Bacillus licheniformis, bacteriostatic agent, method for preparing bacteriostatic active substance and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to bacillus licheniformis, a bacteriostatic agent, a method for preparing bacteriostatic active substances and application. The preservation number of the bacillus licheniformis DCGGN-121 provided by the invention is CCTCC (China Center For Type Culture Collection) NO: M 2024878. The bacteriocin prepared from the strain can resist African swine fever virus (ASFV), Seneca virus (SVA) and enteritis virus 71 (EV71 virus). The results of the embodiment show that the bacteriocin prepared from the bacillus licheniformis DCGGN-121 can obviously reduce the replication level of ASFV (Allergic Sequence Factor Virus), SVA (Seaweed Vector Amplification) and EV-71, and reduce the expression of ASFV-p72, SVA-VP2 and EV71-VP1 proteins. The bacillus licheniformis DCGGN-121 can be used for preparing a natural microecological preparation and is suitable for the food industry and the livestock breeding industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Bacillus licheniformis, an antibacterial agent, and a method for preparing an antibacterial active substance and application thereof. Background Art

[0002] Under the background of the country's proposal of "banning antibiotics in feed, reducing antibiotics in breeding, and making products antibiotic-free or up to standard", it is particularly important to strengthen the production safety of the breeding industry, public health safety and animal-derived food safety, and the exploration of new green natural drugs for the prevention and treatment of animal infectious diseases has attracted widespread attention from researchers. Bacteriocins are small molecule cationic peptides with antibacterial effects produced by bacterial ribosomes. They have the advantages of strong antibacterial properties, safety and no side effects, are not inhibited by antibacterial agents, do not affect the intestinal microbiome, and are not prone to extensive drug resistance. They are at the forefront of research on new microecological preparations in recent years.

[0003] Bacteriocins are antimicrobial peptides synthesized by many bacteria in the ribosome mode during the primary growth stage that have antimicrobial, anticancer and anti-biofilm effects. Studies have found that the protein metabolites with antiviral effects produced by probiotics are various bacteriocins. On the one hand, bacteriocins block host cell receptor sites to prevent viral invasion, and on the other hand, they induce cytopathic effects while reducing viral release; in addition, they can resist viruses by regulating the mucosal system and systemic immune system of humans and animals. For example, bacteriocins secreted by some enterococci can resist influenza virus and polio virus; bacteriocins produced by mucosal Lactobacillus have the activity of killing African swine fever virus and pseudorabies virus; plantarin BN, plantaricin W, etc. 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. All these show the potential of bacteriocins produced by probiotics in the prevention and treatment of viral diseases.

[0004] The prior art reports that Bacillus licheniformis can effectively reduce and inhibit white spot syndrome virus (see CN104388333 A A Bacillus licheniformis and its application), but the antiviral effect is limited. Therefore, it is of great significance to find a new broad-spectrum Bacillus licheniformis to prepare bacteriocins that inhibit broad-spectrum viruses. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus licheniformis, an antibacterial agent, a method for preparing an antibacterial active substance and an application thereof. The bacteriocin prepared by the Bacillus licheniformis DCGGN-121 provided by the present invention can resist African swine fever virus, Seneca virus and EV71 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-121, whose deposit number is CCTCC NO: M 2024878.

[0008] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution in the preparation of antibacterial active substances.

[0009] Preferably, the antibacterial active substance includes bacteriocin; the amino acid sequence of the bacteriocin is shown in SEQ ID NO.1.

[0010] The present invention provides a method for preparing an antibacterial active substance using the Bacillus licheniformis DCGGN-121 described in the above technical solution, comprising:

[0011] The Bacillus licheniformis DCGGN-121 is inoculated into a culture medium for culturing to obtain a cell-free fermentation liquid;

[0012] The cell-free fermentation liquid is mixed with ammonium sulfate and allowed to stand to obtain a crude active substance;

[0013] The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

[0014] The present invention provides an antibacterial agent, comprising the metabolite of Bacillus licheniformis DCGGN-121 described in the above technical solution.

[0015] Preferably, the metabolites include cell-free fermentation broth or antibacterial active substances prepared by the method described in the above technical solution.

[0016] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution or the antibacterial agent described in the above technical solution in the preparation of antiviral drugs.

[0017] Preferably, the antiviral agent includes one or more of anti-African swine fever virus, anti-Seneca virus and anti-EV71 virus.

[0018] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution or the antibacterial active substance prepared by the preparation method described in the above technical solution or the antibacterial agent described in the above technical solution in the preparation of a microecological preparation.

[0019] Preferably, the product form of the probiotic preparation includes feed additives or food additives.

[0020] Beneficial effects of the present invention: The present invention provides a strain of Bacillus licheniformis DCGGN-121, with a deposit number of CCTCCNO: M 2024878. The bacteriocin prepared by the Bacillus licheniformis DCGGN-121 provided by the present invention can resist African swine fever virus (ASFV), Seneca virus (SVA) and enteritis virus 71 (EV71 virus). The results of the embodiments show that the bacteriocin prepared by Bacillus licheniformis DCGGN-121 can significantly reduce the replication level of ASFV, SVA and EV-71, and reduce the expression of ASFV-p72, SVA-VP2, EV71-VP1 proteins, and has significant in vitro anti-ASFV, SVA and EV71 virus activity. Bacillus licheniformis DCGGN-121 can be used to prepare a safe, efficient and green natural microecological preparation, which is suitable for the fields of food industry, animal husbandry, and protection of human public health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony morphology and Gram staining microscopy (1000×) of Bacillus licheniformis DCGGN-121 of swine, where the left side shows the colony morphology and the right side shows the microscopy result;

[0022] Figure 2 This is a phylogenetic analysis diagram of porcine Bacillus licheniformis DCGGN-121;

[0023] Figure 3 This is the identification diagram of the anti-ASFV activity of porcine Bacillus licheniformis DCGGN-121;

[0024] Figure 4 It is the SDS-PAGE electrophoresis diagram of bacteriocin produced by swine Bacillus licheniformis DCGGN-121;

[0025] Figure 5 This is a mass spectrometry analysis of bacteriocin produced by swine Bacillus licheniformis DCGGN-121;

[0026] Figure 6 This is a graph showing the effects of bacteriocins produced by swine Bacillus licheniformis DCGGN-121 on the RNA replication and protein expression of ASFV, SVA and EV71, where A represents the RNA replication and protein expression of EV71, B represents the RNA replication and protein expression of SVA, and C represents the RNA replication and protein expression of ASFV.

[0027] Biological Deposit Description

[0028] Bacillus licheniformis DCGGN-121 was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2024, with the deposit number CCTCC NO: M 2024878. The address of the depository unit is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0029] The present invention provides a strain of Bacillus licheniformis DCGGN-121, with a deposit number of CCTCC NO: M 2024878. The 16s rRNA gene sequence of the Bacillus licheniformis DCGGN-121 of the present invention is shown in SEQ ID NO.25.

[0030]

[0031] The invention uses the gastrointestinal contents of fresh pigs as raw materials to separate Bacillus licheniformis DCGGN-121 with broad-spectrum antiviral effect.

[0032] The colony diameter of the Bacillus licheniformis DCGGN-121 on the LB culture medium plate is 2 to 3 mm, and the colony is white with irregular edges, a rough surface and wrinkles.

[0033] The present invention uses the 16s rRNA sequence of Bacillus licheniformis DCGGN-121 to perform BLAST homologous sequence search in GenBank of NCBI, constructs an evolutionary tree with MEGA8 software, and combines morphological characteristics to identify the strain DCGGN-121 as Bacillus licheniformis. The Bacillus licheniformis DCGGN-121 provided by the present invention is acid-resistant, bile-resistant and high-temperature-resistant, can resist African swine fever virus (ASFV), Seneca virus (SVA) and enteritis virus 71 (EV71 virus), has significant effects, has good antiviral effects, and can use Bacillus licheniformis DCGGN-121 to prepare safe, efficient and green natural antiviral drugs, which are suitable for the fields of food industry, animal husbandry, and protection of human public health.

[0034] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution in the preparation of antibacterial active substances.

[0035] 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. 1. The prepared bacteriocin can resist African swine fever virus (ASFV), Seneca virus (SVA) and enteritis virus 71 (EV71 virus), and the effect is significant.

[0036] The present invention provides a method for preparing antibacterial active substances by using Bacillus licheniformis DCGGN-121 described in the above technical solution, comprising:

[0037] The Bacillus licheniformis DCGGN-121 described in the above technical solution is inoculated into a culture medium for culturing to obtain a cell-free fermentation liquid;

[0038] The cell-free fermentation liquid is mixed with ammonium sulfate and allowed to stand to obtain a crude active substance;

[0039] The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

[0040] As an implementation mode, the present invention inoculates the Bacillus licheniformis DCGGN-121 described in the above technical solution into a culture medium for cultivation to obtain a cell-free fermentation liquid. As an implementation mode, the present invention inoculates the seed liquid of the Bacillus licheniformis DCGGN-121 described in the above technical solution into a culture medium for cultivation, centrifugation and filtration to obtain a cell-free fermentation liquid.

[0041] As an embodiment, the preparation method of the seed liquid of Bacillus licheniformis DCGGN-121 of the present invention can be: inoculate a single colony of the Bacillus licheniformis DCGGN-121 on an LB solid culture medium plate for culturing to obtain the DCGGN-121 seed liquid. The present invention does not specifically limit the parameters of the culture, and conventional methods can be used.

[0042] As an embodiment, the inoculation amount of the seed solution of the present invention can be 1% to 3% of the volume of the culture medium. In a specific embodiment of the present invention, the inoculation amount can be 1%, 2% or 3% of the volume of the culture medium. The culture temperature of the present invention can be 35°C to 40°C; in a specific embodiment of the present invention, the culture temperature can be 35, 36, 37, 38, 39 or 40°C; the culture time can be 22 to 26h, and in a specific embodiment of the present invention, the culture time can be 22h, 23h, 24h, 25h or 26h. The culture medium of the present invention can be LB liquid culture medium, and the composition of the LB liquid culture medium is preferably: 10g peptone, 5g yeast extract, 10g sodium chloride and 1000mL distilled water.

[0043] As an embodiment, after obtaining the culture fluid, the present invention can centrifuge the culture fluid, the centrifugal speed can be 8000 rpm, the centrifugal time can be 30 min, and the centrifugal temperature can be 4°C.

[0044] As an embodiment, a supernatant is obtained after centrifugation, and the present invention filters the supernatant, and the filtration is performed using a filter membrane with a diameter of 0.22 μm. After filtration, a cell-free fermentation broth is obtained, and the function of the filtration is to obtain antibacterial substances in the metabolites. 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.

[0045] The present invention mixes the cell-free fermentation broth with ammonium sulfate and allows it to stand 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; the first stirring can be completed with a stirring rod; the first stirring serves to promote the dissolution of ammonium sulfate. As an embodiment, the second stirring and standing are performed after the ammonium sulfate saturation in the cell fermentation broth reaches 80%, that is, after the addition of ammonium sulfate is completed. The second stirring can be completed using a magnetic stirrer, and the second stirring time can be 1h. The standing temperature can be 4°C, and the standing time can be 12 to 24h, or 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 centrifugal speed can be 10,000rpm, and the time can be 30min. Ammonium sulfate can precipitate and concentrate the protein components in the cell-free fermentation broth.

[0046] 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.

[0047] 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 a protein with a molecular weight between 3kDa and 30kDa is 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, the African swine fever virus (ASFV) CN / GS / 2018 strain can be used to monitor the antiviral activity to ensure that the antibacterial active substances produced by porcine licheniformis DCGGN-121 are successfully separated. The antibacterial active substances prepared by the present invention are the protein components in the cell-free fermentation broth.

[0048] The present invention provides an antibacterial agent, comprising the metabolite of Bacillus licheniformis DCGGN-121 described in the above technical solution.

[0049] In the present invention, as an implementation method, the metabolites of Bacillus licheniformis DCGGN-121 in the antibacterial agent may include a cell-free fermentation broth or an antibacterial active substance prepared by the method described in the above technical solution. The preparation method of the cell-free fermentation broth has been described above and will not be discussed here.

[0050] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution or the antibacterial active substance prepared by the method described in the above technical solution or the antibacterial agent described in the above technical solution in the preparation of antiviral drugs.

[0051] As an implementation method, the virus may include one or more of African swine fever virus, anti-Seneca virus and anti-EV71 virus.

[0052] The present invention provides the use of the Bacillus licheniformis DCGGN-121 described in the above technical solution or the antibacterial active substance prepared by the method described in the above technical solution or the antibacterial agent described in the above technical solution in a microecological preparation.

[0053] As an embodiment, the product form of the probiotic preparation may include feed additives or food additives.

[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-121:

[0056] 1. Sources of materials, strains and cells: Fresh pig gastrointestinal contents were used as samples to screen strains. The samples came from Diebu County, Gannan Prefecture, Gansu Province. The samples were packed in sterile plastic bags and transported to the laboratory at low temperature. Porcine alveolar macrophages PAMs cells, hamster kidney fibroblasts BHK-21 cells, and bovine kidney cells MDBK cells were preserved in this laboratory.

[0057] 2. The indicator strains used in the antiviral experiment were the African swine fever virus (ASFV) CN / GS / 2018 strain, the bovine viral diarrhea virus (BVDV) Oregon C24V strain, and the foot-and-mouth disease virus (FMDV) O-MYA98 strain preserved in our laboratory.

[0058] 3. Culture medium and other biochemical reagents

[0059] LB medium: 10g of peptone, 5g of yeast extract, and 10g of sodium chloride are heated and dissolved in 1000mL of distilled water to obtain LB liquid medium; LB solid medium is prepared by adding 1.5% agar to LB liquid medium. LB medium is sterilized by high pressure at 121℃ for 15min.

[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, and pour into a plate for later use.

[0062] DNA Ligation Kit (Mighty Mix), Taq DNA polymerase, molecular marker, ChamQ Universal qPCR Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; MEM\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 Baoriyi Biological (Beijing) Co., Ltd.; chemiluminescence substrate (enhanced chemiluminescence, ECL) colorimetric solution was purchased from Advansta Company; pre-stained protein marker was purchased from Yazyme Biotechnology, homemade rabbit anti-ASFV-p72, FMDV-VP0, BVDV-Core antibodies were preserved by our laboratory, and HRP goat anti-mouse IgG and HRP goat anti-rabbit IgG were purchased from Beijing Biolong Immunotechnology Co., Ltd.

[0063] Hydrochloric acid, sodium chloride, etc. were all domestically produced analytical grade. The bacterial DNA extraction kit was purchased from Beijing Tiangen Biological Co., Ltd., and the Gram staining kit was purchased from Sigma, USA.

[0064] 4. Experimental Methods

[0065] (1) Isolation of porcine licheniformis DCGGN-121: In a clean bench, take the fresh pig gastrointestinal contents, mark them and take about 1g of each into sterile test tubes, add 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, wait for it to cool, take 100μL and spread it on an LB solid plate, invert it in a 37℃ incubator and culture it for 24h, forming white colonies with a diameter of 2-3mm, with irregular edges, rough surface and wrinkles. Pick a single colony and continue to culture it in LB liquid, repeat the plate streaking until a single colony is obtained.

[0066] (2) Preliminary screening of strains: Resuscitation strains: The strains obtained in step (1) were inoculated into LB liquid culture medium and cultured in a shaking incubator at 37°C until OD 600 was 0.8, and bacterial liquid of each strain was obtained.

[0067] A: Acid resistance screening: prepare LB liquid culture medium with pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, and 6.8 respectively.

[0068] The bacterial suspension of each strain was transferred to 5 mL of culture medium with different pH values ​​at a ratio of 5% of the culture medium volume, and a normal LB liquid culture medium with a pH of 6.8 was used as a blank control.

[0069] After transfer, each culture medium was placed in a 37°C incubator for 2 h, and then 100 μL of the obtained culture solution was spread on a glucose tryptone medium plate. After inverted culture for 24 h, the plate was counted and the survival rate of each strain was calculated. Strains with strong acid resistance that could survive for 2 h in LB liquid culture medium at pH 2.0 and had a survival rate of more than 35% 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% of ox bile salt mass concentration. Take the bacterial liquid of each acid-resistant strain and transfer it to 5mL LB liquid culture medium with different bile salt concentrations at a ratio of 1% (v / v) of the culture medium volume, incubate at 37°C for 12h, take 100μL of the obtained culture liquid and spread it on LB solid plate, incubate for 24h, count the plate, calculate the survival rate of the strain, and preliminarily screen the strains with strong bile salt resistance with a survival rate of more than 50% after being treated in 0.3% bile salt medium for 12h;

[0071] C: High temperature resistance screening: The obtained acid-resistant and bile-resistant strains were inoculated into LB liquid culture medium respectively, and cultured overnight at 37°C to obtain bacterial liquid. The obtained bacterial liquid was placed in a metal bath at 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and 100°C for 10 minutes. After the sample was cooled after treatment, 100 μL of bacterial liquid was taken for plate coating. The bacterial liquid that was not subjected to high temperature treatment was used as a blank control. After 24 hours of culture, the plate counts were performed for the high temperature treatment group and the control group, and the strains with a survival rate of more than 50% were preliminarily screened after being treated at 85°C for 10 minutes.

[0072] After preliminary screening, a strain that is resistant to acid, bile salts and high temperatures was obtained and named DCGGN-121. 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 colony of Bacillus licheniformis and Gram staining microscopy (1000×) showed that strain DCGGN-121 was a Gram-like bacillus.

[0073] (3) Activation, identification and preservation of strains: A single colony of strain DCGGN-121 was selected and transferred to 5 mL of LB liquid culture medium and cultured at 37°C for 48 h. The culture was then subcultured twice with an inoculum volume of 1% (v / v) and preserved after activation.

[0074] The strain DCGGN-121 to be identified was amplified by PCR using primers Bli-man-1F: 5'-AGCCATGGATATTAAATAAC-3' (SEQ ID NO: 21); Bli-man-1R: 5'-TATTCCCTTACAATAAGACG-3' (SEQ ID NO: 22) and 16SrRNA primers: 27F-AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 23), 1492R-TACGGTYACCTTGTTGTTACGACTT (SEQ ID NO: 24), the obtained products were sequenced, the sequences were uploaded to NCBI blast for comparison, and the evolutionary tree was made using the software MEGA8, such as Figure 2 shown. Figure 2 This is the phylogenetic analysis diagram of Bacillus licheniformis DCGGN-121. Figure 2 WBL 009 in represents DCGGN-121. Figure 2 It can be seen that strain DCGGN-121 (ie, WBL 009) is Bacillus licheniformis, which has high similarity with Bacillus licheniformis SR5 strain in the GenBank database and is on the same evolutionary branch.

[0075] (4) Cultivation of indicator virus: PAMs cells were plated in 12-well plates. When the cell density reached 70% to 80%, ASFV cells were infected with a multiplicity of infection (MOI) of 1. ASFV cell samples were collected 24 hours after virus infection and recorded as the first-generation seed virus. The fourth-generation ASFV seed virus was obtained after expansion with PAMs cells, with a titer of 10 6 HAD 50 / 100μL, store at 80℃ for later use.

[0076] Using the same method as ASFV seed virus culture, FMDV was propagated in BHK-21 cells to obtain the fourth generation FMDV seed virus with a titer of 10 6 TCID 50 / mL, store at 80℃ for later use.

[0077] Using the same method as ASFV seed virus culture, BVDV was propagated in MDBK cells to obtain the fourth generation BVDV seed virus with a titer of 10 5 TCID 50 / mL, store at 80℃ for later use.

[0078] Identification of the antiviral activity of Bacillus licheniformis DCGGN-121: A single colony of porcine Bacillus licheniformis DCGGN-121 was transferred to 5 mL and inoculated into LB liquid culture medium and cultured at 37°C for 48 h. It was then inoculated into 1 L of LB liquid culture medium at 1% (v / v). The bacterial solution cultured for 24 h was centrifuged at 8000 rpm and 4°C for 30 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain a cell-free fermentation broth.

[0079] PAMs cells, BHK-21 cells, and MDBK cells were respectively plated in 12-well plates. When the cell density reached 70% to 80%, the cells were infected with a virus at a multiplicity of infection (MOI) of 1 (the 4th generation ASFV seed virus infected PAMs cells, the 4th generation FMDV seed virus infected BHK-21 cells, and the 4th generation BVDV seed virus infected MDBK cells). At the same time, 200 μL of the above-mentioned cell-free fermentation liquid was added to the cell culture medium. Cell samples were collected 24 hours after virus infection, and the effects on the replication of the above-mentioned viruses were analyzed by fluorescent quantitative PCR (primers see Table 1) and Western blotting. It was found that porcine licheniformis DCGGN-121 had anti-ASFV activity.

[0080] Example 2 Isolation, Identification and Antiviral Study of Bacteriocins Produced by Swine Bacillus Licheniformis DCGGN-121:

[0081] 1. Sources of materials, strains and cells: The cell-free fermentation broth isolated in Example 1 was used, and the indicator bacteria for the antibacterial activity detection in the following experiments were all African swine fever virus (ASFV) CN / GS / 2018 strains, and antiviral activity monitoring was performed after each purification step to ensure the successful separation of bacteriocins produced by porcine Bacillus licheniformis DCGGN-121. PAMs cells, BHK-21 cells, MDBK cells, and HEK293T cells were preserved in this laboratory.

[0082] 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-MYA98 strain, enteritis virus 71 (EV-71), and Seneca virus (SVA) CH-FJ-2017 strain preserved in our laboratory.

[0083] 2. Preparation of culture medium, other biochemical reagents and related liquids

[0084] DMEM cell culture medium, PBS solution, 0.25% trypsin-EDTA and newborn bovine serum (FBS) were purchased from BI Company; Trizol reagent was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; ChamQ Universal qPCR MasterMix was purchased from Novozyme Biotech Co., Ltd.; enhanced chemiluminescence (ECL) colorimetric solution was purchased from Advansta; prestained protein marker was purchased from Yazyme Biotech;

[0085] HiTrap Capto S prepacked columns were purchased from Cytiva, Sweden; ultrafiltration tubes were purchased from Merck Millipore;

[0086] 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-ASFV-p72, FMDV-VP0, BVDV-Core, SVA-VP2, and EV71-VP1 antibodies were preserved by our laboratory. HRP goat anti-mouse IgG and HRP goat anti-rabbit IgG were purchased from Beijing Biolong Immunotechnology Co., Ltd.

[0087] The reagents involved in the embodiment are prepared as follows:

[0088] 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 remove particles through filter paper for storage.

[0089] Decolorizing solution: Add 100 mL of glacial acetic acid, 50 mL of ethanol and 850 mL of double distilled water in sequence.

[0090] 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.

[0091] 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.

[0092] 3. Experimental Methods

[0093] (1) Preparation of cell-free fermentation broth of porcine Bacillus licheniformis DCGGN-121: The DCGGN-121 strain stored in a -80°C glycerol tube was streaked on an LB solid culture medium plate for activation. After activation and culture at 37°C for 24 h, a single colony was picked and transferred to 5 mL of LB liquid culture medium and inoculated into the culture medium at 37°C for 48 h. The culture medium was inoculated into 1 L of LB liquid culture medium at 1% (v / v) and cultured for 24 h. The resulting bacterial solution 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 a cell-free fermentation broth.

[0094] (2) Preparation of crude antiviral extract of porcine Bacillus licheniformis DCGGN-121: The cell-free fermentation liquid obtained in step (1) was slowly added with stirring to ground ammonium sulfate powder to make the saturation of ammonium sulfate reach 80%, and the mixture was slowly stirred on a magnetic stirrer for 1 hour, and then placed in a 4°C refrigerator overnight; the mixture was taken out the next day, and centrifuged at 10,000 rpm for 30 minutes 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 crude active substance to dissolve the precipitate, and the precipitate was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the dialysis bag was placed in 2 L of PBS buffer for overnight dialysis to remove the ammonium sulfate and obtain a crude antiviral extract.

[0095] (3) Purification of the antiviral crude extract of porcine Bacillus licheniformis DCGGN-121: The antiviral crude extract obtained in step (2) was centrifuged with ultrafiltration tubes with a molecular weight cutoff of 3 kDa and 30 kDa, and the retained protein was re-dissolved with PBS and filtered with a 0.22 microporous membrane to obtain the protein. The obtained protein was used to monitor the antiviral activity (ASFV), and the method was the same as the "Identification of Antiviral Activity of Bacillus licheniformis DCGGN-121" in Example 1. The 200 μL cell-free fermentation broth added to the infected cells was replaced with the protein. The results showed that the protein could resist ASFV virus.

[0096] (4) Select the protein sample with antiviral activity in step (3) for ion exchange column purification: first equilibrate the pre-loaded column with buffer A, load the sample, rinse with buffer A and collect the flow-through, collect flow-through 1; then use buffer A and buffer B for gradient elution, the NaCl molar concentration in the eluent during gradient elution is 20mM, 40mM, 80mM, 100mM, 200mM (wherein 20mM elution, elution 1 and elution 2 are collected segmentally, 40mM elution, B solution elution 1 and B solution elution 2 are collected segmentally, 80mM elution, B solution elution is collected segmentally, 100mM elution, B solution elution is collected segmentally, and 200mM elution, B solution elution is collected segmentally). The results show that when the NaCl molar concentration in the eluent is 100mM, the collected protein sample has anti-ASFV activity, and the target protein eluted with 100mM NaCl B buffer is bacteriocin.

[0097] (5) PAMs cells were plated in 12-well plates. When the cell density reached 70% to 80%, ASFV was infected at a multiplicity of infection (MOI) of 1. Different concentrations of bacteriocin (0, 25, 100, 500 μg / mL) were added. Cell samples were collected 24 hours after virus infection. The effects of bacteriocin on the replication of the above viruses were analyzed by fluorescence quantitative PCR (primers see Table 1) and Western blotting. The results are shown in Figure 3 It can be seen that bacteriocins have anti-ASFV activity.

[0098] (6) Identification of bacteriocin produced by porcine Bacillus licheniformis DCGGN-121: The protein samples collected above were subjected to SDS-PAGE electrophoresis and the target band was found to be approximately 14Kd. Figure 4 . Figure 4 This is the SDS-PAGE electrophoresis analysis of bacteriocin produced by porcine Bacillus licheniformis DCGGN-121 (1: loading sample; M: Marker; 2: flow-through 1; 3: 20mM B solution elution 1; 4: 20mM B solution elution 2; 5: 40mM B solution elution peak 1; 6: 40mM B solution elution peak 2; 7: 80mM B solution elution; 8: 100mM B elution 1; 9: 100mM B elution 2; 10: 200mM B elution). The loading sample is the protein obtained in step (3).

[0099] according to Figure 4 It can be seen that the target protein (bacteriocin) can be eluted using 100 mM NaCl B buffer, and its size is about 14 Kd.

[0100] (7) The protein concentration of the bacteriocin was determined by BCA, and the amino acid sequence of the purified bacteriocin was determined by LC-MS / MS at Shanghai Baipu Biotechnology Co., Ltd., as shown in SEQ ID NO: 1. The molecular weight of the purified bacteriocin was about 14.2 kDa, and the N-terminal sequence was MKFEESGIEGLTADLNR (SEQ ID NO: 20). The amino acid sequence of the bacteriocin produced by Bacillus licheniformis DCGGN-121 encodes a total of 119 amino acids. Figure 5 Mass spectrometry analysis of bacteriocin produced by porcine Bacillus licheniformis DCGGN-121. Figure 5 This indicates that the corresponding bacteriocin can be identified by the unique peptide corresponding to the N-terminal sequence.

[0101] SEQ ID NO: 1: MKFEESGIEGLTADLNRLDDIMENEGFVRAGQWDYERV TYDRKFDMVEGRFYLRVFGYAIEGDVGARRAVIKLMTPLLGKYYYPHGVEYGEDEHFPPQLLKTSKTVLNSIKEKVSALEV.

[0102] (8) Identification of the antiviral activity of bacteriocins produced by porcine Bacillus licheniformis DCGGN-121: PAMs cells, PK15 cells, BHK-21 cells, MDBK cells, and HEK293T cells were plated in 12-well plates. When the cell density reached 70% to 80%, they were infected with viruses at a multiplicity of infection (MOI) of 1, wherein PAMs cells were infected with ASFV, PK15 cells were infected with SVA, BHK-21 cells were infected with FMDV, MDBK cells were infected with BVDV, and HEK293T cells were infected with EV-71. At the same time, different concentrations of bacteriocins (0, 25, 100, and 500 μg / mL) were added to each infected cell. Cell samples were collected 24 hours after virus infection, and the effects of bacteriocins on the replication of the above viruses were analyzed by fluorescence quantitative PCR (primers see Table 1) and Western blotting. Figure 6 The figures show the effects of bacteriocin produced by swine Bacillus licheniformis DCGGN-121 on RNA replication and protein expression of different viruses, where A is EV71, B is SVA, and C is ASFV. It can be seen that bacteriocin can significantly reduce the replication levels of EV-71, ASFV and SVA, as well as the expression of EV71-VP1, ASFV-p72, and SVA-VP2 proteins, and has significant in vitro activity against ASFV, SVA and EV71 viruses, but has no inhibitory effect on FMDV and BVDV.

[0103] Table 1 Real-time fluorescence quantitative (RT-qPCR) primers

[0104]

[0105]

[0106] In summary, the Bacillus licheniformis DCGGN-121 provided by the present invention can produce bacteriocin, and the molecular weight of the bacteriocin produced by Bacillus licheniformis DCGGN-121 is 14280Da; the bacteriocin has a broad-spectrum antiviral effect, and also has the effects of resisting African swine fever virus (ASFV), Seneca virus (SVA) and EV71 virus. It can be seen that Bacillus licheniformis DCGGN-121 can be used to prepare a safe, efficient and green natural microecological preparation, which is suitable for the fields of food industry, animal husbandry, and protection of human public health.

[0107] 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-121, with a deposit number of CCTCC NO: M2024878.

2. Use of the Bacillus licheniformis DCGGN-121 according to claim 1 in the preparation of antibacterial active substances.

3. The use according to claim 2, characterized in that: The antibacterial active substance includes bacteriocin; the amino acid sequence of the bacteriocin is shown in SEQ ID NO.

1.

4. A method for preparing antibacterial active substances using the Bacillus licheniformis DCGGN-121 according to claim 1, characterized in that: include: The Bacillus licheniformis DCGGN-121 is inoculated into a culture medium for culturing to obtain a cell-free fermentation liquid; The cell-free fermentation liquid is mixed with ammonium sulfate and allowed to stand to obtain a crude active substance; The crude active substance is ultrafiltered and purified to obtain antibacterial active substance.

5. An antibacterial agent, characterized in that The invention relates to a metabolite of Bacillus licheniformis DCGGN-121 according to claim 1.

6. The antibacterial agent according to claim 5, characterized in that: The metabolites include cell-free fermentation broth or antibacterial active substances prepared by the method according to claim 4.

7. Use of the Bacillus licheniformis DCGGN-121 according to claim 1 or the antibacterial active substance prepared by the preparation method according to claim 4 or the antibacterial agent according to claim 5 or 6 in the preparation of antiviral drugs.

8. The use according to claim 7, characterized in that: The antiviral agent includes one or more of anti-African swine fever virus, anti-Seneca virus and anti-EV71 virus.

9. Use of the antibacterial active substance prepared by the Bacillus licheniformis DCGGN-121 of claim 1 or the preparation method of claim 4 or the antibacterial agent of claim 5 or 6 in the preparation of a microecological preparation.

10. The use according to claim 9, characterized in that: The product form of the probiotic preparation includes feed additives or food additives.

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