Brevibacillus laterosporus GBW-M0 and application thereof in aquaculture

Abstract

The invention provides brevibacillus laterosporus GBW-M0 and an application of the brevibacillus laterosporus GBW-M0 in aquaculture. The brevibacillus laterosporus GBW-M0 is classified and named as brevibacillus laterosporus, and the preservation number of the brevibacillus laterosporus GBW-M0 is CGMCC (China General Microbiological Culture Collection Center) No. 32335. The brevibacillus laterosporus GBW-M0 is subjected to seed solution preparation and immersion fermentation, and brevibacillus laterosporus GBW-M0 fermentation liquor is prepared. The brevibacillus laterosporus GBW-M0 fermentation liquor can reduce the concentration of enterocytozoon hepatopenaeus carried in the bodies of penaeus vannamei, is further used for preventing and controlling microsporidiosis of aquaculture animals, and can also inhibit the growth of microcystis aeruginosa, so that the brevibacillus laterosporus GBW-M0 fermentation liquor is used for treating cyanobacterial bloom in aquaculture water, and the success rate of aquaculture is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Brevibacillus laterosporus GBW-M0 and application thereof in aquaculture. Background Art

[0002] In taxonomy, Brevibacillus laterosporus belongs to the Fungal Kingdom, Firmicutes, Bacillaceae, Brevibacillus Genus, and Brevibacillus laterosporus species. It is a Gram-positive bacterium that can produce a variety of extracellular antibacterial substances such as chitinase and gramicidin.

[0003] At present, Brevibacillus laterosporus is widely used in agricultural biological control, such as for the control of plant pathogens such as Phytophthora, Rhizoctonia solani, tobacco mosaic virus and tomato yellow leaf curl virus. It also has application prospects as a biological pesticide for controlling agricultural pests. There are relatively few studies on its application in aquaculture, and there are no related research reports on its use in the control of aquatic animal pathogens.

[0004] Current technologies have not fully utilized the functions of Brevibacillus laterosporus in aquaculture, and the application scenarios need to be further explored. Because it can produce antibacterial substances such as chitinase, gramicidin, and extracellular antibacterial proteins, it has the potential to prevent and control pathogens in aquatic animals, but it is necessary to screen suitable strains, optimize the fermentation process, and combine sufficient effect verification to achieve the ideal application effect. Summary of the invention

[0005] The present invention provides a Brevibacillus laterosporus GBW-M0 and its application in aquaculture. After the fermentation process is optimized, the Brevibacillus laterosporus GBW-M0 can be used to prevent and control enterocytosis (EHP) disease in Penaeus vannamei aquaculture, and can also control the blue algae bloom formed during the aquaculture process.

[0006] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions: The present invention provides a Brevibacillus laterosporus GBW-M0, which is classified and named Brevibacillus laterosporus Brevibacillus laterosporu , the deposit number is CGMCC No.32335, and its 16S rRNA sequence is shown in SEQ ID No.1.

[0007] Furthermore, the Brevibacillus laterosporus GBW-M0 is a Gram-positive bacterium, and its colonies on nutrient agar are small, round, milky white, opaque, and smooth in surface; its bacteria are short rod-shaped and dispersed independently from each other; its spores are oval, lateral, and the cysts are swollen.

[0008] The present invention also provides a Brevibacillus laterosporus fermentation liquid, which is prepared from the Brevibacillus laterosporus GBW-M0.

[0009] Furthermore, the method for preparing the Brevibacillus laterosporus fermentation broth comprises the following steps: (1) Preparation of seed solution: Using Brevibacillus laterosporus as the bacterial species, a single colony is selected and cultured in a seed culture medium to obtain Brevibacillus laterosporus seed solution; (2) Submerged fermentation: The seed solution of step (1) is inoculated into a submerged fermentation medium at an inoculum amount of 5% to 15% to obtain a fermentation liquid of Brevibacillus laterosporus.

[0010] Furthermore, the culture conditions in step (1) are: temperature 30°C ~ 37°C, rotation speed 150 rpm ~ 220 rpm, pH 7 ~ 8, and culture time 24 h; the components of the seed culture medium include glucose 3 g / L ~ 5 g / L, sucrose 3 g / L ~ 5 g / L, tryptone 8 g / L ~ 10 g / L, yeast extract 3 g / L ~ 5 g / L, and NaCl 5 g / L ~ 10 g / L.

[0011] Furthermore, the culture conditions in step (2) are: temperature 30°C ~ 37°C, rotation speed 150 rpm ~ 220 rpm, pH 7 ~ 8, and culture time 24 ~ 48 h; the components of the immersion fermentation medium include corn starch 10 g / L ~ 12 g / L, sucrose 12 g / L ~ 16 g / L, yeast extract 4 g / L ~ 6 g / L, tryptone 9 g / L ~ 11 g / L, MgSO4 0.4 g / L ~ 0.6 g / L, and NaCl 8 g / L ~ 10 g / L.

[0012] After the conditions are optimized, the method for preparing the Brevibacillus laterosporus fermentation broth comprises the following steps: (1) Preparation of seed solution: Using Brevibacillus laterosporus GBW-M0 as the strain, single colonies were picked and placed in a seed culture medium containing 5 g / L glucose, 5 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract powder, and 10 g / L NaCl. The culture was cultured at 37°C, pH 7.5, and 200 rpm for 24 h to obtain Brevibacillus laterosporus GBW-M0 seed solution. (2) Submerged fermentation: The seed liquid of step (1) was inoculated at an inoculum rate of 10% into an immersion fermentation medium containing 10 g / L corn starch, 14 g / L sucrose, 5 g / L yeast extract, 10 g / L tryptone, 0.5 g / L MgSO4, and 10 g / L NaCl, and cultured at 37°C, pH 7.5, and 200 rpm for 36 h to finally obtain a fermentation broth of Brevibacillus laterosporus GBW-M0.

[0013] The present invention also provides the use of the Brevibacillus laterosporus GBW-M0 or the Brevibacillus laterosporus fermentation liquid in preventing and controlling microsporidiosis in aquaculture animals.

[0014] Furthermore, the fermentation liquid of Brevibacillus laterosporus GBW-M0 is added to the feed of aquaculture animals at a weight ratio of 0.5% to 1%, and the aquaculture animals are fed for 7 days to 15 days, thereby reducing the number of microsporidia in the aquaculture animals.

[0015] Furthermore, the aquaculture animals include Penaeus vannamei.

[0016] Furthermore, the microsporidia include Enterocytosis hepatocellularis (EHP).

[0017] The present invention also provides the use of the Brevibacillus laterosporus GBW-M0 or the Brevibacillus laterosporus fermentation liquid in treating blue algae blooms in aquaculture water bodies.

[0018] Further, the fermentation broth of Brevibacillus laterosporus GBW-M0 was heated at 1 mL / min. 3 ~2 mL / m 3 The dosage is sprayed into aquaculture water for 1d~5d after use, which can reduce the density of cyanobacteria cells in aquaculture water.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention obtains a new strain of Brevibacillus laterosporus GBW-M0, and the fermentation liquid prepared by using the strain in combination with the fermentation method of the present invention has a good preventive and therapeutic effect on microsporidiosis of aquatic animals, thereby expanding the application scenarios of Brevibacillus laterosporus in aquaculture.

[0020] 2. The fermentation liquid of Brevibacillus laterosporus GBW-M0 provided by the present invention can control cyanobacterial blooms occurring in aquaculture, reduce the density of cyanobacterial cells in the water body, thereby reducing the harm of cyanobacterial blooms to aquaculture animals and improving the success rate of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony picture of Brevibacillus laterosporus GBW-M0 on NA medium.

[0022] Figure 2 This is a crystal violet staining microscopic examination of the vegetative body of Brevibacillus laterosporus GBW-M0.

[0023] Figure 3 This is a crystal violet staining microscopic examination of Brevibacillus laterosporus GBW-M0 spores.

[0024] Figure 4This is the effect of culture temperature on the growth of Brevibacillus laterosporus GBW-M0.

[0025] Figure 5 This is the effect of culture medium pH on the growth of Brevibacillus laterosporus GBW-M0.

[0026] Figure 6 This is the effect of NaCl concentration in the culture medium on the growth of Brevibacillus laterosporus GBW-M0.

[0027] Figure 7 The concentration of hepatocellular carcinoma carried by shrimp was detected by fluorescence quantitative PCR before treatment with Brevibacillus laterosporus GBW-M0 fermentation broth.

[0028] Figure 8 These are the test results of Brevibacillus laterosporus GBW-M0 inhibiting hepatocellular carcinoma in shrimp.

[0029] Fig. 9 The concentration of enterocytosis in shrimp bodies detected by fluorescence quantitative PCR after treatment with Brevibacillus laterosporus GBW-M0 fermentation broth.

[0030] Fig.10 The double-layer plate method was used to evaluate the inhibitory effect of Brevibacillus laterosporus GBW-M0 on Microcystis aeruginosa.

[0031] Fig.11 The liquid co-culture method was used to evaluate the inhibitory effect of Brevibacillus laterosporus GBW-M0 on Microcystis aeruginosa.

[0032] Fig.12 This is the inhibitory effect of the fermentation broth of Brevibacillus laterosporus GBW-M0 on Microcystis aeruginosa at different algal cell densities.

[0033] Fig.13 Pond experiment on controlling cyanobacterial blooms using fermentation liquid of Brevibacillus laterosporus GBW-M0. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0035] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0036] Example 1: Acquisition of Brevibacillus laterosporus GBW-M0 and preparation of its fermentation broth 1. Acquisition of Brevibacillus laterosporus GBW-M0 The culture water of white shrimp from Southeast China was taken from Jiangsu, and the water was spread on NA medium after gradient dilution. After multiple separation and purification, a single colony was obtained, named GBW-M0, and preserved.

[0037] The colony morphology of strain GBW-M0 is as follows Figure 1 As shown in the figure, the colonies are small, round, milky white, opaque, and smooth in surface; Figure 2 As shown, they are all short rods, scattered independently, and the spores ( Figure 3 ) are oval, lateral, and have swollen spores.

[0038] DNA of strain GBW-M0 was extracted as a template, and 16S rRNA universal primers were used for amplification. The amplified fragments were sequenced, and the 16S rDNA sequencing results of strain GBW-M0 (sequence as SEQ ID No. 1) were compared with the sequences in GenBank. The results showed that strain GBW-M0 and Brevibacillus laterosporus The homology was the highest, so the strain GBW-M0 was determined to be Brevibacillus laterosporus.

[0039] The screened strain GBW-M0 was deposited, and the depository of the Brevibacillus laterosporus GBW-M0 is the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: October 24, 2024; Brevibacillus laterosporus Brevibacillus laterosporus The deposit number is CGMCC No.32335.

[0040] 2. Analysis of growth characteristics of Brevibacillus laterosporus GBW-M0 1. Culture medium: solid culture medium contains 10g / L trypsin powder, 5g / L yeast extract powder, 5g / L NaCl, 20g / L agar powder, prepared with distilled water, and sterilized at 121℃ for 20min before use; growth temperature analysis culture medium contains 10g / L trypsin powder, 5g / L yeast extract powder, 5g / L NaCl, pH7.0, prepared with distilled water, and sterilized at 121℃ for 20min before use; growth pH analysis culture medium contains 10g / L trypsin powder, 5g / L yeast extract powder, 5g / L NaCl, pH3.0~9.5, prepared with distilled water, and sterilized at 121℃ for 20min before use; NaCl tolerance analysis culture medium contains 10g / L trypsin powder, 5g / L yeast extract powder, 5~35g / L NaCl, pH7.0, prepared with distilled water, and sterilized at 121℃ for 20min before use.

[0041] 2. Analysis method: Brevibacillus laterosporus GBW-M0 was used as the strain, and a single colony was picked and placed in the growth temperature analysis medium. After culturing at 37°C, pH 7.5 and 200 rpm for 16 h, it was inoculated into the above three types of liquid culture media at 2% and cultured at 200 rpm for 24 h. The OD600nm of the culture solution was measured. The culture temperatures of the growth temperature analysis group included 20°C, 25°C, 30°C, 35°C, 40°C and 45°C; the culture temperature of the growth pH analysis group was 35°C, and the pH conditions included 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5; the culture temperature of the NaCl tolerance analysis group was 35°C, and the NaCl concentrations included 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L and 35 g / L.

[0042] 3. Analysis results: Brevibacillus laterosporus GBW-M0 can grow and reproduce rapidly in the temperature range of 25~40℃, but its optimal growth temperature is 35℃ ( Figure 4 ), can adapt to pH 4.5~9.0, and the optimal growth pH is 7.5 ( Figure 5 ), the bacteria can tolerate up to 3% NaCl ( Figure 6 ), the bacteria can secrete a variety of substances that inhibit pathogens after fermentation, such as chitinase, bacterium and so on.

[0043] 3. Preparation of Brevibacillus laterosporus GBW-M0 fermentation broth 1. Culture medium: The seed liquid culture medium contains 5 g / L glucose, 5 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract powder, and 10 g / L NaCl. It is prepared with distilled water and sterilized at 121°C for 20 min. It is used after cooling. The immersion fermentation culture medium contains 10 g / L corn starch, 14 g / L sucrose, 5 g / L yeast extract powder, 10 g / L tryptone, 0.5 g / L MgSO4, and 10 g / L NaCl. It is prepared with distilled water and sterilized at 121°C for 20 min. It is used after cooling.

[0044] 2. Preparation of seed solution: Using Brevibacillus laterosporus GBW-M0 as the strain, pick a single colony and place it in a seed culture medium. Culture it at 37°C, pH 7.5 and 200 rpm for 24 h to obtain Brevibacillus laterosporus GBW-M0 seed solution.

[0045] 3. Submerged fermentation: Inoculate the seed solution of Brevibacillus laterosporus GBW-M0 into the submerged fermentation medium at an inoculum size of 10%, and culture at 37°C, pH 7.5, and 200 rpm for 36 h to finally obtain the fermentation solution of Brevibacillus laterosporus GBW-M0.

[0046] Example 2: Pond test for controlling microsporidia In the small-shed culture model of Penaeus vannamei, the concentration of hepatocellular carcinoma carried by shrimp was 7.6×10 6 ( Figure 7 ), with it as the research object, the experiment was carried out in a plastic barrel equipped with an oxygenating device, divided into 2 groups, each group with 6 replicates, each replicate with 20 shrimps, 1% of the fermentation liquid of Brevibacillus laterosporus GBW-M0 prepared in Example 1 was mixed into the feed, and after feeding for 7 days, 1 shrimp was taken from each replicate, and the concentration of shrimp hepatocellular carcinoma carried by the shrimp bodies of the two treatment groups was detected again by fluorescent quantitative PCR.

[0047] result( Figure 8 ) showed that the concentration of the control group remained at 10 6 level, the concentration of hepatocellular carcinoma in the shrimp treated with GBW-M0 fermentation liquid was reduced to about 5.7×10 4 level( Fig. 9 ).

[0048] Example 3: Indoor evaluation test of the algae inhibition effect of Brevibacillus laterosporus 1. Solid double-layer plate method Strain culture method: A total of 5 strains were selected for the experiment, including Brevibacillus laterosporus GBW-M0, Bacillus licheniformis NB2, Bacillus amyloliquefaciens J4, Bacillus Velez J5, and Bacillus subtilis YES1. Single colonies were picked from NA plates and placed in LB test tubes, cultured at 37°C and 200 rpm for 16 h, and then inoculated into 10 mL LB culture tubes at 2%, cultured at 37°C and 200 rpm for 24 h, and refrigerated for later use.

[0049] Algae inhibition test: Prepare BG11 solid culture medium plates, blow dry them in a clean bench for 40-50 minutes, place a sterile Oxford cup on the surface of the culture medium, mix the Microcystis aeruginosa liquid and semi-solid BG11 culture medium (cooled to about 40°C) in a volume ratio of 8:24, slowly pour them on the surface of the solid culture medium, and gently shake them to cover the surface. After the upper layer solidifies, take 100μL of the culture medium of the 5 strains in the previous step and add it to the corresponding Oxford cups, 2 Oxford cups for each strain, and set LB culture medium as the control group. Cultivate them under natural light at room temperature and observe the algae inhibition zone.

[0050] result( Fig.10 ) shows that the diameter of the algae inhibition zone of Brevibacillus laterosporus GBW-M0 culture solution is larger, which means that it has a significant inhibitory effect on Microcystis aeruginosa. In the figure, there are more molds growing around strain YES1, and the algae inhibition zone is produced by the mold, not the effect of YES1.

[0051] 2. Liquid co-culture method The algae solution of Microcystis aeruginosa cultured in BG11 was diluted 6 times with sterile pure water, 10 mL was taken into a sterile glass test tube, and 10 μL of the fermentation solution of Brevibacillus laterosporus GBW-M0 in Example 1 was added and mixed, and the algae solution was cultured for 4 days under the conditions of 12 h of light, 30°C, 12 h of darkness, and 26°C, and the growth of the algae solution in different treatment groups was observed ( Fig.11 ) It can be found that algae liquids with different algae cell densities will become clear or yellow after being treated with GBW-M0 fermentation liquid, while the green color of the algae liquid in the corresponding control group without treatment deepens or there is no obvious change visible to the naked eye. At the same time, the inhibition rate of GBW-M0 fermentation liquid on Microcystis aeruginosa is calculated after counting the algae cell density. This experiment tracks the inhibition rate of GBW-M0 fermentation liquid prepared in Example 1 on algae liquids with different cell densities at different growth stages during the growth of continuously cultured Microcystis aeruginosa ( Fig.12 ).

[0052] The results showed that: in laboratory method evaluation, 0.1% of Brevibacillus laterosporus GBW-M0 fermentation broth had a significant effect on the growth of less than 1×10 7 cells / mL of Microcystis aeruginosa could achieve an inhibition rate of more than 90%. With the increase of algal cell density, the inhibition rate would decrease. 7 cells / mL of algae liquid can achieve an inhibition rate of 25%.

[0053] Example 4: Experiment on treating blue algae in pond In a river crab breeding pond in Jiangsu, the water depth is 50 cm, the water temperature is about 30° C. in July, cyanobacteria multiply in large quantities, and the water color is dark green. The fermentation liquid of Brevibacillus laterosporus GBW-M0 prepared in Example 1 is sprayed into the river crab pond at a dosage of 1-2 mu / L, and the changes in pond water color and cyanobacterial cell density are tracked.

[0054] result( Fig.13 ) showed that 30 hours after using the fermentation broth of Brevibacillus laterosporus GBW-M0, the water color became lighter and the density of cyanobacteria cells decreased.

[0055] The above results show that adding the fermentation liquid of Brevibacillus laterosporus GBW-M0 prepared by the present invention to the feed of white shrimp can reduce the number of microsporidia in white shrimp; spraying the fermentation liquid of Brevibacillus laterosporus GBW-M0 prepared by the present invention in the river crab breeding pond can reduce the density of cyanobacteria cells in the water body and improve the success rate of aquaculture.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A Brevibacillus laterosporus ( Brevibacillus laterosporu )GBW-M0, characterized in that, Its deposit number is CGMCC No.32335.

2. A Brevibacillus laterosporus fermentation broth, characterized in that: The fermentation broth is prepared from the Brevibacillus laterosporus GBW-M0 described in claim 1.

3. The fermentation broth according to claim 2, characterized in that The preparation method comprises the following steps: (1) Preparation of seed solution: Using Brevibacillus laterosporus as the bacterial species, a single colony is selected and cultured in a seed culture medium to obtain Brevibacillus laterosporus seed solution; (2) Submerged fermentation: The seed solution of step (1) is inoculated into a submerged fermentation medium at an inoculum amount of 5% to 15% to obtain a fermentation liquid of Brevibacillus laterosporus.

4. The fermentation broth according to claim 3, characterized in that The culture conditions in step (1) are: temperature 30°C~37°C, rotation speed 150 rpm~220 rpm, pH 7~8, and culture time 24 h; the components of the seed culture medium include glucose 3 g / L~5 g / L, sucrose 3 g / L~5 g / L, tryptone 8 g / L~10 g / L, yeast extract 3 g / L~5 g / L, and NaCl 5 g / L~10 g / L.

5. The fermentation broth according to claim 3, characterized in that The culture conditions in step (2) are: temperature 30°C ~ 37°C, rotation speed 150 rpm ~ 220 rpm, pH 7 ~ 8, and culture time 24 ~ 48 h; the components of the immersion fermentation medium include corn starch 10 g / L ~ 12 g / L, sucrose 12 g / L ~ 16 g / L, yeast extract powder 4 g / L ~ 6 g / L, tryptone 9 g / L ~ 11 g / L, MgSO4 0.4 g / L ~ 0.6 g / L, and NaCl 8 g / L ~ 10 g / L.

6. Use of the Brevibacillus laterosporus GBW-M0 according to claim 1 or the Brevibacillus laterosporus fermentation liquid according to claim 2 in preventing and controlling microsporidiosis in aquaculture animals.

7. The use according to claim 6, characterized in that: The fermentation liquid of Brevibacillus laterosporus GBW-M0 is added to the feed of aquaculture animals at a weight ratio of 0.5% to 1%, and the aquaculture animals are fed for 7 days to 15 days, thereby reducing the number of microsporidia in the aquaculture animals.

8. The use according to claim 6, characterized in that: The aquaculture animals include Penaeus vannamei.

9. Use of the Brevibacillus laterosporus GBW-M0 according to claim 1 or the Brevibacillus laterosporus fermentation liquid according to claim 2 in controlling blue algae blooms in aquaculture water bodies.

10. The use according to claim 9, characterized in that: The fermentation broth of Brevibacillus laterosporus GBW-M0 was heated at 1 mL / min. 3 ~2 mL / m 3 The dosage is sprayed into aquaculture water for 1d~5d after use, which can reduce the density of cyanobacteria cells in aquaculture water.

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