CN114507629B

CN114507629B - Aquaculture probiotics and preparation and application of microbial inoculum thereof

Info

Publication number: CN114507629B
Application number: CN202210407502.0A
Authority: CN
Inventors: 吕剑; 王建华; 武君; 殷璐璐; 王娜
Original assignee: Hailuda Shandong Agricultural Technology Development Co ltd; Yantai Institute of Coastal Zone Research of CAS
Current assignee: Hailuda Shandong Agricultural Technology Development Co Ltd; Yantai Institute of Coastal Zone Research of CAS
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2022-07-22
Anticipated expiration: 2042-04-19
Also published as: CN114507629A

Abstract

The invention relates to the field of aquaculture, in particular to aquaculture probiotics and preparation and application of a microbial inoculum thereof. The microbial inoculum is prepared by respectively pre-culturing and propagating new strains of enterococcus YTLJ-N-SXH1 and Brevibacillus YTLJ-N-SXH2, freeze-drying to obtain corresponding single-bacterium powder, mixing the single-bacterium powder, and then mixing with an auxiliary agent. Enterococcus YTLJ-N-SXH1 and Brevibacillus YTLJ-N-SXH2 are preserved in Guangdong province microorganism strain preservation center, and the preservation numbers are GDMCC No: 62080 and GDMCC No: 62215. the strain and the microbial inoculum thereof have the functions of inhibiting pathogenic bacteria, effectively reducing the use of antibiotics, enhancing the immunity of aquatic animals, reducing the morbidity, simultaneously efficiently removing nitrite, ammonia nitrogen and nitrate, increasing the high (good) oxygen multi-way denitrification strain source of an aquaculture system, and can be used for the maintenance of various aquaculture systems.

Description

Aquaculture probiotics and preparation and application of microbial inoculum thereof

Technical Field

The invention relates to the field of aquaculture, in particular to aquaculture probiotics and preparation and application of a microbial inoculum thereof.

Background

Factory breeding is an important mode of modern fishery breeding. The influence of the large amount of culture wastewater on the water quality of offshore culture water areas is great, and the problems of water environment pollution, frequent occurrence of aquatic diseases and the like caused by large-scale industrial culture are seriously troubled and hinder the development of the aquaculture industry in China. The in-situ purification treatment technology of the aquaculture water has the advantages of small damage to the ecological environment, less tail water discharge and the like, can protect the offshore ecological environment, and can obtain high-yield and high-quality aquatic products. Therefore, in-situ purification treatment of aquaculture water is one of the key development directions in the aquaculture field. With the rapid development of economy and the enhancement of human activities, environmental pollution has become an important factor limiting the development of coastal ecologically vulnerable areas. The circulating water culture is the inevitable choice for sustainable development of land-based mariculture in China. Therefore, the development of the aquaculture water in-situ advanced treatment and purification technology with low cost and simple operation has important significance and application value.

The popularization and application of the engineering culture are restricted by the double pressure of nitrite stress and pathogenic bacteria increase brought by the industrial high-density culture. The high culture density can reduce the growth performance of cultured fishes, cause the change of the biochemical composition of the fish body, trigger the excessive consumption of energy of the fish body and the activation of an antioxidant system, inhibit the immune system to a certain extent and reduce the nutritional value of muscles. Meanwhile, under the condition of high-density industrial culture, when the feeding is increased, the content of ammonia nitrogen and nitrite is increased, pathogenic bacteria are activated or amplified, and influence is generated on aquatic animals. Aquatic animals raised in a pond at high density frequently suffer from the stimulation of ammonia nitrogen and nitrite, so that the immunity is reduced, and the aquatic animals are more susceptible to various infectious diseases. Nitrate tends to accumulate in industrial farming systems, and nitrate tends to convert to toxic nitrite when the system is anoxic. Therefore, the control of ammonia nitrogen, nitrite and nitrate can effectively prevent fish diseases.

The development and utilization of probiotics are an important technical means for solving aquatic diseases. Through the addition and application of probiotics, a large amount of pathogenic bacteria are killed or inhibited, and the aim of protecting aquatic animals is further fulfilled. However, the dual pressure of nitrite stress and pathogenic bacteria increase caused by industrial high-density culture restricts the popularization and application of the engineering culture. At present, no report exists for screening and obtaining probiotics with compound efficacy which can remove ammonia nitrogen and nitrite and can inhibit various pathogenic bacteria simultaneously. The invention develops the composite probiotic with the effects of removing ammonia nitrogen, nitrite and nitrate and simultaneously inhibiting various pathogenic bacteria, then reasonably mixes the probiotic to prepare the composite probiotic agent, the advantages of the mixed strains are complemented to form a high-efficiency microbial community, so that various pathogenic bacteria are inhibited and killed, most of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in an aquaculture system are promoted to be converted into nitrogen to be removed, the in-situ maintenance of the aquaculture water body is realized, and the invention has important practical significance for the quality improvement and synergy of industrial aquaculture.

Disclosure of Invention

The invention aims to provide aquaculture probiotics and preparation and application of a microbial inoculum thereof aiming at the defects of the prior art.

In order to realize the purpose, the invention adopts the technical scheme that:

aquatic productThe aquaculture probiotic is enterococcusEnterococcussp, YTLJ-N-SXH1 or Brevibacillus spBrevibacillussp.YTLJ-N-SXH2；

The enterococcusEnterococcussp, YTLJ-N-SXH1, taxonomic designationEnterococcussp. deposited at the Guangdong province culture Collection center at 26.11 months in 2021, Guangzhou, China, with the deposit number GDMCC No: 62080;

the Bacillus brevisBrevibacillusYTLJ-N-SXH2, taxonomic nameBrevibacillussp.And has been preserved in Guangdong province culture collection center at 17.1.2022, Guangzhou, China, with the preservation number GDMCC No: 62215.

the application of aquaculture probiotics, namely enterococcus YTLJ-N-SXH1 and/or bacillus brevis YTLJ-N-SXH2 in inhibiting aquatic pathogenic bacteria or improving immunity of aquatic animals.

The enterococcus YTLJ-N-SXH1 is used for inhibiting pathogenic bacteria of Enterobacteriaceae, and specifically is one or more of salmonella, Edwardsiella and Serratia.

The bacillus brevis YTLJ-N-SXH2 shows a wide inhibition effect on aquatic pathogenic bacteria, and is specifically applied to inhibition of one or more of vibrio, aeromonas, pseudomonas, flavobacterium, acinetobacter, saprolegnia, cotton mold, myceliophthora killing, gill mold, fusarium, streptozochytrium and chytrium.

The application of the aquaculture probiotic enterococcus YTLJ-N-SXH1 and/or Bacillus brevis YTLJ-N-SXH2 in purification of aquaculture environment.

Further, the aquaculture probiotics YTLJ-N-SXH1 and/or Brevibacillus YTLJ-N-SXH2 are applied to removing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen in a culture system.

The enterococcus YTLJ-N-SXH1 has an anammox function, can remove ammonia nitrogen and nitrite and generate nitrogen, thereby reducing the accumulation of ammonia nitrogen and nitrite in an aquaculture system. The Bacillus brevis YTLJ-N-SXH2 has a denitrification function, can remove nitrate through denitrification, generates nitrogen, and reduces accumulation of nitrate in an aquaculture system.

The probiotic strains (enterococcus YTLJ-N-SXH1 and Brevibacillus YTLJ-N-SXH 2) are mixed, and the advantages of the two strains are complemented to form a symbiotic system. The Brevibacillus YTLJ-N-SXH2 can generate antibacterial peptide with broad-spectrum antipathogenic bacteria, and can improve the inhibition effect of enterococcus YTLJ-N-SXH1 on various enterobacteriaceae pathogenic bacteria; meanwhile, enterococcus YTLJ-N-SXH1 effectively removes ammonia nitrogen and nitrite through anaerobic ammoxidation, but cannot effectively remove nitrate in a culture system; the Bacillus brevis YTLJ-N-SXH2 can remove nitrate through denitrification, and overcomes the defect that the accumulation of nitrate in a culture system cannot be effectively relieved by enterococcus YTLJ-N-SXH 1.

An aquaculture probiotic agent contains enterococcus YTLJ-N-SXH1 and/or Bacillus brevis YTLJ-N-SXH 2.

The microbial inoculum contains enterococcus YTLJ-N-SXH1 and Brevibacillus YTLJ-N-SXH2, wherein the enterococcus YTLJ-N-SXH1 and the Brevibacillus YTLJ-N-SXH2 after propagation are mixed according to the proportion (mass ratio) of 10-50:1-10, and the total effective viable count in the microbial inoculum is more than or equal to 1.0 multiplied by 10⁹One per mL.

The preparation method comprises

1) And (3) strain propagation: respectively inoculating enterococcus YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 by 5-10wt% to an improved propagation culture medium, culturing at 28-35 ℃ for 24-72 hours, and separating and collecting precipitated bacteria;

2) preparing mixed bacterial powder: respectively freeze-drying the single enterococcus YTLJ-N-SXH1 and the single Bacillus brevis YTLJ-N-SXH2 after propagation to obtain single strain dry powder, and mixing the single strain dry powder according to the mass ratio of 10-50:1-10 to obtain mixed strain powder;

3) preparing an auxiliary agent: mixing the feces residual bait, the corn flour, the soybean meal and the like to obtain a mixture, adding the mixed bacterial powder obtained in the step 2) with the mass of the mixture being 0.1-1.0%, and adding NH with the mass of the mixture being 0.05-0.5%₄NO₃Adding Cu (NO) with the mass of the mixture being 0.05-0.5 percent₃)₂Mixing the components evenly to mixThe water content of the combined system is 40-60%, the pH value is 6.5-7.5, the combined system is fermented and cultured for 5-10 days at the temperature of 28-35 ℃, the precipitate fermentation product is fermented and collected, magnesium modified short bacillus antibacterial peptide with the mass of 0.1-0.5% and inulin with the mass of 0.5-1.0% are added into the dried fermentation product, and the auxiliary agent is obtained after mixing;

the breeding excrement residual bait is fish and shrimp breeding excrement residual bait;

4) preparation of a microbial inoculum: mixing the mixed bacteria powder obtained in the step 2) with the auxiliary agent obtained in the step 3) according to a mass ratio of 1:1-100 to prepare the aquaculture probiotic agent.

The culture medium for propagation in the step 1) is 0.05-0.1% of NH by mass ratio₄NO₃、0.05-0.1% NH₄NO₂0.2-1% of sodium acetate and 2-5% of K₂HPO₄、1-3% MgSO₄、1-3% NaCl，0.01-0.05% FeSO₄、0.01-0.05% MnSO₄And 0.01-0.05% La (NO)₃)₃The balance being water;

when the strain in the step 1) is propagated, nitroimidazole antibiotics with the mass of 0.02-0.2% and paranitroaniline with the mass of 0.001-0.01% are added into a propagation medium, and the improved propagation medium is obtained.

The antibiotic is nitroimidazole antibiotic, including metronidazole, ornidazole and tinidazole. During propagation of the strain, the strain is subjected to tolerance domestication by using antibiotics, and meanwhile, the nitrogen metabolism capability of the strain can be enhanced, and the survival capability and activity of the strain in an aquaculture system can be improved.

The magnesium-doped Brevibacillus brevis antibacterial peptide in the step 3) is prepared by filtering a Brevibacillus brevis YTLJ-N-SXH2 culture obtained by propagation in the step 1), harvesting bacteria, adding 70-75% by mass of ammonium sulfate and 10-12% by mass of magnesium salt into the collected bacteria, mixing, adjusting the pH of the system to 9.0-10.0, standing for 1-2 days, standing, freeze-drying to obtain a crude product of the Brevibacillus brevis antibacterial peptide (slowly adjusting the pH of the solution to be more than 10.0, modifying and precipitating the antibacterial peptide, improving the activity of the precipitated antibacterial peptide, standing to fully precipitate the antibacterial peptide), adding 0.1-1% by mass of nano magnesium oxide, 0.1-1% by mass of calcium carbonate and 0.01-0.1% by mass of lanthanum nitrate into the crude product, uniformly mixing to obtain the magnesium-doped Brevibacillus brevis antibacterial peptide, and storing the magnesium-doped Brevibacillus brevis antibacterial peptide in a drying place for later use.

The magnesium salt is magnesium chloride, magnesium sulfate or magnesium nitrate, and is doped with 1-10% by mass of lithium nitrate and 0.1-1.0% by mass of lanthanum nitrate.

The magnesium-doped Brevibacillus brevis antibacterial peptide is obtained by modifying a large amount of antibacterial peptide generated by a strain (Brevibacillus brevis YTLJ-N-SXH 2) in the propagation culture process after coarse purification, has the effect of inhibiting various pathogenic bacteria, and can improve the immunity of aquatic animals after ingestion.

An application of aquaculture probiotic agent, and an application of the agent in aquaculture purification.

Furthermore, the microbial inoculum is applied to inhibiting pathogenic bacteria and biological denitrification in an aquaculture system.

The method specifically comprises the following steps: the prepared microbial inoculum is put into aquaculture feed or aquaculture systems according to the mass ratio of 1-10% to play the role of killing and inhibiting pathogenic bacteria, the morbidity of aquatic animals and the usage amount of antibiotics are reduced by more than 50%, and ammonia nitrogen and nitrite nitrogen are removed simultaneously (the effective removal rate is more than 30%).

The invention has the advantages that:

1) the aquaculture bacterial strain enterococcus YTLJ-N-SXH1 can resist high salt (6% NaCl) and high pH (pH is more than 9), can form a colony with a smooth surface after being cultured for 12-24 hours at 35 ℃, and can inhibit one or more of salmonella, Edwardsiella and Serratia. The bacillus brevis YTLJ-N-SXH2 has stable property, is not easy to stain, is high temperature resistant, can survive in gastric juice environment, and has broad-spectrum inhibition on one or more of vibrio, aeromonas, pseudomonas, flavobacterium, acinetobacter, saprolegnia, cotton mold, myceliophthora, gill mold, fusarium, streptochytrium and chytrium. The advantages of the two bacteria are complementary, the short bacillus YTLJ-N-SXH2 can generate antibacterial peptide with broad spectrum antipathogenic bacteria, and the inhibition effect of enterococcus YTLJ-N-SXH1 on various enterobacteriaceae pathogenic bacteria can be improved; meanwhile, enterococcus YTLJ-N-SXH1 effectively removes ammonia nitrogen and nitrite through anaerobic ammoxidation, but cannot effectively remove nitrate in a culture system; the short bacillus YTLJ-N-SXH2 can remove nitrate through denitrification, and overcomes the defect that the enterococcus YTLJ-N-SXH1 cannot effectively relieve the accumulation of nitrate in a culture system.

2) The composite microbial inoculum is prepared by mixing indigenous strains separated from a culture system, and is easy to colonize and propagate in the culture system after being used. Has the economic and ecological benefits of less microbial inoculum consumption, wide strain source and no secondary pollution.

3) The strains in the composite microbial inoculum can form probiotic communities with complementary advantages, the microbial inoculum has stable properties, has no strict requirements on the pH, temperature, salinity and oxygen content of the putting environment, can realize the simultaneous prevention and control of pathogenic bacteria and toxic nitrogen pollutants, increases the strain sources of high (good) oxygen multi-way denitrification in aquaculture, and belongs to a multipurpose environment-friendly microbial inoculum.

Drawings

FIG. 1 is a diagram of the cluster analysis of enterococcus YTLJ-N-SXH1 according to the present invention.

FIG. 2 is a cluster analysis diagram of Bacillus brevis YTLJ-N-SXH2 of the present invention.

FIG. 3 is a scanning electron microscope image of the magnesium-modified Brevibacillus brevis antimicrobial peptide provided by the invention.

FIG. 4 is an EDS (electron Desorption) energy spectrum of the magnesium-modified Brevibacillus brevis antimicrobial peptide provided by the invention.

Detailed Description

The present invention is further illustrated by the following examples, which, however, are not intended to limit the invention thereto.

Example 1

Isolation and characterization of enterococcus YTLJ-N-SXH1

1. Isolation of the Strain

Enterococcus YTLJ-N-SXH1 was isolated from the fecal residue of a shrimp mariculture pond by the following procedure: 5 g of feces residual bait is put into 50 mL of sterilized seawater to be cultured for 72 h under the condition of 28 ℃, the bacterial liquid is centrifuged for 10 min under 10000 rpm, the lower layer bacterial liquid is put into a sterilized propagation culture medium to be cultured for 48 h under the condition of 35 ℃, then the diluted culture is carried out for 48 h in an LB culture medium, and single bacterial colony is selected to be subjected to streak separation in an agar culture medium, so that the strain YTLJ-N-SXH1 is obtained.

2. Identification of strains

2.1 morphological identification

Analyzing and comparing the strain separated and purified in the step 1, wherein the strain belongs to enterococcus (A)Enterococcussp.）。

2.2, 16S rDNA sequence homology analysis

And (2) amplifying the 16S rDNA fragment of the strain obtained in the step (1) by adopting a colony PCR method, amplifying the 16S rDNA gene fragment, and cloning and sequencing to show that the 16S rDNA of the strain has a nucleotide sequence of a sequence 1 in a sequence table.

Meanwhile, the enterococcus YTLJ-N-SXH1 is compared with other congeneric strains to further confirm that the enterococcus YTLJ-N-SXH1 is a new strain.

The sequence 116S rDNA gene sequence is:

GAACGCTTCTTTCCTCCCGAGTGCTTGCACTCAATTGGAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTACCCATCAGAGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAACAGTTTATGCCGCATGGCATAAGAGTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGACGTTAGTAACTGAACGTCCCCTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCTAGACCGCGAGGTCATGCAAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGG

the enterococcus isEnterococcussp, YTLJ-N-SXH1 taxonomic nomenclatureEnterococcussp. deposited at the Guangdong province culture Collection center at 26.11 months in 2021, Guangzhou, China, with the deposit number GDMCC No: 62080;

effect experiment using the obtained strains: enterococcus faecalisEnterococcusInoculating the sp, YTLJ-N-SXH1 pure bacterial liquid to the nitrogen-containing wastewater according to the inoculation amount of 5wt%, wherein the initial concentration of nitrite nitrogen is 15 mg/L, the initial concentration of ammonia nitrogen is 2 mg/L, reacting for 72 h at 28 ℃, and respectively reducing the concentrations of nitrite nitrogen and ammonia nitrogen to 4 mg/L and 0.3 mg/L.

Example 2

Separation and identification of brevibacillus YTLJ-N-SXH2

1. Isolation of the Strain

The method for separating the bacterial strain from the culture water of the tilapia mariculture system comprises the following specific steps: sucking 0.1 mL of culture water, pumping into a sterilized amplification culture medium, uniformly coating a flat plate by using an aseptic coating rod, culturing for 7 days at 30 ℃, selecting a single colony, and culturing for 48 hours at 35 ℃ in an agar solid culture medium to obtain a strain YTLJ-N-SXH 2.

2. Identification of strains

2.1 morphological identification

Analyzing and comparing the strains separated and purified in the step 1, wherein the strains belong to the genus Brevibacillus (C.) (Brevibacillussp.)。

2.2, 16S rDNA sequence homology analysis

And (2) amplifying the 16S rDNA fragment of the strain obtained in the step (1) by adopting a colony PCR method, and amplifying the 16S rDNA gene fragment and cloning and sequencing to show that the 16S rDNA of the strain has a nucleotide sequence of a sequence 2 in the sequence table.

Meanwhile, the cluster analysis chart of the Brevibacillus YTLJ-N-SXH2 and other strains of the same genus is compared to further confirm that the strain is a new strain.

Sequence 216S rDNA gene sequence:

AGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGGGTTTTCGGTCCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACCGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATTGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGTGCCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGAGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGAGGACGCCAATCTCTGAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGA

the Bacillus brevisBrevibacillussp, YTLJ-N-SXH2, taxonomic nameBrevibacillussp. deposited at the Guangdong province culture Collection center at 17.1.2022, Guangzhou, China, with the deposit number GDMCC No: 62215.

effect experiment using the obtained strains: bacillus brevisBrevibacillusInoculating the bacterial liquid of sp.YTLJ-N-SXH2 to the culture tail water with the inoculation amount of 10wt%, wherein the concentration of nitrite nitrogen is 0.9 mg/L, the concentration of nitrate nitrogen is 3.2 mg/L, and the nitrite nitrogen concentration is treated at 30 ℃ for 3 days0.5 mg/L and the concentration of nitrate nitrogen is 1.8 mg/L.

Example 3

1) And (3) strain propagation: enterococcus YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 obtained in the above examples were inoculated into modified propagation media in an amount of 10wt%, respectively, and then cultured at 35 ℃ for 24 hours, after the completion of the culturing period, the bacteria (precipitate) were collected by filtration.

The propagation culture medium is 0.05 percent of NH by mass ratio₄NO₃、0.05%NH₄NO₂0.2% sodium acetate, 2% K₂HPO₄、1% MgSO₄、1% NaCl，0.01% FeSO₄、0.01% MnSO₄And 0.01% La (NO)₃)₃The balance being water;

the improved multiplication culture medium is prepared by adding nitroimidazole antibiotics (ornidazole) and 0.001% of paranitroaniline into the multiplication culture medium by mass of 0.02%.

2) Preparing mixed bacterial powder: respectively carrying out freeze drying on single enterococcus YTLJ-N-SXH1 and short bacillus YTLJ-N-SXH2 bacteria obtained by propagation (the pre-freezing temperature is 80 ℃ below zero, the pre-freezing time is 2 hours, and the freeze drying time is 4 hours), and then mixing single strain dry powder according to the mass ratio of 10:1 (enterococcus: short bacillus) to prepare mixed bacteria powder.

3) Preparing an auxiliary agent: sterilizing the prawn mariculture excrement residual bait by hydrogen peroxide, and mixing the disinfected prawn excrement residual bait with corn flour and soybean meal, wherein the mass mixing ratio of the excrement residual bait to the corn flour to the soybean meal is 1:1: 1; then adding the mixed bacterial powder obtained in the step 2) with the mass of 0.1 percent of the mixture and NH with the mass of 0.05 percent of the mixture₄NO₃And 0.05% by mass of Cu (NO)₃)₂. After the addition, the reaction system was stirred to control the water content to 40% and the pH to 6.5, and the fermentation was carried out for 5 days. And drying the obtained fermentation product after fermentation, adding 0.1% of magnesium modified Brevibacillus brevis antibacterial peptide and 0.5% of inulin by mass, and uniformly mixing to obtain the auxiliary agent.

The magnesium-modified antibacterial peptide of the brevibacillus brevis is prepared by inoculating the brevibacillus brevis YTLJ-N-SXH2 into a propagation culture medium according to the inoculation amount of 5 percent, carrying out propagation culture at 28 ℃ (culture condition), filtering and collecting the culture obtained by culture to obtain bacteria, then adding ammonium sulfate with the mass of 70 percent and magnesium salt with the mass of 10 percent into the collected bacteria, mixing, slowly adjusting the pH of the solution to 10.0 by 0.1 mol/LNaOH solution, modifying and precipitating the antibacterial peptide and improving the activity of the antibacterial peptide, standing for 48 hours to fully precipitate the antibacterial peptide, carrying out freeze drying to obtain a crude product of the antibacterial peptide of the brevibacillus brevis, adding nano magnesium oxide with the mass of 0.1 percent, calcium carbonate with the mass of 0.1 percent and lanthanum nitrate into the crude product, uniformly mixing to obtain the magnesium-doped brevibacillus brevis antibacterial peptide, and storing the magnesium-doped brevibacillus brevis antibacterial peptide in a drying place for later use (see figures 3 and 4).

The magnesium salt is magnesium chloride, and is doped with lithium nitrate accounting for 1% of the mass ratio of the magnesium salt and lanthanum nitrate accounting for 0.1% of the mass ratio of the magnesium salt.

The scanning electron micrograph of fig. 3 shows that the obtained antimicrobial peptide is coated with magnesium oxide particles on the surface, and the EDS energy spectrum of fig. 4 confirms that the magnesium oxide particles are successfully doped on the surface of the material.

4) Preparation of a microbial inoculum: and (3) mixing the mixed bacteria powder obtained in the step (2) with the auxiliary agent obtained in the step (3) according to a mass ratio of 1:1 to obtain the aquatic probiotic preparation.

5) The application of the aquatic probiotic comprises the following steps: the prepared microbial inoculum is added into the prawn aquaculture feed according to the mass ratio of 1 percent, and is applied to feeding a certain prawn mariculture farm on a smoke table to realize killing and inhibiting of pathogenic bacteria, and meanwhile, non-microbial inoculum addition contrast culture is set. The frequency of feeding the feed is 2 times a day, the feeding amount of each feeding is about 0.5 percent of the weight of the prawns, and corresponding antibiotics (florfenicol) are applied according to the morbidity (vibriosis) of the prawns. After 3 months of culture, the morbidity of a control group is 5%, the application amount of antibiotics is 10% of the feed mass, the morbidity of a microbial inoculum group is 2%, the application amount of antibiotics is 4% of the feed mass, and the morbidity of aquatic animals and the application amount of antibiotics are reduced by over 50%. The initial concentrations of ammonia nitrogen and nitrite nitrogen in the culture water of the control group are respectively 3.8 mg/L and 0.9 mg/L, and the water quality is not changed during the culture period. The initial concentrations of ammonia nitrogen and nitrite nitrogen in the bacteria agent group culture water are 3.5 mg/L and 0.7 mg/L, the concentrations of ammonia nitrogen and nitrite nitrogen after 3 months of culture are respectively 2.3 mg/L and 0.48 mg/L, and the removal rate is more than 30%.

Example 4

1) And (3) strain propagation: enterococcus YTLJ-N-SXH1 and Brevibacillus YTLJ-N-SXH2 obtained in the above examples were inoculated to the propagation medium in an amount of 10wt% respectively. After inoculation, nitroimidazole antibiotics (ipronidazole, 0.1 (wt)%) and paranitroaniline (0.005 (wt)%) are added into the propagation culture medium. The culture was carried out at 30 ℃ for 48 hours, and after the completion of the culture period, the bacteria (precipitate) were collected by filtration.

The propagation culture medium is 0.08 percent of NH₄NO₃、0.08%NH₄NO₂0.5% sodium acetate, 4% K₂HPO₄、2% MgSO₄、2% NaCl，0.03% FeSO₄、0.03% MnSO₄And 0.03% La (NO)₃)₃And the balance being water.

2) Preparing mixed bacterial powder: respectively carrying out freeze drying on single enterococcus YTLJ-N-SXH1 and single Brevibacillus YTLJ-N-SXH2 obtained by propagation (-80 ℃ for 2 h, freeze drying for 4 h), and mixing the single strain dry powder according to a mass ratio of 15:5 (enterococcus: Brevibacillus) to prepare mixed strain powder.

3) Preparing an auxiliary agent: sterilizing the grouper mariculture excrement residual bait by hydrogen peroxide, and mixing the sterilized excrement residual bait with corn flour and soybean meal, wherein the mass mixing ratio of the excrement residual bait to the corn flour to the soybean meal is 1:1: 1; then adding the mixed bacterial powder obtained in the step 2) with the mass of the mixture being 0.5 percent, and adding NH with the mass of the mixture being 0.2 percent respectively₄NO₃And Cu (NO)₃)₂. After the addition, the reaction system was stirred to control the water content to 50% and the pH to 7.5, and the fermentation was carried out for 10 days. And drying the obtained fermentation product after fermentation, adding 0.2 mass percent of magnesium modified Brevibacillus brevis antibacterial peptide and 0.8 mass percent of inulin into the dried fermentation product, and uniformly mixing to obtain the auxiliary agent.

The magnesium-modified short bacillus antibacterial peptide is prepared by inoculating short bacillus YTLJ-N-SXH2 into a propagation culture medium according to the inoculation amount of 3 percent, carrying out propagation culture at 30 ℃ (culture condition), filtering and collecting the culture obtained by culture to obtain bacteria, adding 72 percent of ammonium sulfate and 11 percent of magnesium salt into the collected bacteria, mixing, slowly adjusting the pH of the solution to 10.0 through 0.1 mol/LNaOH, modifying and precipitating the antibacterial peptide, improving the activity of the antibacterial peptide, standing for 48 h to fully precipitate the antibacterial peptide, freeze-drying to obtain a short bacillus antibacterial peptide crude product, adding nano magnesium oxide with the mass of 0.5 percent, calcium carbonate with the mass of 0.5 percent and lanthanum nitrate into the crude product, uniformly mixing to obtain the magnesium-doped short bacillus antibacterial peptide, and storing the magnesium-doped short bacillus antibacterial peptide in a dry place for later use.

The magnesium salt is magnesium nitrate, lithium nitrate with the mass ratio of 5% and lanthanum nitrate with the mass ratio of 0.5%.

4) Preparation of a microbial inoculum: and (3) mixing the mixed bacterial powder obtained in the step (2) with the auxiliary agent obtained in the step (3) according to a mass ratio of 1:10 to obtain the aquatic probiotic.

5) The application of the aquatic probiotic comprises the following steps: the prepared microbial inoculum is put into a certain grouper culture system of a cigarette table according to the mass ratio of 5 percent to realize the killing and inhibition of pathogenic bacteria, and a control group without the addition of the microbial inoculum is set. The frequency of feeding the feed is 2 times a day, the feeding amount of the feed is about 0.5 percent of the weight of the garrupa every time, and corresponding antibiotic (doxycycline) is applied according to the pathogenesis of the garrupa bacterial diseases. After 3 months of culture, the morbidity of the control group is 7%, the application amount of the antibiotics is 50 mg/kg (fish weight), the morbidity of the microbial inoculum group is 1%, the application amount of the antibiotics is 10 mg/kg (fish weight), and the morbidity of the aquatic animals and the application amount of the antibiotics are reduced by more than 80%. The initial concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the culture water of the control group are respectively 0.95 mg/L, 0.11 mg/L and 4.90 mg/L, and the water quality is not changed during the culture period. The initial concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the water cultured by the microbial inoculum group are 0.92 mg/L, 0.11 mg/L and 5.00 mg/L, the concentrations of the ammonia nitrogen, the nitrite nitrogen and the nitrate nitrogen after 3 months of culture are respectively 0.18 mg/L, 0.02 mg/L and 0.51 mg/L, and the removal rate is more than 80%.

Example 5

1) And (3) strain propagation: enterococcus YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 obtained in the above examples were inoculated into the respective propagation medium in an amount of 10 wt%. After inoculation, nitroimidazole antibiotics (ornidazole, 0.2 wt%) and paranitroaniline (0.01 wt%) were added to the medium. The culture was carried out at 28 ℃ for 72 hours, and after the end of the culture period, the bacteria (precipitate) were collected by filtration.

The propagation culture medium is 0.1 percent of NH₄NO₃、0.1%NH₄NO₂1% sodium acetate, 5% K₂HPO₄、3% MgSO₄、3% NaCl，0.05% FeSO₄、0.05% MnSO₄And 0.05% La (NO)₃)₃And the balance being water.

2) Preparing mixed bacterial powder: respectively carrying out freeze drying on single enterococcus YTLJ-N-SXH1 and single Brevibacillus YTLJ-N-SXH2 obtained by propagation (-80 ℃ for 2 h, freeze drying for 4 h), and mixing the single strain dry powder according to a mass ratio of 50:10 (enterococcus: Brevibacillus) to prepare mixed strain powder.

3) Preparing an auxiliary agent: sterilizing the prawn culture excrement residual bait by hydrogen peroxide, and mixing the disinfected prawn culture excrement residual bait with corn flour and soybean meal in a mass mixing ratio of 1:1: 1; then adding the mixed bacterial powder obtained in the step of adding 1.0% of the mixture by mass, and respectively adding NH with the mass of 0.5% of the mixture by mass₄NO₃And Cu (NO)₃)₂. After the addition, the reaction system was stirred to control the water content to 60% and the pH to 7.5, and the fermentation was carried out for 10 days. And drying the obtained fermentation product after fermentation, adding 0.5 mass percent of magnesium modified short bacillus antibacterial peptide and 1.0 mass percent of inulin, and uniformly mixing to obtain the auxiliary agent.

The magnesium modified antibacterial peptide of the brevibacillus brevis is prepared by inoculating the brevibacillus brevis YTLJ-N-SXH2 into a propagation culture medium according to the inoculation amount of 5 percent, carrying out propagation culture at 30 ℃ (culture condition), filtering and collecting the culture obtained by culture to obtain bacteria, then adding ammonium sulfate accounting for 75 percent of the mass of the bacteria and magnesium salt accounting for 12 percent of the mass of the bacteria into the collected bacteria, mixing, slowly adjusting the pH of the solution to 9.0 through 0.1 mol/LNaOH after mixing, modifying and precipitating the antibacterial peptide and improving the activity of the antibacterial peptide, standing for 24 h to fully precipitate the antibacterial peptide, freezing and drying to obtain a crude product of the antibacterial peptide of the brevibacillus brevis, adding nano magnesium oxide accounting for 1.0 percent of the mass of the crude product, calcium carbonate accounting for 1.0 percent of the mass of the calcium carbonate and lanthanum nitrate into the crude product, uniformly mixing to prepare the magnesium doped antibacterial peptide of the brevibacillus brevis, and storing the magnesium doped antibacterial peptide in a dry place for later use.

The magnesium salt is magnesium sulfate, lithium nitrate with the mass ratio of 10% and lanthanum nitrate with the mass ratio of 1.0% are doped.

4) Preparation of a microbial inoculum: and (3) mixing the mixed bacterial powder obtained in the step (2) with the auxiliary agent obtained in the step (3) according to a mass ratio of 1:100 to obtain the aquatic probiotic.

5) The application of the aquatic probiotic comprises the following steps: in a certain shrimp culture base in yellow river delta, the prepared microbial inoculum is put into an aquaculture feed and a culture system according to the mass ratio of 10 percent to realize the killing and inhibition of pathogenic bacteria, and meanwhile, non-microbial inoculum is set to be added for contrast culture. The feed feeding frequency is 1 time per day, the feeding amount is about 1 percent of the weight of the prawn every time, and the florfenicol is applied according to the disease condition of the prawn vibriosis. The morbidity of a control group is 6 percent after 3 months of culture, the antibiotics are applied according to 30 mg per kg of shrimps, the microbial inoculum group has no morbidity, the application amount of the antibiotics is 0, and the morbidity of aquatic animals and the usage amount of the antibiotics are reduced by 100 percent. The initial concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the culture water of the control group are respectively 3.6 mg/L, 1.1mg/L and 30.05 mg/L, and the water quality is not changed during the culture period. The initial concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the water cultured by the microbial inoculum group are 3.6 mg/L, 0.95 mg/L and 29.90 mg/L, the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen after 3 months of culture are respectively 1.7 mg/L, 0.42 mg/L and 5.11 mg/L, and the removal rate is more than 50%.

Sequence listing

<110> institute of tobacco pipe coastal zone of Chinese academy of sciences

<120> aquaculture probiotics and preparation and application of microbial inoculum thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1394

<212> DNA

<213> Enterococcus sp.

<400> 1

gaacgcttct ttcctcccga gtgcttgcac tcaattggaa agaggagtgg cggacgggtg 60

agtaacacgt gggtaaccta cccatcagag ggggataaca cttggaaaca ggtgctaata 120

ccgcataaca gtttatgccg catggcataa gagtgaaagg cgctttcggg tgtcgctgat 180

ggatggaccc gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gccacgatgc 240

atagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300

gggaggcagc agtagggaat cttcggcaat ggacgaaagt ctgaccgagc aacgccgcgt 360

gagtgaagaa ggttttcgga tcgtaaaact ctgttgttag agaagaacaa ggacgttagt 420

aactgaacgt cccctgacgg tatctaacca gaaagccacg gctaactacg tgccagcagc 480

cgcggtaata cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg 540

cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg 600

ggagacttga gtgcagaaga ggagagtgga attccatgtg tagcggtgaa atgcgtagat 660

atatggagga acaccagtgg cgaaggcggc tctctggtct gtaactgacg ctgaggctcg 720

aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg 780

ctaagtgttg gagggtttcc gcccttcagt gctgcagcaa acgcattaag cactccgcct 840

ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900

gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcctttga 960

ccactctaga gatagagctt tcccttcggg gacaaagtga caggtggtgc atggttgtcg 1020

tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattgttag 1080

ttgccatcat ttagttgggc actctagcga gactgccggt gacaaaccgg aggaaggtgg 1140

ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatgggaa 1200

gtacaacgag tcgctagacc gcgaggtcat gcaaatctct taaagcttct ctcagttcgg 1260

attgcaggct gcaactcgcc tgcatgaagc cggaatcgct agtaatcgcg gatcagcacg 1320

ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380

acacccgaag tcgg 1394

<210> 2

<211> 1403

<212> DNA

<213> Brevibacillus sp.

<400> 2

agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcga 60

gggttttcgg tccctagcgg cggacgggtg agtaacacgt aggcaacctg cctctcagac 120

cgggataaca tagggaaact tatgctaata ccggataggt ttttggattg catgatccga 180

aaagaaaaga tggcttcggc tatcactggg agatgggcct gcggcgcatt agctagttgg 240

tggggtaacg gcctaccaag gcgacgatgc gtagccgacc tgagagggtg accggccaca 300

ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat tttccacaat 360

ggacgaaagt ctgatggagc aacgccgcgt gaacgatgaa ggtcttcgga ttgtaaagtt 420

ctgttgtcag ggacgaacac gtgccgttcg aatagggcgg taccttgacg gtacctgacg 480

agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540

ccggatttat tgggcgtaaa gcgcgcgcag gcggctatgt aagtctggtg ttaaagcccg 600

gagctcaact ccggttcgca tcggaaactg tgtagcttga gtgcagaaga ggaaagcggt 660

attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcggc 720

tttctggtct gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag gattagatac 780

cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg ggggtttcaa taccctcagt 840

gccgcagcta acgcaataag cactccgcct ggggagtacg ctcgcaagag tgaaactcaa 900

aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960

agaaccttac caggtcttga catcccgctg accgctctgg agacagagct tcccttcggg 1020

gcagcggtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080

tcccgcaacg agcgcaaccc ttatctttag ttgccagcat tcagttgggc actctagaga 1140

gactgccgtc gacaagacgg aggaaggcgg ggatgacgtc aaatcatcat gccccttatg 1200

acctgggcta cacacgtgct acaatggttg gtacaacggg atgctacctc gcgagaggac 1260

gccaatctct gaaaaccaat ctcagttcgg attgtaggct gcaactcgcc tacatgaagt 1320

cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380

acaccgcccg tcacaccacg gga 1403

Claims

1. An aquaculture probiotic, characterized in that: the aquaculture probiotic is enterococcusEnterococcussp, YTLJ-N-SXH1 and Brevibacillus spBrevibacillus sp. YTLJ-N-SXH2；

The enterococcus isEnterococcussp, YTLJ-N-SXH1, taxonomic nameEnterococcussp. has been deposited at 26.11.2021 in the culture collection of microorganisms of Guangdong province, at Guangzhou, China, with the deposit number GDMCC No: 62080;

the Bacillus brevisBrevibacillussp, YTLJ-N-SXH2, taxonomic nameBrevibacillussp. deposited in the Guangdong province culture Collection, Guangzhou, China, at 17.1.2022, with the deposit number GDMCC No: 62215.

2. the use of an aquaculture probiotic as claimed in claim 1, characterized in that: the application of the aquaculture probiotics YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 in purification of aquaculture environment.

3. An aquaculture probiotic, characterized in that: the microbial preparation comprises enterococcus YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 according to claim 1.

4. An aquaculture probiotic as claimed in claim 3 wherein: the microbial inoculum contains intestinal spheresBacteria YTLJ-N-SXH1 and Brevibacillus brevis YTLJ-N-SXH2, wherein the expanded enterococcus YTLJ-N-SXH1 and Brevibacillus brevis YTLJ-N-SXH2 are mixed according to the mass ratio of 10-50:1-10, and the total effective viable count in the microbial inoculum is more than or equal to 1.0 multiplied by 10⁹One per mL.

5. A method of preparing an aquaculture probiotic according to claim 4, comprising the steps of:

1) and (3) strain propagation: respectively inoculating 5-10wt% of enterococcus YTLJ-N-SXH1 and Bacillus brevis YTLJ-N-SXH2 to an improved propagation culture medium, culturing at 28-35 ℃ for 24-72 hours, and separating and collecting precipitated bacteria;

3) preparing an auxiliary agent: mixing the fecal residual bait, the corn flour, the soybean meal and the like to obtain a mixture, adding the mixed bacterial powder obtained in the step 2) with the mass of the mixture being 0.1-1.0%, and adding NH with the mass of the mixture being 0.05-0.5%₄NO₃Adding Cu (NO) with the mass of the mixture being 0.05-0.5 percent₃)₂Uniformly mixing until the water content of a mixing system is 40-60 percent and the pH value is 6.5-7.5, fermenting and culturing for 5-10 days at the temperature of 28-35 ℃, fermenting and collecting precipitated fermentation products, adding magnesium modified short bacillus antibacterial peptide with the mass of 0.1-0.5 percent of the dried fermentation products and inulin with the mass of 0.5-1.0 percent of the dried fermentation products, and mixing to obtain the auxiliary agent;

4) preparation of a microbial inoculum: mixing the mixed bacterial powder obtained in the step 2) with the auxiliary agent obtained in the step 3) according to the mass ratio of 1:1-100 to prepare the aquaculture probiotic agent;

when the strain in the step 1) is propagated, nitroimidazole antibiotics accounting for 0.02-0.2% of the mass of the strain and paranitroaniline accounting for 0.001-0.01% of the mass of the strain are added into a propagation culture medium, so that an improved propagation culture medium is obtained;

the magnesium-modified short bacillus antibacterial peptide in the step 3) is prepared by filtering a short bacillus YTLJ-N-SXH2 culture obtained by propagation in the step 1) to obtain bacteria, adding ammonium sulfate accounting for 70-75% of the mass of the bacteria and magnesium salt accounting for 10-12% of the mass of the bacteria into the collected bacteria, adjusting the pH of a system to 9.0-10.0 after mixing, standing for 1-2 days, freeze-drying after standing to obtain a short bacillus antibacterial peptide crude product, adding nano magnesium oxide accounting for 0.1-1% of the mass of the magnesium oxide, calcium carbonate accounting for 0.1-1% of the mass of the magnesium oxide and lanthanum nitrate accounting for 0.01-0.1% of the mass of the magnesium oxide into the crude product, uniformly mixing to obtain the magnesium-modified short bacillus antibacterial peptide, and storing the magnesium-modified short bacillus antibacterial peptide in a drying place for later use.

6. Use of an aquaculture probiotic according to claim 3, characterized in that: the application of the microbial inoculum in aquaculture purification.