Microbial agent based on bacillus velezensis and application thereof

Abstract

The invention relates to a microbial agent prepared based on a new strain of bacillus velezensis, which has the function of broad-spectrum antagonism of common pathogenic bacteria of aquatic products and can effectively prevent and / or treat bacterial diseases and / or fungal diseases of aquatic organisms including crustaceans and fishes. Meanwhile, the content of nitrite and ammonia nitrogen in aquaculture water can be effectively reduced, and the usage amount and residual quantity of antibiotics and chemical drugs in aquaculture are reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to a strain of Bacillus velezensis, a microbial inoculant prepared therefrom, and applications thereof. BACKGROUND

[0002] Aquaculture, as an important pillar industry of global food supply chain, faces severe challenges in sustainable development. In the process of intensive aquaculture, frequent outbreaks of diseases cause economic losses of up to billions of dollars each year. Pathogenic factor analysis shows that waterborne pathogens (such as Vibrio, Aeromonas, Streptococcus and Fusarium) with multiple pathogenicity, rapid proliferation ability and extensive transmission characteristics are the main culprits of large-scale epidemics. In current prevention and control practices, broad-spectrum antibiotics can quickly control the disease, but their non-selective bacteriostatic mechanism leads to an increase in drug-resistant strains. Drug-resistant genes transmitted through the food chain have become a public health safety hazard. Under this background, the development of environmentally friendly biological control agents has become an urgent need in the industry. It is worth noting that the microbial resource library contains a wealth of antibacterial active substances, and their natural antagonistic properties and targeted inhibitory effects provide an ideal solution for building a new "bacteria-to-bacteria" prevention and control system.

[0003] In addition, with the expansion of aquaculture scale, the amount of leftover feed, aquaculture animal excrement and other organic pollutants in the aquaculture water body is increasing. These organic pollutants consume a large amount of dissolved oxygen in the water body during decomposition. At the same time, a large amount of toxic and harmful substances such as ammonia nitrogen and nitrite are also produced during the decomposition process. When these substances accumulate to a certain extent in the water body, they will have a serious impact on the growth and reproduction of aquatic animals. Moreover, the proliferation of pathogenic microorganisms in the water body is positively correlated with the concentration of ammonia nitrogen and nitrite in the water body.

[0004] In order to solve the problem of pollution in aquaculture water bodies, the current commonly used methods all have drawbacks. Physical treatment methods can remove some larger particles of pollutants, but the removal effect of dissolved ammonia nitrogen and nitrite is limited. The use of chemical agents not only easily causes secondary pollution, but also may be left in the bodies of aquatic animals, endangering human health. Existing biological treatment methods have become a research hotspot because of their environmental protection and high efficiency.

[0005] Among the numerous microorganisms used for water purification, Bacillus has been widely concerned due to its unique advantages. Bacillus can maintain high activity in complex and variable water environment of aquaculture, and has strong colonization ability, can continuously play a role, and is not prone to rebound phenomenon, which can fundamentally improve the water environment. At the same time, Bacillus has fast reproduction speed, and can rapidly increase population number in a short time under suitable conditions, thereby improving the efficiency of degrading ammonia nitrogen and nitrite. In addition, as a microbial preparation, Bacillus has less impact on the ecological environment of water body, will not cause secondary pollution, and will not cause drug residues and other hazards to aquatic animals, which meets the development trend of "green and safe" of modern aquaculture.

[0006] As an important member of Bacillus, B. velezensis has shown great application potential in the field of aquaculture. However, at present, the systematic research and application of B. velezensis in the field of aquaculture for synergistically inhibiting pathogenic bacteria and removing nitrite and ammonia nitrogen are relatively few. Most studies only focus on the exploration of single function, and lack of in-depth analysis of the comprehensive action mechanism and actual application effect. Moreover, the existing microbial agents also have problems such as poor stability and large influence of environmental factors on the effect in the actual application process.

[0007] Therefore, it has important practical significance to develop a compound microbial agent based on B. velezensis, which is efficient, stable and can synergistically inhibit pathogenic bacteria and remove nitrite and ammonia nitrogen, for promoting the healthy and sustainable development of aquaculture. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application discloses a microbial agent based on B. velezensis and its application in inhibiting pathogenic bacteria and removing nitrite and ammonia nitrogen in aquaculture. The microbial agent has the function of broad-spectrum antagonism to common pathogenic bacteria in aquaculture, can effectively inhibit the growth of waterborne pathogenic bacteria such as Escherichia coli, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, Vibrio parahaemolyticus, Fusarium oxysporum and Streptococcus agalactiae, and can effectively reduce the content of nitrite and ammonia nitrogen in aquaculture water, reduce the use amount and residue amount of chemical drugs in aquaculture, and is expected to be developed into a natural antibiotic product, which has potential application value and huge market demand in the field of aquaculture and feed antibiotic replacement.

[0009] The application is implemented through the following technical scheme, and provides a microbial inoculant based on Bacillus velezensis, which comprises at least one of the following Bacillus velezensis strains: a. a Bacillus velezensis strain with a preservation number of CCTCC M 2025378; b. a Bacillus velezensis strain with a preservation number of CCTCC M 2025379; c. a Bacillus velezensis strain with a preservation number of CCTCC M 2025380; and d. a Bacillus velezensis strain with a preservation number of CCTCC M 2025381.

[0010] Bacillus velezensis belongs to Firmicutes, Bacillales, Bacillaceae and Bacillus. It is a kind of gram-positive bacteria producing spores. Most Bacillus has the advantages of wide antibacterial spectrum, rapid growth, easy isolation and culture, strong stress resistance and high biological safety, and is widely studied as probiotics in agriculture, food, industry, medicine, metallurgy, forestry, environmental protection and military, etc. Bacillus velezensis inhibits the growth of pathogenic bacteria by synthesizing secondary metabolites with antibacterial activity, mainly including bacteriocins, antibacterial proteins, lipopeptides and polyketides. Their main modes of action include destroying cell walls, controlling key DNA and RNA, and inhibiting enzyme activity. Among them, antibacterial lipopeptides play an important role. The lipopeptides produced by Bacillus velezensis include surfactin, iturin, formosin and bacillomycin, etc.

[0011] The four Bacillus velezensis strains of the application all have typical biological characteristics of Bacillus velezensis: gram-positive bacteria, short rod-shaped bacteria, single arrangement, non-motile; colony 3-4 mm, round, convex, milky white, relatively viscous, regular edge, surface convex with wrinkles.

[0012] The microbial inoculant refers to a live bacteria preparation processed from fermentation liquor containing target microorganisms (effective bacteria). After industrialized production and expansion of the target microorganisms, porous materials such as grass carbon and vermiculite can be used as adsorbents to adsorb the target microorganisms, or the fermentation bacteria liquor or powder can be used.

[0013] As certain embodiments of the application, the microbial inoculant comprises the Bacillus velezensis strain with a preservation number of CCTCC M 2025378, the Bacillus velezensis strain with a preservation number of CCTCC M 2025379, the Bacillus velezensis strain with a preservation number of CCTCC M 2025380 and the Bacillus velezensis strain with a preservation number of CCTCC M 2025381.

[0014] As certain embodiments of the application, the microbial inoculant comprises live bacteria of the Bacillus velezensis strain.

[0015] As certain embodiments of the present application, the viable bacteria of the Bacillus velezensis strain include a solid-state fermentation viable bacteria preparation and a liquid-state fermentation viable bacteria preparation.

[0016] As certain embodiments of the present application, the dosage form of the microbial inoculant includes a powder, a liquid, and a solid preparation.

[0017] As certain embodiments of the present application, the ratio of the Bacillus velezensis strain with the accession number CCTCC M 2025378: the Bacillus velezensis strain with the accession number CCTCC M 2025379: the Bacillus velezensis strain with the accession number CCTCC M 2025380: the Bacillus velezensis strain with the accession number CCTCC M 2025381 in the microbial inoculant is 2-5:0.1-3:0.5-4:0.5-3.

[0018] The ratio of the Bacillus velezensis strain with the accession number CCTCC M 2025378 is selected from 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0.

[0019] The ratio of the Bacillus velezensis strain with the accession number CCTCC M 2025379 is selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0.

[0020] The ratio of the Bacillus velezensis strain with the accession number CCTCC M 2025380 is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0.

[0021] The ratio of the Bacillus velez strain with the deposit number CCTCC M 2025381 is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

[0022] As certain embodiments of the present application, the ratio of the Bacillus Velez strain with the preservation number of CCTCC M 2025378: the Bacillus Velez strain with the preservation number of CCTCC M 2025379: the Bacillus Velez strain with the preservation number of CCTCC M 2025380: the Bacillus Velez strain with the preservation number of CCTCC M 2025381 in the microbial agent is 3-4:0.5-2:1-3:1-2.

[0023] As certain embodiments of the present application, the ratio of the Bacillus Velez strain with the preservation number of CCTCC M 2025378: the Bacillus Velez strain with the preservation number of CCTCC M 2025379: the Bacillus Velez strain with the preservation number of CCTCC M 2025380: and the Bacillus Velez strain with the preservation number of CCTCC M 2025381 in the microbial agent is 4:2:3:2.

[0024] As certain embodiments of the present application, the bacterial concentration in the microbial agent is ≥10 6 CFU / g.

[0025] The bacterial concentration in the microbial agent is selected from 1.0×10 6 CFU / g, 2.0×10 6 CFU / g, 3.0×10 6 CFU / g, 4.0×10 6 CFU / g, 5.0×10 6 CFU / g, 6.0×10 6 CFU / g, 7.0×10 6 CFU / g, 8.0×10 6 CFU / g, 9.0×10 6 CFU / g, 1.0×10 7 CFU / g, 2.0×10 7 CFU / g, 3.0×10 7 CFU / g, 4.0×10 7 CFU / g, 5.0×10 7 CFU / g, 6.0×10 7CFU / g, 7.0 x 10 7 CFU / g, 8.0 x 10 7 CFU / g, 9.0 x 10 7 CFU / g, 1.0 x 10 8 CFU / g, 2.0 x 10 8 CFU / g, 3.0 x 10 8 CFU / g, 4.0 x 10 8 CFU / g, 5.0 x 10 8 CFU / g, 6.0 x 10 8 CFU / g, 7.0 x 10 8 CFU / g, 8.0 x 10 8 CFU / g, 9.0 x 10 8 CFU / g, 1.0 x 10 9 CFU / g, 2.0 x 10 9 CFU / g, 3.0 x 10 9 CFU / g, 4.0 x 10 9 CFU / g, 5.0 x 10 9 CFU / g, 6.0 x 10 9 CFU / g, 7.0 x 10 9 CFU / g, 8.0 x 10 9 CFU / g, 9.0 x 10 9 CFU / g, 1.0 x 10 10 CFU / g, 2.0 x 10 10 CFU / g, 3.0 x 10 10 CFU / g, 4.0 x 10 10 CFU / g, 5.0 x 10 10 CFU / g, 6.0 x 10 10 CFU / g, 7.0 x 10 10 CFU / g, 8.0 x 10 10 CFU / g, 9.0 x 10 10 CFU / g, 1.0 x 10 11 CFU / g, 2.0 x 10 11 CFU / g, 3.0 x 10 11 CFU / g, 4.0 x 10 11 CFU / g, 5.0 x 10 11 CFU / g, 6.0 x 10 11 CFU / g, 7.0 x 10 11 CFU / g, 8.0 x 10 11 CFU / g, 9.0 x 10 11 CFU / g, 1.0 x 10 12CFU / g, 1.0 x 10 12 CFU / g, 3.0 x 10 12 CFU / g, 4.0 x 10 12 CFU / g, 5.0 x 10 12 CFU / g, 6.0 x 10 12 CFU / g, 7.0 x 10 12 CFU / g, 8.0 x 10 12 CFU / g, 9.0 x 10 12 CFU / g, 1.0 x 10 13 CFU / g, 2.0 x 10 13 CFU / g, 3.0 x 10 13 CFU / g, 4.0 x 10 13 CFU / g, 5.0 x 10 13 CFU / g, 6.0 x 10 13 CFU / g, 7.0 x 10 13 CFU / g, 8.0 x 10 13 CFU / g, 9.0 x 10 13 CFU / g, 1.0 x 10 14 CFU / g, 2.0 x 10 14 CFU / g, 3.0 x 10 14 CFU / g, 4.0 x 10 14 CFU / g, 5.0 x 10 14 CFU / g, 6.0 x 10 14 CFU / g, 7.0 x 10 14 CFU / g, 8.0 x 10 14 CFU / g, 9.0 x 10 14 CFU / g, 1.0 x 10 15 CFU / g, 2.0 x 10 15 CFU / g, 3.0 x 10 15 CFU / g, 4.0 x 10 15 CFU / g, 5.0 x 10 15 CFU / g, 6.0 x 10 15 CFU / g, 7.0 x 10 15 CFU / g, 8.0 x 10 15 CFU / g, 9.0 x 10 15 CFU / g, 1.0 x 10 16 CFU / g.

[0026] The second aspect of the present application provides a feed additive comprising the microbial inoculant.

[0027] The application of the feed additive is expected to replace one or more of anti-Escherichia coli drugs, anti-Aeromonas hydrophila drugs, anti-Aeromonas veseri drugs, anti-Vibrio harveyi drugs, anti-Vibrio parahaemolyticus drugs, anti-common Fusarium drugs, and anti-Streptococcus agalactiae drugs.

[0028] The third aspect of the present application is to provide a feed comprising the feed additive.

[0029] The fourth aspect of the present application is to provide a water quality improver, which includes the microbial agent.

[0030] Water quality improvers may include a variety of complex biological enzymes, probiotics, vitamins, trace elements, mineral enhancers and salts. Through biological and chemical methods, they work together to improve the poor water quality and bottom quality caused by organisms such as leftover bait and feces caused by high temperature and heat.

[0031] The fifth aspect of the present application is to provide a method for preparing the microbial agent, which comprises inoculating the Bacillus Velez strain into a culture medium and fermenting and culturing the culture medium to obtain the microbial agent.

[0032] As certain embodiments of the present application, the method comprises inoculating the Bacillus Velez strains into a culture medium, fermenting and culturing the culture medium, and mixing the fermentation products in proportion to obtain the microbial agent.

[0033] As certain embodiments of the present application, the culture medium comprises: 5-15 g / L corn starch, 20-30 g / L flour, 5-15 g / L corn flour, 5-15 g / L bran, 10-20 g / L corn steep liquor powder, 5-10 g / L yeast extract, 2-5 g / L peptone, 6-12 g / L soybean meal, 0.1-0.5 g / L anhydrous calcium chloride, 0.1-0.5 g / L manganese chloride tetrahydrate, 1.0-2.0 g / L magnesium sulfate heptahydrate, 1.0-2.0 g / L sodium dihydrogen phosphate dihydrate, and 1-10 g / L sodium chloride, and the initial pH of the culture medium is 4.0-9.0.

[0034] As certain embodiments of the present application, the culture medium comprises: 10 g / L corn starch, 30 g / L flour, 10 g / L corn flour, 10 g / L bran, 17 g / L corn steep liquor powder, 8 g / L yeast extract, 4 g / L peptone, 9 g / L soybean meal, 0.1 g / L anhydrous calcium chloride, 0.2 g / L manganese chloride tetrahydrate, 1.5 g / L magnesium sulfate heptahydrate, 1 g / L sodium dihydrogen phosphate dihydrate, and 5 g / L sodium chloride, and the initial pH of the culture medium is 7.0.

[0035] As certain embodiments of the present application, the culture condition of the fermentation culture is a culture temperature of 20-42℃, a fermentation time of 12-72h, and an inoculation amount of the Bacillus velezensis strain of 1%-10%.

[0036] As certain embodiments of the present application, the culture condition of the fermentation culture is a culture temperature of 37℃, a fermentation time of 37h, and an inoculation amount of the Bacillus velezensis strain of 5%.

[0037] A sixth aspect of the present application is to provide a method for improving the water quality of a water body, comprising administering the water quality improver to the water body, thereby improving the water quality of the water body.

[0038] As certain embodiments of the present application, the water body is an aquaculture water body.

[0039] A seventh aspect of the present application is to provide the use of the microbial agent or the feed or the feed additive in the preparation of an agent for inhibiting pathogenic bacteria.

[0040] As certain embodiments of the present application, the pathogenic bacteria include pathogenic bacteria in an aquaculture water body.

[0041] As certain embodiments of the present application, the pathogenic bacteria include any one or more of Escherichia coli, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, Vibrio parahaemolyticus, Fusarium oxysporum, and Streptococcus agalactiae.

[0042] As certain embodiments of the present application, the pathogenic bacteria include any one or more of Escherichia coli, Aeromonas hydrophila, Fusarium oxysporum, and Vibrio parahaemolyticus.

[0043] The microbial agent is expected to replace antibacterial chemical drugs, which are antibacterial drugs or / and antifungal drugs. Further, the antibacterial chemical drugs include one or more of anti-Escherichia coli drugs, anti-Aeromonas hydrophila drugs, anti-Aeromonas veronii drugs, anti-Vibrio harveyi drugs, anti-Vibrio parahaemolyticus drugs, anti-Fusarium oxysporum drugs, and anti-Streptococcus agalactiae drugs.

[0044] As certain embodiments of the present application, the animal includes aquatic organisms.

[0045] As certain embodiments of the present application, the animal includes crustacean organisms and fish organisms.

[0046] As certain embodiments of the present application, the preparation of an agent for inhibiting pathogenic bacteria includes the preparation of an agent for inhibiting bacteria and / or fungi.

[0047] The eighth aspect of the present application is to provide the use of the microbial agent, the feed, the feed additive or the water quality improver in improving the water quality of water bodies.

[0048] As certain embodiments of the present application, the water quality of the water body includes the water quality of aquaculture water body.

[0049] As certain embodiments of the present application, the improvement of water quality includes reducing the content of nitrite and / or ammonia nitrogen in the water.

[0050] As described above, the Bacillus velezensis strain of the present application and its application have the following beneficial effects:

[0051] The present application is based on a new microbial agent of Bacillus Velez, which has the function of broad-spectrum antagonism against common aquatic pathogens and can effectively inhibit the growth of aquatic pathogens such as Escherichia coli, Aeromonas hydrophila, Aeromonas welchii, Vibrio harveyi, Vibrio parahaemolyticus, Fusarium spp. and Streptococcus agalactiae. Compared with the Bacillus Velez DH82, Bacillus Velez WLYS39, Bacillus Velez WLYS39, Bacillus Velez RS6-14 used to inhibit aquatic pathogens in the prior art, the antibacterial spectrum is wider.

[0052] The microbial agent of the present application can effectively reduce the content of nitrite and ammonia nitrogen in aquaculture water, reduce the usage and residue of antibiotics and chemical drugs in aquaculture, and is expected to be developed into a natural antibiotic product. It has potential application value and huge market demand in the fields of aquaculture and feed antibiotic replacement.

[0053] The microbial agent based on the new Bacillus Velez subtilis of the present application is applied to aquaculture water bodies, which can simultaneously antagonize common aquatic pathogens in a broad spectrum and reduce the nitrite and ammonia nitrogen content in aquaculture water bodies. It can reduce nitrite and ammonia nitrogen pollution in water bodies while preventing and / or treating bacterial diseases and fungal diseases of aquatic organisms. DETAILED DESCRIPTION

[0054] In order to make the technical means, creative features, objectives and effects achieved by this application easier to understand, the following will be combined with the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of this application.

[0055] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. The various terms and phrases used in this application have the general meanings known to those skilled in the art.

[0056] Example 1. Isolation and identification of Bacillus velezensis

[0057] 1. Isolation and screening of strains

[0058] Water samples were collected from the culture ponds in the culture area. The water samples from the culture ponds were serially diluted 10 times for 6 times with sterile normal saline, 100 μL of solution was taken from each concentration gradient dilution with a pipette gun onto LB solid plates, evenly coated with a coating rod, numbered, and three repeats were made. After uniform coating, the culture medium was placed in a clean bench for 5-10 min to allow the bacterial solution on the surface to be fully absorbed. Finally, the plates were inverted and incubated in a constant temperature incubator at 30°C for 24 h. Different morphological colonies were selected and inoculated on LB plates for isolation and purification.

[0059] The bacteria obtained by isolation and purification were subjected to antibacterial performance determination by Oxford cup plate antibacterial method. After a large number of isolation and screening, 4 strains of bacteria showing good inhibition effect on pathogenic bacteria such as Escherichia coli, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, and Fusarium oxysporum were obtained. Detection showed that the 4 strains of microorganisms were gram-positive bacteria, the bacterial bodies were short rods, arranged singly, and were not motile; the colonies were 3-4 mm, round, convex, milky white, relatively viscous, with neat edges and a convex surface with wrinkles.

[0060] 2. Molecular biological identification of strains

[0061] The pure cultures of the above 4 strains of microorganisms were inoculated in LB liquid medium and cultured at 30°C with shaking at 180 r / min for 24 h, and the bacterial bodies were collected by centrifugation; the total DNA of the 4 strains of microorganisms was extracted by using a centrifugal column type bacterial genomic DNA extraction kit; the 16S rRNA gene of the strains was amplified by using universal primers, and the 4 strains of microorganisms were identified as Bacillus velezensis by 16S rRNA sequence homology analysis. Bacillus velezensis is a gram-positive bacterium and is strictly aerobic. The colonies formed after 48 h of culture in LB medium at 30°C are milky white and translucent, with neat edges and a convex surface with wrinkles.

[0062] The 4 strains of microorganisms were named as Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4, respectively in this application.

[0063] Bacillus velezensis LTS-AF1, classified as Bacillus velezensis, accession number CCTCC M 2025378, preserved in China Center for Type Culture Collection (CCTCC), preservation date: March 5, 2025.

[0064] Bacillus velezensis LTS-AF2, classified as Bacillus velezensis, accession number CCTCC M 2025379, preserved in China Center for Type Culture Collection (CCTCC), preservation date: March 5, 2025.

[0065] Bacillus velezensis LTS-AF3, classified as Bacillus velezensis, accession number CCTCC M 2025380, preserved in China Center for Type Culture Collection (CCTCC), preservation date: March 5, 2025.

[0066] Bacillus velezensis LTS-AF4, classified as Bacillus velezensis, accession number CCTCC M 2025381, preserved in China Center for Type Culture Collection (CCTCC), preservation date: March 5, 2025.

[0067] Example 2 Determination of the antibacterial function of Bacillus velezensis

[0068] Four strains of Bacillus velezensis with antibacterial function and pathogenic bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, Fusarium oxysporum were inoculated into LB liquid medium respectively, and cultured at 30°C with 180r / min shaking for 24h. The bacterial liquid was resuspended with sterile normal saline for counting, so that the final concentration of the four strains of Bacillus velezensis, Bacillus velezensis complex bacterial liquid (LTS-AF1:LTS-AF2:LTS-AF3:LTS-AF4 was 2:1:1:1) and pathogenic bacteria liquid were all 1×10 6 CFU / ml; 100μl of pathogenic bacteria liquid was taken and spread on different LB solid plates, and placed in a sterile operation table for 30min to allow the culture medium surface liquid to be fully absorbed. Two sterile Oxford cups (inner diameter 6mm) were placed on the pathogenic bacteria plate, 50μl of Bacillus velezensis liquid was added to one Oxford cup, and 50μl of PBS was added to the other as a blank control. After 24h of culture, the diameter of the inhibition zone was measured. The antibacterial circles of the four strains of Bacillus velezensis are shown in Table 1, while no antibacterial circle appeared on the PBS blank control.

[0069] Table 1

[0070]

[0071] As shown in Table 1, the Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 all have good inhibitory effect on E. coli, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi and Fusarium graminearum. The inhibitory effect of the compound microbial inoculum of Bacillus velezensis on various pathogenic microorganisms is better than that of the single strain under the condition that the viable cell count of the compound microbial inoculum is equivalent to that of the single strain, indicating that the compound microbial inoculum produces a synergistic inhibitory effect on pathogenic microorganisms.

[0072] Example 3 Preparation of compound microbial inoculum based on Bacillus velezensis

[0073] The compound microbial inoculum of the present embodiment is prepared by culturing the Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 through primary seed, secondary seed, fermentation, centrifugal concentration and drying, respectively. The effective viable cell count ratio of the Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 is 4:2:3:2, and the effective bacterial concentration of the prepared compound microbial inoculum is 10 10 CFU / g.

[0074] The medium used for the primary seed and secondary seed culture of the Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 in the present embodiment is LB broth, and the formula is as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 ml of distilled water, with the initial pH adjusted to 7.0.

[0075] The medium formula used for the fermentation culture of the Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 in the present embodiment is as follows: 10 g / L of corn starch, 30 g / L of flour, 10 g / L of corn flour, 10 g / L of bran, 17 g / L of corn syrup dry powder, 8 g / L of yeast extract, 4 g / L of proteose peptone, 9 g / L of soybean meal, 0.1 g / L of anhydrous calcium chloride, 0.2 g / L of manganese chloride tetrahydrate, 1.5 g / L of magnesium sulfate heptahydrate, 1 g / L of sodium phosphate dibasic dihydrate, and 5 g / L of sodium chloride, with the initial pH of the medium being 7.0.

[0076] The drying method used in the present embodiment is as follows: the bran, diatomite and corn flour are mixed uniformly according to a weight ratio of 1:1:1, and the obtained mixture is used as a carrier for the auxiliary materials; the obtained concentrated bacterial solution is added to the carrier for the auxiliary materials, and the amount of the auxiliary materials added is 1000 g per 100 ml of the concentrated bacterial solution. After mixing, the mixture is dried at 55°C to obtain a powdery preparation.

[0077] Example 4 Safety evaluation of compound microbial inoculum based on Bacillus velezensis

[0078] The complex microbial agent was resuspended with 0.85% sterile normal saline for counting, and the model animals zebrafish and daphnia magna were respectively immersed in 1x10 5 CFU / ml, 1x10 6 CFU / ml, 1x10 7 CFU / ml, 1x10 8 CFU / ml of bacteria solution for 2h, and the control group was immersed with the same volume of PBS, and the incidence and death of zebrafish and daphnia magna within 10d were observed.

[0079] The results showed that zebrafish and daphnia magna grew well and no death occurred within 10d, indicating that the complex microbial agent based on bacillus beljainus had no pathogenicity.

[0080] Example 5 Application of complex microbial agent based on bacillus beljainus in breeding ponds

[0081] The test site was selected in the breeding ponds of eriocheir sinensis, lateolabrax japonicus and procambarus clarkii in Gaochun, Nanjing.

[0082] The test set 3 breeding varieties as eriocheir sinensis, lateolabrax japonicus and procambarus clarkii respectively, and 6 breeding ponds were selected for test in each group. 3 ponds were sprayed with complex microbial agent based on bacillus beljainus as treatment group, and 3 ponds were used as blank control group. The feeding conditions were unchanged during the test period.

[0083] The use method of the complex microbial agent: 1kg of complex microbial agent (10 10 CFU / g) was dissolved in 50L water, and evenly sprayed in the pond. Each 1kg of complex microbial agent was used for 10 mu of pond.

[0084] At 0h of the test, the water samples and intestinal tract of breeding animals in each pond were taken for detection of aeromonas veronii and aeromonas hydrophila, and the levels of ammonia nitrogen and nitrite in the pond water were detected. After 72h, the number of aeromonas hydrophila and aeromonas veronii in the water samples and intestinal tract of breeding animals in all test ponds was determined again, and the levels of ammonia nitrogen and nitrite in the pond water were detected. The test results are shown in Tables 2-4.

[0085] Among them, the inhibition rate = (F-F’) / F’x100%

[0086] Among them, F’: 0h bacterial number; F: 72h bacterial number.

[0087] The determination of ammonia nitrogen and nitrite was carried out by Nessler’s method and diazo azo oxidation method respectively.

[0088] Reduction rate = (P-P’) / P’x100%

[0089] P': 0h ammonia nitrogen and nitrite level; P: 72h ammonia nitrogen and nitrite level.

[0090] Table 2

[0091]

[0092] Table 3

[0093]

[0094] Table 4

[0095]

[0096] It can be seen that for fish, crustaceans and other aquatic organisms, the microbial agent of the present application can effectively inhibit harmful microorganisms in the outside and inside (intestine) of the organism, and the inhibition effect on harmful microorganisms in the outside and inside of the organism is quite.

[0097] For fish, crustaceans and other aquatic organisms, the microbial agent of the present application can effectively inhibit the ammonia nitrogen and nitrite content in the aquaculture water, and can effectively purify the aquaculture water in a short time.

[0098] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed methods and technical contents without departing from the scope of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the present application.

Claims

1. A microbial agent based on Bacillus velezensis, characterized in that: The microbial agent comprises at least any one of the following Bacillus Velez strains: a. Bacillus velez strain, whose deposit number is CCTCC M 2025378; b. Bacillus velez strain, whose deposit number is CCTCC M 2025379; c. Bacillus velez strain, whose deposit number is CCTCC M 2025380; d. Bacillus velez strain, whose deposit number is CCTCC M 2025381; Preferably, the microbial agent comprises the Bacillus Velezii strains described in a to d.

2. The microbial agent based on Bacillus Velezii according to claim 1, characterized in that The microbial agent includes live bacteria of the Bacillus Velez strain; Preferably, the live bacteria of the Bacillus Velez strain include solid-state fermentation live bacteria preparations and liquid-state fermentation live bacteria preparations; Preferably, the dosage forms of the microbial agent include powder, liquid and solid preparations.

3. The microbial agent based on Bacillus Velezii according to claim 1, characterized in that The ratio of the Bacillus velez strain with the deposit number of CCTCC M 2025378: the Bacillus velez strain with the deposit number of CCTCC M 2025379: the Bacillus velez strain with the deposit number of CCTCC M 2025380: the Bacillus velez strain with the deposit number of CCTCC M 2025381 in the microbial agent is 2-5:0.1-3:0.5-4:0.5-3; Alternatively, the bacterial concentration in the microbial agent is ≥10 6 CFU / g.

4. A feed or feed additive, characterized in that The feed additive comprises the microbial agent based on Bacillus velezensis according to any one of claims 1 to 3; Alternatively, the feed comprises the feed additive.

5. A water quality improver, characterized in that: The water quality improver comprises the microbial agent based on Bacillus velezensis according to any one of claims 1 to 3.

6. A method for preparing the microbial agent based on Bacillus velezensis according to claims 1 to 3, characterized in that: The method comprises inoculating the Bacillus velezensis strain described in claim 1 into a culture medium, and fermenting and culturing the culture medium to obtain the microbial agent.

7. Use of the microbial agent based on Bacillus velezensis according to any one of claims 1 to 3 or the feed or feed additive according to claim 4 in the preparation of an agent for inhibiting pathogenic bacteria; Preferably, the pathogenic bacteria include pathogenic bacteria in aquaculture water; Preferably, the animals comprise aquatic organisms.

8. The use according to claim 7, characterized in that The agent that inhibits pathogens includes an agent that inhibits bacteria and / or fungi.

9. A method for improving water quality, characterized in that: The method comprises administering the water quality improver according to claim 5 into a water body, thereby improving the water quality of the water body; Preferably, the water body is an aquaculture water body.

10. Use of the microbial agent based on Bacillus Velezii according to any one of claims 1 to 3, the feed or feed additive according to claim 4, or the water quality improver according to claim 5 in improving water quality; Preferably, the water quality includes aquaculture water quality; Preferably, the improvement of water quality includes reducing the nitrite and / or ammonia nitrogen content in the water.

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