Vibrio was not detected.

[0122] Depend on Figure 5 Combining the data in Table 4, we can see that:

[0123] On May 25, the number of Vibrio detected in shrimp bodies was 2000+CFU / mL, which was seriously exceeded;

[0124] On May 27, the detection showed that the Vibrio parahaemolyticus in the shrimp had been eliminated, but there were still Vibrio parahaemolyticus in the water;

[0125] On May 28, the Vibrio parahaemolyticus in the shrimps had disappeared, leaving only Vibrio alginolyticus;

[0126] Vibrio alginolyticus

[0127] Vibrio parahaemolyticus

[0128] As of May 29, the number of Vibrio in the breeding water and shrimps has been effectively controlled.

[0129] Application Case 3

[0130] (1) Breeding species and cycle: Whiteleg shrimp, 30 days after stocking, stocking specification 700 heads / catties, 45,000 shrimps stocked.

[0131] (2) Case: After Mr. Zhang conducted a simple Vibrio test on the pond in the southeast of his village, he found that the number of Vibrio in the water was 2625 CFU / mL, and that in the shrimp bodies it was 198 CFU / mL. The shrimps' eating speed was slightly slowed down.

[0132] (3) Treatment plan: Since the number of Vibrio in the water and shrimps of Master Zhang is relatively high, it is recommended to use "Vibrio One Knife" for spraying and mixing with feed, with a spraying amount of 200g / time. The mixing ratio is 1% of the weight of the feed. In view of the slow feeding of shrimp, butyrate milk is used in combination, and the mixing ratio is adjusted to 2% of the weight of the feed.

[0133] (4) Application effect:

[0134] Table 5 Effects of Master Zhang’s “Arc Knife”

[0135] date May 22 May 23 May 24 May 25 May 27 Vibrio count in water (CFU / mL) 2625 575 350 ＜10 ＜10 Vibrio count in shrimp (CFU / mL) 198 556 380 ＜10 ＜10

[0136] Depend on Figure 6 , Figure 7 From Table 5, we can see that:

[0137] On May 23, it was detected that the number of Vibrio in the aquaculture water had decreased, but the number of Vibrio in the shrimp was still increasing. The reason was analyzed. Since the number of Vibrio in the shrimp was not very large on May 22, only 198CFU / mL, the proportion of "Hu Yidao" mixed feed was 1% of the weight of the feed. However, on May 23, it was detected that the number of Vibrio in the shrimp increased instead of decreasing. It was speculated that the amount of "Hu Yidao" used was too little, so the amount of "Hu Yidao" mixed feed was increased to 2% of the weight of the feed. On May 24, the number of Vibrio in the shrimp dropped to 380CFU / mL, which also indirectly proved that the inhibitory effect of "Hu Yidao" on Vibrio was very significant.

[0138] On May 27, the number of Vibrio in the breeding water and shrimp bodies was effectively controlled and dropped to 0; at the same time, the feeding speed of the southern pond was 1 hour faster than that of other ponds, and the butyrate milk had a significant effect in attracting food.

[0139] Application Case 4

[0140] (1) Breeding species and cycle: Whiteleg shrimp, 14 days after seeding, 1,500 heads / catties, 80,000 seedlings;

[0141] (2) Case details: On May 22, Mr. Li felt that the feed feeding speed of the feed table had slowed down. That evening, he conducted a Vibrio plate test on the aquaculture water and shrimp. The test results showed that the number of Vibrio in the water was 325 CFU / mL, and the number of Vibrio in the shrimp was 175 CFU / mL.

[0142] (3) Treatment plan: According to the test results of Mr. Li, there are a few Vibrio in the breeding water and shrimp bodies. It is recommended to use "Vibrio Knife" for spraying and mixing with feed. The spraying amount is 200g / time, and the mixing ratio is 1% of the weight of the feed.

[0143] (4) Application effect:

[0144] Table 6 Effect of Master Li’s “Arc Knife”

[0145] date May 22 May 23 May 24 May 28 Vibrio count in water (CFU / mL) 325 25 ＜10 ＜10 Vibrio count in shrimp (CFU / mL) 175 16 ＜10 ＜10

[0146] Depend on Figure 8 , Fig. 9 From Table 6, we can see that:

[0147] On May 23, it was detected that the number of Vibrio in the aquaculture water and shrimp bodies had been greatly reduced;

[0148] On May 24, the Vibrio in the breeding water and shrimp bodies had been completely controlled. After that, Master Li reduced the proportion of mixed feed to 0.5% of the weight of the feed and only mixed it once a day. By May 28, the Vibrio in the shrimp bodies was only 2 CFU / mL, and the prevention effect was very significant.

Claims

1. A strain of Brevibacillus laterosporus, characterized in that: The Bacillus laterosporus was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 22, 2024, with the deposit number: GDMCC NO: 64668.

2. Use of Brevibacillus laterosporus as claimed in claim 1 in inhibiting aquatic pathogenic Vibrio.

3. A bacterial agent containing the Brevibacillus laterosporus according to claim 1.

4. Use of the bacterial agent as claimed in claim 3 in inhibiting aquatic pathogenic Vibrio.

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