Compound microbial degerming agent as well as preparation method and application thereof

Abstract

The invention discloses a compound microbial degerming agent as well as a preparation method and application thereof, and relates to the technical field of microorganisms, and the technical key points are as follows: the degerming agent is formed by compounding bacillus subtilis and bacillus pumilus according to the viable count ratio of 1: (10-20); the preparation method comprises the three steps of single bacterium culture, mixed seed solution preparation and fermentation amplification, through culture and amplification under specific conditions, the total number of viable bacteria in the degerming agent can be effectively increased to be larger than or equal to 10 < 10 > CFU / mL, and the degerming effect is guaranteed; the components of the LB liquid culture medium are clear, sampling is carried out every 12 hours in the preparation process to detect the density of thalli and the proportion of bacillus pumilus, and the stable product quality is ensured; the degerming agent can be used for degrading vibrio alginolyticus, vibrio parahaemolyticus and aeromonas hydrophila in aquaculture water, the dosage is 1-5 mg / L, the content of harmful bacteria in the water can be effectively reduced, the water quality is improved, and the degerming agent has remarkable technical effects and application value.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite microbial sterilizing agent, a preparation method and application thereof. Background Art

[0002] Vibriosis, a collective term for diseases in aquaculture caused by various Vibrio species, including Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi, causes economic losses to the aquaculture industry annually. Furthermore, some Vibrio species are also human pathogens, causing not only gastroenteritis but also extraintestinal infections. The most common method for combating bacterial pathogens like Vibrio is antibiotics, but long-term use can lead to bacterial resistance.

[0003] With the expansion of marine aquaculture and the deterioration of the marine environment, aquaculture disease outbreaks have become frequent, posing a serious threat to the health of marine aquaculture, the marine ecosystem, and food safety. For example, a new shrimp disease has emerged in recent years, and a newly discovered, highly virulent, highly pathogenic Vibrio species has been identified as the infectious agent responsible for this outbreak. As an emerging aquaculture disease, highly pathogenic Vibrio species are more virulent than common Vibrio species, with higher rates of infection, morbidity, and mortality. Within two days of infection, they can cause widespread mortality in shrimp and other aquatic animals, with a mortality rate as high as 75%, severely impacting the healthy development of the aquaculture industry. As an emerging pathogen, the mechanism of the highly pathogenic Vibrio species' virulence is unclear, and effective and precise control products are lacking.

[0004] Currently, aquaculture disease control still relies primarily on medication, most of which are relatively basic fishery medicine products such as chemical disinfectants, antibiotics, and traditional Chinese medicine powders. There is a lack of specialized, highly effective, and ecologically sound control drugs, which fall far short of meeting the industry's needs for disease control. The emergence of new and frequent diseases and the lag in green fishery medicines and precision control technologies have become bottlenecks restricting the high-quality development of the industry. With the increasing scale of marine aquaculture and the continued promotion of intensive farming methods, outbreaks of aquaculture diseases have become a constraint on the healthy and sustainable development of the aquaculture industry. When Vibrio disease first emerged, farmers primarily used disinfectants, iodine preparations, and potassium persulfate complexes for treatment. However, this led to the gradual development of drug resistance in bacteria, and the effectiveness of disinfectants in killing bacteria gradually weakened. Current prevention and control measures are not only unable to control the spread of the disease, but the misuse and abuse of drugs caused by the disease also pose a significant threat to the quality and safety of aquatic products and the ecological environment. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a composite microbial bactericide, a preparation method and its application, which can degrade highly pathogenic Vibrio in aquaculture.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention discloses a composite microbial fungicide, which is composed of Bacillus subtilis and Bacillus putrefaciens.

[0008] Preferably, the ratio of the number of live bacteria of Bacillus subtilis to that of Bacillus putida is 1:10-20.

[0009] A method for preparing a composite microbial sterilizing agent comprises the following steps:

[0010] a. Single bacterial culture: Bacillus subtilis culture: Inoculate the purified strain into LB liquid medium and culture at 36±1℃ and 180rpm for 18-24h until the bacterial concentration reaches 10 8 ~10 9 CFU / mL; Bacillus putida culture: inoculate the purified strain into LB liquid medium, shake and culture at 30±1℃ and 150rpm for 24-36h until the bacterial concentration reaches 10 7 ~10 8 CFU / mL;

[0011] b. Preparation of mixed seed solution: The two bacterial solutions obtained in step a were mixed in proportion and co-cultured at 30°C and 120rpm for 12h to form a symbiotic system;

[0012] c. Fermentation expansion: The mixed seed solution was inoculated into a fermenter (containing sterilized LB medium) at a 5% inoculum volume, and cultured at 30°C, with a ventilation volume of 1.0 vvm and a stirring speed of 100 rpm for 48-72 hours. The total number of viable cells at the end point was ≥10 10 CFU / mL.

[0013] Preferably, the LB liquid culture medium comprises 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0±0.2, and is sterilized at 121°C for 20 min.

[0014] Preferably, in step c, sampling and testing are performed every 12 hours:

[0015] Spectrophotometry (OD 600 ) Monitoring bacterial density;

[0016] The proportion of Bacillus putida was quantitatively detected by qPCR (primer F: 5'-GCTGGATCACCTCCTTTCTA-3'; R: 5'-TCCGATACGGTGAGGTAGAA-3'), with an annealing temperature of 60°C.

[0017] The invention discloses an application of a composite microbial fungicide for degrading Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila in aquaculture water, with a dosage of 1-5 mg / L.

[0018] The present invention has the following beneficial effects:

[0019] 1. Bactericidal Effect of Bacillus subtilis: Bacillus subtilis, a common probiotic, has demonstrated powerful antibacterial capabilities in aquaculture. It produces a variety of substances with antimicrobial activity, such as antimicrobial peptides and bacteriocins. These substances can directly target highly pathogenic Vibrio spp., disrupting their cell membranes and interfering with intracellular physiological and biochemical processes, thereby inhibiting their growth and reproduction. For example, antimicrobial peptides can penetrate into the cell membrane, forming pores that allow the cell contents to leak out and render the bacteria inactive. Furthermore, Bacillus subtilis exhibits a strong competitive inhibitory effect. It can rapidly grow and multiply in water, occupying an ecological niche and competing with highly pathogenic Vibrio spp. for nutrients and living space, thereby limiting their survival and growth. In this way, Bacillus subtilis effectively reduces the number of highly pathogenic Vibrio spp. in water, reducing the susceptibility of aquatic animals to disease and providing a strong guarantee for the healthy growth of aquatic organisms.

[0020] 2. The Bacterial Elimination Effect of Bacillus putida: Bacillus putida also plays an important role in the sterilization of aquaculture water. It secretes a variety of enzymes, such as proteases and lipases. These enzymes break down organic matter in the water, reducing eutrophication and thus altering the habitat of highly pathogenic Vibrio. Highly pathogenic Vibrio thrive in eutrophic waters, and Bacillus putida regulates the nutrient status of the water, inhibiting its growth. Furthermore, Bacillus putida has the ability to adsorb and immobilize Vibrio. Through its cell surface structure, it can adsorb and immobilize highly pathogenic Vibrio in the water, rendering it inactive and preventing it from harming aquatic animals. Furthermore, Bacillus putida can stimulate the immune system of aquatic animals, enhancing their resistance to highly pathogenic Vibrio, further improving their health.

[0021] 3. Unique Bacterial Effects of the Combination: A microbial agent composed of Bacillus subtilis and Bacillus putida in a specific ratio exhibits unique bactericidal effects unmatched by either agent alone. First, their mechanisms of action complement each other. Bacillus subtilis primarily eliminates bacteria through the production of antimicrobial substances and competitive inhibition, while Bacillus putida focuses on regulating the water environment and adsorbing and immobilizing Vibrio spp. The combination of the two attacks highly pathogenic Vibrio spp. from multiple angles, significantly improving the efficiency and effectiveness of bactericidal treatment. Second, in terms of ecological adaptability, both Bacillus subtilis and Bacillus putida perform effectively under diverse environmental conditions. Bacillus subtilis grows best under higher temperatures and rapid shaking conditions, while Bacillus putida can also reproduce effectively under relatively low temperatures and slow shaking conditions. The combination of the two agents is adaptable to a wider range of aquaculture environments, providing stable bactericidal effects in both high and low temperature seasons. Furthermore, the symbiotic system they form exhibits synergistic effects. During the co-cultivation process of the mixed seed solution, the two bacteria promote each other's growth, enhancing the activity and stability of the inoculum. This synergistic effect enables the composite microbial inoculum to be more efficient and durable in degrading highly pathogenic Vibrio spp. in aquaculture, providing a reliable guarantee for the healthy development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a schematic diagram of the qPCR amplification results of Bacillus putida in Experiment 1 of an embodiment of the present invention.

[0024] Figure 2 This is a schematic diagram of the qPCR amplification results of Bacillus putida in Experiment 5 of an embodiment of the present invention.

[0025] Figure 3 This is a schematic diagram of the specific experimental results of the real-time fluorescence method for Bacillus putida in Experiment 5 of an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] The present invention mixes Bacillus subtilis and Bacillus putida in different ratios (1:10, 1:15, and 1:20) as three examples, and a series of experimental projects are conducted to verify the feasibility, significant technical effects, and application value of these examples. The specific experimental projects are as follows:

[0028] Experiment 1: Basic preparation of Bacillus subtilis and Bacillus putida composite agent

[0029] 1. Purpose

[0030] This experiment aims to comprehensively and systematically verify the feasibility of the basic formula and preparation process of the composite inoculant of Bacillus subtilis and Bacillus putida, laying a solid foundation for subsequent in-depth research and large-scale application.

[0031] 2. Materials and Methods

[0032] 2.1 Host bacteria (Bacillus subtilis) culture

[0033] Bacillus subtilis was streaked and purified to ensure the purity of the strain.

[0034] Inoculate the purified Bacillus subtilis into 50 mL of LB medium. LB medium contains 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. Adjust the pH to 7.0 ± 0.2 and sterilize at 121°C for 20 min.

[0035] The inoculated culture medium was placed at 36±1℃ and shaken at 180rpm for 18-24h, and samples were taken regularly for testing. 8 -10 9 CFU / mL, the culture was stopped and the obtained bacterial solution was used as the subsequent experimental material.

[0036] 2.2 Preparation of Bacillus putida seed solution

[0037] Preparation of primary seed solution: Take 1 mL of Bacillus putida bacterial solution and inoculate it into 5 mL of LB medium. Incubate with shaking at 30±1°C and 150 rpm for 2 h to allow Bacillus putida to initially proliferate.

[0038] Preparation of secondary seed solution: Inoculate 5 mL of the above-mentioned primary seed solution into a 200 mL conical flask containing 50 mL of fresh LB medium, and culture at 30±1°C and 150 rpm for 24-36 hours to obtain more active and larger numbers of Bacillus putida strains.

[0039] 2.3 Fermentation culture

[0040] The Bacillus subtilis liquid and the secondary seed liquid of Bacillus putida were mixed in a ratio of 1:10, and the total bacterial concentration after mixing was accurately adjusted to 10 9 CFU / mL (this is the initial ratio, and subsequent experiments will be adjusted according to different ratios).

[0041] 50 mL of the mixed seed solution was inoculated into a 5 L fermenter containing 1 L of host bacterial solution (Bacillus subtilis bacterial solution, used to maintain a suitable growth environment), and cultured at 30°C, 1.0 vvm aeration, and 100 rpm stirring speed for 48-72 h.

[0042] During the fermentation process, samples were taken every 12 h and the OD 600 ) monitor bacterial density; quantitatively detect the proportion of Bacillus putida by qPCR (primer F: 5'-GCTGGATCACCTCCTTTCTA-3'; R: 5'-TCCGATACGGTGAGGTAGAA-3'). The reaction system volume is 25μL: TB Green Premix Ex Taq II (TliRNaseH Plus) (2X) 12.5μL, 1.0μL each primer (10μmol / L), 2μL template DNA, and sterile deionized water to 25μL. The amount of each reagent in the reaction system can be appropriately adjusted according to the specific situation or different total reaction volumes. Two parallel reactions should be set up for each reaction system. Reaction conditions: 95℃ pre-denaturation for 30s; 95℃ annealing for 5s, 60℃ extension for 34s, and 40 cycles. This can be used to preliminarily judge the growth of the bacteria and changes in their proportion.

[0043] 2.4 Results

[0044] After qPCR detection, the results showed that Bacillus putida grew in the mixed system according to the expected proportion, and the bacterial concentration reached 10 10 CFU / mL or above, indicating that the basic preparation process can effectively obtain the expected composite bacterial agent.

[0045] like Figure 1 The figure shows qPCR amplification results for Bacillus putida SYBG. Six amplification curves were obtained for this bacterium during a 72-hour fermentation period with samples taken every 12 hours. The results show that the ct value decreases with increasing incubation time, as the concentration of the bacterium increases.

[0046] Experiment 2: Verification of the inhibitory effect of different bacterial ratios on Vibrio parahaemolyticus

[0047] 1. Purpose

[0048] The purpose of this experiment is to deeply explore the inhibitory effect of Bacillus subtilis and Bacillus putida on Vibrio parahaemolyticus at different ratios (1:10, 1:15, 1:20), so as to determine the optimal bacterial ratio and provide a scientific basis for the ratio of composite bacterial agents in practical applications.

[0049] 2. Materials and Methods

[0050] 2.1 Preparation of inoculants

[0051] Bacillus subtilis and Bacillus putida were mixed according to different set ratios (1:10, 1:15, and 1:20), and the total bacterial concentration after mixing was accurately adjusted to 109 CFU / mL by adjusting the culture conditions, etc., to ensure comparability between bacterial agents in different ratios.

[0052] 2.2 Pathogen challenge test

[0053] Dilute Vibrio parahaemolyticus to a concentration of 10 4 CFU / mL, and then mixed with the prepared bacterial agents in different proportions at a volume ratio of 1:1. The mixed system was placed in an environment of 25°C for incubation.

[0054] During the incubation process, samples were collected every 12 hours and streaked onto TCBS medium, with two parallel plates plated each time. The growth of V. parahaemolyticus was observed and viable bacteria were counted. Based on the changes in viable bacteria counts at different time points, the inhibition rates at different bacterial ratios were calculated.

[0055] 2.3 Results

[0056] The experimental results, as shown in Table 1, show that when the ratio of Bacillus subtilis to Bacillus putida was 1:10, the inhibition rate reached 88% after 96 hours of incubation. At a ratio of 1:15, the inhibition rate increased to 95%, and at a ratio of 1:20, the inhibition rate reached 92%. A comprehensive comparison of the antibacterial effects of different ratios concluded that the optimal ratio was 1:15.

[0057] Table 1 Pathogen challenge test results

[0058]

[0059]

[0060] Experiment 3: Practical application of microbial agents in aquaculture ponds

[0061] 1. Purpose

[0062] The purpose of this experiment is to verify the on-site control effect of the composite bacterial agent of Bacillus subtilis and Bacillus putida on highly pathogenic Vibrio (such as Vibrio parahaemolyticus, Vibrio alginolyticus, etc.) in aquaculture water through tests in actual aquaculture environments, and to evaluate its practical application value in aquaculture.

[0063] 2. Materials and Methods

[0064] 2.1 Experimental design

[0065] Experimental group: The composite bacterial agent was added to the shrimp culture pond at a dosage of 1-5 mg / L per cubic meter of water (according to the application part of the claims), wherein the ratio of Bacillus subtilis to Bacillus putida was 1:15.

[0066] Control group: No bacterial agent was added, serving as a blank control to observe the changes in Vibrio in aquaculture water under natural conditions.

[0067] During the experiment, the concentration of Vibrio in the water was measured every 48 hours using the TCBS plate count method. The control effect of the microbial agent on Vibrio was evaluated by comparing the changes in Vibrio concentrations at different time points in the experimental and control groups.

[0068] 2.2 Breeding objects

[0069] A shrimp breeding pond was selected as the test site. The water temperature of the breeding pond was controlled at 28±1℃ and the salinity was 15‰. These environmental conditions are consistent with the actual breeding environment and can better reflect the effect of the microbial agent in actual application.

[0070] 2.3 Results

[0071] After 72 hours of observation, the experimental group's Vibrio concentration decreased by 88%, and the shrimp mortality rate was less than 6%. In contrast, the Vibrio concentration in the control group's pond increased by 18%, and the shrimp mortality rate was as high as 33%. This result demonstrates that the composite bacterial agent can effectively control the number of Vibrio in the aquaculture water and reduce the mortality rate of shrimp caused by Vibrio infection. It has good practical application results and complies with the relevant provisions of Claim 8.

[0072] Experiment 4: Preparation and stability test of solid inoculant

[0073] 1. Purpose

[0074] The purpose of this experiment was to develop a solid inoculant in the form of a wettable powder to meet the demand for easy transportation and storage of inoculants in practical applications, and to evaluate the stability of the solid inoculant under different storage conditions.

[0075] 2. Materials and Methods

[0076] 2.1 Preparation process

[0077] The fermentation broth was centrifuged to collect the bacterial cells, and then the collected bacterial cells were fully mixed with diatomaceous earth (as a carrier) at a ratio of 1:3.

[0078] The mixed materials are dried under low temperature conditions to remove moisture, and finally a solid bacterial agent in the form of a wettable powder is obtained.

[0079] 2.2 Stability test

[0080] The prepared solid inoculum was stored at 37°C for 30 days. Samples were taken every five days during storage to determine the viable bacterial count and antibacterial efficacy. The stability of the solid inoculum was evaluated by comparing the changes in viable bacterial count and antibacterial efficacy before and after storage.

[0081] 2.3 Results

[0082] As shown in Table 2, after 30 days of storage, the survival rate of live bacteria in the solid inoculum was greater than 83%, and the antibacterial rate remained above 88%. This result demonstrates the excellent stability of the solid inoculum, effectively extending its shelf life and facilitating its transportation and storage in practical applications.

[0083] Table 2 Composite bacterial agent stability measurement data

[0084]

[0085]

[0086] Experiment 5: Optimization and Verification of qPCR Detection Method (for Bacillus putida)

[0087] 1. Purpose

[0088] The experiment aims to confirm the reliability of specific primers used to detect Bacillus putida, ensure that Bacillus putida can be accurately and specifically detected through qPCR technology, and avoid detection errors caused by nonspecific amplification of primers.

[0089] 2. Materials and Methods

[0090] 2.1 DNA extraction Take an appropriate amount of fermentation broth sample and extract DNA from the sample using the boiling method.

[0091] 2.2 qPCR amplification

[0092] The extracted DNA was used as a template and primers F / R were used to perform a qPCR amplification reaction. The amplification curve and melting curve were analyzed to determine whether the product was a Bacillus putida-specific band.

[0093] 2.3 Specificity Verification

[0094] DNA from Escherichia coli and Staphylococcus aureus was used as negative controls for qPCR amplification and analysis alongside the Bacillus putida sample. The specificity of the primers was determined by comparing the amplification results from different samples.

[0095] 2.4 Specificity Verification Results

[0096] like Figure 2 and Figure 3 As shown in the figure, the qPCR amplification results showed that only the Bacillus putida sample showed a typical amplification curve and a single melting curve peak, while samples of other strains such as Escherichia coli and Staphylococcus aureus did not show amplification signals. This result fully demonstrates that the primers used have good specificity and can accurately detect Bacillus putida.

[0097] Experiment 6: Environmental Compatibility Test

[0098] 1. Purpose

[0099] The purpose of this experiment is to comprehensively evaluate the safety of the combined bacterial agent of Bacillus subtilis and Bacillus putida on non-target organisms and the environment, to ensure that the agent will not have a negative impact on the ecological environment in actual application, and to provide a scientific basis for environmental safety for the large-scale promotion and application of the combined bacterial agent.

[0100] 2. Methods

[0101] 2.1 Acute toxicity test

[0102] Test materials

[0103] Test subjects: Healthy, vigorous, and uniformly sized zebrafish (3.0 ± 0.2 cm in length and 0.3 ± 0.05 g in weight) were selected. Zebrafish were housed in the laboratory for 7 days prior to the experiment. The water temperature was maintained at 25 ± 1°C, the pH was 7.0-7.5, and the dissolved oxygen content was no less than 5 mg / L. An appropriate amount of brine shrimp larvae was fed daily. The mortality rate during the housed period did not exceed 5%.

[0104] Test agent: Bacillus subtilis and Bacillus putida were mixed in the optimal ratio of 1:15 to prepare a composite agent. The bacterial concentration was adjusted to 10 10 CFU / mL, used as the stock solution of the test bacteria.

[0105] Test water: tap water aerated for more than 48 h, with water quality parameters meeting the requirements for zebrafish breeding, pH value of 7.2 ± 0.2, dissolved oxygen content of 6.5 ± 0.5 mg / L, and hardness of 150-200 mg / L (calculated as CaCO3).

[0106] Test method

[0107] Pre-test: five concentration gradients (100 mg / L, 500 mg / L, 1000 mg / L, 2000 mg / L, and 5000 mg / L) were set, with one parallel group for each concentration, and 10 zebrafish were placed in each group. After 24 h of observation, the death of zebrafish was recorded, and the concentration range for the formal test was preliminarily determined.

[0108] Formal test: according to the results of the pre-test, seven concentration gradients (100 mg / L, 300 mg / L, 500 mg / L, 700 mg / L, 1000 mg / L, 1500 mg / L, and 2000 mg / L) were set, with three parallel groups for each concentration, and 10 zebrafish were placed in each group. A blank control group was also set, in which an equal amount of test water was added without adding the bacterial agent. The zebrafish were randomly assigned to each test group and control group, and the test container was a 5 L glass tank, with 3 L of test water added to each glass tank. During the test, the water temperature was controlled at (25 ± 1) °C, and the light cycle was 12 h light / 12 h dark. Appropriate amounts of brine shrimp larvae were fed to the zebrafish every day, but observation and recording were not performed within 2 h after feeding to avoid the influence of food residues on the test results. The death of zebrafish was observed and recorded every 24 h, and the dead individuals were removed in time.

[0109] Data processing

[0110] The 96 h median lethal concentration (LC50) of the bacterial agent on zebrafish was calculated by the method of probability units. When the 96 h LC50 was > 1000 mg / L, it indicated that the bacterial agent had no obvious acute toxicity effect on zebrafish.

[0111] 2.2 Ecological impact test

[0112] Test materials

[0113] Simulation of water body environment: six simulation water body ecosystems of the same specifications were constructed, each system being a 50 L glass tank, with 10 cm thick sediment (taken from a local unpolluted pond) laid in the tank and appropriate aquatic plants (such as golden algae, cattail, etc.) planted, to simulate the ecological environment of natural water bodies. 40 L of tap water aerated for more than 48 h was added to the glass tank, and the water temperature was controlled at (25 ± 1) °C, with a pH value of 7.0-7.5 and a dissolved oxygen content not less than 5 mg / L.

[0114] Test bacterial agent: the same compound bacterial agent stock solution prepared in the acute toxicity test.

[0115] Test methods

[0116] Bacterial agent administration: Six simulated water ecosystems were randomly divided into two groups, with three replicates in each group. In the experimental group, 10 mL of bacterial agent stock solution was administered to each glass tank to make the bacterial agent concentration in the water reach 2.5 × 10 7 CFU / mL; the control group did not add any bacterial agent, but only added an equal amount of test water.

[0117] Environmental parameter monitoring: The pH value and dissolved oxygen content of the water were measured at 0, 12, 24, 48, 72, 96, 168, and 336 hours after the inoculant was added. The pH value was measured using a portable pH meter, and the dissolved oxygen content was measured using a dissolved oxygen meter.

[0118] Analysis of microbial community structure: Water and sediment samples were collected before (0 h) and 96 h and 336 h after the inoculum was added. For water sampling, 50 mL of water was collected from the middle layer of the glass tank using a sterile 50 mL centrifuge tube. For sediment sampling, 10 g of sediment from the surface layer (0-5 cm) was collected using a sterile sampler. The collected samples were quickly brought back to the laboratory, and high-throughput sequencing technology was used to analyze changes in the microbial community structure. The specific steps are as follows:

[0119] DNA extraction: Soil DNA extraction kit was used to extract total microbial DNA from water and sediment samples.

[0120] PCR amplification: Using the extracted DNA as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene (e.g., 338F: 5'-ACTCCTACGGGAGGCAGCAG-3'; 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction system (25 μL) contained 12.5 μL of 2× TaqPCR MasterMix, 1 μL of forward primer (10 μmol / L), 1 μL of reverse primer (10 μmol / L), 2 μL of template DNA, and 8.5 μL of sterile deionized water. The PCR reaction conditions were: initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; and a final extension at 72°C for 10 min.

[0121] High-throughput sequencing: After purification and quantification of the PCR amplification products, a professional sequencing company was commissioned to perform high-throughput sequencing. After sequencing, the sequencing data was analyzed using bioinformatics software to compare the diversity, richness, and changes in the dominant bacterial communities in the experimental and control groups at different time points.

[0122] 2.3 Results

[0123] Results of acute toxicity test

[0124] After 96h of observation and recording, the 96h LC50 of the bacterial agent to zebrafish was calculated by the method of probability unit to be >2000mg / L, which was much greater than the judgment standard of 1000mg / L, indicating that the bacterial agent had no toxic effect on zebrafish. During the test, the activity and surface characteristics of zebrafish at each concentration gradient did not appear obvious abnormalities, and only a few zebrafish in the high concentration group (1500mg / L and 2000mg / L) showed a short-term slow swimming phenomenon, but then recovered to normal, and the final mortality was low.

[0125] Results of ecological impact test

[0126] Changes in environmental parameters: Within 336h after the bacterial agent was put in, the pH value and dissolved oxygen content of the water body in the test group and the control group did not change significantly. The pH value of the water body in the test group was always maintained between 7.0-7.5, and the dissolved oxygen content was not less than 5mg / L, and compared with the control group, the difference was not statistically significant (P>0.05).

[0127] Changes in microbial community structure: High-throughput sequencing results showed that before the bacterial agent was put in, the microbial community structure in the water and sediment in the test group and the control group was similar, and the dominant bacterial flora mainly included Proteobacteria, Bacteroidetes, Firmicutes, etc. Within 96h and 336h after the bacterial agent was put in, the microbial community diversity and richness in the water and sediment in the test group and the control group did not appear obvious fluctuations (P>0.05). Although a small amount of Bacillus subtilis and Bacillus odoriferous was detected in the test group, it did not cause significant influence on the original microbial community structure, and the relative abundance of the dominant bacterial flora changed little.

[0128] Based on the results of the above acute toxicity test and ecological impact test, it can be concluded that the complex bacterial agent meets the environmental safety requirements, has good compatibility to the ecological environment in actual application, and will not cause obvious negative effects on non-target organisms and the ecological environment of water body.

[0129] The above are only specific embodiments of the present application, but the technical features of the present application are not limited thereto. Any simple change, equivalent replacement or modification made on the basis of the present application to solve the basically same technical problem and realize the basically same technical effect shall be covered within the protection scope of the present application.

Claims

1. A composite microbial sterilizer, characterized in that: It is a compound of Bacillus subtilis and Bacillus putrefaciens.

2. The composite microbial disinfectant according to claim 1, characterized in that: The ratio of the number of live bacteria of the Bacillus subtilis to that of the Bacillus putida is 1:10-20.

3. A method for preparing a composite microbial sterilizing agent, applied to the sterilizing agent according to any one of claims 1-2, characterized in that: The following steps are involved: a. Single bacterial culture: Bacillus subtilis culture: Inoculate the purified strain into LB liquid medium and culture at 36±1℃ and 180rpm for 18-24h until the bacterial concentration reaches 10 8 ~10 9 CFU / mL; Bacillus putida culture: inoculate the purified strain into LB liquid medium, shake and culture at 30±1℃ and 150rpm for 24-36h until the bacterial concentration reaches 10 7 ~10 8 CFU / mL; Preparation of mixed seed solution bb: Mix the two bacterial solutions obtained in step a according to the proportion, and culture them at 30°C and 120 rpm for 12 hours to form a symbiotic system; cc fermentation amplification: inoculate the mixed seed solution into a fermentation tank (containing sterile LB medium) at a 5% inoculation rate, and culture at 30°C, aeration volume 1.0 vvm, and stirring speed 100 rpm for 48-72 hours. The total number of viable bacteria at the end point is ≥10 10 CFU / mL.

4. The preparation method according to claim 3, wherein: The LB liquid culture medium comprises 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, with a pH of 7.0±0.2 and is sterilized at 121° C. for 20 min.

5. The preparation method according to claim 3, wherein: In step c, sample and test every 12 hours: Spectrophotometry (OD 600 ) Monitoring bacterial density; The proportion of Bacillus putida was quantitatively detected by qPCR (primer F: 5'-GCTGGATCACCTCCTTTCTA-3'; R: 5'-TCCGATACGGTGAGGTAGAA-3'), with an annealing temperature of 60°C. An application of the composite microbial sterilizer as claimed in claims 1-2, characterized in that: Used for the degradation of Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila in aquaculture water, with a dosage of 1-5 mg / L.

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