Brevibacillus parvus GBW-M0 and application thereof in aquaculture
By optimizing the fermentation process of Bacillus lateralis GBW-M0, fermentation broth was prepared for aquaculture, solving the problems of pathogens and cyanobacterial blooms in aquatic animals. This enabled the prevention and control of enterocytozoonosis in Litopenaeus vannamei and the management of cyanobacterial blooms, thereby improving aquaculture efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINGDAO SHANGDE BIOTECH
- Filing Date
- 2024-12-23
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies do not fully utilize the functions of Bacillus laterosporus in aquaculture and lack effective methods for controlling aquatic animal pathogens and cyanobacterial blooms.
The fermentation process of Bacillus lateralis GBW-M0 was optimized to prepare a fermentation broth for the prevention and control of hepatocystis disease and the treatment of cyanobacterial blooms in Litopenaeus vannamei in aquaculture. This was achieved by adding the fermentation broth to the feed or by sprinkling it into the water.
It effectively reduces the number of microsporidia in aquatic animals and the density of cyanobacteria in the water, thereby improving the success rate of aquaculture.
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Figure CN119875879B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus lateralis GBW-M0 and its application in aquaculture. Background Technology
[0002] In taxonomy, *Bacillus laterosporus* belongs to the kingdom Fungi, phylum Firmicutes, family Bacillusaceae, genus *Bacillus*, and species *Bacillus laterosporus*. It is a Gram-positive bacterium that can produce various extracellular antibacterial substances such as chitinase and bacitracin.
[0003] Currently, Bacillus laterosporus is widely used in agricultural biocontrol, such as for the control of plant pathogens like Phytophthora, Rhizoctonia solani, tobacco mosaic virus, and tomato yellow leaf curl virus. It also shows promise as a biological pesticide for controlling agricultural pests. However, its application in aquaculture is relatively limited, and there are no reports on its use in controlling pathogens in aquatic animals.
[0004] Current technology does not fully utilize the functions of Bacillus laterosporus in aquaculture, and its application scenarios need further exploration. Because it can produce antimicrobial substances such as chitinase, bacitracin, and extracellular antimicrobial proteins, it has the potential to control pathogens in aquatic animals. However, achieving ideal application results requires screening suitable strains, optimizing fermentation processes, and conducting thorough efficacy verification. Summary of the Invention
[0005] This invention provides a Bacillus laterosporus GBW-M0 and its application in aquaculture. After optimization of the fermentation process, the Bacillus laterosporus GBW-M0 can be used to control enterohepatocellular carcinoma (EHP) disease in Litopenaeus vannamei shrimp farming, and can also control cyanobacterial blooms that occur during the farming process.
[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:
[0007] This invention provides a *Bacillus laterosporus* GBW-M0, which is classified as *Bacillus laterosporus*. Brevibacillus laterosporus The accession number is CGMCC No. 32335, and its 16S rRNA sequence is shown in SEQ ID No. 1.
[0008] Furthermore, the *Bacillus lateralis* GBW-M0 is a Gram-positive bacterium, with small, round, milky-white, opaque colonies on nutrient agar that have a smooth surface; its cells are short rod-shaped and dispersed independently; its spores are elliptical, lateral, and have enlarged sporangia.
[0009] The present invention also provides a Bacillus retroflexus fermentation broth, which is prepared from the aforementioned Bacillus retroflexus GBW-M0.
[0010] Furthermore, the preparation method of the Bacillus lateralis fermentation broth includes the following steps:
[0011] (1) Seed liquid preparation: Using Bacillus laterosporus as the strain, single colonies were picked and cultured in seed culture medium to obtain Bacillus laterosporus seed liquid;
[0012] (2) Submerged fermentation: The seed liquid from step (1) is inoculated into the submerged fermentation medium at an inoculation rate of 5% to 15% to obtain the fermentation broth of Bacillus lateralis.
[0013] Furthermore, the culture conditions in step (1) are: temperature 30℃~37℃, 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.
[0014] Furthermore, the culture conditions in step (2) are: temperature 30℃~37℃, 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.
[0015] After optimization, the preparation method of the Bacillus lateralis fermentation broth includes the following steps:
[0016] (1) Seed culture preparation: Using Bacillus 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 and 10 g / L NaCl. The culture was carried out at 37℃, pH 7.5 and 200 rpm for 24 h to obtain Bacillus laterosporus GBW-M0 seed culture.
[0017] (2) Submerged fermentation: The seed liquid from step (1) was inoculated at a rate of 10% into a submerged 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. The medium was cultured at 37°C, pH 7.5, and 200 rpm for 36 h to finally obtain the fermentation broth of Bacillus retroflexus GBW-M0.
[0018] The present invention also provides the application of the aforementioned Bacillus laterosporus GBW-M0 or the fermentation broth of the aforementioned Bacillus laterosporus in the prevention and control of microsporidia diseases in aquatic animals.
[0019] Furthermore, by adding the fermentation broth of *Bacillus laterosporus* GBW-M0 to the feed of aquaculture animals at a weight ratio of 0.5% to 1% and feeding the aquaculture animals for 7 to 15 days, the number of microsporidia in the aquaculture animals can be reduced.
[0020] Furthermore, the aquatic farmed animals include the Pacific white shrimp.
[0021] Furthermore, the microsporidia include shrimp hepatocellular carcinoma (EHP).
[0022] The present invention also provides the application of the aforementioned Bacillus laterosporus GBW-M0 or the fermentation broth of the aforementioned Bacillus laterosporus in the treatment of cyanobacterial blooms in aquaculture water bodies.
[0023] Furthermore, the fermentation broth of the *Bacillus laterosporus* GBW-M0 was diluted at 1 mL / m³. 3 ~2 mL / m 3 The dosage is applied to the aquaculture water and applied for 1 to 5 days after application, which can reduce the density of cyanobacteria cells in the aquaculture water.
[0024] Compared with the prior art, the advantages and beneficial effects of the present invention are:
[0025] 1. This invention yielded a new strain of Bacillus laterosporus GBW-M0, and the fermentation broth prepared by combining this bacterium with the fermentation method of this invention has a good preventive and control effect on microsporidiasis in aquatic animals, thus expanding the application scenarios of Bacillus laterosporus in aquaculture.
[0026] 2. The Bacillus retroflexus GBW-M0 fermentation broth provided by this invention can control cyanobacterial blooms in aquaculture, reduce the density of cyanobacterial cells in the water, thereby reducing the harm of cyanobacterial blooms to aquaculture animals and improving the success rate of aquaculture. Attached Figure Description
[0027] Figure 1This is a colony diagram of Bacillus laterosporus GBW-M0 on NA medium.
[0028] Figure 2 This is a crystal violet-stained microscopic image of a vegetative body of Bacillus laterosporus GBW-M0.
[0029] Figure 3 This is a crystal violet-stained microscopic image of Bacillus retroflexus GBW-M0 spores.
[0030] Figure 4 To investigate the effect of culture temperature on the growth of Bacillus lateralis GBW-M0.
[0031] Figure 5 The effect of culture medium pH on the growth of Bacillus lateralis GBW-M0.
[0032] Figure 6 The effect of NaCl concentration in the culture medium on the growth of Bacillus lateralis GBW-M0.
[0033] Figure 7 The concentration of shrimp hepatocellular carcinoma parasites carried by shrimp was detected by real-time PCR before treatment with Bacillus lateralis GBW-M0 fermentation broth.
[0034] Figure 8 The results of the experiment on the inhibition of shrimp hepatocellular carcinoma by Bacillus lateralis GBW-M0.
[0035] Figure 9 The concentration of *Bacillus lateralis* GBW-M0 in shrimp was detected by real-time quantitative PCR after treatment with the fermentation broth.
[0036] Figure 10 To evaluate the inhibitory effect of Bacillus lateralis GBW-M0 on Microcystis aeruginosa using the double-layer plate method.
[0037] Figure 11 To evaluate the inhibitory effect of Bacillus lateralis GBW-M0 on Microcystis aeruginosa using a liquid co-culture method.
[0038] Figure 12 The study investigated the inhibitory effect of Bacillus laterosporus GBW-M0 fermentation broth on Microcystis aeruginosa at different algal cell densities.
[0039] Figure 13 Pond experiment on the treatment of cyanobacterial blooms using Bacillus laterosporus GBW-M0 fermentation broth. Detailed Implementation
[0040] The technical solution of the present invention will be further described in detail with reference to the following specific examples.
[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0042] Example 1: Obtaining Bacillus laterosporus GBW-M0 and preparing its fermentation broth
[0043] I. Acquisition of Bacillus laterosporus GBW-M0
[0044] Water from the cultivation of Litopenaeus vannamei in Jiangsu Province was serially diluted and spread onto NA medium. After multiple separations and purifications, a single colony was obtained, named GBW-M0, and stored.
[0045] The colony morphology of strain GBW-M0 is as follows Figure 1 As shown, the colonies are small and round, milky white, opaque, and have a smooth surface; their cells are as follows: Figure 2 As shown, they are all short rods, independently dispersed, with spores ( Figure 3 The sporangia are oval, lateral, and enlarged.
[0046] DNA was extracted from strain GBW-M0 as a template and amplified using universal 16S rRNA primers. The amplified fragment was sequenced, and the 16S rDNA sequencing results of strain GBW-M0 (sequence as shown in SEQ ID No. 1) were compared with sequences in GenBank. The results showed that strain GBW-M0 and... Brevibacillus laterosporus The strain GBW-M0 was identified as Bacillus lateralis due to the highest homology.
[0047] The selected strain GBW-M0 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on October 24, 2024. Brevibacillus laterosporus Its accession number is CGMCC No. 32335.
[0048] II. Growth Characteristics Analysis of Bacillus Laterosporus GBW-M0
[0049] 1. Culture media: Solid culture media contain 10 g / L trypsin powder, 5 g / L yeast extract, 5 g / L NaCl, and 20 g / L agar powder, prepared with distilled water, and sterilized at 121℃ for 20 min before use; Culture media for growth temperature analysis contain 10 g / L trypsin powder, 5 g / L yeast extract, and 5 g / L NaCl, pH 7.0, prepared with distilled water, and sterilized at 121℃ for 20 min before use; Culture media for growth pH analysis contain 10 g / L trypsin powder, 5 g / L yeast extract, and 5 g / L NaCl, pH 3.0~9.5, prepared with distilled water, and sterilized at 121℃ for 20 min before use; Culture media for NaCl tolerance analysis contain 10 g / L trypsin powder, 5 g / L yeast extract, and 5~35 g / L NaCl, pH 7.0, prepared with distilled water, and sterilized at 121℃ for 20 min before use.
[0050] 2. Analytical Methods: Using *Bacillus laterosporus* GBW-M0 as the strain, single colonies were picked and cultured in a culture medium for growth temperature analysis at 37℃, pH 7.5, and 200 rpm for 16 h. Then, 2% of the culture was inoculated into the three types of liquid culture media mentioned above and cultured at 200 rpm for 24 h. The OD600nm of the culture was then measured. The growth temperature analysis group included cultures at 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃; the growth pH analysis group included cultures at 35℃ with pH values of 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; and the NaCl tolerance analysis group included cultures at 35℃ with NaCl concentrations of 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L.
[0051] 3. Analysis Results: Bacillus laterosporus GBW-M0 can grow and reproduce rapidly in the temperature range of 25~40℃, but its optimal growth temperature is 35℃. Figure 4 It can adapt to conditions with a pH of 4.5 to 9.0, and the optimal growth pH is 7.5. Figure 5 This bacterium can tolerate up to 3% NaCl ( Figure 6 This bacterium can secrete various substances that inhibit pathogens through fermentation, such as chitinase and bacitracin.
[0052] III. Preparation of Fermentation Broth of Bacillus Laterosporus GBW-M0
[0053] 1. Culture medium: The seed culture medium contains 5 g / L glucose, 5 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, prepared with distilled water, sterilized at 121℃ for 20 min by moist heat, and used after cooling; the immersion fermentation medium contains 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, prepared with distilled water, sterilized at 121℃ for 20 min by moist heat, and used after cooling.
[0054] 2. Seed culture preparation: Using Bacillus laterosporus GBW-M0 as the strain, single colonies were picked and cultured in seed culture medium at 37℃, pH 7.5 and 200 rpm for 24 h to obtain Bacillus laterosporus GBW-M0 seed culture.
[0055] 3. Submerged fermentation: The seed culture of Bacillus laterosporus GBW-M0 was inoculated into the submerged fermentation medium at an inoculation rate of 10%, and cultured at 37℃, pH 7.5, and 200 rpm for 36 h to finally obtain the fermentation broth of Bacillus laterosporus GBW-M0.
[0056] Example 2: Pond Experiment for the Control of Microsporidia
[0057] In the small-scale greenhouse culture of Litopenaeus vannamei, the concentration of Enterocytozoa hepatica carried by the shrimp was 7.6 × 10⁻⁶, as detected by quantitative real-time PCR. 6 ( Figure 7 Using shrimp as the research object, the experiment was conducted in a plastic bucket equipped with an oxygenation device, divided into 2 groups, with 6 replicates in each group and 20 shrimp in each replicate. 1% of the fermentation broth of Bacillus lateralis prepared in Example 1 GBW-M0 was mixed into the feed. After feeding for 7 days, 1 shrimp was taken from each replicate, and the concentration of Enterocytozoa hepatis carried by the shrimp in the two treatment groups was detected again by real-time quantitative PCR.
[0058] result( Figure 8 This indicates that the concentration in the control group remained at 10. 6 At the level of GBW-M0 fermentation broth, the concentration of Enterocytozoa in shrimp liver cells decreased to approximately 5.7 × 10⁻⁶. 4 level( Figure 9 ).
[0059] Example 3: Indoor evaluation test of the algae-inhibiting effect of Bacillus laterosporus
[0060] 1. Solid double-layer plate method
[0061] Culture methods for bacterial strains: Five bacterial strains were selected for the experiment, including Bacillus laterosporus GBW-M0, Bacillus licheniformis NB2, Bacillus amyloliquefaciens J4, Bacillus belye J5, and Bacillus subtilis YES1. Single colonies were picked from NA plates and transferred to LB tubes. The culture was carried out at 37°C and 200 rpm for 16 h with shaking. The culture was then inoculated at 2% into 10 mL LB medium tubes and cultured at 37°C and 200 rpm for 24 h with shaking. The culture was then stored in the refrigerator for later use.
[0062] Algae inhibition test: Prepare BG11 solid culture medium plates, dry them in a clean bench for 40-50 min, place sterile Oxford cups on the surface of the culture medium, mix Microcystis aeruginosa algal solution with semi-solid BG11 culture medium (cooled to about 40℃) at a volume ratio of 8:24, slowly pour the mixture onto the surface of the solid culture medium, and gently shake to cover the surface. After the upper layer solidifies, take 100 μL 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 up LB medium as a control group. Incubate at room temperature under natural light and observe the algae inhibition zone.
[0063] result( Figure 10 The results show that the *Bacillus laterosporus* GBW-M0 culture medium exhibits a large algae inhibition zone, indicating a significant inhibitory effect on *Microcystis aeruginosa*. In contrast, strain YES1 in the figure shows abundant mold growth around it, suggesting the algae inhibition zone was generated by the mold, not by the effect of YES1 itself.
[0064] 2. Liquid co-culture method
[0065] The Microcystis aeruginosa cultured in BG11 was diluted 6 times with sterile pure water, and 10 mL was placed in a sterile glass test tube. 10 μL of Bacillus retroflexus GBW-M0 fermentation broth from Example 1 was added and mixed thoroughly. After culturing for 4 days under conditions of 12 h light at 30°C and 12 h darkness at 26°C, the growth of the algae in different treatment groups was observed. Figure 11 It was found that algal solutions with different cell densities became clear or yellow after treatment with GBW-M0 fermentation broth, while the control group, which received no treatment, showed a deeper green color or no obvious changes visible to the naked eye. The inhibition rate of GBW-M0 fermentation broth on *Microcystis aeruginosa* was calculated after statistical analysis of algal cell density. This experiment tracked the inhibition rate of the GBW-M0 fermentation broth prepared in Example 1 on algal solutions with different cell densities at different growth stages during the continuous growth of *Microcystis aeruginosa*. Figure 12 ).
[0066] Results showed that, according to laboratory methods, 0.1% of the *Bacillus laterosporus* GBW-M0 fermentation broth was effective against strains below 1×10⁻⁶. 7 The inhibition rate of Microcystis aeruginosa cells / mL can reach over 90%. However, the inhibition rate decreases with increasing algal cell density. For 3.5 × 10⁻⁶ cells / mL, the inhibition rate is lower.7 An algal solution with cells / mL can achieve an inhibition rate of 25%.
[0067] Example 4: Pond Blue-green Algae Control Experiment
[0068] In crab farming ponds in Jiangsu Province, with a water depth of 50cm and a water temperature of about 30℃ in July, cyanobacteria proliferate in large quantities, turning the water a dark green color. The fermentation liquid of Bacillus retroflexus GBW-M0 prepared in Example 1 was applied to the crab ponds at a rate of 1~2 mu / L, and the changes in pond water color and cyanobacteria cell density were monitored.
[0069] result( Figure 13 The results showed that after 30 hours of using Bacillus lateralis GBW-M0 fermentation broth, the water color became lighter and the density of cyanobacterial cells decreased.
[0070] The above results indicate that adding the GBW-M0 fermentation broth of Bacillus laterosporus prepared in this invention to the feed of Litopenaeus vannamei can reduce the number of microsporidia in the shrimp; and spraying the GBW-M0 fermentation broth of Bacillus laterosporus prepared in this invention into crab farming ponds can reduce the density of cyanobacteria in the water and improve the success rate of aquaculture.
[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.
Claims
1. A type of Laterospora brevispera ( Brevibacillus laterosporus GBW-M0, characterized in that, Its accession number is CGMCC No.32335.
2. A fermentation broth of Bacillus laterosporus, characterized in that, The fermentation broth is prepared from Bacillus lateralis GBW-M0 as described in claim 1.
3. The fermentation broth according to claim 2, characterized in that, Its preparation method includes the following steps: (1) Seed liquid preparation: Using Bacillus laterosporus as the strain, single colonies were picked and cultured in seed culture medium to obtain Bacillus laterosporus seed liquid; (2) Submerged fermentation: The seed liquid from step (1) is inoculated into the submerged fermentation medium at an inoculation rate of 5% to 15% to obtain the fermentation broth of Bacillus lateralis.
4. The fermentation broth according to claim 3, characterized in that, The culture conditions in step (1) are: temperature 30℃~37℃, 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℃~37℃, rotation speed 150 rpm~220 rpm, pH 7~8, 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.
6. The use of Bacillus retroflexus GBW-MO as described in claim 1 or the fermentation broth of Bacillus retroflexus as described in claim 2 in the preparation of a formulation for reducing the concentration of Enterocytozoa hepatis in aquaculture animals.
7. The application according to claim 6, characterized in that, Adding the fermentation broth of *Bacillus laterosporus* GBW-M0 to the feed of aquaculture animals at a weight ratio of 0.5% to 1% and feeding the aquaculture animals for 7 to 15 days can reduce the number of microsporidia in the aquaculture animals.
8. The application according to claim 6, characterized in that, The aquatic animals mentioned include whiteleg shrimp.
9. The application of Bacillus retroflexus GBW-MO as described in claim 1 or the fermentation broth of Bacillus retroflexus as described in claim 2 in the treatment of cyanobacterial blooms in aquaculture water bodies.
10. The application according to claim 9, characterized in that, The fermentation liquor of the Brevibacillus parvus GBW-M0 is sprayed into the water body for aquaculture in an amount of 1 mL / m 3 2 mL / m 3 After 1-5 days, the cyanobacterial cell density in the water body for aquaculture is reduced.
Citation Information
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