Bacillus Velez GY1 and its application

Abstract

The present invention discloses a Bacillus velezensis GY1, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 28, 2024, with a deposit number of GDMCC No: 64792. The Bacillus velezensis GY1 provided by the present invention can degrade 100% and 56.4% of nitrogen in M9 culture medium containing 200 mg / L and 1000 mg / L ammonia nitrogen, respectively, and the ammonia reduction rate in the actual production of aquaculture water reaches 53.3%. It also has a strong inhibitory effect on common pathogens in aquatic products: Vibrio alginolyticus, Salmonella and Vibrio parahaemolyticus.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and more specifically relates to Bacillus Velez GY1 and application thereof. Background Art

[0002] Aquaculture accounts for a large proportion of the agricultural economic structure in my country. In recent years, it has brought good economic benefits to many farmers and improved people's living standards. However, with the development of the economy, the unreasonable breeding scale and management mode of some aquaculture farmers, such as the abuse of chemical agents and high-density intensive breeding, have caused the organic matter, nitrogen, phosphorus and other pollutants in large areas of water such as lakes and aquaculture ponds to exceed the standard, resulting in eutrophication of water bodies, discoloration and odor of water bodies, and causing disturbances in the aquatic ecosystem. Among them, ammonium nitrogen (NH4 + -N), nitrite nitrogen (N02 - -N) and other harmful nitrogen content has a serious stress effect on aquatic organisms. In addition, the abuse of antibiotics and other drugs has led to drug resistance in pathogenic bacteria in the water. Once the immunity of fish is reduced or physically damaged, a large number of pathogenic bacteria will invade, causing heavy losses to farmers. Therefore, finding a technology that is efficient and environmentally friendly for the removal of nitrogen from aquaculture water and has a protective effect on farmed animals is one of the key difficulties in the development of aquaculture. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0004] One object of the present invention is to provide a Velez subtilis GY1, which can efficiently degrade ammonia nitrogen and inhibit the growth of aquatic pathogens.

[0005] In order to achieve these purposes and other advantages of the present invention, a Bacillus velezensis GY1 is provided, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC for short), with a deposit number of GDMCC No: 64792, a deposit date of June 28, 2024, and an address of the depository unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] A microbial preparation for degrading ammonia nitrogen, comprising the Bacillus Velez GY1.

[0007] A microbial preparation for antagonizing pathogenic bacteria, comprising the Bacillus Velez GY1 and / or its metabolites.

[0008] Preferably, the pathogenic bacteria are Vibrio alginolyticus, Salmonella and Vibrio parahaemolyticus.

[0009] The preparation method of the microbial preparation is as follows: specifically, Bacillus Velez GY1 is inoculated into LB medium or M9 inorganic salt medium for a period of time to obtain a microbial preparation capable of degrading ammonia nitrogen and antagonizing pathogens.

[0010] Preferably, the culture temperature is 37°C, the rotation speed is 120-200 rpm, and the culture time is 48 hours.

[0011] Application of Bacillus Velez GY1 for degrading ammonia nitrogen and / or antagonizing aquatic pathogens.

[0012] The present invention at least has the following beneficial effects: the Velez subtilis GY1 provided by the present invention can degrade 100% and 56.4% of nitrogen in M9 culture medium containing 200 mg / L and 1000 mg / L ammonia nitrogen respectively, and the ammonia reduction rate in the actual production of aquaculture water reaches 53.3%. It also has a strong inhibitory effect on common pathogens in aquatic products: Vibrio alginolyticus, Salmonella and Vibrio parahaemolyticus.

[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a photo of the colony morphology of Bacillus Velez GY1 of the present invention on LB medium;

[0015] Figure 2 This is a diagram showing the antibacterial effect of Bacillus Velez GY1 of the present invention on Vibrio parahaemolyticus;

[0016] Figure 3 This is a diagram showing the antibacterial effect of the Bacillus Velez GY1 of the present invention on Salmonella;

[0017] Figure 4 This is a diagram showing the antibacterial effect of the Bacillus Velez GY1 of the present invention on Vibrio alginolyticus. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0019] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0020] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0021] The present invention provides a Bacillus velezensis, which is classified and named Bacillus velezensis GY1. The bacterium is deposited in Guangdong Microbiological Culture Collection Center (GDMCC for short), with a deposit number of GDMCC No: 64792, a deposit date of June 28, 2024, and an address of the deposit unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0022] The embodiments of the present application will be described in detail below in conjunction with the embodiments. If no specific conditions are specified, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0023] 1. Screening and Isolation of Strain

[0024] 5 mL of sewage sample was collected from a breeding farm in Tianhe District, Guangzhou, and vortexed with 45 mL of sterile water. After standing for 10 min, 100 μL of supernatant was aspirated and placed in M9 plate culture medium with potassium nitrate as the only nitrogen source, and cultured at 37°C for 2 days. A single colony was picked and inoculated into M9 medium with ammonium sulfate as the only nitrogen source, and cultured at 37°C, 150 rpm for 24 h. The culture solution was streaked onto potassium nitrate M9 plate culture medium and cultured at 37°C for 2 days. The liquid culture medium and plate culture medium were repeatedly inoculated for 3 times, and several strains capable of degrading ammonia nitrogen were isolated. One of the strains with stronger ability was named GY1. The colony morphology characteristics of this strain are as follows: Figure 1 As shown, the single colony on LB medium was milky white, opaque, with irregular wrinkled edges, and was Gram-positive.

[0025] 2. Identification of the 16S rDNA gene sequence of the genome of strain GY1

[0026] The ammonia nitrogen degradation strain GY1 was inoculated into LB liquid culture medium, cultured overnight at 37°C, and the bacteria were collected. The genomic DNA was extracted using the CTAB method, and then the genomic samples that passed the preliminary inspection were sent to Bio-Tech for genome third-generation sequencing. The strain GY1 was finally determined to be Bacillus velezsima through ANI analysis, and its 16S rDNA gene sequence SEQ ID No: 1 was:

[0027] aaaggaggtgatccagccgcaccttccgatacggctaccttgttacgacttcaccccaatcatctgtcccaccttcggcggctggctccata

[0028] aaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgc

[0029] tgatccgcgattactagcgattccagcttcacgcagtcgagttgcagactgcgatccgaactgagaacagatttgtgggattggcttaacctc

[0030] gcggtttcgctgccctttgttctgtccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccg

[0031] gtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaagatcaagggttgcgctcgttgcgggacttaacccaacatct

[0032] cacgacacgagctgacgacaaccatgcaccacctgtcactctgcccccgaaggggacgtcctatctctaggattgtcagaggatgtcaag

[0033] acctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcagtcttgcgac

[0034] cgtactccccaggcggagtgcttaatgcgttagctgcagcactaaggggcggaaaccccctaacacttagcactcatcgtttacggcgtgg

[0035] actaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctc

[0036] cacatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctccccggttgagcc

[0037] gggggctttcacatcagacttaagaaaccgcctgcgagccctttacgcccaataattccggacaacgcttgccacctacgtattaccgcgg

[0038] ctgctggcacgtagttagccgtggctttctggttaggtaccgtcaaggtgccgccctatttgaacggcacttgttcttccctaacaacagagct

[0039] ttacgatccgaaaaccttcatcactcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagt

[0040] ctgggccgtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgtcgccttggtgagccgttacctcaccaactagct

[0041] aatgcgccgcgggtccatctgtaagtggtagccgaagccaccttttatgtctgaaccatgcgattcagacaaccatccggtattagccccgg

[0042] tttcccggagttatcccagtcttacaggcaggttacccacgtgttcacccgtccgccgctaacatcagggagcaagctcccatctgtccgctcgacttgcatgtattaggcacgccgccagcgttcgtcctgagccaggatcaaactctccgataa 1550.

[0043] The bacteria is deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short), with the deposit number GDMCCNo:64792 and the deposit date June 28, 2024. The address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0044] 3. Physiological and biochemical tests of strain GY1

[0045] Pick the colonies to be tested on the plate and dilute them to about 10% with sterile saline. 8 CFU / mL, 0.05-0.08mL of bacterial suspension was pipetted into each microvial, the inoculated vials were then stoppered, placed upright in a bottle rack, and cultured in a 37°C incubator. The results are shown in Table 1 below.

[0046] Table 1 Physiological and biochemical test table

[0047]

[0048] The bacteria can utilize β-galactoside, D-ribose, and arabinose; can grow in 3% NaCl peptone water and 3% NaCl sucrose; but cannot utilize 3% NaCl glucose phosphate peptone water, amygdalin, and 3% hydrogen sulfide.

[0049] 4. Ammonium ion content detection

[0050] 1. Detection reagent configuration

[0051] 1) Phenol solution: weigh 10 g phenol and 100 mg sodium nitroprusside, dilute to 1 L, and store in a brown bottle at 4°C.

[0052] 2) Sodium hypochlorite alkaline solution, 10g NaOH, 7.06g Na2HPO4·7H2O, 31.8g Na3PO4·12H2O and 10mL sodium hypochlorite, dilute to 1L, and store in a brown bottle at 4℃.

[0053] 3) Masking squeeze: Weigh 400 g / L potassium sodium tartrate and 100 g / L EDTA disodium salt solution in equal proportions and add 0.5 mL NaOH (10 mol / L) per 100 mL of the mixture.

[0054] 2. Drawing of standard curve

[0055] The mother solution (1 mg / mL ammonia nitrogen) was diluted to the following concentrations of standard: 0, 2, 4, 6, 8, 10 μg / mL. 20 μL of each concentration was taken into a 96-well microplate, 100 μL of phenol solution and 100 μL of sodium hypochlorite alkaline solution were added, mixed and placed at 25°C for 1 hour, and finally 20 μL of masking agent was added, and the absorbance was measured at 625 nm, and a standard curve was drawn based on the absorbance.

[0056] 3. After the sample to be tested is reasonably diluted with sterile water, the color developing reagent is added, the absorbance value is measured at 625nm, and the concentration of the sample to be tested is calculated using the drawn standard curve.

[0057] 5. Ammonia nitrogen degradation ability of Bacillus velez GY1

[0058] The culture fluid obtained by LB liquid culture was inoculated into M9 medium with nitrogen content of 200 mg / L and 1000 g / L at a ratio of 2% (v / v), with 3 parallels per group, cultured at 37°C for 48 hours, centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken. The supernatant was diluted according to the detection concentration range of 2-10 μg / mL, and the ammonia degradation rate of each bacterial sample was compared using the indophenol blue colorimetric method, with the 0 hour culture medium as the control.

[0059] Table 2 Ammonia nitrogen degradation ability of Bacillus velez GY1

[0060]

[0061] M9 inorganic salt medium: (NH4)2SO4 (0.2g or 1g), KH2PO4 3g, Na2HPO4 11.68g, NaCl 0.5g, add water to 1L, sterilize at 120℃ for 20min, add 250μL trace elements (FeCl3·6H2O 0.27g, ZnCl4·4H2O0.02g, CoCl2·6H2O 0.02g, Na2MoO4·2H2O 0.02g, CaCl2·2H2O 0.01g, CuCl26H2O 0.013g, H3BO30.005g, Concentrated HCl 1mL, add water to 100mL, filter), 1M CaCl2 100μL, 1M MgSO42mL, and glucose (according to the carbon-nitrogen ratio of 10:1).

[0062] The results are shown in Table 2. Bacillus Velez GY1 has a strong ability to degrade ammonia nitrogen.

[0063] VI. Preparation of ammonia-reducing and antibacterial preparations and their application in purifying aquaculture water and inhibiting pathogenic bacteria

[0064] 1. Preparation of microbial agents

[0065] Bacillus Velez GY1 was inoculated into LB medium and cultured at 37°C and 150 rpm for 12 h to obtain a microbial agent for reducing ammonia and inhibiting bacteria. The OD value of the microbial agent prepared in LB medium was measured by an enzyme marker. 600 The viable count can reach 1.8, and the viable count can reach 2×10 9 CFU / mL. The bacterial preparation prepared in M9 inorganic salt medium was detected by microplate reader OD 600 It can exceed 1.2, and the viable count calculated by the dilution plate method can reach 1×10 9CFU / mL. Bacillus Velezii has a fast growth rate and strong environmental adaptability. It is easy to form dominant bacteria when applied and thus exert its functions. It is suitable for application in various complex natural environments.

[0066] 2. Application of ammonia-reducing and antibacterial agents in purifying aquaculture wastewater

[0067] Inoculate 1 mL of Bacillus Velezii GY1 into 100 mL of LB medium, add 100 g of culture pellets, and culture at 37°C overnight. Discard the LB medium and place the culture pellets into the aquaculture circulating water system with a stocking density of 750 fish / m 3 Tilapia, the amount of bacteria added is about 10 5 CFU / L. The control group was 100g of culture pellets without bacterial agent. 10mL of water samples were collected every day. After 5 days of collection, the above method was used to detect the content of ammonium ions and nitrite. The results are shown in Table 3. After 5 days of treatment, the ammonium ion content in the aquaculture water decreased from 0.22mg / L to 0.1mg / L, a decrease of 53.3%, and the nitrite content decreased from 3.18mg / L to 1.44mg / L, a decrease of 54.7%.

[0068] Table 3 Ammonia reduction effect of Bacillus velez GY1

[0069]

[0070] The results showed that the ammonia-reducing microbial preparation Bacillus Velez GY1 had a good effect in controlling ammonia nitrogen and nitrite in water bodies.

[0071] 3. Antibacterial effect of antibacterial agents on aquatic disease bacteria

[0072] The activated GY1 was inoculated into LB and M9 culture media respectively, cultured at 37°C, 150 rpm for 48 h, and then centrifuged at 12000×g for 10 min. The obtained bacterial supernatant was added dropwise to a plate containing pathogenic Vibrio parahaemolyticus, Salmonella and Vibrio alginolyticus, and placed in a 37°C incubator to observe the inhibition zone of GY1 against the pathogens and the antibacterial effect.

[0073] Table 4 Inhibition zone diameter of Bacillus velez GY1 (cm)

[0074]

[0075] like Figure 2-4As shown in Table 4, the culture supernatants (M9 and LB) have the strongest antibacterial effect on Vibrio alginolyticus, and the antibacterial effect is stronger than kanamycin and ampicillin; followed by Vibrio parahaemolyticus, the antibacterial effect of the culture supernatant (M9) is stronger than kanamycin, the antibacterial effect of the culture supernatant (LB) is equivalent to kanamycin, and ampicillin has no antibacterial effect on Vibrio parahaemolyticus; the culture supernatants (M9 and LB) also have significant antibacterial effects on Salmonella, while kanamycin and ampicillin have no antibacterial effect on Salmonella.

[0076] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Bacillus velezensis GY1, characterized in that The bacteria is deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short), with the deposit number GDMCC No: 64792 and the deposit date June 28, 2024. The address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial preparation for degrading ammonia nitrogen, characterized in that: The method comprises the Bacillus Velez GY1 according to claim 1.

3. A microbial preparation for antagonizing pathogenic bacteria, characterized in that: The method comprises the Bacillus Velez GY1 and / or its metabolites as described in claim 1.

4. The microbial preparation for antagonizing pathogens according to claim 3, characterized in that: The pathogenic bacteria are Vibrio alginolyticus, Salmonella and Vibrio parahaemolyticus.

5. A method for preparing a microbial preparation, characterized in that: Bacillus Velez GY1 is inoculated into LB medium or M9 inorganic salt medium for a period of time to obtain a microbial preparation capable of degrading ammonia nitrogen and antagonizing pathogenic bacteria.

6. The method for preparing the microbial preparation according to claim 5, characterized in that: The culture temperature is 37°C, the rotation speed is 120-200 rpm, and the culture time is 48 hours.

7. Use of Bacillus Velez GY1, characterized in that: Used to degrade ammonia nitrogen and / or antagonize aquatic pathogens.

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