Complex microbial inoculant for water quality purification and vibrio prevention and control and preparation method of complex microbial inoculant

Abstract

The invention relates to the technical field of aquaculture, and particularly discloses a complex microbial inoculant for water purification and vibrio prevention and control and a preparation method of the complex microbial inoculant. The complex microbial inoculant for water quality purification and vibrio prevention and control comprises bacillus subtilis, bacillus natto and bacillus pumilus in a viable count ratio of (1-3): (1-2): (1-2). The bacillus subtilis is a strain capable of producing protease and amylase; the bacillus natto is a bacterial strain capable of generating nattokinase and protease; the bacillus pumilus is a bacterial strain capable of generating antibacterial small peptide for inhibiting vibrio. The total viable count of the complex microbial inoculant can be increased, the COD degradation rate of the complex microbial inoculant within a short time and the vibrio inhibition rate of the complex microbial inoculant within a long time can be increased, and the rapid water quality purification performance and the lasting vibrio prevention and control performance of the complex microbial inoculant can be improved.

Description

Technical Field

[0001] This invention relates to the technical field of aquaculture, and in particular to a compound bacterial agent for water purification and Vibrio control, and its preparation method. Background Technology

[0002] With the rapid development of intensive aquaculture, water pollution and frequent diseases have become key issues restricting the industry's sustainable development. The accumulation of organic pollutants such as uneaten feed, feces, and biological remains during the aquaculture process leads to eutrophication, excessive ammonia nitrogen and nitrite levels, and deterioration of the bottom sediment, providing a breeding ground for pathogenic microorganisms. Vibrio disease is one of the most common and damaging bacterial diseases in aquaculture, often causing large-scale mortality of economically important species such as shrimp and fish, resulting in huge economic losses.

[0003] Currently, the main methods for addressing these problems include the use of chemical disinfectants and antibiotics. However, these methods have many drawbacks: while chemical disinfectants kill harmful bacteria, they also disrupt the beneficial microecological balance in the water and may produce toxic byproducts; the overuse of antibiotics can easily lead to drug residues, increased bacterial resistance, and environmental pollution. Therefore, developing safe, efficient, and environmentally friendly microecological preparations has become a research hotspot in aquaculture.

[0004] In related technologies, microecological preparations are mostly single-species or simple compound bacterial agents, which have shortcomings such as single function, slow onset of action, and short duration of effect. For example, some water purification bacterial agents have a strong ability to decompose organic matter, but have no inhibitory effect on pathogenic bacteria; some antibacterial agents can temporarily reduce the number of Vibrio bacteria, but cannot solve the fundamental problem of organic pollution in water bodies, and because the strains cannot effectively colonize in water bodies, the antibacterial effect is not lasting.

[0005] Therefore, there is an urgent need in this field for a novel composite microbial agent that can simultaneously achieve the dual functions of rapid water purification and long-term control of Vibrio. Summary of the Invention

[0006] This application provides a compound bacterial agent for water purification and Vibrio control, and a method for preparing the same. The bacterial agent can rapidly purify water and provide long-lasting control of Vibrio.

[0007] Firstly, this application provides a compound bacterial agent for water purification and Vibrio control, employing the following technical solution: A compound bacterial agent for water purification and Vibrio control includes Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of (1-3):(1-2):(1-2).

[0008] In one specific implementation, the Bacillus subtilis strain is capable of producing protease and amylase; the Bacillus natto strain is capable of producing nattokinase and protease; and the Bacillus pumilus strain is capable of producing antimicrobial peptides that inhibit Vibrio.

[0009] In one specific feasible implementation, the total viable count of the compound microbial agent used for water purification and Vibrio control is ≥2.0 × 10⁻⁶. 10 CFU / g.

[0010] In one specific implementation, the ratio of viable Bacillus subtilis, Bacillus natto, and Bacillus pumilus is 2:1:1.

[0011] Secondly, this application provides a method for preparing a compound bacterial agent for water purification and Vibrio control, which adopts the following technical solution: A method for preparing a compound bacterial agent for water purification and Vibrio control includes the following steps: S1. Single strains of Bacillus subtilis, Bacillus natto and Bacillus pumilus were cultured in liquid seed culture to obtain Bacillus subtilis liquid seed liquid, Bacillus natto liquid seed liquid and Bacillus pumilus liquid seed liquid. S2. Mix Bacillus subtilis liquid seed solution, Bacillus natto liquid seed solution and Bacillus pumilus liquid seed solution at a live cell count ratio of (1-3):(1-2):(1-2), and then inoculate them into a solid fermentation medium for co-solid fermentation. After fermentation, the fermentation product is obtained. S3. The fermentation product is dried and pulverized at low temperature to obtain a compound microbial agent for water purification and Vibrio control.

[0012] In one specific implementation, the solid fermentation medium comprises wheat bran, soybean meal, and corn flour in a mass ratio of (4-6):(2.5-3.5):2.

[0013] In a specific feasible implementation, step S2 is as follows: Bacillus subtilis liquid seed liquid, Bacillus natto liquid seed liquid and Bacillus pumilus liquid seed liquid are mixed at a live cell count ratio of (1-3):(1-2):(1-2), and then inoculated into a solid fermentation medium. Co-solid fermentation is carried out at a fermentation temperature of 35-37℃. After fermentation for 36-72 hours, the fermentation is completed and the fermentation product is obtained.

[0014] In one specific implementation, the Bacillus subtilis strain is screened according to the following steps: Collect humus soil, add the humus soil to physiological saline, shake in a constant temperature shaker at 30℃ until evenly dispersed, take the supernatant, dilute it and spread it on casein-containing medium and starch-containing medium respectively, and incubate upside down at 30-37℃ for 24-48 hours. Observe whether a clear zone appears around the colonies on a casein-containing culture medium, measure the ratio of the diameter of the clear zone to the diameter of the colony, and collect strains of colonies with a clear zone diameter to colony diameter ratio ≥2.0 to obtain Bacillus subtilis producing protease; pour Lugol's iodine solution onto a starch-containing culture medium, let it stand, observe whether a clear zone appears around the colonies on the starch-containing culture medium, measure the ratio of the diameter of the clear zone to the diameter of the colony, and collect strains of colonies with a clear zone diameter to colony diameter ratio ≥1.5 to obtain Bacillus subtilis producing amylase; identify the Bacillus subtilis producing protease and the Bacillus subtilis producing amylase, and retain the qualified strains to obtain the Bacillus subtilis of this application.

[0015] In one specific implementation, the *Bacillus pumilus* strain is screened according to the following steps: The contents of the shrimp intestine were removed, dispersed in physiological saline, and shaken in a constant temperature shaker at 30℃ until evenly dispersed. The supernatant was diluted and spread on nutrient agar plates and incubated at 30-37℃ for 24-48 hours. Individual colonies with different morphology, color and size were picked and purified to obtain 23 pure cultures. Vibrio parahaemolyticus was inoculated onto liquid culture medium and cultured in a shaker at 30°C until mid-log phase to obtain indicator bacterial suspension. The indicator bacterial suspension was mixed with soft agar at 45-50°C and poured onto a bottom plate formed by solidification of nutrient agar. After solidification, sterile Oxford cups were placed at equal intervals on the bottom plate. Twenty-three pure cultures were dispersed separately and added to the wells of sterile Oxford cups. The cultures were pre-diffused at 4°C for 2-4 hours and then transferred to a 30°C incubation chamber for 18-24 hours. The presence of inhibition zones was observed, and the diameter of the inhibition zones was measured. Strains with inhibition zone diameters ≥15 mm were collected. After identification, the qualified strains were retained to obtain Bacillus pumilus.

[0016] Thirdly, the application of the compound bacterial agent for water purification and Vibrio control provided in this application adopts the following technical solution: An application of a compound microbial agent for water purification and Vibrio control in aquaculture.

[0017] In summary, this application has the following beneficial effects: 1. This application uses Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of (1-3):(1-2):(1-2) and employs a defined preparation method to increase the total live bacteria count of the compound bacterial agent, thereby improving the short-term COD degradation rate and long-term Vibrio inhibition rate of the compound bacterial agent. This helps to improve the rapid water purification performance and long-term Vibrio control performance of the compound bacterial agent.

[0018] 2. In this application, the preferred solid fermentation culture medium includes wheat bran, soybean meal and corn flour in a mass ratio of (4-6):(2.5-3.5):2, and Bacillus subtilis, Bacillus natto and Bacillus pumilus in a live bacteria ratio of 2:1:1. This can further increase the total live bacteria count of the compound microbial agent and improve its rapid water purification performance and long-lasting Vibrio control performance. Detailed Implementation

[0019] Unless otherwise specified, all raw materials used in this application are commercially available.

[0020] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0021] Example 1 This embodiment provides a compound bacterial agent for water purification and Vibrio control, comprising Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of 2:1:1, with a total live bacteria count of 3.5 × 10⁻⁶. 10 CFU / g.

[0022] Bacillus subtilis strains were screened using the following steps: Humus soil was collected and added to physiological saline. The mixture was shaken in a constant temperature shaker at 30°C until it was evenly dispersed. The supernatant was diluted and spread onto casein-containing and starch-containing culture media, respectively. The media were incubated upside down at 33°C for 36 hours.

[0023] Observe whether a clear zone appears around the colonies on a casein-containing medium, measure the ratio of the clear zone diameter to the colony diameter, and collect colonies with a clear zone diameter ≥ 2.0 to obtain Bacillus subtilis strains that produce protease. Pour Lugol's iodine solution onto a starch-containing medium, let it stand for 1 minute, observe whether a clear zone appears around the colonies on the starch-containing medium, measure the ratio of the clear zone diameter to the colony diameter, and collect colonies with a clear zone diameter ≥ 1.5 to obtain Bacillus subtilis strains that produce amylase.

[0024] The Bacillus subtilis that produces protease was subjected to Gram staining to observe its morphology. The strains confirmed to be Gram-positive bacilli were subjected to 16S rDNA sequencing, and the measured sequences were BLAST-aligned in the NCBI database. Those with a similarity > 99% were regarded as qualified for identification, and Bacillus subtilis was obtained.

[0025] The screening of Bacillus pumilus strains was carried out according to the following steps: The contents of the shrimp intestine were taken out, dispersed in physiological saline, shaken in a constant temperature shaker at 30 °C until evenly dispersed, and the supernatant was taken, diluted, and spread on a nutrient agar plate, and cultured at 33 °C for 36 hours. Single colonies with different morphologies, colors, and sizes were picked for purification, and 23 pure cultures were obtained.

[0026] Vibrio parahaemolyticus was inoculated on a liquid medium and cultured in a constant temperature shaker at 30 °C until the mid-log phase to obtain an indicator bacteria solution. The indicator bacteria solution was mixed with soft agar at 48 °C and poured onto a bottom plate solidified from nutrient agar. After solidification, sterile Oxford cups were placed equidistantly on the bottom plate. After the 23 pure cultures were separately dispersed, they were added to the wells of the sterile Oxford cups, pre-diffused at 4 °C for 3 hours, transferred to 30 °C for upright culture for 21 hours, and observed whether an inhibition zone appeared around the colonies. The diameter of the inhibition zone was measured, and the strains of the colonies with an inhibition zone diameter ≥ 15 mm were collected. The strains were subjected to 16S rDNA sequencing, and the measured sequences were BLAST-aligned in the NCBI database. Those with a similarity > 99% were regarded as qualified for identification, and Bacillus pumilus was obtained.

[0027] Bacillus natto was purchased from the China Center for Industrial Culture Collection, and the strain number is CICC 10244.

[0028] This embodiment also provides a preparation method of a compound bactericide for water purification and vibrio prevention and control, including the following steps: S1. At 37 °C, the single strains of Bacillus subtilis, Bacillus natto, and Bacillus pumilus were respectively inoculated onto a liquid seed medium and cultured in a constant temperature shaker at 37 °C and 180 rpm for 12 hours to complete the liquid seed expansion culture, and the liquid seed solutions of Bacillus subtilis, Bacillus natto, and Bacillus pumilus were respectively obtained. The liquid seed medium includes 10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, 5 g / L of glucose, and the pH is 7.0.

[0029] S2. A mixture of Bacillus subtilis liquid seed culture, Bacillus natto liquid seed culture, and Bacillus pumilus liquid seed culture at a viable count ratio of 2:1:1 is inoculated into a solid-state fermentation medium. Fermentation is carried out at 36℃ for 54 hours, after which the co-solid-state fermentation is completed, yielding the fermentation product. The solid-state fermentation medium comprises wheat bran, soybean meal, and corn flour in a mass ratio of 5:3:2. The wheat bran, soybean meal, and corn flour are mixed evenly in the specified proportions to obtain the solid-state fermentation medium.

[0030] S3. The fermentation product is dried at 43°C until the moisture content is ≤10%, then pulverized to obtain a compound microbial agent for water purification and Vibrio control.

[0031] Example 2 The only difference between this embodiment and Embodiment 1 is that the compound bacterial agent used for water purification and Vibrio control includes Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of 1:1:1.

[0032] Example 3 The only difference between this embodiment and Embodiment 1 is that the compound bacterial agent used for water purification and Vibrio control includes Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of 3:2:2.

[0033] Example 4 The only difference between this embodiment and Embodiment 1 is that, in step S2 of the method for preparing the compound microbial agent for water purification and Vibrio control, the solid fermentation culture medium includes wheat bran, soybean meal, and corn flour in a mass ratio of 3:2:2.

[0034] Example 5 The only difference between this embodiment and Embodiment 1 is that, in step S2 of the method for preparing the compound microbial agent for water purification and Vibrio control, the solid fermentation culture medium includes wheat bran, soybean meal, and corn flour in a mass ratio of 4:2.5:2.

[0035] Example 6 The only difference between this embodiment and Embodiment 1 is that, in step S2 of the method for preparing the compound microbial agent for water purification and Vibrio control, the solid fermentation culture medium includes wheat bran, soybean meal, and corn flour in a mass ratio of 6:3.5:2.

[0036] Example 7 The only difference between this embodiment and Embodiment 1 is that, in step S2 of the method for preparing the compound microbial agent for water purification and Vibrio control, the solid fermentation culture medium includes wheat bran, soybean meal, and corn flour in a mass ratio of 7:4:2.

[0037] Example 8 The difference between this embodiment and Embodiment 1 is that, in step S2 of the preparation method of the compound bacterial agent for water purification and Vibrio control, Bacillus subtilis liquid seed liquid, Bacillus natto liquid seed liquid and Bacillus pumilus liquid seed liquid are mixed at a live cell count ratio of 2:1:1 and then inoculated into a solid fermentation medium. Fermentation is carried out at a fermentation temperature of 35°C for 36 hours, and the fermentation is then stopped to complete the co-solid fermentation and obtain the fermentation product.

[0038] Example 9 The difference between this embodiment and Embodiment 1 is that, in step S2 of the preparation method of the compound bacterial agent for water purification and Vibrio control, the liquid seed liquid of Bacillus subtilis, liquid seed liquid of Bacillus natto, and liquid seed liquid of Bacillus pumilus are mixed at a live cell count ratio of 2:1:1 and then inoculated into a solid fermentation medium. The mixture is fermented at a fermentation temperature of 37°C for 72 hours, and the fermentation is then completed to obtain the fermentation product.

[0039] Example 10 The only difference between this embodiment and Example 1 is that in the strain screening step of Bacillus subtilis: humus soil is collected, the humus soil is added to physiological saline, and shaken in a constant temperature shaker at 30°C until it is evenly dispersed. The supernatant is taken, diluted, and then spread on a medium containing casein and a medium containing starch, respectively, and incubated upside down at 30°C for 24 hours.

[0040] Example 11 The only difference between this embodiment and Example 1 is that in the strain screening step of Bacillus subtilis: humus soil is collected, added to physiological saline, and shaken in a constant temperature shaker at 30°C until evenly dispersed. The supernatant is taken, diluted, and spread on culture media containing casein and starch, respectively, and incubated upside down at 37°C for 48 hours.

[0041] Example 12 The only difference between this embodiment and Example 1 is that in the strain screening step of Bacillus pumilus: the contents of the shrimp intestine were taken out, dispersed in physiological saline, and shaken in a constant temperature shaker at 30°C until evenly dispersed. The supernatant was diluted and spread on a nutrient agar plate, and cultured at 30°C for 24 hours. Individual colonies with different morphology, color and size were picked and purified to obtain 23 pure cultures.

[0042] Vibrio parahaemolyticus was inoculated onto liquid culture medium and cultured in a shaker at 30°C until mid-log phase to obtain indicator bacterial suspension. This suspension was mixed with soft agar at 45°C and poured onto a solidified nutrient agar plate. After solidification, sterile Oxford cups were placed at equal intervals on the plate. Twenty-three pure cultures were dispersed and added to the wells of sterile Oxford cups. Pre-diffusion was performed at 4°C for 2 hours, followed by incubation at 30°C for 18 hours. The presence of inhibition zones was observed, and their diameter was measured. Colonies with inhibition zones ≥15 mm in diameter were collected. The strains underwent 16S rDNA sequencing, and the sequences were compared against the NCBI database using BLAST. A similarity >99% was considered acceptable, yielding *Bacillus pumilus*.

[0043] Example 13 The only difference between this embodiment and Example 1 is that in the strain screening step of Bacillus pumilus: the contents of the shrimp intestine were taken out, dispersed in physiological saline, and shaken in a constant temperature shaker at 30°C until evenly dispersed. The supernatant was diluted and spread on a nutrient agar plate, and cultured at 37°C for 48 hours. Individual colonies with different morphology, color and size were picked and purified to obtain 23 pure cultures.

[0044] Vibrio parahaemolyticus was inoculated onto liquid culture medium and cultured in a shaker at 30°C until mid-log phase to obtain indicator bacterial suspension. The indicator bacterial suspension was mixed with soft agar at 50°C and poured onto a solidified nutrient agar plate. After solidification, sterile Oxford cups were placed at equal intervals on the plate. Twenty-three pure cultures were dispersed and added to the wells of sterile Oxford cups. Pre-diffusion was performed at 4°C for 4 hours, followed by incubation at 30°C for 24 hours. The presence of inhibition zones was observed, and their diameter was measured. Colonies with inhibition zones ≥15 mm in diameter were collected. The strains underwent 16S rDNA sequencing, and the sequences were compared with the NCBI database using BLAST. A similarity >99% was considered acceptable, yielding *Bacillus pumilus*.

[0045] Comparative Example Comparative Example 1 The only difference between this comparative example and Example 1 is that the compound bacterial agent used for water purification and Vibrio control includes Bacillus subtilis, Bacillus natto, and Bacillus pumilus with a live bacteria ratio of 0.5:0.5:1.

[0046] Comparative Example 2 The only difference between this comparative example and Example 1 is that the compound bacterial agent used for water purification and Vibrio control includes Bacillus subtilis, Bacillus natto, and Bacillus pumilus in a live bacteria ratio of 4:3:1.

[0047] Comparative Example 3 The only difference between this comparative example and Example 1 is that, in the raw materials and preparation method of the compound bacterial agent used for water purification and Vibrio control, an equal amount of Bacillus subtilis is used to replace Bacillus natto.

[0048] Comparative Example 4 The only difference between this comparative example and Example 1 is that, in the raw materials and preparation method of the compound bacterial agent used for water purification and Vibrio control, an equal amount of Bacillus subtilis is used to replace Bacillus pumilus.

[0049] Performance testing The following performance tests were conducted on Examples 1-13 and Comparative Examples 1-4: Total viable count test: According to GB / T 26428-2010 Detection of Bacillus subtilis in feed microbial preparations, the total viable count of the compound microbial agents prepared in each example and comparative example for water purification and Vibrio control was tested.

[0050] Rapid water purification performance test: Glucose, peptone, and ammonium chloride were added to dechlorinated tap water to obtain simulated polluted water with a COD concentration of 100 mg / L. 500 mL of the simulated polluted water sample was taken, and a compound bacterial agent was added at a dosage of 10 mg per liter of water to obtain the experimental group. The experimental group was placed in a constant temperature shaker at 30℃ and 150 rpm. Samples were taken at 72 hours to determine the COD concentration. The COD degradation rate at 72 hours was calculated using the formula: COD degradation rate = (initial COD concentration - COD concentration at 72 hours) / initial COD concentration × 100%.

[0051] Durability test for Vibrio control: The Oxford cup method was used, with cell-free fermentation extracts of the compound bacterial agents prepared for water purification and Vibrio control in each example and comparative example as samples. Samples were added to plates containing Vibrio parahaemolyticus, incubated, and the initial inhibition zone diameter (D0) was measured at time 0. The remaining sample solution was stored at 25°C or 4°C. On day 28 after storage, the above test was repeated on the same indicator bacterial plate using the same batch of sample solution, and the inhibition zone diameter (D28) was measured. The Vibrio inhibition rate on day 28 was calculated according to the formula: Vibrio inhibition rate = (D0 - D28) / D0.

[0052] The test results are shown in Table 1.

[0053] Table 1 Combining Example 1 and Comparative Examples 1-4 with Table 1, it can be seen that compared to Example 1, the total viable bacteria count of the composite bacterial agents prepared in Comparative Examples 1-4 for water purification and Vibrio control was significantly reduced, and the COD degradation rate at 72 hours and the Vibrio inhibition rate at 28 days were significantly lower. This indicates that using the raw material ratio and preparation method of Example 1 can increase the total viable bacteria count of the composite bacterial agent, as well as improve the short-term COD degradation rate and long-term Vibrio inhibition rate, thus contributing to improved rapid water purification performance and long-term Vibrio control performance of the composite bacterial agent.

[0054] As can be seen from Examples 1-13 and Table 1, the total viable count of the compound bacterial agents prepared in Examples 1-13 for water purification and Vibrio control is greater than 3.0 × 10⁻⁶. 10 The CFU / g COD degradation rate at 72 h was >50%, and the Vibrio inhibition rate at 28 days was ≥57%. This indicates that the raw material ratios and preparation methods within the range of Examples 1-13 can all increase the total viable count of the compound bacterial agent, thereby improving its rapid water purification performance and long-term Vibrio control performance.

[0055] Comparing the test data of each embodiment, it can be seen that the solid fermentation culture medium, which includes wheat bran, soybean meal and corn flour in a mass ratio of (4-6):(2.5-3.5):2, and Bacillus subtilis, Bacillus natto and Bacillus pumilus in a live bacteria ratio of 2:1:1, can further increase the total live bacteria count of the compound microbial agent, and improve the rapid water purification performance and long-term Vibrio control performance of the compound microbial agent.

[0056] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A preparation method for water purification and Vibrio control, characterized in that, Includes the following steps: The compound microbial agent for water purification and Vibrio control comprises Bacillus subtilis, Bacillus natto, and Bacillus pumilus with a viable count ratio of (1-3):(1-2):(1-2); wherein the Bacillus subtilis strain is capable of producing protease and amylase; the Bacillus natto strain is capable of producing nattokinase and protease; and the Bacillus pumilus strain is capable of producing antimicrobial peptides that inhibit Vibrio; the total viable count of the compound microbial agent for water purification and Vibrio control is ≥2.0 × 10⁻⁶. 10 CFU / g; S1. Single strains of Bacillus subtilis, Bacillus natto and Bacillus pumilus were cultured in liquid seed culture to obtain Bacillus subtilis liquid seed liquid, Bacillus natto liquid seed liquid and Bacillus pumilus liquid seed liquid. S2. Mix Bacillus subtilis liquid seed solution, Bacillus natto liquid seed solution and Bacillus pumilus liquid seed solution at a live cell count ratio of (1-3):(1-2):(1-2), and then inoculate them into a solid fermentation medium for co-solid fermentation. After fermentation, the fermentation product is obtained. S3. The fermentation product is dried and pulverized at low temperature to obtain a compound microbial agent for water purification and Vibrio control.

2. The method for preparing the compound bacterial agent for water purification and Vibrio control according to claim 1, characterized in that: The solid fermentation medium comprises wheat bran, soybean meal, and corn flour in a mass ratio of (4-6):(2.5-3.5):

2.

3. The method for preparing the compound bacterial agent for water purification and Vibrio control according to claim 1, characterized in that, Step S2 is as follows: Mix Bacillus subtilis liquid seed liquid, Bacillus natto liquid seed liquid and Bacillus pumilus liquid seed liquid at a live cell count ratio of (1-3):(1-2):(1-2), then inoculate into a solid fermentation medium and carry out co-solid fermentation at a fermentation temperature of 35-37℃. After fermentation for 36-72 hours, the fermentation is completed and the fermentation product is obtained.

4. The method for preparing the compound bacterial agent for water purification and Vibrio control according to claim 1, characterized in that, The Bacillus subtilis strain was screened according to the following steps: Collect humus soil, add the humus soil to physiological saline, shake in a constant temperature shaker at 30℃ until evenly dispersed, take the supernatant, dilute it and spread it on casein-containing medium and starch-containing medium respectively, and incubate upside down at 30-37℃ for 24-48 hours. Observe whether a clear zone appears around the colonies on a casein-containing culture medium, measure the ratio of the diameter of the clear zone to the diameter of the colony, and collect strains of colonies with a clear zone diameter to colony diameter ratio ≥2.0 to obtain Bacillus subtilis producing protease; pour Lugol's iodine solution onto a starch-containing culture medium, let it stand, observe whether a clear zone appears around the colonies on the starch-containing culture medium, measure the ratio of the diameter of the clear zone to the diameter of the colony, and collect strains of colonies with a clear zone diameter to colony diameter ratio ≥1.5 to obtain Bacillus subtilis producing amylase; identify the Bacillus subtilis producing protease and the Bacillus subtilis producing amylase, and retain the qualified strains to obtain the Bacillus subtilis of this application.

5. The method for preparing the compound bacterial agent for water purification and Vibrio control according to claim 1, characterized in that, The *Bacillus pumilus* strains were screened according to the following steps: The contents of the shrimp intestine were removed, dispersed in physiological saline, and shaken in a constant temperature shaker at 30℃ until evenly dispersed. The supernatant was diluted and spread on nutrient agar plates and incubated at 30-37℃ for 24-48 hours. Individual colonies with different morphology, color and size were picked and purified to obtain 23 pure cultures. Vibrio parahaemolyticus was inoculated onto liquid culture medium and cultured in a shaker at 30°C until mid-log phase to obtain indicator bacterial suspension. The indicator bacterial suspension was mixed with soft agar at 45-50°C and poured onto a bottom plate formed by solidification of nutrient agar. After solidification, sterile Oxford cups were placed at equal intervals on the bottom plate. Twenty-three pure cultures were dispersed separately and added to the wells of sterile Oxford cups. The cultures were pre-diffused at 4°C for 2-4 hours and then transferred to a 30°C incubation chamber for 18-24 hours. The presence of inhibition zones was observed, and the diameter of the inhibition zones was measured. Strains with inhibition zone diameters ≥15 mm were collected. After identification, the qualified strains were retained to obtain Bacillus pumilus.

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