Lipopeptide mixture with antibacterial and antiviral activity and application thereof

Abstract

The invention relates to a lipopeptide mixture with broad-spectrum antibacterial activity, and belongs to the technical field of microorganisms. The lipopeptide mixture is prepared from a derivative of C13-19 surfactin A and a derivative of C17 surfactin A, and the lipopeptide mixture is prepared from a derivative of Bacillus pumilus FJAT-56819, and the derivative of C13-19 surfactin A and a derivative of C17 surfactin A is prepared from Bacillus pumilus FJAT-56819. The lipopeptide mixture prepared by the preparation method disclosed by the invention has a relatively strong inhibition effect on dairy cow mastitis staphylococcus aureus and duck viruses, and has a relatively good production and application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus brevis and an antibacterial and antiviral lipopeptide mixture produced by the strain, and application of the strain in livestock and poultry breeding. Background Art

[0002] Cow mastitis is a common disease in the dairy farming industry. It is one of the important factors restricting the healthy breeding of dairy cows and has caused huge economic losses to the dairy farming industry. Cow mastitis is usually caused by infection with one or more pathogenic microorganisms, among which Staphylococcus aureus is one of the most important pathogens. Staphylococcus aureus is very easy to develop drug resistance, and cow mastitis infected by it often shows subclinical symptoms, which is difficult to prevent and control. In production, most antibiotics are used for treatment. However, excessive use of antibiotics leads to problems such as drug residues and bacterial resistance. There is an urgent need to develop new drugs.

[0003] Currently, duck viral diseases are mainly prevented by vaccines and treated with Western medicines. However, vaccines have limitations in practical applications, and long-term use of chemical antiviral drugs can cause animal poisoning, drug residues, viral resistance or mutation, etc. Moreover, chemical drugs such as ribavirin, amantadine, and morphine biguanide have been banned in my country. Natural products are rich in sources, diverse in structure, and novel in mechanism of action. Finding natural antiviral compounds from plants and microorganisms for the prevention and treatment of duck viral diseases is an effective way.

[0004] Lipopeptide compounds are important antibacterial active substances produced by Bacillus, which can resist bacteria, fungi, viruses, mycoplasma and tumors, etc. They have the advantages of high yield, simple production process, good stability, not easy to produce drug resistance, low toxicity to humans and animals, and environmental friendliness. Therefore, lipopeptide compounds can be used as a substitute for chemical drugs and have great research value and application potential in the field of aquaculture.

[0005] The types and characteristics of viruses that infect livestock and poultry are diverse, and the transmission routes are also diverse, which determines that livestock and poultry prevention and control measures must be targeted. Muscovy duck parvovirus (MPV) is a member of the Parvoviridae family and the Parvovirus genus. The virus has a diameter of 20 to 24 nm, and the nucleic acid is single-stranded DNA. It is insensitive to acid, enzymes and heat. The virus mainly causes disease in 1- to 3-week-old Muscovy ducklings, characterized by diarrhea, soft feet, and panting, with an incidence rate of 27% to 62%; the case fatality rate is 22% to 43%. Duck short beak syndrome virus is a new type of goose parvovirus or goose parvovirus variant of the Parvoviridae genus, which is different from the classic goose parvovirus and other waterfowl parvoviruses. The disease is characterized by short beak and growth disorder, which mostly occurs at 7 to 40 days of age, with an incidence rate of 10% to 100%, a mortality rate of 2% to 10%, and a rate of culling or crippled ducks of 20% to 80%. Before being marketed, the weight of ducks that survived the disease was about 1 kg lower than that of healthy ducks on average, causing serious economic losses to the duck industry. At present, these two types of viral diseases of Muscovy ducks are mainly humoral immunity, and maternal antibodies can provide passive protection for young Muscovy ducklings. Vaccines are used for breeding ducks in large-scale Muscovy duck farms, but some duck farms often have unsatisfactory vaccine immunization effects or immune failure. Therefore, lipopeptides can effectively prevent and treat Muscovy duck virus diseases, which can provide a new drug for the prevention and treatment of Muscovy duck parvovirus disease. The reported antiviral application of lipopeptide surfactin is only for pig, chicken and canine viruses, including porcine parvovirus, porcine epidemic diarrhea virus, Newcastle disease virus and pseudorabies virus. The lipid composition of the virus envelope is different, and the sensitivity to surfactin is also different. So far, there have been no reports on the use of Bacillus lipopeptides to prevent and treat duck viruses. In addition, the lipopeptides produced by Bacillus are diverse in structure, and the lipopeptide composition of different strains is significantly different. Therefore, screening out drugs against Muscovy duck parvovirus from Bacillus lipopeptides has important application value for the prevention and treatment of duck virus diseases. Summary of the invention

[0006] In order to solve the problem of preventing and treating cow mastitis and duck viral diseases, the inventors provided a strain of Bacillus and a mixture of antibacterial and antiviral lipopeptides produced by the strain. The technical solution is as follows:

[0007] A lipopeptide mixture with antibacterial and antiviral activity, the lipopeptide mixture is composed of C 13-19 surfactin A and C 17 The composition is composed of surfactinA derivatives.

[0008] The lipopeptide mixture is produced by Bacillus pumilus strain FJAT-56819. The Bacillus pumilus strain FJAT-56819 was deposited in the China Center for Type Culture Collection on September 9, 2024, with a deposit number of CCTCC NO.M20241940, and the deposit address is: Wuhan University, Wuhan, China.

[0009] The preparation method of the lipopeptide mixture comprises the following steps:

[0010] (1) Activation of strain FJAT-56819: The Bacillus pumilus strain FJAT-56819 was streaked onto NA medium using an inoculation loop and cultured in a constant temperature incubator for 36-60 h at a temperature of 25-35° C. The components of the NA medium are: 0.2-0.5% beef extract, 0.2-0.8% peptone, 0.5-1.5% glucose, 1.5-2.0% agar, prepared with water, pH 7.0-7.2, and the percentages of the components in the medium are by weight;

[0011] (2) Preparation of seed solution: A single colony of the Bacillus pumilus strain FJAT-56819 obtained in step (1) is inoculated into a flask containing PDB medium, and the flask is placed in a constant temperature shaker for shaking culture at a speed of 150-200 rpm and a temperature set at 25-35° C. After culturing for 20-30 hours, the seed solution is obtained; the PDB medium comprises: 0.2-0.8% potato extract powder, 0.5-1.5% peptone, 0.2-0.8% sodium chloride, and 1.0-2.0% glucose, prepared with water, with a pH of 7.0-7.2, and the percentages in the medium components are all weight ratios;

[0012] (3) Preparation of fermentation broth: The seed liquid of Bacillus pumilus strain FJAT-56819 obtained in step (2) was inoculated into sterilized PDB medium at an inoculum size of 0.8-1.2%, a stirring rate of 150-200 rpm, and a temperature of 25-35° C. After culturing for 36-60 h, the bacterial concentration was measured to be 4.0×10 8 -6.0×10 8 CFU / mL, the desired fermentation broth is obtained;

[0013] (4) Preparation of a lipopeptide mixture: centrifuge the fermentation broth obtained in step (3) at a speed of 8000-10000 r / min; discard the cells to obtain a supernatant after centrifugation, add 1.5-2.5 mol / L hydrochloric acid to the supernatant until the pH is less than 2, let stand at 2-8°C for 15-30 hours, and centrifuge to obtain a precipitate; dry the precipitate using low-temperature vacuum freeze drying to obtain a crude lipopeptide powder; add methanol to the powder at a mass ratio of 1:(40-60) for extraction by vibration, and centrifuge to obtain a supernatant; after rotary evaporation of the supernatant, add 3-10 mL of water, and dry the precipitate using low-temperature vacuum freeze drying to obtain a lipopeptide mixture powder.

[0014] Furthermore, the lipopeptide mixture is used in the prevention and treatment of cow mastitis.

[0015] Furthermore, the application of the lipopeptide mixture in the prevention and treatment of viral diseases in ducks is as follows: dissolving the lipopeptide mixture in drinking water at a concentration of 0.02 to 0.325 mg / mL and giving it to ducks to drink directly; or mixing the lipopeptide mixture in feed and adding it at a mass percentage of 0.2% to 3% and giving it to ducks to eat.

[0016] The beneficial effects of the present invention are:

[0017] (1) The present invention is the first to obtain a Bacillus pumilus strain that has an inhibitory effect on Staphylococcus aureus, a pathogenic bacterium of dairy cow mastitis, and can be used for the prevention and treatment of dairy cow mastitis.

[0018] (2) The lipopeptide mixture obtained by the present invention has antiviral activity and has a strong inhibitory effect on Muscovy duck parvovirus (MPV) and short-beaked duck dwarf syndrome virus (SBDSV), and can be used for the prevention and treatment of duck viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention relates to the inhibitory effect of Bacillus FJAT-56819 and its supernatant on Staphylococcus aureus described in the specific implementation manner.

[0020] Figure 2 This is a circle map of the genome of strain FJAT-56819 described in the specific implementation method.

[0021] Figure 3 The present invention is a specific embodiment of the present invention showing the inhibitory effect of the Bacillus pumilus FAJT-56819 lipopeptide on Staphylococcus aureus.

[0022] Figure 4 The hemolytic activity assay of the Bacillus pumilus FJAT-56819 lipopeptide described in the specific implementation method.

[0023] Figure 5 The invention relates to the effect of the Bacillus pumilus FJAT-56819 lipopeptide described in the specific implementation manner on the normal growth of duck embryo fibroblast cell line.

[0024] Figure 6 The present invention is a specific embodiment of the present invention, which shows the inhibitory effect of the Bacillus pumilus FJAT-56819 lipopeptide on the replication of Muscovy duck parvovirus (MPV).

[0025] Figure 7 The present invention is a specific embodiment of the inhibitory effect of the Bacillus pumilus FJAT-56819 lipopeptide on the replication of duck short-beak dwarf syndrome virus (SBDSV).

[0026] Figure 8 This is the effect of different concentrations of lipopeptides on the expression of viral mRNA in DEF cells as described in the specific implementation method. DETAILED DESCRIPTION

[0027] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0028] Example 1

[0029] Bacillus pumilus FJAT-56819 was isolated and screened from rapeseed root soil in Wuwei City, Gansu Province, and had good biological control effect.

[0030] 1. Isolation of strains

[0031] (1) Weigh 10 g of soil sample and add it to 90 mL of sterile water to make a concentration of 10 -1 The mother liquor was diluted to 10 -2 Up to 10 -4 , take 100 μL of the dilution and spread it on the NA medium plate, and spread 3 plates for each concentration. Place the plate in a 30℃ constant temperature box and culture for 2-3 days, pick a single colony and streak it on the NA plate, and culture it at 30℃.

[0032] (2) Pick a single colony obtained by culturing in step (1) for smearing, stain with alkaline fuchsin and examine under a microscope, select a strain capable of producing spores and transfer it to a slant medium for storage as the test strain, then streak the test strain on a NA solid plate, and place the NA solid plate in a constant temperature incubator at 30° C. for 48 h; wherein the components of the NA liquid medium are: 0.5% peptone, 0.3% beef extract, 1.0% glucose, 1.8% agar, prepared with distilled water, pH 7.2, and the percentages in the components are all weight ratios.

[0033] (3) Using dairy bovine Staphylococcus aureus FJAT-2544 (preserved in Fujian Bacillus Collection Center) as an indicator bacterium, the strains to be tested collected on the plate in step (2) were screened by the double-layer plate method. The specific operation was as follows: Staphylococcus aureus was cultured in LB liquid medium (5g / L yeast powder 0.5%, peptone 1.0%, NaCl 1.0%, prepared with distilled water, pH value 7.0-7.2), the stirring rate was 170rpm, the temperature was set to 30°C, and after 48h of culture, the Staphylococcus aureus bacterial liquid was added to the melted LB semi-solid medium at a ratio of 1%, shaken well, and poured onto the pre-solidified LB solid medium (1.8% agar). Use a sterile inoculation loop to dip the fresh bacillus colony on the solid plate and streak it on the surface of the double-layer plate. Then, the plate was placed in a 30°C constant temperature incubator and cultured for 24-48h, thereby screening out strains with antibacterial effects on Staphylococcus aureus.

[0034] (4) Using Staphylococcus aureus as the indicator bacteria, the antibacterial zone method was used to further screen the strains to be tested collected on the plate in step (3). The specific operation was as follows: the Bacillus strain to be screened was cultured in LB liquid medium, and the fermentation liquid was obtained after 48 hours of culture. The fermentation liquid was centrifuged to obtain the supernatant. Staphylococcus aureus was cultured in LB liquid medium, with a stirring rate of 170 rpm and a temperature set to 30°C. After 48 hours of culture, the pathogen was cultured at 30°C and 170 r / min for 2 days, and then the pathogen was evenly spread on the solid culture medium. Then, a hole with a diameter of 7 mm was made on the plate, 80 μL of fermentation liquid and fermentation supernatant were added, and a sterile aqueous solution was used as a blank control. The plate was placed in a 30°C incubator for 24 to 48 hours. The plate was photographed with a culture dish intelligent camera and the diameter of the antibacterial zone was measured.

[0035] A Bacillus strain with good inhibitory effect on Staphylococcus aureus was screened and obtained ( Figure 1 ), the strain was named Bacillus strain FJAT-56819. The diameters of the inhibition zones of the fermentation broth and supernatant of the strain were 25.68±0.47 mm and 21.66±0.55 mm, respectively.

[0036] 2. Identification of strains

[0037] DNA of strain FJAT-56819 was extracted and sequenced by Nanopore PromethION and IlluminaNovaSeqPE150 to obtain the whole genome data of FJAT-56819 strain. The gene bank number is SAMN45898837. The genome circle map is as follows Figure 2 The G+C content of the strain was 41.89%. The ANI value of strain FJAT-56819 and Bacillus pumilus model strain CBMB205 was calculated by ChunLab's online Average Nucleotide Identity (ANI) calculator to be 95.76%, which exceeded the Bacillus species classification threshold (95%). Therefore, strain FJAT-56819 was identified as Bacillus pumilus FJAT-56819.

[0038] Example 2

[0039] 1. Preparation of Lipopeptide Mixture

[0040] (1) Activation of Bacillus pumilus strain FJAT-56819: Bacillus pumilus strain FJAT-56819 was streaked onto the strain's NA medium using an inoculation loop and cultured in a constant temperature incubator for 48 h at a temperature of 30° C. The components of the NA medium are: 0.3% beef extract, 0.5% peptone, 1.0% glucose, 1.8% agar, prepared with water, pH 7.0-7.2, and the percentages in the medium components are weight ratios.

[0041] (2) Preparation of seed solution: A single colony of the Bacillus pumilus strain FJAT-56819 obtained in step (1) was inoculated into a flask containing seed culture medium (bottled volume: 50 mL / 250 mL flask), and the flask was placed in a constant temperature shaker for shaking culture at a speed of 170 rpm and a temperature set to 30° C. After culturing for 24 h, the seed solution was obtained.

[0042] (3) Preparation of fermentation broth: The seed liquid of Bacillus pumilus strain FJAT-56819 obtained in step (2) was inoculated into PDB liquid medium (potato dextrose nutrient broth) with an inoculum size of 1%, a stirring rate of 170 rpm, and a temperature of 30°C. After culturing for 48 h, the cell concentration was measured to be 5.0 × 10 8 CFU / mL, the desired fermentation broth is obtained.

[0043] Wherein, the seed culture medium in step (2) and the fermentation culture medium in step (3) are both PDB culture medium, whose components are: 0.5% potato extract powder, 1% peptone, 0.5% sodium chloride, 1.5% glucose, prepared with water, pH 7.0-7.2; the percentages in the culture medium components are all weight ratios.

[0044] (4) Preparation of lipopeptide mixture: The fermentation liquid obtained in step (3) is centrifuged at a speed of 9000 r / min; after centrifugation, the bacteria are discarded to obtain the supernatant, 2 mol / L hydrochloric acid is added to the supernatant until the pH is <2, and the mixture is allowed to stand at 4°C for 24 hours, and then centrifuged to obtain a precipitate; the precipitate is dried by low-temperature vacuum freeze drying to obtain a crude lipopeptide powder. Methanol is added to the powder at a mass ratio of 1:50 for vibration extraction, and the supernatant is obtained by centrifugation. After the supernatant is rotary evaporated, an appropriate amount of water is added, and the precipitate is dried by low-temperature vacuum freeze drying to obtain a lipopeptide mixture powder.

[0045] 2. Plate antagonism test of lipopeptides against Staphylococcus aureus

[0046] (1) Test materials

[0047] The pathogen (the pathogen of bovine mastitis: Staphylococcus aureus FJAT-2544) was cultured on the plate for 2-7 days in advance.

[0048] (2) Test methods

[0049] The inhibition zone method was used to determine the inhibitory effect of different concentrations of Bacillus pumilus FJAT-56819 lipopeptides on Staphylococcus aureus. After the pathogen was cultured at 30°C and 170r / min for 2 days, the pathogen was evenly spread on the solid culture medium. Then, a 7mm diameter hole was punched on the plate, and 80μL of the solution of the lipopeptide mixture of different concentrations was injected respectively. The sterile methanol solution was used as the blank control. Each treatment was repeated 3 times. After culturing at 30°C for 2-5 days, the diameter of the inhibition zone was measured.

[0050] (3) Test results

[0051] The diameters of the inhibition zones produced by Bacillus pumilus FJAT-56819 lipopeptide (40 mg / mL and 20 mg / mL) against Staphylococcus aureus FJAT-2544 were 18.27±0.25 mm and 14.94±0.22 mm, respectively. Figure 3 ).

[0052] Example 3

[0053] 1. Determination of the composition of the lipopeptide mixture of strain FJAT-56819

[0054] (1) Test materials

[0055] 30 mg / mL lipopeptide mixture (prepared according to Example 2)

[0056] (2) Test methods

[0057] The lipopeptides produced by strain FJAT-54560 were determined using LC-QTOF-MS / MS technology. The test conditions were as follows:

[0058] Liquid chromatography conditions: Chromatographic column: Agilent ZORBAX Extend-C 18 Chromatographic column (2.1×150 mm, 1.8-Micron), flow rate 0.3 mL / min; mobile phase A is 0.1% formic acid in water; mobile phase B is methanol; elution procedure 0, 60% B; 60 min, 100% B; 65 min, 60% B.

[0059] Mass spectrometry conditions: ESI (+ / -), drying gas temperature 350°C, drying gas flow rate 8 L / min, nebulizer pressure (nebulizer) 30 psig, Fragmentor 175 V, Collision Energy 100 V, Skimmer 65 V, scanning mode auto MS / MS; ion scanning range: 100-3000 m / z.

[0060] (3) Test results

[0061] The lipopeptides produced by strain FJAT-56819 were determined by LC-QTOF-MS / MS technology, and the lipopeptide composition is shown in Table 3. The results showed that the lipopeptides produced by strain FJAT-56819 consisted of C 13-19 surfactin A and C 17 The composition is composed of surfactinA derivatives.

[0062] Table 3 Lipopeptide composition of strain FJAT-56819

[0063]

[0064]

[0065] 2. Determination of hemolytic activity of lipopeptides of strain FJAT-56819

[0066] (1) Experimental methods

[0067] Prepare 2 mg / mL FJAT-56819 lipopeptide, use sterile water as negative control, and 0.7% TritonX-100 as positive control. Soak the drug-sensitive tablet in the solution of lipopeptide, sterile water and 0.7% TritonX-100 for 15 minutes, place it on a blood agar plate, and culture the blood agar plate at 30°C for 48 hours to observe whether a transparent circle appears around the drug-sensitive tablet.

[0068] (2) Experimental results

[0069] Blood agar plate results showed ( Figure 4 ), there was no transparent ring around the drug-sensitive tablets treated with lipopeptide and sterile water, which was γ hemolysis, that is, non-hemolytic. An obvious transparent ring was seen around the drug-sensitive tablets treated with 0.7% TritonX-100, which was β hemolysis. The above experimental results show that 2mg / mL FJAT-56819 lipopeptide is non-hemolytic.

[0070] Example 4

[0071] 1. Effects of strain FJAT-56819 lipopeptide on duck embryo fibroblast cell line

[0072] (1) Experimental Materials

[0073] Lipopeptide mixture (prepared according to the method of Example 2)

[0074] The cell line DEF (SPDC-CCL141) immortalized duck embryo fibroblast cell line was purchased from American type culture collection (ATCC) and stored in the Animal Virus Research Laboratory, Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0075] (2) Experimental methods

[0076] According to 1×10 4 Cells were added to a 96-well plate and placed in a 5% CO 2 Culture in a 37℃ incubator. When the cell density reaches more than 90%, discard the culture medium, wash twice with PBS and discard. Dilute the lipopeptide with maintenance medium at an initial concentration of 1.25 mg / mL in 6 steps of 2-fold dilutions, add 100 μL / well to the cell plate, and set 3 replicates for each concentration. Set up a blank group with only 100 μL PBS and a cell control group with only 100 μL maintenance medium. Place in a 5% CO 2 Culture in a 37°C incubator for 48 h; discard the culture medium and wash twice with PBS.

[0077] The OD value of each well was measured by CCK-8 method: 100 μL of new maintenance medium and 10 μL of CCK-8 solution were added to each well, gently shaken to mix, and placed in a 5% CO 2 The cells were cultured in a 37°C incubator for 2 h, and then the absorbance at 450 nm was measured using an ELISA reader. The cell viability was calculated using the following formula: (Cell viability / % = (OD 样品 -OD 空白 ) / OD 对照 -OD 空白 )×100%, CV), and the modified Koch method was used to calculate the 50% safe concentration of the drug that can cause 50% of the cells to develop pathological changes (50% cytotoxic concentration, CC 50 ) and the maximum safe concentration (maximum no-cytotoxic concentration, MNTC) of the drug that can keep more than 90% of the cells alive.

[0078] (3) Experimental results

[0079] CCK8 results showed ( Figure 5 ), when the lipopeptide concentration was lower than 0.3120 mg / mL, it did not affect the normal growth of immortalized duck embryo fibroblasts.

[0080] 2. Inhibitory effect of strain FJAT-56819 lipopeptide on duck virus

[0081] (1) Experimental Materials

[0082] The cell line DEF (SPDC-CCL141) immortalized duck embryo fibroblast cell line was purchased from American type culture collection (ATCC), Muscovy Duck parvovirus (MPV) and Shortbeak and dwarfism syndrome virus (SBDSV) were stored in the Animal Virus Laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0083] (2) Experimental methods

[0084] After 24 hours of exposure to lipopeptide at a starting concentration of 0.312 mg / ml in 5 gradients of 2-fold dilution, Muscovy duck parvovirus (MPV) and duck short beak dwarf syndrome virus (SBDSV) were infected with 2 μL of virus MOI (Multiplicity of Infection) at 0.2 concentrations in 96-well DEF cells. A virus control group (only virus without lipopeptide) and a normal cell control group (no virus) were set up. Four days after infection, the virus proliferation in the cells was observed by indirect immunofluorescence test.

[0085] Laser confocal fluorescence imaging: Wash the adherent cells to be tested twice with PBS, then cover the cell layer with 4% polymethanol, fix at room temperature for 30 minutes, incubate with broad-spectrum monoclonal antibody of waterfowl parvovirus at room temperature for 30 minutes, and then rinse the cell plate three times with T-PBS. Further, incubate with sheep anti-mouse IFIT diluted with T-PBS 1:100 at room temperature for 30 minutes, then rinse the cell plate three times with T-PBS, add DAPI solution (1×) and incubate at room temperature in the dark for 5 minutes, then rinse the cell plate three times with T-PBS. Finally, image and take pictures in a Keyence laser confocal microscope.

[0086] The relative expression of viral mRNA was detected by fluorescence quantitative PCR method: the drug with a starting concentration of 0.312 mg / ml was diluted 5 times in a 2-fold gradient and acted on 12-well DEF cells for 24 hours, and then two waterfowl parvoviruses (MPV and SBDSV) were inoculated at an MOI of 0.2, and a virus control group (only virus without drug) and a normal cell control group (no virus) were set up. Four days after infection, total cell RNA was extracted for reverse transcription. The above reverse transcription product was diluted to 100 μL and then subjected to fluorescence quantitative PCR test. The reaction system is shown in Table 1:

[0087] Table 1 qPCR reaction system

[0088]

[0089] Use the two-step PCR standard amplification program as shown in Table 2:

[0090] Table 2 Two-step amplification procedure

[0091]

[0092] GAPDH was used as an internal reference, and the mRNA expression level of the virus-infected control group was set to 1.0. The virus mRNA expression levels of the virus-infected cells at each drug concentration were compared with those of the virus-infected control cells at the same time. A total of three independent experiments were performed, and each sample group was repeated three times. The experimental data were subjected to independent sample t-tests using SPSS20.0, and the relative expression of target gene mRNA was expressed as fold change. The results are shown as mean ± standard error (mean ± SE) ( * P<0.05 has statistical difference. ** P<0.01, significant difference *** P<0.001, extremely significant difference).

[0093] (3) Experimental results

[0094] Laser confocal microscopy results ( Figure 6 , Figure 7 , the green fluorescence in the cells is the viral protein, and the control is the normal cell control) showed that under the action of different concentrations of lipopeptides, the number of green fluorescence in the cells decreased. As the concentration of lipopeptides increased, the number of green fluorescence decreased, that is, the antiviral effect was more significant, and the inhibition process was dose-dependent. The above results show that high concentrations of lipopeptides can significantly inhibit the replication of Muscovy duck parvovirus (MPV) and duck short beak dwarf syndrome virus (SBDSV). The relative expression of viral mRNA in each group is shown in Figure 8 As shown, the results showed that there was no significant difference in the viral expression amount under the action of the drug concentration of 0.0195 mg / ml and the infected control. With the increase of lipopeptide concentration, the relative expression of viral mRNA gradually decreased, showing a dose-dependency. The inhibition rate of MPV and SBDSV at a concentration of 0.3120 mg / mL lipopeptide reached up to 80%. The above results indicate that lipopeptides can significantly inhibit the proliferation and expression of viruses.

[0095] In summary, the lipopeptide prepared by the Bacillus pumilus strain FJAT-56819 of the present invention has a strong inhibitory effect on bovine mastitis Staphylococcus aureus and duck virus. The lipopeptide can be dissolved in water and added to livestock and poultry drinking water at a concentration of 0.02-0.325 mg / mL, or the lipopeptide can be added to livestock and poultry feed at a mass percentage of 0.2%-3% and stirred before consumption, which can prevent and treat bovine mastitis and duck viral diseases.

[0096] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. A lipopeptide mixture having antibacterial and antiviral activity, characterized in that: The lipopeptide mixture is composed of C 13- 19 surfactin A and C 17 The composition is composed of surfactinA derivatives.

2. The lipopeptide mixture with antibacterial and antiviral properties according to claim 1, characterized in that: The lipopeptide mixture is produced by Bacillus pumilus strain FJAT-56819; the Bacillus pumilus strain FJAT-56819 was deposited in the China Center for Type Culture Collection on September 9, 2024, with the deposit number CCTCCNO.M20241940, and the deposit address is: Wuhan University, Wuhan, China.

3. The lipopeptide mixture with antibacterial and antiviral properties according to claim 1 or 2, characterized in that: The preparation method of the lipopeptide mixture comprises the following steps: Activation of strains: Bacillus pumilus strain FJAT-56819 was streaked on NA medium and cultured in a constant temperature incubator for 36-60 hours, and the culture temperature was set at 25-35° C. The components of the NA medium are: 0.2-0.5% beef extract, 0.2-0.8% peptone, 0.5-1.5% glucose, 1.5-2.0% agar, prepared with water, pH 7.0-7.2, and the percentages in the medium components are weight ratios; Preparation of seed solution: inoculate a single colony of the Bacillus pumilus strain FJAT-56819 obtained in the previous step into a PDB medium, and place it in a constant temperature shaker for shaking culture at a speed of 150-200 rpm and a temperature set at 25-35° C. After culturing for 20-30 hours, the seed solution is obtained; the PDB medium components are: 0.2-0.8% potato extract powder, 0.5-1.5% peptone, 0.2-0.8% sodium chloride, 1.0-2.0% glucose, prepared with water, pH 7.0-7.2, and the percentages in the medium components are all weight ratios; Preparation of fermentation broth: The seed solution of Bacillus pumilus strain FJAT-56819 obtained in the previous step was inoculated into PDB medium with an inoculum amount of 0.8-1.2%, a stirring rate of 150-200 rpm, and a temperature of 25-35°C. After culturing for 36-60 hours, the measured bacterial concentration was 4.0×10 8 -6.0×10 8 CFU / mL, the desired fermentation broth is obtained; Preparation of lipopeptide mixture: centrifuge the fermentation liquid obtained in the previous step at a speed of 8000-10000 r / min; discard the bacteria to obtain the supernatant after centrifugation, add 1.5-2.5 mol / L hydrochloric acid to the supernatant until the pH is less than 2, let stand at 2-8°C for 15-30 hours, and centrifuge to obtain a precipitate; dry the precipitate by low-temperature vacuum freeze drying to obtain a crude lipopeptide powder; add methanol to the powder at a mass ratio of 1: (40-60) for vibration extraction, centrifuge to obtain a supernatant, rotary evaporate the supernatant, add 3-10 mL of water, dry the precipitate by low-temperature vacuum freeze drying, and obtain a lipopeptide mixture powder.

4. Use of the lipopeptide mixture with antibacterial and antiviral activity as claimed in any one of claims 1 to 3 in the prevention and treatment of bovine mastitis.

5. Use of the lipopeptide mixture with antibacterial and antiviral activity as claimed in any one of claims 1 to 3 in the prevention and treatment of viral diseases in ducks.

6. Use of the lipopeptide mixture with antibacterial and antiviral activity in the prevention and treatment of duck viral diseases according to claim 5, characterized in that: The application method is to dissolve the lipopeptide mixture in drinking water with a concentration of 0.02-0.325 mg / mL and feed it to ducks; or to mix the lipopeptide mixture in feed at a mass percentage of 0.2%-3% and feed it to ducks.

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