Bacillus licheniformis YHD2 and application thereof in degrading nitrite and inhibiting aquatic pathogenic bacteria

Abstract

The invention discloses bacillus licheniformis YHD2 and application thereof in degrading nitrite and inhibiting aquatic pathogenic bacteria, and belongs to the technical field of microorganisms. The preservation number of the bacillus licheniformis YHD2 is CCTCC NO: M 20251259, and the bacterial colony of the bacillus licheniformis YHD2 is milk white, opaque, irregular in edge and rough in surface; the viable count of the prepared bacterial powder is at least 1 * 10 < 9 > CFU / g, and the bacillus licheniformis YHD2 has the capability of rapidly degrading nitrite and the capability of inhibiting aquatic pathogenic bacteria. Experiments prove that the novel bacillus licheniformis YHD2 has important significance on development of aquaculture water quality purification and preparation of a bacteriostatic agent for aquatic pathogenic bacteria, the application range of the bacillus licheniformis YHD2 is further widened, and the bacillus licheniformis YHD2 has wide economic value and development prospects in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus licheniformis YHD2 and its application in degrading nitrite and inhibiting aquatic pathogens. Background Technology

[0002] With the development of aquaculture, various farming models currently suffer from problems such as excessively high stocking density and large feeding amounts, leading to excessive levels of harmful substances such as ammonia nitrogen and nitrite. This not only directly affects the health of farmed animals, but also results in pollutant concentrations in the wastewater discharged from these farms being far higher than those in natural water bodies, threatening the ecological balance of the natural environment.

[0003] Vibrio, a common aquatic animal pathogen in aquaculture, is characterized by its strong survival ability, rapid infection and disease progression, and high mortality rate. Furthermore, its high residual rate in aquaculture water means that outbreaks can severely impact the aquaculture industry. Currently, the industry primarily treats Vibrio in water through chemical disinfection or by adding antibiotics to feed. However, disinfection methods indiscriminately disinfect beneficial bacteria, disrupting the microecological balance; the extensive use of antibiotics easily leads to bacterial resistance, and the government has already banned the use of many antibiotics in the aquaculture industry.

[0004] Bacillus species are widely used in aquaculture for water quality improvement and inhibiting harmful bacteria due to their strong ability to decompose organic matter, high environmental tolerance, and the antibacterial properties of some varieties. Among them, Bacillus licheniformis (… Bacillus licheniformis Bacillus licheniformis can efficiently decompose organic matter such as uneaten feed and feces in water by secreting functional enzymes such as proteases and amylases, reducing pollution caused by the decay of organic matter. At the same time, some strains have good antibacterial effects against aquatic pathogens and can be used to develop antibiotic alternatives. Therefore, discovering new multifunctional Bacillus licheniformis strains, combined with their denitrification and antibacterial properties, is an important direction for the green and healthy development of the aquaculture industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a strain of Bacillus licheniformis YHD2 and its application in degrading nitrite and inhibiting aquatic pathogens. The Bacillus licheniformis YHD2 can efficiently degrade nitrite, reduce pollution from organic matter decay, and inhibit the growth of Vibrio in aquaculture. It has a good antibacterial effect on aquatic pathogens and can be used to develop antibiotic alternatives. It is of great significance for developing the comprehensive value of the strain.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a strain of Bacillus licheniformis YHD2, which is classified and named as follows: Bacillus licheniformis, and is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M 20251259.

[0007] Further, the colony of the Bacillus licheniformis YHD2 is milky white, opaque, with irregular edge and rough surface.

[0008] The Bacillus licheniformis YHD2 Bacillus licheniformis The 16S rDNA sequence of the Bacillus licheniformis YHD2 is shown in SEQ NO. 1.

[0009] The present application also provides a bacterial agent prepared from the Bacillus licheniformis YHD2, which comprises bacterial powder, and the viable bacterial count of the bacterial powder is at least 1×10 9 CFU / g.

[0010] Further, the preparation method of the bacterial powder is as follows: (1) inoculating the Bacillus licheniformis YHD2 into LB culture medium for activation culture to prepare seed liquid; (2) inoculating the seed liquid into a fermentation tank at an inoculation amount of 2-10%, and performing constant temperature culture, centrifuging the fermentation bacterial liquid, discarding the supernatant, washing and resuspending the bacterial sludge precipitate with sterile water, adding heat-resistant protective agent thereto and mixing uniformly, and then performing spray drying to obtain the Bacillus licheniformis YHD2 bacterial powder.

[0011] Further, the components and content of the heat-resistant protective agent are as follows: 10-15 g / L skimmed milk powder, 10-15 g / L malt dextrin and 10-15 g / L glucose.

[0012] Further, the preparation method of the Bacillus licheniformis YHD2 bacterial powder is as follows: inoculating the Bacillus licheniformis YHD2 into LB culture medium for activation culture for 24 h to prepare seed liquid, inoculating the seed liquid into a fermentation tank at an inoculation amount of 5%, and performing constant temperature culture at 37℃ and 120 r / min for 26 h; centrifuging the fermentation bacterial liquid at 4000 r / min for 15 min, discarding the supernatant, washing and resuspending the bacterial sludge precipitate with sterile water, adding heat-resistant protective agent thereto, and mixing uniformly, wherein the heat-resistant protective agent specifically comprises 10 g / L skimmed milk powder, 10 g / L malt dextrin and 10 g / L glucose, and then performing spray drying to obtain the Bacillus licheniformis YHD2 bacterial powder.

[0013] The present application also provides application of the Bacillus licheniformis YHD2 and / or the bacterial agent in degradation of nitrite.

[0014] Further, when the bacterial agent is bacterial powder, the bacterial powder is diluted 80-120 times with sterile water, and the diluted bacterial liquid is added into the aquatic environment containing 0.1-1 g / L nitrite at an addition amount of 2-8%.

[0015] The application also provides application of the bacillus licheniformis YHD2 and / or the microbial agent in inhibiting aquatic pathogenic bacteria.

[0016] Further, the aquatic pathogenic bacteria include Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0017] Compared with the prior art, the application has the following beneficial effects: The application screens a bacillus licheniformis YHD2 with nitrite degradation effect from a breeding pond sediment, the strain can reduce waste nitrogen emission and has antagonism to common vibrio, and can be used for developing functional microecological preparation in the aquaculture industry, the application mines the function of the bacillus licheniformis YHD2, and experiment verifies that the new bacillus licheniformis YHD2 with multifunction has important significance for the development of water quality purification in aquaculture and the preparation of antibacterial agents for aquatic pathogenic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the 24h and 48h degradation rate of 0.2g / L NaNO2 for 5 strains; Figure 2 is the colony characteristics of the bacillus licheniformis YHD2; Figure 3 is the phylogenetic tree of the bacillus licheniformis YHD2 based on 16S rDNA gene sequences; Figure 4 is the bacillus licheniformis YHD2 powder; Figure 5 is the inhibition effect of the bacillus licheniformis YHD2 on different vibrio. DETAILED DESCRIPTION

[0019] In order to further illustrate the technical means adopted by the application and its effects, the application will be further described below in combination with technical schemes, drawings and application examples. It can be understood that the specific embodiments described herein are only used to explain the application, and not to limit the application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between any document incorporated by reference and the present specification, the present specification will control.

[0021] Example 1: Screening, identification and preservation of the strain (1) Strain enrichment The black mud from the bottom of the pond was collected using a mud sampler and placed in a sterile reagent bottle, sealed and stored at low temperature. The sample was transported back to the laboratory for strain enrichment. Under sterile conditions, 5 g of the sample was weighed into 95 mL of sterile water and shaken at 37°C and 200 rpm for 30 min. 5 mL of the mixed solution was added to 95 mL of liquid tryptone soy broth (purchased from HIBIO) and incubated at 37°C and 200 rpm for 24 h. The previous enrichment culture was inoculated into the liquid tryptone soy broth at a 5% inoculation rate and incubated for 3 consecutive times.

[0022] (2) Isolation and purification The enrichment culture was diluted with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 8 gradients, respectively. 100 μL of bacterial suspension was taken from 10 -6 , 10 -7 , 10 -8 3 gradients and spread on solid tryptone soy broth, and incubated at 37°C for 24-48 h. Three parallel experiments were set up for each group. Single colonies with spore-producing ability of different morphologies were selected from the plates and streaked on new solid tryptone soy broth plates for 3-5 times until pure Bacillus strains with consistent morphology were obtained.

[0023] (3) Strain screening The purified Bacillus strains were inoculated into LB medium and incubated at 37°C and 200 rpm for 24 h. The activated bacterial solution was inoculated into the screening medium containing 0.2 g / L NaNO2 (purchased from MACKLIN) at a 5% inoculation rate, and the control group did not contain the strain. The culture was incubated at 37°C and 200 rpm for 48 h. Samples were taken every 24 h during the incubation period, and the nitrite concentration of different Bacillus strains was determined by the naphthyl ethylenediamine hydrochloride method (reference GB / T7493-1987 Determination of Nitrite Nitrogen in Water). The nitrite degradation rate was calculated. Several strains capable of degrading nitrite were isolated (see Figure 1 ), and one of the strains with strong ability, C3, was selected for further study. The colony morphology of this strain is shown in Figure 2 , which appeared as a milky white, opaque, irregular edge, and rough surface on LB medium.

[0024] Screening medium: Proteose peptone 10 g, NaCl 10 g, yeast extract 5 g, purified water 1 L, sterilized at 121 ℃ for 30 min. After cooling, 0.2 g of NaNO2 was added.

[0025] (4) Strain identification The purified Bacillus strain C3 was inoculated in LB liquid medium for activation culture, and bacterial genomic DNA extraction kit (Beijing Tiangeng) was used to extract bacterial DNA, and 16S rDNA sequence amplification was performed. The primers used were bacterial universal primers with sequences 27F: 5'-AGAGTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system (25 μL) contained PCR Mixture 21 μL, upstream and downstream primers 1 μL each, and DNA template 2 μL. The PCR amplification program was set as 96 ℃ pre-denaturation for 5 min, 96 ℃ denaturation for 20 s, 57 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 10 min. The PCR product of strain C3 was sent to Qingdao Plushang Biotechnology Co., Ltd. for sequence determination. The 16S rDNA sequence of strain C3 is shown in SEQ ID NO. 1.

[0026] After BLAST comparison of the sequence results in the NCBI database, it was found that strain C3 had the highest sequence similarity of 100% with Bacillus licheniformis (NCBI number: MF871790) in the genus Bacillus (NCBI number: MF871790). To further determine the phylogenetic relationship of YH033, the software MEGA 12.0 was used to construct a phylogenetic tree (Neighbor-Joining method) for the screening strains, with Bootstrap set to 1000. It was found that strain C3 was clustered with Bacillus licheniformis (NCBI number: MF871790), indicating that strain C3 had the closest phylogenetic relationship with Bacillus licheniformis (NCBI number: MF871790), so the strain was determined to be Bacillus licheniformis, and the C3 strain was named Bacillus licheniformis YHD2, with the classification name Bacillus Bacillus licheniformis Figure 3 Bacillus licheniformis G Bacillus licheniformis Bacillus licheniformis

[0027] (5) Strain preservation The screened Bacillus licheniformis YHD2 strain was preserved, and the preservation unit was China Center for Type Culture Collection; address: Wuhan, China. Wuhan University; preservation date: June 4, 2025; Bacillus licheniformis YHD2 Bacillus licheniforis ​​​​​​The accession number of YHD2 is CCTCC NO: M 20251259.

[0028] Example 2: Preparation of Bacillus licheniformis YHD2 powder Bacillus licheniformis YHD2 frozen in a glycerol tube was inoculated in LB medium and activated for 24 h to prepare a seed solution. The seed solution was inoculated in a fermenter at an inoculation amount of 5%, and incubated at 37°C and 120 r / min for 26 h. The fermentation broth was centrifuged at 4000 r / min for 15 min, and the supernatant was discarded. The bacterial slurry was washed with sterile water and resuspended. Anti-heat protective agents (10 g / L skimmed milk powder, 10 g / L malt dextrin, and 10 g / L glucose) were added and mixed uniformly, followed by spray drying to obtain Bacillus licheniformis YHD2 powder. Figure 4 1.00 g of Bacillus licheniformis YHD2 powder collected by spray drying was accurately weighed, added with sterile water to make a total volume of 10 mL, and viable cell count was performed by dilution plating method. It was found that the viable cell count of Bacillus licheniformis YHD2 powder was 1.12 x 10 9 CFU / g.

[0029] Example 3: Nitrite degradation ability of Bacillus licheniformis YHD2 Bacillus licheniformis YHD2 powder was diluted 100 times with sterile water, and the diluted bacterial solution was inoculated into a screening medium containing 0.5 g / L NaNO2 at an inoculation amount of 5%, and incubated at 37°C and 200 rpm for 48 h. Samples were taken every 24 h during the incubation, and the nitrite concentration of the sample bacterial solution was determined by the naphthalene ethylenediamine hydrochloride method.

[0030] Table 1: Nitrite degradation ability of Bacillus licheniformis YHD2

[0031] As shown in Table 1, Bacillus licheniformis YHD2 has strong nitrite degradation ability.

[0032] Example 4: Inhibition test of Bacillus licheniformis YHD2 on common Vibrio Bacillus licheniformis YHD2 powder was diluted 10 times, 100 times, 500 times, and 1000 times with sterile water, respectively, and the in vitro bacteriostatic effect of the above dilutions on Vibrio parahaemolyticus and Vibrio alginolyticus was determined by the Oxford cup method. The inhibition zones were observed and the diameters were measured. (The Vibrio parahaemolyticus and Vibrio alginolyticus involved in this example were obtained by commercial channels) Table 2: Bacteriostatic ability of Bacillus licheniformis YHD2

[0033] As shown in Table 2, Bacillus licheniformis YHD2 has strong bacteriostatic ability. Figure 5As shown in Table 2, the bacillus licheniformis YHD2 has strong bacteriostatic ability to the two common vibrio.

[0034] The above examples only illustrate the technical solutions of the present application, but do not limit the present application; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features thereof can be replaced by equivalent replacements; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A strain of Bacillus licheniformis YHD2, characterized in that, The classification name is Bacillus licheniformis , and the preservation number is CCTCC NO: M 20251259.

2. The Bacillus licheniformis YHD2 according to claim 1, characterized in that, The colony of the Bacillus licheniformis YHD2 is milky white, opaque, with irregular edge and rough surface.

3. The bacterial agent prepared from Bacillus licheniformis YHD2 according to claim 1, characterized in that, The bacterial agent includes bacterial powder, the viable bacterial count of the bacterial powder is at least 1 x 10 9 CFU / g.

4. The bacterial agent of claim 3, characterized in that, The preparation method of the bacterial powder is as follows: (1) inoculating the Bacillus licheniformis YHD2 of claim 1 into LB culture medium for activation culture to prepare seed liquid; (2) inoculating the seed liquid into a fermenter at an inoculation amount of 2-10%, and culturing at constant temperature, centrifuging the fermentation bacterial liquid, discarding supernatant, washing and resuspending the bacterial sludge with sterile water, adding heat-resistant protective agent thereto and mixing uniformly, and then performing spray drying to obtain the Bacillus licheniformis YHD2 bacterial powder.

5. The bacterial powder according to claim 3, characterized in that, The components and content of the heat-resistant protective agent are as follows: 10-15 g / L skimmed milk powder, 10-15 g / L malt dextrin and 10-15 g / L glucose.

6. Application of the Bacillus licheniformis YHD2 of claim 1 and / or the bacterial agent of claim 3 in degrading nitrite.

7. Use according to claim 6, characterized in that, When the bacterial agent is bacterial powder, it is diluted 80-120 times with sterile water, and the diluted bacterial liquid is added into the water product environment containing 0.1-1 g / L nitrite at an addition amount of 2-8% based on the volume of the water body containing nitrite.

8. Application of the Bacillus licheniformis YHD2 of claim 1 and / or the bacterial agent of claim 3 in inhibiting aquatic pathogenic bacteria.

9. Use according to claim 8, characterized in that, The aquatic pathogenic bacteria include Vibrio parahaemolyticus and / or Vibrio alginolyticus.

Images