Bacillus licheniformis DCGGN-06 and application thereof in resisting multiple pathogens

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus licheniformis DCGGN-06 and application of the bacillus licheniformis DCGGN-06 in resisting various pathogens. The invention provides a bacillus licheniformis DCGGN-06 and application of the bacillus licheniformis DCGGN-06 in resisting various pathogens, the preservation number of the bacillus licheniformis DCGGN-06 provided by the invention is CCTCCNO: M 2024879, and bacteriocin prepared from the bacillus licheniformis DCGGN-06 can resist staphylococcus aureus, methicillin-resistant staphylococcus aureus and foot and mouth disease viruses. The result of the embodiment shows that the bacteriocin prepared from the bacillus licheniformis DCGGN-06 can obviously reduce the FMDV replication level and the expression of VP0 protein, and has obvious in-vitro anti-FMDV virus activity; the bacteriocin can generate an obvious inhibition zone on staphylococcus aureus and methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Bacillus licheniformis DCGGN-06 and its use in combating multiple pathogens. Background Art

[0002] With the continuous emergence of antibiotic residues and drug-resistant bacteria in the fields of health, food, and breeding, many countries in the world have banned the use of antibiotics as feed additives in animal production, and probiotics as a microecological feed additive have attracted widespread attention. Probiotics are mainly divided into two categories: Bacillus and lactic acid bacteria. Bacillus plays an important role in the field of human health due to its strong resistance and stability, promoting the growth of lactic acid bacteria, inhibiting the growth of pathogens, producing hydrolases, and producing vitamins. In addition, it has low requirements for external nutrition and is more suitable for mass production.

[0003] Bacteriocins are small molecule cationic peptides with antibacterial effects produced by bacterial ribosomes. They are naturally non-toxic and have strong antibacterial properties. They are at the forefront of research on new microecological preparations in recent years. The important role of bacteriocins is not only reflected in the removal of specific pathogens in the body and the inhibition of multiple pathogens in vitro, but also in regulating intestinal flora, anti-inflammatory, anti-tumor, and antioxidant reactions. Bacteriocins inhibit bacterial infections through multiple mechanisms, including blocking the synthesis of cell walls, destroying the integrity of cell membranes, and inhibiting the synthesis of biological macromolecules such as proteins and nucleic acids, making them a good substitute for antibiotics. For example, Nisin has antibacterial activity against drug-resistant Streptococcus pneumoniae, enterococci, and drug-resistant Clostridium difficile; Curvaticin 13 produced by Lactobacillus curvatus combined with Nisin has antibacterial activity against Listeria monocytogenes; Bacillus subtilis bacteriocin BLIS has antibacterial activity against Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Clostridium perfringens. In addition, bacteriocins can resist viruses by regulating the mucosal system and systemic immune system of humans and animals, blocking host cell receptor sites to prevent virus invasion on the one hand, and inducing cell lesions while reducing virus release on the other hand. For example, plantaricin BN and plantaricin W can fight against the infection of the new coronavirus by blocking the cell receptor binding region of the new coronavirus S protein domain or the interaction between the S protein and the host cell angiotensin-converting enzyme 2 (ACE2) receptor protein.

[0004] The prior art reports that Bacillus licheniformis can effectively reduce and inhibit white spot syndrome virus (see CN104388333A A Bacillus licheniformis and its application), but the antiviral effect is limited. Therefore, it is of great significance to find Bacillus licheniformis that has both antibacterial and antiviral effects. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus licheniformis DCGGN-06 and its use in resisting multiple pathogens. The bacteriocin prepared by the Bacillus licheniformis DCGGN-06 provided by the present invention can resist Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and foot-and-mouth disease virus.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention provides a strain of Bacillus licheniformis DCGGN-06, whose deposit number is CCTCC NO:M 2024879.

[0008] The present invention provides a bacterial agent, comprising the Bacillus licheniformis DCGGN-06 described in the above technical solution.

[0009] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution in the preparation of antibacterial active substances.

[0010] Preferably, the antibacterial active substance includes bacteriocin; the amino acid sequence of the bacteriocin is shown in SEQ ID NO.2.

[0011] The present invention provides a method for preparing an antibacterial active substance, characterized in that it comprises:

[0012] Cultivating the Bacillus licheniformis DCGGN-06 described in the above technical solution, and obtaining a cell-free fermentation broth by centrifuging and filtering the obtained culture broth;

[0013] mixing the cell-free fermentation broth with ammonium sulfate to obtain a crude active substance;

[0014] The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

[0015] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution in the preparation of antiviral and / or antibacterial drugs.

[0016] Preferably, the virus includes foot-and-mouth disease virus; and the bacteria include Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

[0017] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution in the preparation of a microecological preparation.

[0018] Preferably, the product form of the probiotic preparation includes feed additives and / or antibacterial agents.

[0019] Beneficial effects of the present invention: The present invention provides a strain of Bacillus licheniformis DCGGN-06, with a deposit number of CCTCCNO: M 2024879. The bacteriocin prepared by the Bacillus licheniformis DCGGN-06 provided by the present invention can resist foot-and-mouth disease virus, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. The results of the embodiments show that the bacteriocin prepared by Bacillus licheniformis DCGGN-06 can significantly reduce the replication level of FMDV and the expression of VP0 protein, and has significant in vitro anti-FMDV virus activity; the bacteriocin can produce obvious inhibition zones against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. It can be seen that the bacteriocin has antibacterial and antiviral effects, is a safe, efficient, green natural anti-infection drug, and is suitable for the fields of food industry, animal husbandry, and protection of human public health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology and Gram staining microscopy (1000×) of Bacillus licheniformis DCGGN-06 of swine, where the left side shows the colony morphology and the right side shows the microscopy result;

[0021] Figure 2 This is the phylogenetic analysis diagram of porcine Bacillus licheniformis DCGGN-06;

[0022] Figure 3 This is the plate image of the culture medium for identifying the antibacterial activity of porcine Bacillus licheniformis DCGGN-06;

[0023] Figure 4 This is a plate image of the culture medium for identifying the antibacterial activity of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06;

[0024] Figure 5 It is the SDS-PAGE electrophoresis diagram of bacteriocin produced by swine Bacillus licheniformis DCGGN-06;

[0025] Figure 6 This is a mass spectrometry analysis of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06;

[0026] Figure 7 This graph shows the effect of bacteriocin produced by swine Bacillus licheniformis DCGGN-06 on FMDV RNA replication and protein expression.

[0027] Biological Deposit Description

[0028] Bacillus licheniformis DCGGN-06 was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2024, with the deposit number CCTCCNO: M 2024879. The address of the depository unit is: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0029] The present invention provides a strain of Bacillus licheniformis DCGGN-06, with a deposit number of CCTCC NO: M 2024879. The 16srRNA gene sequence of the Bacillus licheniformis DCGGN-06 of the present invention is shown in SEQ ID NO.1.

[0030]

[0031] The present invention provides a bacterial agent, comprising the Bacillus licheniformis DCGGN-06 described in the above technical solution.

[0032] The invention uses the gastrointestinal contents of fresh pigs as raw materials to separate Bacillus licheniformis DCGGN-06 with antiviral and antibacterial effects.

[0033] The present invention uses the 16s rRNA sequence of Bacillus licheniformis DCGGN-06 to perform BLAST homologous sequence search in NCBI, constructs an evolutionary tree with the obtained sequences with high homology using MEGA8 software, and combines bacterial morphological characteristics to identify the strain DCGGN-06 as Bacillus licheniformis.

[0034] The colony diameter of the Bacillus licheniformis DCGGN-06 on the LB medium plate of the present invention is 2 to 3 mm, and the colony is white, the colony is wrinkled, the colony surface is not smooth, and the edges are irregular. The Bacillus licheniformis DCGGN-06 provided by the present invention is acid-resistant, bile-resistant, and high-temperature-resistant, and can resist Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and foot-and-mouth disease virus, and has good antiviral and antibacterial effects, and Bacillus licheniformis DCGGN-06 can be used to prepare safe, efficient, and green natural antiviral and antibacterial drugs, which are suitable for the fields of food industry, animal husbandry, and protection of human public health.

[0035] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution in the preparation of antibacterial active substances.

[0036] In the present invention, as an implementation mode, the antibacterial active substance includes bacteriocin; the amino acid sequence of the bacteriocin is shown in SEQ ID NO. 2. The prepared bacteriocin can resist Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and foot-and-mouth disease virus. The bacteriocin amino acid sequence can encode 95 amino acids and is about 11 kDa in size.

[0037] The present invention provides a method for preparing an antibacterial active substance, comprising:

[0038] Cultivating the Bacillus licheniformis DCGGN-06 described in the above technical solution, and obtaining a cell-free fermentation broth by centrifuging and filtering the obtained culture broth;

[0039] mixing the cell-free fermentation broth with ammonium sulfate to obtain a crude active substance;

[0040] The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

[0041] As an implementation mode, the present invention cultivates the Bacillus licheniformis DCGGN-06 described in the above technical solution, and obtains a cell-free fermentation liquid after filtering and centrifuging the obtained culture liquid. As another implementation mode, the present invention inoculates a single colony of the Bacillus licheniformis DCGGN-06 described in the above technical solution into a culture medium for cultivation, and obtains a cell-free fermentation liquid after filtering and centrifuging the obtained culture liquid.

[0042] As an embodiment, the preparation method of a single colony of Bacillus licheniformis DCGGN-06 of the present invention can be: streak separation and activation culture of the preservation solution of Bacillus licheniformis DCGGN-06 on an LB solid culture medium plate to obtain a single colony of DCGGN-06. The present invention does not specifically limit the parameters of the streak separation and activation culture, and conventional methods can be used.

[0043] A single colony is obtained. The present invention can inoculate the single colony into a culture medium for culture. The obtained culture solution is centrifuged and filtered to obtain a cell-free fermentation solution. The culture temperature of the present invention can be 34°C to 40°C; in a specific embodiment of the present invention, the culture temperature can be 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; the culture time can be 23 to 27h, and in a specific embodiment of the present invention, the culture time can be 23h, 24h, 25h, 26h or 27h. The culture medium of the present invention can be an LB liquid culture medium, and the composition of the LB liquid culture medium can be: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water.

[0044] As an embodiment, to obtain the culture solution, the present invention can centrifuge the culture solution, the centrifugal speed can be 8000 rpm, the centrifugal time can be 30 minutes, and the centrifugal temperature can be 4°C.

[0045] As an embodiment, a supernatant is obtained after centrifugation, and the present invention filters the supernatant, and the filtration can be performed using a filter membrane with a diameter of 0.22 μm. After filtration, a cell-free fermentation broth is obtained. The function of the filtration is sterilization. The cell-free fermentation broth of the present invention is a fermentation broth containing only the metabolites of Bacillus licheniformis DCGGN-121, and does not contain Bacillus licheniformis DCGGN-121 bacteria.

[0046] The present invention mixes the cell-free fermentation broth with ammonium sulfate to obtain a crude active substance. As an embodiment, the present invention can add ammonium sulfate to the cell-free fermentation broth until the ammonium sulfate saturation in the cell-free fermentation broth reaches 80%. The ammonium sulfate can be added in the form of powder. The mixing method can include a first stirring and a second stirring; the addition can be accompanied by a first stirring, and the addition needs to be added slowly; the first stirring is stopped after the addition of ammonium sulfate is completed. As an embodiment, the second stirring and standing are started after the ammonium sulfate in the cell fermentation broth is added. The second stirring can be completed using a magnetic stirrer, and the second stirring time can be 1h. The temperature of the standing can be 4°C, and the standing time can be 12 to 24h, or it can be overnight. As an embodiment, after the standing is completed, the obtained product is centrifuged to obtain a precipitate, and the precipitate is the crude active substance. The speed of the centrifugation can be 10000rpm, and the time can be 30min. The role of ammonium sulfate is to precipitate and purify the protein components in the cell-free fermentation broth.

[0047] As an embodiment, the crude active substance is ultrafiltered and purified to obtain an antibacterial active substance. As another embodiment, the crude active substance is dialyzed and then ultrafiltered and purified to obtain an antibacterial active substance. Before the dialysis, the crude active substance is mixed with a phosphate buffered saline (PBS) and then dialyzed. The dialysis bag used in the dialysis can have a molecular weight cutoff of 3.5 kDa, and the dialysate used in the dialysis can be a PBS buffer. The role of the dialysis is to remove ammonium sulfate.

[0048] As an embodiment, the ultrafiltration can be completed using an ultrafiltration centrifuge tube, the molecular weight cutoff of the ultrafiltration centrifuge tube can be 3kDa and 30kDa, and proteins with a molecular weight between 3kDa and 30kDa are obtained after ultrafiltration centrifugation. As an embodiment, the protein retained by the ultrafiltration centrifuge tube is reconstituted with PBS and filtered through a filter membrane before purification. The PBS reconstitution can be performed using conventional parameters. The diameter of the filter membrane can be 0.22μm. The purification method can be an ion exchange column. During the purification process, Staphylococcus aureus ATCC6538 can be used to monitor the antibacterial activity to ensure that the antibacterial active substance produced by porcine Bacillus licheniformis DCGGN-06 is successfully separated. The antibacterial active substance prepared by the present invention is the protein component in the cell-free fermentation broth.

[0049] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution in the preparation of antiviral and / or antibacterial drugs.

[0050] As an implementation mode, the virus may include foot-and-mouth disease virus; the bacteria may include Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

[0051] The present invention has no special limitation on the preparation method of the drug, and a conventional method can be used. The dosage form of the drug of the present invention can be a conventional dosage form.

[0052] The present invention provides the use of the Bacillus licheniformis DCGGN-06 described in the above technical solution or the bacterial agent described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution in the preparation of a microecological preparation.

[0053] As an embodiment, the product form of the probiotic preparation may include feed additives and / or antibacterial agents.

[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1 Isolation and Identification of Porcine Bacillus licheniformis DCGGN-06:

[0056] 1. Sources of materials, strains and cells: Fresh pig gastrointestinal contents were used as samples for strain isolation. The samples came from the slaughterhouse in Yongjing County, Gansu Province. After collection, the samples were packaged in sterile plastic bags and transported to the laboratory at low temperature. PK-15 cells, BHK-21 cells and MDBK cells were preserved in this laboratory.

[0057] The indicator bacteria used in the antibacterial test were Salmonella Typhimurium CVCC541, Escherichia coli ATCC25922, Staphylococcus aureus ATCC6538, Staphylococcus aureus MRSA, Klebsiella pneumoniae ATCC700603, Enterococcus Faecium, and Pseudomonas aeruginosa ATCC27853 preserved in our laboratory.

[0058] 2. Culture medium and other biochemical reagents

[0059] LB liquid medium: 10 g of peptone, 5 g of yeast extract and 10 g of sodium chloride were heated and dissolved in 1000 mL of distilled water; LB solid medium was prepared by adding 1.5% agar to the liquid medium; both the liquid medium and the solid medium were sterilized by high pressure at 121°C for 15 min;

[0060] Nutrient broth medium: 10 g peptone, 3 g beef extract powder, 5 g sodium chloride, heat and dissolve in 1000 mL distilled water, and sterilize at 121°C for 15 min.

[0061] Glucose tryptone medium: 10 g tryptone, 5 g glucose, 0.04 g bromocresol purple, 15 g agar, heat and dissolve in 1000 mL distilled water to adjust the pH to 6.9±0.2, sterilize by high pressure at 121°C for 15 min, pour into a plate for later use.

[0062] DNA Ligation Kit (MightyMix), Taq DNA polymerase, and molecular markers were purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0063] HiTrap Capto S prepacked columns were purchased from Cytiva, Sweden; ultrafiltration centrifuge tubes were purchased from Merck Millipore;

[0064] Hydrochloric acid, sodium chloride, etc. were all domestically produced analytical grade; bacterial DNA extraction kit was purchased from Beijing Tiangen Biological Co., Ltd., and Gram staining kit was purchased from Sigma, USA.

[0065] 3. Experimental methods:

[0066] (1) Isolation of strains: In a clean bench, collect fresh pig gastrointestinal contents, mark them, and take about 1g of each into sterile test tubes, adding 5mL of nutrient broth medium to each tube; culture the test tubes in a 37℃ constant temperature shaker for 2h, then in an 80℃ water bath for 15min to kill non-spore-forming bacteria. After cooling, take 100μL and spread it on an LB solid plate, invert it in a 37℃ incubator for 24h to form a single colony. Pick a single colony and continue culturing in LB liquid, repeat the plate streaking until a single colony is obtained, and save multiple strains.

[0067] (2) Preliminary screening of strains:

[0068] Resuscitation strain: The single colony obtained in step (1) was inoculated into LB liquid culture medium and cultured in a shaking incubator at 37°C to obtain a bacterial solution. The OD of the bacterial solution was 600 =0.8.

[0069] A: Acid resistance screening: LB liquid culture medium with pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, and 6.8 were prepared respectively, and the bacterial solution was transferred to 5mL LB liquid culture medium with different pH values ​​of 2.0, 2.5, 3.0, 3.5, and 4.0 at a ratio of 5% of the volume of the culture medium as the treatment group, and the bacterial solution was transferred to a normal LB liquid culture medium with a pH of 6.8 as a blank control. After the treatment group and the blank control were transferred and mixed, they were placed in a 37°C incubator for static culture for 2 hours, and 100μL of the culture solution was spread on a glucose tryptone medium plate, and the plate was counted after inverted culture for 24 hours, and the survival rate of each strain was calculated. The strains with strong acid resistance that can still survive in the LB liquid culture medium with pH 2.0 for 2 hours with a survival rate of 35.8% were preliminarily screened.

[0070] B: Bile salt resistance screening: Prepare LB liquid culture medium containing 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ox bile salt concentrations, take bacterial liquid of strains with strong acid resistance, transfer to 5mL LB liquid culture medium with different bile salt concentrations at a culture medium volume ratio of 1% (v / v), culture at 37°C for 12h, take 100μL of the obtained culture liquid and spread it on LB solid culture medium plate, culture for 24h, count the plate, calculate the survival rate of the strain, and preliminarily screen the strain with strong bile salt resistance after being treated in 0.3% bile salt medium for 12h with a survival rate of 51.6%;

[0071] C: High temperature resistance screening: The bile salt-resistant strain was inoculated into LB liquid culture medium, and cultured overnight at 37°C in a shaking table to obtain a bacterial solution. 5 mL of the overnight cultured bacterial solution was respectively taken and placed in metal baths at 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and 100°C for treatment for 10 min. After the sample was cooled after treatment, 100 μL was taken for coating on LB solid culture medium plates as the treatment group, and the bacterial solution that was not subjected to high temperature treatment was used as the blank control and coated on LB solid culture medium plates. After culturing the treatment group and the blank control for 24 hours, the plate counts were performed, and the survival rate of the strains with a survival rate of more than 50% after treatment at 85°C for 10 min was calculated.

[0072] After preliminary screening, one of the strains that was resistant to acid, bile salt and high temperature was named DCGGN-06. The colony morphology on LB medium was: white colonies with a diameter of 2 to 3 mm, irregular edges, rough surface and wrinkles; Figure 1 The colonies of Bacillus licheniformis and Gram staining microscopy (1000×) showed that strain DCGGN-06 was a Gram-negative, short rod.

[0073] (3) Activation identification and preservation of strains: A single colony that was initially identified as strain DCGGN-06 was selected and transferred to 5 mL of LB liquid medium and cultured at 37°C for 48 h, and then subcultured twice with an inoculum volume of 1% (v / v). After subculture, the culture was stored in -20°C low-temperature glycerol.

[0074] Use primers specific for Bacillus licheniformis:

[0075] Bli-man-1F: 5'-AGCCATGGATATTAAATAAC-3' (SEQ ID NO: 3),

[0076] Bli-man-1R: 5'-TATTCCCTTACAATAAGACG-3' (SEQ ID NO: 4);

[0077] 16S rRNA primers: 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 5), 1492R: TACGGTYACCTTGTTGTTACGACTT (SEQ ID NO: 6).

[0078] The strain to be identified was amplified by PCR and sequenced using Bacillus licheniformis specific primers and 16S rRNA primers. The resulting sequence is shown in SEQ ID NO: 1. The sequence was uploaded to NCBIblast for comparison, and the phylogenetic tree was constructed using the software MEGA8. Figure 2 . Figure 2 This is a phylogenetic analysis of Bacillus licheniformis DCGGN-06. WBL 006 is strain DCGGN-06. Figure 2 It can be seen that the strain DCGGN-06 has a high similarity with the Bacillus licheniformis B5 strain in the GenBank database, and the strain DCGGN-06 is on the same evolutionary branch.

[0079] (4) Cultivation of indicator bacteria: Various indicator bacteria strains (see Table 1) stored in -80°C glycerol tubes were streaked onto LB solid culture medium plates, and distinct single colonies were cultured at the optimal temperature. Single colonies were picked and inoculated into the corresponding LB liquid culture medium and cultured on a shaker until the logarithmic phase. The bacterial concentration was adjusted to OD 0. 600 =1.0, and the bacterial solution was obtained, which was stored at 4°C for later use.

[0080] (5) Antibacterial identification of porcine Bacillus licheniformis DCGGN-06: Porcine Bacillus licheniformis DCGGN-06 was transferred to LB medium and cultured for 24 h. It was then inoculated into 1L LB liquid medium at 1% (v / v). The bacterial solution was obtained by culturing for 24 h. The bacterial solution was centrifuged at 8000 rpm and 4°C for 30 min. The obtained supernatant was filtered through a 0.22 μm filter membrane to obtain a cell-free fermentation broth.

[0081] Treatment group: Inoculate the indicator bacteria cultured in step (4) at 1% (v / v) into the corresponding agar medium sterilized at 40°C, mix gently and quickly, and pour into plates. After the agar medium solidifies, place an Oxford cup on the surface. Add 200 μL of the above cell-free fermentation liquid to each Oxford cup. After diffusing for 3 hours in a biological clean bench, culture at 37°C overnight, detect whether there is an antibacterial ring and measure the diameter of the antibacterial ring with a vernier caliper. The control group is the same as the treatment group, with the only difference being that 200 μL of PBS is added to the Oxford cup. The results of the control group and the treatment group are shown in Tables 1 and Figure 3 . Figure 3 The plate diagram for identifying the antibacterial activity of porcine Bacillus licheniformis DCGGN-06 shows that the porcine Bacillus licheniformis DCGGN-06 of the present invention has an inhibitory effect on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).

[0082] Table 1 Antibacterial spectrum of porcine Bacillus licheniformis DCGGN-06

[0083] Indicator bacteria type Indicator bacteria name Indicator bacteria type Inhibition zone diameter Salmonella Typhimurium CVCC541 <![CDATA[G - ]]> - Escherichia coli ATCC25922 <![CDATA[G - ]]> - Staphylococcus aureus ATCC6538 <![CDATA[G + ]]> ++ Staphylococcus aureus (MRSA) Clinical isolates <![CDATA[G + ]]> ++ Klebsiella pneumoniae ATCC700603 <![CDATA[G - ]]> - Enterococcus faecium Clinical isolates <![CDATA[G + ]]> - Pseudomonas aeruginosa ATCC27853 <![CDATA[G - ]]> -

[0084] Note: “+” means 5-10mm; “++” means 10-15mm; “+++” means 15-20mm; “-” means no inhibition zone; G - Represents Gram-negative bacteria, G + Represents Gram-positive bacteria.

[0085] Example 2 Isolation, Identification and Antiviral Study of Bacteriocins Produced by Swine Bacillus Licheniformis DCGGN-06

[0086] 1. Sources of materials, strains and cells: The cell-free fermentation broth separated in Example 1 was used, and the indicator bacteria for the antibacterial activity detection in the following experiments were all Staphylococcus aureus ATCC6538, and antibacterial activity monitoring was performed after each purification step to ensure the successful separation of bacteriocins produced by porcine Bacillus licheniformis DCGGN-06. PK-15 cells, BHK-21 cells, and MDBK cells were preserved in this laboratory.

[0087] The indicator strains used in the antiviral experiment were African swine fever virus (ASFV) CN / GS / 2018 strain, bovine viral diarrhea virus (BVDV) Oregon C24V strain, foot-and-mouth disease virus (FMDV) O type MYA98 strain, classical swine fever virus (CSFV) C strain, and Seneca virus (SVA) CH-FJ-2017 strain preserved in our laboratory.

[0088] 2. Preparation of culture medium, other biochemical reagents and related liquids:

[0089] DMEM cell culture medium, PBS solution, 0.25% EDTA trypsin and newborn bovine serum (FBS) were purchased from BI Company; Trizol reagent was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; ChamQ Universal qPCR Master Mix was purchased from Novozyme Biotech Co., Ltd.; enhanced chemiluminescence (ECL) colorimetric solution was purchased from Advansta; prestained protein marker was purchased from Yazyme Biotech;

[0090] HiTrap Capto S prepacked columns were purchased from Cytiva, Sweden; ultrafiltration centrifuge tubes were purchased from Merck Millipore;

[0091] Ethanol, ammonium sulfate, hydrochloric acid, sodium chloride, etc. were all domestic analytical grade. Dimethyl sulfoxide (DMSO) was purchased from AMRESCO. Mouse anti-β-actin monoclonal antibody (ARM2001) was purchased from Shanghai Nolai Biotechnology. Homemade rabbit anti-FMDV-VP0 antibody was preserved by our laboratory. HRP goat anti-mouse IgG and HRP goat anti-rabbit IgG were purchased from Beijing Biolong Immunotechnology Co., Ltd.

[0092] The reagents involved in the embodiment are prepared as follows:

[0093] Saturated ammonium sulfate solution: Add excess ammonium sulfate to ultrapure water at 50-60°C, filter and place at room temperature for 24 hours. When crystals precipitate, the saturation is 100%.

[0094] Staining solution: weigh 1g of R-250, add 250mL of isopropanol, 100mL of glacial acetic acid, and 600mL of double distilled water. Stir and dissolve each step, and finally filter paper to remove particles and store for use.

[0095] Decolorizing solution: Add 100 mL of glacial acetic acid, 50 mL of ethanol and 850 mL of double distilled water in sequence and mix.

[0096] Buffer A: 50 mM sodium acetate (add 4.1 g sodium acetate to 1 L buffer A), adjust the pH to 5.0 with concentrated hydrochloric acid, and filter through a 0.22 μm filter membrane before use.

[0097] Buffer B: 50 mM sodium acetate, 1 M NaCl (add 58.5 g NaCl and 4.1 g sodium acetate to 1 L buffer B), adjust the pH to 5.0 with concentrated hydrochloric acid, and filter through a 0.22 μm filter membrane before use.

[0098] 3. Experimental Methods

[0099] (1) Preparation of antibacterial fermentation broth of porcine Bacillus licheniformis DCGGN-06: The DCGGN-06 strain stored in a -80°C glycerol tube was streaked on an LB solid culture medium plate for activation. After culturing at 37°C for 24 h, a single colony was picked and transferred to LB culture medium for 24 h, and then inoculated into a new 1L LB liquid culture medium at 1% (v / v). The bacterial solution obtained after culturing for 24 h was centrifuged at 8000 rpm and 4°C for 30 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the antibacterial fermentation broth.

[0100] (2) Preparation of antibacterial crude extract of porcine Bacillus licheniformis DCGGN-06: The antibacterial fermentation liquid obtained in step (1) was slowly added with ground ammonium sulfate powder while stirring, so that the saturation of ammonium sulfate in the antibacterial fermentation liquid reached 80%, and then slowly stirred on a magnetic stirrer for 1 hour, and then placed in a 4°C refrigerator overnight; the next day, the extract was taken out and centrifuged at 10000 rpm for 30 min to obtain a precipitate, and the obtained precipitate was the crude active substance; 2 mL of 0.01 M phosphate buffer (PBS) was added to the above crude active substance to dissolve the precipitate, and the dissolved solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in 2 L of PBS buffer overnight to remove the ammonium sulfate and obtain a dialyzed sample.

[0101] (3) Purification of the antibacterial crude extract of porcine Bacillus licheniformis DCGGN-06: The dialyzed sample obtained in step (2) was centrifuged using ultrafiltration centrifuge tubes with a molecular weight cutoff of 3 kDa and 30 kDa, respectively, to retain proteins with a molecular weight cutoff of 3 kDa to 30 kDa. The retained proteins were reconstituted with PBS and filtered with a 0.22 μm microporous membrane to obtain a protein sample.

[0102] The obtained protein sample was purified by an ion exchange column, and the ion exchange column was a HiTrap Capto S cation exchange column. The purification was specifically as follows: balancing the pre-loaded column with buffer A, loading the sample, rinsing with buffer A and collecting the flow-through; gradient elution was performed by mixing buffer A and buffer B to obtain an eluent, and the NaCl molar concentration in the eluent was 20 mM, 40 mM, 80 mM, 100 mM, and 200 mM, respectively. The NaCl molar concentration was gradient eluted from low to high, and the spectrophotometer was used to detect at a wavelength of 280 nm and collect the eluted peak protein. Among them, the elution peak proteins of flow-through 1 and flow-through 2 were collected by rinsing with buffer A. When the molar concentration of NaCl in the eluent was 20mM, the elution peak proteins of 20mM elution a and 20mM elution b were collected; when the molar concentration of NaCl in the eluent was 40mM, the elution peak proteins of 40mM elution were collected; when the molar concentration of NaCl in the eluent was 80mM, the elution peak proteins of 80mM elution were collected; no absorption peak appeared when eluting at 100mM and 200mM. The collected elution peak proteins were subjected to antibacterial identification and SDS-PAGE electrophoresis identification.

[0103] 1) Antibacterial identification: The elution peak protein collected above was used to detect the antibacterial activity of the indicator bacteria Staphylococcus aureus ATCC6538, and the method was the same as (5) in Example 1. The results showed that the 20mM gradient elution peak had the activity of inhibiting Staphylococcus aureus, as shown in Figure 4 . Figure 4 The antibacterial activity identification diagram of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06 is shown in Figure 1. The 20 mM elution peak protein fraction with antibacterial activity is collected, freeze-dried, reconstituted with deionized water, and filtered with a microporous filter membrane to obtain a purified bacteriocin solution, which is the obtained antibacterial active substance.

[0104] 2) SDS-PAGE electrophoresis identification of bacteriocin produced by swine Bacillus licheniformis DCGGN-06: The protein samples collected above were subjected to SDS-PAGE electrophoresis identification, and the target band was found to be approximately 11 kDa. Figure 5 , Figure 5 The SDS-PAGE electrophoresis diagram of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06 is shown in the figure; wherein, M: Marker, 1: loading sample; 2: flow-through 1; 3: flow-through 2; 4: 20mM elution a; 5: 20mM elution b; 6: 40mM elution; 7: 80mM elution. The loading sample is the protein sample collected in step (3).

[0105] (4) The protein concentration collected from the 20 mM elution peak in step (3) was determined by BCA and sent to the company for LC-MS / MS determination of the amino acid sequence of the purified bacteriocin. The results showed that the molecular weight of the purified bacteriocin was about 11 kDa. The amino acid sequence of the bacteriocin was shown in SEQ ID NO: 2, encoding a total of 95 amino acids, and the N-terminal sequence was MHVQAQELSKTENLSS (SEQ ID NO: 7).

[0106] SEQ ID NO:2: MHVQAQELSKTENLSSHAFAQGIIAPQAIVINRQMRVGQEDQYVGISNVSYSG SAITVRITSDRVYVKAVSPGKATFSYLNSLGQLVINNITVTR.

[0107] The protein collected from the 20 mM elution peak after purification was analyzed by mass spectrometry. Figure 6 It can be seen that the corresponding bacteriocin can be identified by identifying the unique peptide N-terminal sequence corresponding to the protein.

[0108] (5) Identification of the antiviral activity of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06: PK15 cells, BHK-21 cells, and MDBK cells were plated in 12-well plates, and DMEM medium was added. When the cell density reached 70% to 80%, the cells were infected with viruses (ASFV, CSFV, SVA, FMDV, and BVDV) at a multiplicity of infection (MOI) of 1. At the same time, different concentrations of bacteriocin (0, 25, 100, and 500 μg / mL) were added to the treatment group of each virus. Cell samples were collected 24 hours after virus infection, and the effects of bacteriocin on the replication of the above viruses were analyzed by fluorescence quantitative PCR (primers see Table 2) and Western blotting. Figure 7 The effect of bacteriocin produced by porcine Bacillus licheniformis DCGGN-06 on FMDV RNA replication and protein expression was studied. The results showed that bacteriocin could significantly reduce the FMDV replication level and the expression of VP0 protein, and had significant in vitro antiviral activity against FMDV virus. Bacteriocin had no antiviral effect on ASFV, CSFV, SVA and BVDV.

[0109] Table 2 Real-time fluorescence quantitative (RT-qPCR) primers

[0110] Primer name Primer sequences ASFV-qPCR-F GATACCACAAGATCAGCCGT(SEQ ID NO:8) ASFV-qPCR-R CTGCTCATGGTATCAATCTATCGA(SEQ ID NO:9) CSFV-qPCR-F GACACAAGCGCAGGCAATAG(SEQ ID NO:10) CSFV-qPCR-R AGTGGGTTCCAGGARTACAT(SEQ ID NO:11) SV-qPCR-F GAATTTGGAAGCCATGCTC(SEQ ID NO:12) SV-qPCR-R AGCCAACATAGARACCAGATTGC(SEQ ID NO:13) FMDV-qPCR-F CACTGGTGACAGGCTAAGG(SEQ ID NO:14) FMDV-qPCR-R CCCTTCTCAGATTCCGAGT(SEQ ID NO:15) BVDV-qPCR-F GCCATGCCCTTAGTAGGACT(SEQ ID NO:16) BVDV-qPCR-R CACCCTATCAGGCTGTRTYC(SEQ ID NO:17) Pig-GAPDH-qPCR-F ACATGGCCTCCAAGGAGTAAGA(SEQ ID NO:18) Pig-GAPDH-qPCR-R GATCGAGTTGGGGCTGTGACT(SEQ ID NO:19) Mouse-GAPDH-qPCR-F GTGGCAAAGTGGAGATTGTTG(SEQ ID NO:20) Mouse-GAPDH-qPCR-R CGTTGAATTTGCCGTGAGTG(SEQ ID NO:21) Cattle-GAPDH-qPCR-F AGCGAGATCCTGCCAACATCAAG(SEQ ID NO:22) Cattle-GAPDH-qPCR-R GCAGGAGGCATTGCTGACAATCT(SEQ ID NO:23)

[0111] In summary, the present invention provides a strain of porcine Bacillus licheniformis DCGGN-06 and the bacteriocin produced by it. As a well-known technology, the bacteriocin can be basically completely decomposed in the body without any residue and will not lead to the production of drug-resistant strains. At the same time, porcine Bacillus licheniformis DCGGN-06 has antibacterial (Staphylococcus aureus) and antiviral (foot-and-mouth disease virus) effects, laying the foundation for the exploration of new antibacterial molecules with low toxicity and harmless to animal husbandry and a brand-new structure.

[0112] The bacteriocin produced by the porcine Bacillus licheniformis DCGGN-06 of the present invention has antibacterial and antiviral effects, is a safe, efficient, green natural anti-infection drug, and is suitable for the fields of food industry, animal husbandry, and protection of human public health.

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis DCGGN-06, with a deposit number of CCTCC NO: M2024879.

2. A bacterial agent, characterized in that It includes the Bacillus licheniformis DCGGN-06 described in claim 1.

3. Use of the Bacillus licheniformis DCGGN-06 according to claim 1 or the bacterial agent according to claim 2 in the preparation of antibacterial active substances.

4. The use according to claim 3, characterized in that: The antibacterial active substance includes bacteriocin; the amino acid sequence of the bacteriocin is shown in SEQ ID NO.

2.

5. A method for preparing an antibacterial active substance, characterized in that: include: Cultivating the Bacillus licheniformis DCGGN-06 according to claim 1, and centrifuging and filtering the obtained culture solution to obtain a cell-free fermentation solution; mixing the cell-free fermentation broth with ammonium sulfate to obtain a crude active substance; The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

6. Use of the Bacillus licheniformis DCGGN-06 according to claim 1, the bacterial agent according to claim 2, or the antibacterial active substance prepared by the preparation method according to claim 5 in the preparation of antiviral and / or antibacterial drugs.

7. The use according to claim 6, characterized in that: The virus includes foot-and-mouth disease virus; the bacteria include Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

8. Use of the Bacillus licheniformis DCGGN-06 according to claim 1, the bacterial agent according to claim 2, or the antibacterial active substance prepared by the preparation method according to claim 5 in the preparation of microecological preparations.

9. The use according to claim 8, characterized in that: The product form of the microecological preparation includes feed additives and / or antibacterial agents.

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