Bacillus subtilis and application thereof

Abstract

The invention discloses a bacillus subtilis and application thereof, and particularly provides the bacillus subtilis and a bacillus subtilis culture supernatant obtained through the bacillus subtilis, the bacillus subtilis is classified and named as Bacillus subtilis SD24, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO.M20242253, and the preservation time is October 21, 2024. Experiments prove that when the bacillus subtilis and the culture supernatant thereof are used in aquaculture, reproduction of harmful bacteria (aquatic pathogenic vibrio) can be well inhibited, so that the water quality is improved. Experimental results also prove that the bacillus subtilis culture supernatant still has good antibacterial stability after being placed for 30 days, and is convenient to store.

Description

Technical Field

[0001] The invention belongs to the technical field of Bacillus subtilis, and particularly relates to Bacillus subtilis and application thereof. Background Art

[0002] Bacillus subtilis is a type of aerobic, endogenous, stress-resistant Gram-positive rod-shaped bacteria. Bacillus subtilis is widely found in soil, lakes, oceans, and on the surface of animals and plants. Bacillus subtilis is a probiotic that produces a large amount of active substances during its growth, such as subtilisin, polymyxin, nystatin, gramicidin, etc., which can effectively inhibit the growth and reproduction of pathogenic bacteria; subtilisin, polymyxin, nystatin, gramicidin and other active substances produced during the growth of Bacillus subtilis have a significant inhibitory effect on pathogenic bacteria or conditional pathogens of endogenous infection. Bacillus subtilis rapidly consumes free oxygen in the intestine, causing intestinal hypoxia and promoting the growth of beneficial anaerobic bacteria; Bacillus subtilis has a strong inhibitory effect on harmful microorganisms such as Vibrio, Escherichia coli and baculovirus in aquatic products.

[0003] Since different strains of Bacillus subtilis have different performances, it is of great significance to screen and obtain a strain of Bacillus subtilis that has excellent inhibitory effect on aquatic pathogenic Vibrio. Summary of the invention

[0004] In view of the technical problems in the background technology, the object of the present invention is to provide a Bacillus subtilis and applications thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a Bacillus subtilis, the classification name of the Bacillus subtilis is Bacillus subtilis SD24, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO.M 20242253, and the preservation time is October 21, 2024.

[0007] The second aspect of the present invention provides the use of the above-mentioned Bacillus subtilis in aquaculture.

[0008] The third aspect of the present invention provides a method for obtaining a Bacillus subtilis culture supernatant by the above-mentioned Bacillus subtilis, comprising the following steps: inoculating Bacillus subtilis SD24 (i.e., Bacillus subtilis SD24 strain) into a liquid culture medium, culturing at 25-28° C. and shaking on a shaker at 250-300 rpm / min, centrifuging, removing the bacteria, and collecting the Bacillus subtilis culture supernatant.

[0009] Preferably, the liquid culture medium is selected from any one of LB medium, YEB medium and BHI medium, and more preferably YEB medium.

[0010] Preferably, the shaking culture time is 1 to 4 days, more preferably 2 days.

[0011] Preferably, the centrifugal speed is 5500-6500 rpm / min, and the centrifugal time is 3-10 min.

[0012] The fourth aspect of the present invention provides the Bacillus subtilis culture supernatant obtained by the above method.

[0013] A fifth aspect of the present invention provides the use of the above-mentioned Bacillus subtilis culture supernatant in aquaculture.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a Bacillus subtilis and a culture supernatant thereof with excellent antibacterial effect, which can be used in aquaculture to effectively inhibit the reproduction of harmful bacteria (aquatic pathogenic Vibrio: Vibrio harveyi and Vibrio fluvii) in aquaculture and improve water quality. In addition, experimental results show that the culture supernatant of Bacillus subtilis provided by the present invention still has good antibacterial stability after being placed for 30 days, and is easy to store.

[0016] Collection Information

[0017] Deposit date: October 21, 2024;

[0018] Depository: China Center for Type Culture Collection;

[0019] Deposit number: CCTCC NO.M 20242253;

[0020] Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University;

[0021] Postal code: 430072;

[0022] Taxonomic nomenclature: Bacillus subtilis SD24. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a diagram showing the screening results of aquatic Vibrio inhibitory strains;

[0025] Figure 2 This is a graph showing the results of the sterile supernatant of Bacillus subtilis SD24 strain inhibiting the growth of Vibrio aquatica;

[0026] Figure 3 The antibacterial effect result diagram of the supernatant of the culture solution of Bacillus subtilis SD24 strain (wherein, Y represents YEB; L represents LB; B represents BHI);

[0027] Figure 4 This is the relationship between the production of antibacterial substances and the number of days of Bacillus subtilis SD24 strain;

[0028] Figure 5 This is the result diagram of the stability of the sterile antibacterial supernatant of Bacillus subtilis SD24 strain;

[0029] Figure 6 This is the complete genome DNA map of Bacillus subtilis SD24 strain. DETAILED DESCRIPTION

[0030] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0031] Example 1

[0032] 1. Experimental methods

[0033] 1.1 Screening of soil bacterial strains with antibacterial activity

[0034] Bacterial co-culture technology was applied. Specifically, aquatic pathogenic bacteria Vibrio harveyi and Vibrio rivulosa were uniformly mixed with melted TSA solid culture medium and then poured into a plate. After the culture medium solidified, a 200 μl pipette tip with the tip cut off was used to punch holes. 5 μl of freshly resuspended test strain cells were added to the holes. Each three wells were repeated and cultured in a 28°C incubator for 23 days. Then, bacterial strains that can inhibit Vibrio were screened. A total of 200 soil bacterial strains were screened, and 4 bacterial strains that can effectively inhibit Vibrio harveyi and Vibrio rivulosa were obtained. The results are shown in Table 1. Figure 1 , and the strain with the best inhibition effect will be the target strain.

[0035] 1.2 Obtaining the culture supernatant of Bacillus subtilis SD24 and its antibacterial test

[0036] The selected Bacillus subtilis SD24 strain was inoculated into 5 ml YEB liquid medium, and cultured at 28°C in a shaker at 250 rpm / min for 2 days. After centrifugation at 6000 rpm / min for 5 minutes, the bacteria were removed and the supernatant was collected. The bacteria were then filtered through a 0.2 μm pore filter, and the sterile supernatant was stored in a 4°C refrigerator. The antibacterial activity was analyzed by the punching method (the method is the same as the punching content in 1.1). The results are shown in Figure 2 .

[0037] Depend on Figure 2 The results showed that 5 μl of culture supernatant could effectively inhibit the growth and reproduction of Vibrio harveyi and Vibrio fluvibrios.

[0038] 1.3 Selection of the best culture medium

[0039] The cells of the target strain were inoculated into 5 ml of LB medium, YEB medium and BHI medium respectively, and cultured on a shaker at 28°C. The sterile supernatant filtrate was collected and then the antibacterial activity was analyzed. Figure 3 .Depend on Figure 3 The results showed that the antibacterial activity of the filtrate in YEB medium was significantly greater than that in LB medium or BHI medium.

[0040] 1.4 Optimal culture time for target strains to produce antibacterial substances

[0041] Equal amounts of target strain cells were inoculated into 5 ml YEB (liquid) medium and cultured on a shaker at 28°C for 1, 2, 3 and 4 days. The sterile supernatant filtrate was collected for antibacterial activity analysis. Figure 4 .

[0042] Figure 4 The results showed that the antibacterial activity of each filtrate obtained under different culture time was: 2 days (highest) > 1 day > 3 days > 4 days. That is, the optimal culture time for the target strain to produce antibacterial substances was 2 days.

[0043] 1.5 Stability of the supernatant of the target strain (sterile) culture fluid

[0044] The culture supernatant (liquid) obtained by culturing the target strain for 2 days was placed at room temperature for 6 months and then the antibacterial activity was analyzed. Figure 5 .

[0045] Figure 5 The results showed that the supernatant of the culture fluid stored at room temperature for less than three months had strong antibacterial activity, which indicated that the supernatant of the culture fluid of the target strain (sterile) had excellent antibacterial stability.

[0046] 1.6 Whole genome sequencing of target strains

[0047] Sequencing was performed based on the Nanopore third-generation sequencing technology platform and the second-generation sequencing technology platform to complete bacterial map analysis, with a sequencing depth of ≥100X and 0 gap in assembly results. After filtering out adapters, short fragments, and low-quality data, the data measured by the third-generation Nanopore sequencer obtained a total of 1,816,707,611bp of clean data for assembly, and the second-generation Illumina sequencing obtained a total of 2,593,269,162bp of clean data; the size of the complete genome obtained after assembly was 3.908M (see Figure 6 ). Relevant information can be found on the PubMLST (https: / / pubmlst.org / ) website through allele spectrum or sequence type. Multi-locus sequence typing was performed using mlst (Version: .23.0) software, and it was determined to be a new strain of Bacillus subtilis. The species was identified as Bacillus subtilis. The whole genome encoded 3918 genes, including 3721 protein-coding genes. It was classified and named Bacillus subtilis SD24.

[0048] 1.7 Antibacterial substance synthesis gene cluster in the genome of Bacillus subtilis SD24 strain

[0049] The whole genome encodes 7 types of gene clusters for the synthesis of antimicrobial substances: cyclic lipopeptide surfactants, butyric acid, midecamycin, dithiothioate, fengycin, damicin, bacillus and bacterial lysin (Table 1). It is the Bacillus strain with the most gene clusters encoding antimicrobial substances found so far.

[0050] Table 1.

[0051]

[0052] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A Bacillus subtilis, characterized in that The classification name of the Bacillus subtilis is Bacillus subtilis SD24, the preservation unit is the China Center for Type Culture Collection, the preservation number is CCTCC NO.M 20242253, and the preservation time is October 21, 2024.

2. Use of the Bacillus subtilis as claimed in claim 1 in aquaculture.

3. A method for obtaining a culture supernatant of Bacillus subtilis based on the Bacillus subtilis according to claim 1, comprising the following steps: The Bacillus subtilis SD24 strain was inoculated into a liquid culture medium, cultured at 25-28° C. and at 250-300 rpm / min on a shaker, centrifuged, the bacteria were removed, and the Bacillus subtilis culture supernatant was collected.

4. The method according to claim 3, characterized in that: The liquid culture medium is selected from any one of LB culture medium, YEB culture medium and BHI culture medium.

5. The method according to claim 3, characterized in that: The shaking culture time is 1 to 4 days.

6. A Bacillus subtilis culture supernatant obtained by the method according to any one of claims 2 to 5.

7. Use of the Bacillus subtilis culture supernatant as claimed in claim 6 in aquaculture.

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