Bacillus licheniformis and application thereof

Abstract

The present invention provides a Bacillus licheniformis AQ19-11, which is non-toxic to water bodies and can hydrolyze starch in water bodies to reduce the ammonia nitrogen content in water bodies; the Bacillus licheniformis AQ19-11 of the present invention can be used as a probiotic to improve water quality, for example, for water environment management, to improve the water quality of natural water bodies, or for aquaculture, to improve the water quality of aquaculture water bodies.

Description

Technical Field

[0001] The invention relates to a bacillus licheniformis and application of the bacillus licheniformis as a probiotic in improving water quality, in particular to application of the bacillus licheniformis in improving aquaculture water. Background Art

[0002] With the environmental protection and the ecological development of agriculture, forestry, animal husbandry and fishery, people try to develop pollution-free and harmless microbial products to meet the requirements of maintaining ecological balance and rationally transforming nature.

[0003] Although the mechanisms of action of microorganisms are diverse, the basic consensus that has been revealed and reached so far is that the functions of microorganisms are not single, but often have multiple functions at the same time, such as improving animal / plant nutrition, stimulating growth, inhibiting pathogens, preventing and controlling pests and diseases, improving water quality, etc.

[0004] Specifically in the field of aquaculture, intensive high-density farming is currently the main aquaculture model in my country, but this model often causes the accumulation of residual bait due to excessive feeding, which in turn leads to deterioration of water quality. Among them, the residual starch in the residual bait is the main water pollutant. Therefore, timely hydrolysis of residual bait (especially starch-based organic pollutants) is an important way to improve the water quality of aquaculture water bodies.

[0005] Therefore, it is desirable in the art to be able to screen for microorganisms that improve water quality, especially water quality in aquaculture environments. Summary of the invention

[0006] In view of the above-mentioned problems and / or other problems of the related art, the first aspect of the present invention provides a Bacillus licheniformis AQ19-11, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: M 2022012.

[0007] The second aspect of the present invention provides a microbial agent, wherein the microbial agent is the fermentation broth of the above-mentioned Bacillus licheniformis AQ19-11, or a bacterial liquid preparation prepared from the fermentation broth, or a bacterial powder preparation prepared by drying the fermentation broth.

[0008] The third aspect of the present invention provides the above-mentioned Bacillus licheniformis AQ19-11, which is used as a probiotic in improving water quality.

[0009] Preferably, the application is to improve aquaculture water.

[0010] The fourth aspect of the present invention provides the above-mentioned microbial agent, which is used as a probiotic to improve water quality.

[0011] Preferably, the application is to improve aquaculture water.

[0012] A fifth aspect of the present invention provides a product for improving water quality, wherein the product for improving water quality comprises the above-mentioned Bacillus licheniformis AQ19-11 or comprises the above-mentioned microbial agent.

[0013] The present invention provides a Bacillus licheniformis AQ19-11, which is non-toxic to water bodies and can hydrolyze starch in water bodies to reduce the ammonia nitrogen content in water bodies; the Bacillus licheniformis AQ19-11 of the present invention can be used as a probiotic to improve water quality, for example, for water environment management, to improve the water quality of natural water bodies, or for aquaculture, to improve the water quality of aquaculture water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a bacterial morphology of Bacillus licheniformis AQ19-11 of the present invention after staining under a 100-fold optical microscope (oil lens);

[0015] Figure 2 This is a colony morphology diagram of Bacillus licheniformis AQ19-11 formed on a nutrient agar plate;

[0016] Figure 3 The results of the amylase hydrolysis test of the Bacillus licheniformis AQ19-11 of the present invention and various control bacteria are shown in FIG.

[0017] Figure 4 This is a result diagram showing the effect of the Bacillus licheniformis AQ19-11 of the present invention on the ammonia nitrogen content in aquaculture water. DETAILED DESCRIPTION

[0018] The present invention is further described below through specific implementation modes, but the present invention is not limited to these specific implementation modes.

[0019] Unless otherwise specified, the materials, reagents, etc. used in the following specific embodiments can be obtained from commercial sources. Where no specific techniques or conditions are specified, the techniques or conditions described in the literature in the field (e.g., refer to the third edition of Molecular Cloning Experiment Guide by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or the product instructions are used.

[0020] 1. The following is an introduction to the operation process of obtaining Bacillus licheniformis AQ19-11 (Bacillus licheniformis AQ19-11) CCTCC No: M 2022012 of the present invention by separation and screening from the natural environment by the inventors of the present application.

[0021] 1) Preliminary screening: Weigh 1.0 g of aquaculture sludge (from Haifeng Aquaculture Co., Ltd., Changyi City, Shandong Province), suspend it in 9 mL of sterile water, shake and mix well, and place it in a water bath at 80°C for 10 min. Then, use the dilution plate method and streak plate method to separate and purify the strains on nutrient agar medium. Aseptically select single colonies with good growth as candidate strains, inoculate them into sterile nutrient broth, and culture them on a shaker at 30°C and 200 r / min for 18 to 24 hours to make a final concentration of 1.0×10 6 CFU / mL of bacterial suspension;

[0022] 2) Further screening was performed using amylase production ability as a screening evaluation index; 5 μL of the bacterial suspension of the candidate strains was aseptically pipetted onto a sterile circular filter paper, and then pasted onto an amylase hydrolysis medium plate. After culturing at 30°C for 24 hours, a layer of iodine solution was covered on the surface, and the diameter of the amylase hydrolysis circle produced by each candidate strain was observed and measured.

[0023] The amylase-producing strain (Bacillus subtilis BYK201031) already in the inventor's laboratory was used as a control strain, and the strain with the largest diameter of the amylase hydrolysis circle among the candidate strains was selected as the excellent strain with amylase production ability obtained by screening in the present invention, and was named AQ19-11.

[0024] 2. The identification process of Bacillus licheniformis AQ19-11 of the present invention is as follows:

[0025] (1) Molecular biological identification

[0026] Based on the bacterial 16S rRNA sequence, phylogenetic analysis was used to perform molecular biological identification of the screened strain AQ19-11.

[0027] Specifically, the strain AQ19-11 obtained by screening was inoculated into 100 mL of sterile nutrient broth, and after shaking culture at 30°C and 200 r / min for 24 hours, it was centrifuged at 4°C and 12000 r / min for 1 min, the supernatant was discarded, and then the genomic DNA of the strain was extracted using the Ezup column genomic DNA extraction kit; then, the genomic DNA was used as a template to perform PCR amplification on the DNA corresponding to its 16S rRNA. Among them, the forward primer was 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, and the reverse primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′; the PCR reaction system was 50.0 μL: 10×Easy taq Buffer (Mg 2+) 5.0μL, High Purified NTP 4.0μL, forward and reverse primers 0.5μL each, Easy Taq enzyme 1.0μL, double distilled water to 50.0μL; PCR amplification conditions: 95℃ pre-denaturation 3min; 95℃1min, 60℃1min, 72℃1min, 35 cycles in total. The amplified fragments were detected by 1.0% agarose gel electrophoresis, and the DNA purification and recovery kit of Shanghai Tiangen Biochemical Technology Co., Ltd. was used for gel excision and recovery; the recovered samples were sent to Shanghai Maipu Biotechnology Co., Ltd. for sequencing.

[0028] The gene sequence corresponding to the 16S rRNA of AQ19-11 was determined (as shown in SEQ ID NO.1).

[0029] The 16S rRNA sequences of strain AQ19-11 were aligned for homology using NCBI BLAST in the GenBank database, and a phylogenetic tree was constructed using the neighbor-joining method using Mega 5.0 software (Replications = 1000, bootstrap value in percentage). From the alignment results, strain AQ19-11 naturally clustered with Bacillus licheniformis strains in the database (97% to 99% sequence homology), and was most closely related to the Bacillus licheniformis strain with GenBank accession number MZ959528.

[0030] (2) Morphological identification

[0031] Strain AQ19-11 is a Gram-positive bacterium. Its bacterial morphology under a 100x optical microscope (oil objective) after staining is shown in Figure 1 ; The colony morphology formed on the nutrient agar plate, see Figure 2 , Colony morphological characteristics: white, round, with irregular edges; strain AQ19-11 is morphologically similar to the common rod-shaped Bacillus licheniformis.

[0032] (3) Physiological and biochemical identification

[0033] Physiological and biochemical test traits were selected with reference to the relevant contents of identification of corresponding genera and species in the Manual of Systematic Identification of Common Bacteria (first edition) compiled by Dong Xiuzhu et al., and bacterial microbiochemical identification tubes were used to conduct physiological and biochemical identification of strain AQ19-11.

[0034] From the physiological and biochemical results, strain AQ19-11 can hydrolyze starch and gelatin, reduce nitrate, and is positive for catalase reaction, VP, and nitrate reduction reaction. It can ferment glucose, rhamnose, sorbitol, D-salicin and arabinose, and can hydrolyze gelatin and starch. It cannot ferment mannitol, xylose and D-raffinose, cannot utilize malonate, does not produce urease and hydrogen sulfide, and can survive in an environment of 20-100g / LNaCl. These characteristics are basically consistent with the physiological and biochemical characteristics of Bacillus licheniformis in the Manual of Systematic Identification of Common Bacteria.

[0035] Based on the identification results of parts (1) to (3), the strain AQ19-11 obtained by screening in the present invention was finally determined to be Bacillus licheniformis.

[0036] III. Preservation of strains

[0037] The strain AQ19-11 of the present invention belongs to Bacillus licheniformis, has been deposited in the China Center for Type Culture Collection, the preservation date is January 5, 2022, its preservation number is CCTCC No: M2022012, and the preservation unit address is Wuhan University, Wuhan, China.

[0038] 4. Fermentation and culture method of Bacillus licheniformis AQ19-11 of the present invention

[0039] Aseptically scrape the bacterial moss of Bacillus licheniformis AQ19-11 preserved on the slant of the present invention and inoculate it into 20mL fermentation medium (commercially available conventional nutrient broth medium can be used, such as the Wokai brand nutrient broth purchased from Sinopharm Group (Shanghai) Chemical Reagent Co., Ltd.; or the fermentation medium with patent number ZL 201110346583.X can be used), and culture at 30°C, 200r / min constant temperature shaker for 48 hours until the medium becomes turbid; then transfer it to 100mL fermentation medium, culture at 30°C, 200r / min constant temperature shaker for 48 hours until turbidity appears, and then transfer it to 400mL fermentation medium, and culture at 30°C, 200r / min constant temperature shaker for 72 hours to obtain the fermentation liquid of Bacillus licheniformis AQ19-11 of the present invention. After testing, the bacterial concentration of the fermentation liquid is about 10 8 ~10 9 CFU / ml.

[0040] Regarding the bacterial agent of Bacillus licheniformis AQ19-11 of the present invention, the fermentation liquid obtained by fermentation production can be directly used or the fermentation liquid can be processed to make a bacterial liquid preparation for use, or the obtained fermentation liquid can be dried, such as microencapsulated or freeze-dried, to make a bacterial powder preparation for use.

[0041] 5. Safety of Bacillus licheniformis AQ19-11 of the present invention

[0042] The safety of the Bacillus licheniformis AQ19-11 of the present invention was evaluated using zebrafish, an aquatic model animal, as a test subject.

[0043] The experiment set up 1 control group and 4 experimental groups, each with 3 parallel groups. 3 Ten healthy zebrafish were placed in a glass aquarium (22 cm×18 cm×25 cm) filled with standard dilution water. The Bacillus licheniformis AQ19-11 of the present invention was added to the four experimental groups to a final concentration of 5.0×10 6 , 5.0×10 7 , 5.0×10 8 , 5.0×10 9 CFU / mL, and the control group did not add any substance. During the experiment, the water temperature was controlled at 23℃, DO>5mg / L, and pH was 7.2; the zebrafish were immersed for 7 days continuously, and the number of deaths of zebrafish in each group was recorded. The Bliss method was used to calculate the median lethal concentration (LC50) of zebrafish. 50 ). The results are shown in Table 1 below.

[0044] Table 1

[0045]

[0046]

[0047] The results of the four experimental groups showed that when the final concentration of strain AQ19-11 was 5.0×10 6 ~5.0×10 9 CFU / mL in an aquaculture environment for 7 consecutive days, the zebrafish grew well, had normal swimming and breathing abilities, and did not show abnormal phenomena such as flipping over, slow reaction, or death. The zebrafish in the control group also grew well, with no death.

[0048] This indicates that the Bacillus licheniformis AQ19-11 of the present invention has an effect on the LC of zebrafish. 50 >5.0×10 9 CFU / mL. According to Cutting (2010)'s evaluation criteria for strain safety: LC 50 >4.7×10 8 CFU / g is a non-toxic strain, and it can be concluded that the Bacillus licheniformis AQ19-11 of the present invention is a non-toxic strain and has no toxicity to the aquaculture environment.

[0049] VI. Amylase hydrolysis test of Bacillus licheniformis AQ19-11 of the present invention

[0050] Known amylase-producing bacteria: Bacillus subtilis BYK201031, Bacillus amyloliquefaciens AQ19-3, Bacillus pumilus AQ-8, Bacillus cereus AQ19-15 (provided by the National Aquatic Animal Pathogen Bank) were used as control bacteria.

[0051] 5 μL of the final concentration of 1.0×10 6 CFU / mL of Bacillus licheniformis AQ19-11 and each control strain were placed on a sterile circular filter paper, then pasted on an amylase hydrolysis medium plate, and cultured at 30°C for 24 hours. A layer of iodine solution was covered on the surface, and the diameter (mm) of the amylase hydrolysis zone produced by each strain was observed and measured.

[0052] Results Figure 3 It can be seen that the diameter of the hydrolysis circle of amylase produced by Bacillus licheniformis AQ19-11 increased by 28.93% (P<0.05), 35.65% (P<0.05), 22.83% (P<0.05), and 51.46% (P<0.05) respectively compared with the known amylase producing bacteria: Bacillus subtilis BYK201031, Bacillus amyloliquefaciens AQ19-3, Bacillus pumilus AQ-8, and Bacillus cereus AQ19-15, indicating that Bacillus licheniformis AQ19-11 of the present invention has good ability to produce amylase and hydrolyze starch.

[0053] VII. Effect of Bacillus licheniformis AQ19-11 of the present invention on ammonia nitrogen in aquaculture water

[0054] Before the experiment, the Bacillus licheniformis AQ19-11 of the present invention was inoculated into the fermentation medium and fermented at 32°C for 48 hours to obtain a bacterial concentration of about 5.0×10 8 CFU / mL of liquid bacterial agent.

[0055] The experiment set up 1 control group and 1 experimental group. Each group included 2 whiteleg shrimp breeding ponds (8 mu / pond, water depth 1.5m). The stocking density of whiteleg shrimp seedlings in each breeding pond was 50,000 / mu.

[0056] The experimental group used the above-mentioned liquid bacterial agent of Bacillus licheniformis AQ19-11, and the control group used EM bacterial agent (Hongbao EM liquid purchased from Wuxi Hongbao Microbiological Technology Co., Ltd.); both the experimental group and the control group were treated with 1.0 g / m 3The dosage was evenly sprinkled throughout the pond, once every 10 days, and the test period was 120 days. During the test, each group was fed with normal white shrimp feed 3 times / day. The water was not changed during the culture period. The oxygenation time was flexibly adjusted every day to ensure that the dissolved oxygen was greater than 5 mg / L (the oxygenation time of each group was kept consistent). Water samples were taken every 10 days (at the same time point, 3 different areas of each culture pond were sampled), and the ammonia nitrogen content (mg / L) in the water samples was determined using the ammonia nitrogen detection kit produced by Hangzhou Luheng Biotechnology Co., Ltd., and the average ammonia nitrogen content in the culture ponds of the control group and the test group at each time point was calculated. The results are shown in Figure 4 .

[0057] from Figure 4 It can be seen from the results that during the entire test period, the use of Bacillus licheniformis AQ19-11 of the present invention can better control the ammonia nitrogen content in the aquaculture water, which is significantly better than the EM bacterial agent in the control group; at the end of 120 days of the aquaculture test, the ammonia nitrogen content of the test pool where the strain AQ19-11 was sprayed throughout the pool was reduced by 42.11% compared with the EM bacterial agent in the control group.

[0058] In addition, based on the characteristics of Bacillus licheniformis itself and the physiological and biochemical identification results of subsection (3) of the above-mentioned Part II, a person skilled in the art, after knowing the results of the above-mentioned Part V (proving that the Bacillus licheniformis AQ19-11 of the present invention is safe and non-toxic to aquaculture water bodies), the above-mentioned Part II, Part VI (proving the ability of strain AQ19-11 to hydrolyze starch), and the above-mentioned Part VII (proving the ability of strain AQ19-11 to reduce the ammonia nitrogen content in aquaculture water bodies), can conceive of applying the Bacillus licheniformis AQ19-11 of the present invention to improve water quality, for example, for water environment management, to improve the water quality of natural water bodies, or for aquaculture, to improve the water quality of aquaculture water bodies.

[0059] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0060] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus licheniformis ( Bacillus licheniformis ) AQ19-11, the Bacillus licheniformis ( Bacillus licheniformis ) AQ19-11 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M2022012, the deposit date is January 05, 2022, and the depository address is Wuhan University, Wuhan, China.

2. A microbial agent, characterized in that: The microbial agent is the fermentation liquid of Bacillus licheniformis AQ19-11 according to claim 1, or a liquid preparation made from the fermentation liquid, or a powder preparation made by drying the fermentation liquid.

3. The use of Bacillus licheniformis AQ19-11 as claimed in claim 1 as a probiotic in improving aquaculture water, characterized in that: The Bacillus licheniformis AQ19-11 can hydrolyze starch in water and reduce the ammonia nitrogen content in the water.

4. The use of the microbial agent as a probiotic in improving aquaculture water as claimed in claim 2, characterized in that: The Bacillus licheniformis AQ19-11 can hydrolyze starch in water and reduce the ammonia nitrogen content in the water.

5. A product for improving water quality, characterized in that: The product for improving water quality comprises the Bacillus licheniformis AQ19-11 as claimed in claim 1 or comprises the microbial agent as claimed in claim 2.

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