CN112661814B - Antibacterial peptide compound and antibacterial peptide extract as well as preparation method and application thereof - Google Patents
Antibacterial peptide compound and antibacterial peptide extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an antibacterial peptide compound which has a structure shown in a formula (I). The invention also provides a preparation method of the extract taking the compound as the main component and application of the extract in the aspects of resisting bacteria and fungi. The compound has the advantages of novel structure, simple preparation process of the extract, low energy consumption and high product purity, and the obtained antibacterial peptide compound and the extract have good antibacterial effect.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and relates to an antibacterial peptide compound and an extract thereof, and a preparation method and application thereof.
Technical Field
In the food, medicine and livestock breeding industries, due to the use of a large amount of traditional antibiotics, a large amount of drug-resistant strains and drug residues are caused, and great threat is caused to human health. Along with the promotion of health consumption consciousness of people, the development of novel natural antibacterial substances which are not easy to generate drug resistance, have no residue and have no potential safety hazard is urgently needed. The natural antibacterial peptide becomes a hotspot of research in recent years and has wide application in food, medicine and livestock breeding industries.
The antibacterial peptide is derived from various organisms and is a component of a natural defense system in the organisms in the nature. The antibacterial peptide is a small molecular polypeptide with various biological activities, is not only used for gram-negative bacteria, but also acts on fungi, insects, certain viruses and tumors. Compared with antibiotics, the antibacterial agent has no harm to normal cells of human and animals, no side effect and no residue, is hopeful to become a new generation antibacterial agent after the national issue of the restriction order, and is the best choice for replacing antibiotics.
Disclosure of Invention
The invention aims to provide a novel antibacterial peptide compound and an antibacterial peptide extract, and provides a preparation method and application in the aspects of resisting bacteria, fungi and the like.
In order to realize the purpose of the invention, the invention provides a novel antibacterial peptide compound A, the structural formula of which is shown as formula (I), the molecular weight of which is 1556Da, and the polypeptide sequence of which is shown as
Leu-Val-Dhb-Orn-Val-Val-Val-Lys-Val-Leu-Lys-Tyr-Leu-Thr-ol
The inventors searched the whole sequence structure of the antibacterial peptide of the present invention with a protein database and did not find the same polypeptide.
The invention also provides an antibacterial peptide extract which takes the compound A as the main component, other active components are analogues of the compound A, the amino acid composition is basically the same as that of the compound in the claim 1, only the difference between individual amino acid and partial modification position exists, and the molecular weight range of the antibacterial peptide analogues is 1542-1602 Da.
The preparation method of the antibacterial peptide extract provided by the invention comprises the following steps:
1) taking Brevibacillus laterosporus CGMCC No.16337 fermentation liquor, adjusting pH to 2.5-3.5, adding filter aid, and filtering to obtain antibacterial peptide filtrate;
2) adjusting the pH of the antibacterial peptide filtrate to 5.5-6.5, adsorbing the filtrate by macroporous resin, and then resolving by a resolving agent to obtain an antibacterial peptide resolving solution;
3) decolorizing the analytic solution by a decolorizing resin column to obtain a decolorized solution;
4) concentrating the decolorized solution to small volume, adjusting pH of the solution to 2.5-3.5, adding precipitant, filtering, collecting precipitate, and dissolving with solvent to obtain solution;
5) concentrating the dissolved solution to obtain antibacterial peptide extract.
The preparation method of the antibacterial peptide A provided by the invention comprises the following steps:
1) taking Brevibacillus laterosporus CGMCC No.16337 fermentation liquor, adjusting pH to 2.5-3.5, adding filter aid, and filtering to obtain antibacterial peptide filtrate;
2) adjusting the pH of the antibacterial peptide filtrate to 5.5-6.5, adsorbing the filtrate by macroporous resin, and then resolving by a resolving agent to obtain an antibacterial peptide resolving solution;
3) decolorizing the analytic solution by a decolorizing resin column to obtain a decolorized solution;
4) concentrating the decolorized solution to small volume, adjusting pH of the solution to 2.5-3.5, adding precipitant, filtering, collecting precipitate, and dissolving with solvent to obtain solution;
5) concentrating the dissolved solution to obtain an antibacterial peptide extract, performing ODS preparative chromatography, performing gradient elution with 0.5-1% trifluoroacetic acid-acetonitrile and 25-35% acetonitrile, performing liquid phase detection, collecting chromatographic peak eluate with peak time of 12.5min, concentrating, and drying to obtain antibacterial peptide A.
The following is further preferable for any of the above production methods:
wherein the resin in the step 2) is any one of XR925CS, XR925J, D312 and HD-2 resin.
Wherein the resolving agent in the step 2) is 70-80% of ethanol, acetone or methanol aqueous solution.
Wherein the decolorizing resin in the step 3) is any one of XRTS-3, TS-4 and D304.
Wherein the precipitator in the step 4) is any one of calcium chloride, sodium sulfate, ammonium sulfate and magnesium sulfate. The addition amount of the precipitant is 5-15% of the volume of the solution.
Wherein the solvent in the step 4) is 70-80% of ethanol, acetone or methanol aqueous solution.
In one embodiment, the antimicrobial activity of antimicrobial peptide compound a and antimicrobial peptide extracts is shown. The compound A and the antibacterial peptide extract have particularly outstanding antibacterial activity on staphylococcus aureus, and also have obvious antibacterial activity on bacillus subtilis, sarcina lutea, bacillus cereus, shigella dysenteriae, escherichia coli, salmonella enteritidis, salmonella paratyphi b, fusarium, wheat root rot, apple rot, rhizoctonia solani, cucumber gray mold and the like, so the compound A and the antibacterial peptide extract can be widely applied to feed additives, preservatives, cosmetics and medical products. In particular, the antibacterial peptide compound A and the antibacterial peptide extract can be mixed with pharmaceutically acceptable carriers to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations and mucosal absorption preparations.
The invention has the beneficial effects that:
the antibacterial peptide compound provided by the invention has the advantages of novel structure, strong activity and definite extract effective components, and can be widely applied to feed additives, preservatives, cosmetics and medical products.
The antibacterial peptide compound and the extract have simple preparation process, low energy consumption and high product purity, effectively remove pigments, polysaccharides, foreign proteins and the like generated in microbial fermentation metabolism, and have remarkable antibacterial and antifungal effects.
Drawings
FIG. 1 HPLC chromatogram of antibacterial peptide fermentation broth
FIG. 2 HPLC chromatogram of antibacterial peptide extract
FIG. 3 mass spectrum of antibacterial peptide extract
FIG. 4 HPLC chromatogram of antimicrobial peptide A
Detailed Description
The following examples and experimental examples are only for confirming the present invention and should not be construed as limiting the present invention. The methods used in the following examples are conventional methods unless otherwise specified.
The Brevibacillus laterosporus CGMCC No.16337 antibacterial peptide fermentation liquid used by the invention is produced by the drug research and development company Limited liability of North China pharmaceutical group. Resins XR925CS, XR925J, HD-2, D312, manufactured by Shanghai-son technologies, Inc.; XRTS-3, XRTS-4, D315, D304, manufactured by Shanghai Sen technology Inc.; the high performance liquid chromatograph is Shimadzu LC-2010A; INVOA 500 model nuclear magnetic resonance spectrometer, Varian Corp; q active (Thermo Fisher), Easy-nLC 1000(Thermo Fisher).
The fermentation medium formula comprises: 20-50g of glucose, 5-20g of glycerol, 2-5g of inorganic ammonium salt, 0.05-0.5g of dipotassium phosphate, 3-10g of yeast powder or corn pulp powder, 5-15g of industrial peptone, 5-10g of sodium chloride, 1-3g of calcium chloride, 1-4g of Val, 1000ml of water and pH 6.0-7.0. Taking the fermentation broth of Brevibacillus laterosporus CGMCC No.16337 with the fermentation time of 12-25 h.
EXAMPLE 1 preparation of antibacterial peptide fermentation broth
1) Slant strain activation: the brevibacillus laterosporus cold-storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 35 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, and culturing at 35 deg.C and 200rpm for 8hr in shake flask to obtain seed solution, shaped Brevibacterium, and thallus concentration of 7 × 107 cfu/ml;
the seed culture medium is prepared as follows: 10g of yeast extract, 15g of corn steep liquor powder, 30g of glucose, 5g of sodium chloride, 5g of potassium dihydrogen phosphate and 1g of calcium carbonate, 1000ml of tap water, 6.0 of PH, 121 ℃, and sterilizing for 30 min;
3) fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by the inoculation amount of 2%.
The fermentation medium was prepared as follows:
30g of glucose, 15g of glycerol, 2g of ammonium chloride, 2g of yeast powder, 5g of industrial peptone, 15g of calcium chloride, 1g of sodium chloride, 10g of dipotassium hydrogen phosphate, 0.5g of Val, 3g of tap water, 1000ml of pH 6.0, 121 ℃, and sterilizing for 30 min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions were 35 degrees, agitation speed was 300rpm, air flow rate was 1:1.2, the tank pressure is 0.05 MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20 hr, FD101 tryptone 0.2g is added into each liter of fermentation liquid, and water-soluble composite antifoaming agent T31 is used to control foam in the foam period, the addition is controlled below 0.5%, and the total fermentation culture time is 25 hr. The titer of fermentation liquor in a tank is 3540ug/ml by HPLC measurement.
EXAMPLE 2 preparation of antimicrobial peptide fermentation broth
1) Activating strains: the brevibacillus laterosporus cold-storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 32 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 32 deg.C and 200rpm for 16hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 5 × 107 cfu/ml;
the seed culture medium is prepared as follows: 20g of yeast extract, 5g of corn steep liquor powder, 20g of glucose, 3g of sodium chloride, 0.1g of potassium dihydrogen phosphate, 2g of calcium carbonate, 1000ml of tap water, 7 of PH (potential of hydrogen) and 121 ℃, and sterilizing for 30 min;
3) fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
20g of glucose, 20g of glycerol, 4g of ammonium sulfate, 10g of yeast powder, 8g of industrial peptone, 2g of calcium chloride, 7g of sodium chloride, 0.05g of dipotassium phosphate, 0.05g of Val 2g, 1000ml of tap water, 7.0 pH (potential of Hydrogen), 121 ℃, and sterilizing for 30 min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions 32 degrees, stirring speed 400rpm, air flow 1:1.2 tank pressure 0.05 MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-12 hr, FD101 tryptone 1g is added to each liter of fermentation liquid, and water-soluble composite antifoaming agent T31 is used to control foam in the foam high-foaming period, the addition is controlled below 0.5%, and the total fermentation culture time is 12 hr. The titer of fermentation liquor in a tank is 3576ug/ml by HPLC measurement.
EXAMPLE 3 preparation of antimicrobial peptide fermentation broth
1) Activating strains: the strain of the Brevibacillus laterosporus cold storage tube is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 8hr to obtain seed solution, shaped Brevibacterium with thallus concentration of 8.4 × 107 cfu/ml;
the seed culture medium is prepared as follows: 5g of yeast extract, 20g of corn steep liquor powder, 20g of glucose, 10g of sodium chloride, 0.5g of potassium dihydrogen phosphate, 3g of calcium carbonate and 1000ml of tap water, wherein the pH value is 6.5 and the temperature is 121 ℃, and the mixture is sterilized for 30 min;
3) seed tank culture: the volume of the tank is 300L, and the filling amount is 150L; the culture condition is 37 ℃, the rotating speed is 40hz, the air flow is 1:1.2, and the tank pressure is 0.05 MPa. Culture medium the same kind of seed bottle culture medium. Sterilizing the first-stage seeding tank culture medium, inoculating the seed solution obtained in step 2), culturing at an inoculum size of 0.3% and 37 deg.C for 16hr to obtain seed solution;
4) fermentation culture:
inoculating the seed solution obtained in the step 3) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
40g of glucose, 10g of glycerol, 3g of ammonium sulfate, 7g of yeast powder, 10g of industrial peptone, 3g of calcium chloride, 7g of sodium chloride, 0.2g of dipotassium hydrogen phosphate, 0.2g of Val 2g, 1000ml of tap water, 6.5 of PH, 121 ℃, and sterilization for 30 min.
5) The fermentation tank culture process comprises the following steps: the volume of the tank is 2T, and the filling amount is 1.2T; culture conditions at 37 degrees, stirring speed at 50hz, air flow rate at 1:1.2 the pot pressure is 0.05 MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20 hr, FD101 tryptone 0.3g is added into each liter of fermentation liquid, and water-soluble composite antifoaming agent T31 is used to control foam in the foam period, the addition is controlled below 0.5%, and the total fermentation culture time is 25 hr. The titer of the fermentation liquor in the tank is 3880ug/ml by HPLC measurement.
Example 4 preparation and analysis of antimicrobial peptide extract and antimicrobial peptide A
Taking 10L of the antibacterial peptide fermentation liquor obtained in the example 1 and a fermentation unit of 3540ug/ml, adjusting the pH value to 3.0, filtering to obtain an antibacterial peptide filtrate, and obtaining an HPLC spectrogram shown in an attached figure 1; adjusting pH of the antibacterial peptide filtrate to 6.0, adsorbing the filtrate with macroporous resin XR925CS at a resin loading of 1.5L and a flow rate of 1.3L/hr, then resolving with 75% ethanol-water at a flow rate of 1.0L/ml, and when antibacterial peptide flows out from the outlet of the resolving solution, serially connecting TS-3 decolorizing resin column for decolorizing at a resin loading of 0.5L and a flow rate of 0.5L/hr to obtain decolorized solution; concentrating the decolorized solution, concentrating under reduced pressure at 50 deg.C to obtain 300ml concentrated solution, adjusting pH of the concentrated solution to 3.0, adding 10% of anhydrous sodium sulfate as precipitant, stirring, filtering to obtain precipitate, dissolving with 200ml 70% ethanol-water, filtering to obtain filtrate, concentrating the filtrate under reduced pressure at 50 deg.C to obtain antibacterial peptide extract 35.39g, with HPLC content 75.40% calculated by antibacterial peptide A and yield 75.38%. The HPLC spectrum is shown in figure 2, and the mass spectrum is shown in figure 3.
Dissolving the antibacterial peptide extract with methanol, performing ODS preparative chromatography gradient elution with acetonitrile-1 ‰ trifluoroacetic acid water solution (25% -35%), collecting chromatographic peak eluate with peak time of 12.5min, concentrating, and drying to obtain antibacterial peptide A with HPLC content of 98.2%, and the HPLC chromatogram is shown in figure 4. High resolution mass spectrum display of [ M + H ] of antibacterial peptide A]+1557.0611Da, combined with MS-MS,1H-NMR、13C-NMR、HMQC、HMBC，1H-1HCOSY, determining the molecular formula of the compound as C77H137N17O16The amino acid sequence is leucine-valine-2-amino-2-butenoic acid-ornithine-valine-lysine-valine-leucine-lysine-tyrosine-leucine-threoninol. The structural formula is as follows:
the secondary mass spectral data are shown in Table 1, the hydrogen spectral data are shown in Table 2, and the carbon spectral data are shown in Table 3.
TABLE 1 Secondary Mass Spectrometry data
| b ion | ΔM | Amino acid residue | y ion | ΔM | Amino acid residue |
| L | 1557.06 | M+H | |||
| 115.09 | 99 | V | / | / | L |
| 214.15 | 83 | Dhb | / | / | V |
| 297.18 | 114 | O | / | / | Dhb |
| 411.26 | 99 | V | / | / | O |
| 510.33 | 99 | V | / | / | V |
| 609.4 | 227 | V+K | / | / | V |
| 836.56 | 99 | V | 849.6 | 99 | V |
| 935.63 | 113 | L | 721.52 | 128 | K |
| 1048.72 | 128 | K | / | / | V |
| 1176.81 | 164 | Y | / | / | L |
| 1340.88 | 113 | L | / | / | K |
| 1453.96 | 103 | Thr(ol) | / | / | Y |
| 1557.06 | M+H | / | / | L | |
| Thr(ol) |
TABLE 2 Hydrogen Spectrum (500MHz, CD3OD) data
TABLE 3 carbon spectra (125MHz, CD)3OD) data
EXAMPLE 5 preparation of antibacterial peptide extract
Taking 12L of the antibacterial peptide fermentation liquor in the example 1, adjusting the pH to 3.2 by a fermentation unit of 3540IU/ml, and filtering to obtain antibacterial peptide filtrate; adjusting pH of the antibacterial peptide filtrate to 6.5, adsorbing the filtrate with macroporous resin XR925J at a resin loading of 1.8L and a flow rate of 1.5L/hr, resolving with 70% acetone-water at a flow rate of 1.0L/ml, and decolorizing with TS-4 decolorizing resin column when antibacterial peptide flows out from the outlet of the resolving solution at a resin loading of 600ml and a flow rate of 0.5L/hr to obtain decolorized solution; concentrating the decolorized solution, concentrating under reduced pressure at 55 deg.C to obtain 350ml concentrated solution, adjusting pH of the concentrated solution to 3.3, adding 10% of precipitant sodium chloride, stirring, filtering to obtain precipitate, dissolving with 250ml of 70% acetone-water, filtering to obtain filtrate, concentrating the filtrate under reduced pressure at 55 deg.C to obtain antibacterial peptide extract with HPLC content of 78.90% and yield of 71.13%.
Example 6 preparation of antibacterial peptide extract
Taking 6.6L of the antibacterial peptide fermentation liquor in the embodiment 2, adjusting the pH to 2.8 by a fermentation unit of 3576IU/ml, and filtering to obtain antibacterial peptide filtrate; adjusting pH of the antibacterial peptide filtrate to 6.5, adsorbing the filtrate with macroporous resin HD-2 with resin content of 1.0L and flow rate of 0.8L/hr, eluting with 75% ethanol-water with flow rate of 0.7L/hr, and decolorizing with TS-3 decolorizing resin column in series when antibacterial peptide flows out from the eluate outlet, wherein the resin content is 400ml and flow rate is 0.3L/hr to obtain decolorized solution; concentrating the decolorized solution, concentrating under reduced pressure at 50 deg.C to obtain 200ml concentrated solution, adjusting pH of the concentrated solution to 2.8, adding 10% ammonium sulfate as precipitant, stirring, filtering to obtain precipitate, dissolving in 100ml 70% acetone-water, filtering to obtain filtrate, concentrating the filtrate under reduced pressure at 50 deg.C to obtain antibacterial peptide extract 25.16g with HPLC content of 74.20% calculated by antibacterial peptide A and yield of 75.97%.
Example 7 preparation of antibacterial peptide extract
Taking 11L of the antibacterial peptide fermentation liquor in the example 2, adjusting the pH to 2.5 by a fermentation unit of 3576IU/ml, and filtering to obtain antibacterial peptide filtrate; adjusting pH of the antibacterial peptide filtrate to 5.5, adsorbing the filtrate with macroporous resin HD-2 at a resin loading of 1500L and a flow rate of 1.2L/hr, desorbing with 70% acetone-water at a flow rate of 1.0L/hr, and decolorizing with a series D304 decolorizing resin column when antibacterial peptide flows out from the outlet of the desorption solution at a resin loading of 500ml and a flow rate of 0.5L/hr to obtain decolorized solution; concentrating the decolorized solution, concentrating under reduced pressure at 50 deg.C to obtain 280ml concentrated solution, adjusting pH of the concentrated solution to 3.2, adding precipitant calcium chloride 15% of the volume of the solution, stirring, filtering to obtain precipitate, dissolving with 120ml 80% methanol-water, filtering to obtain filtrate, concentrating the filtrate under reduced pressure at 50 deg.C to obtain antibacterial peptide extract 39.96g, with HPLC content of 72.10% calculated by antibacterial peptide A and yield of 73.24%.
EXAMPLE 8 preparation of antibacterial peptide extract
Taking 300L of the antibacterial peptide fermentation liquor obtained in the embodiment 3, wherein the fermentation unit is 3880IU/ml, adjusting the pH value to 3.5, and filtering to obtain antibacterial peptide filtrate; adjusting pH of the antibacterial peptide filtrate to 6.0, adsorbing the filtrate with macroporous resin XR925CS, loading the resin at a flow rate of 35L/hr, eluting with 80% ethanol-water at a flow rate of 30L/hr, and decolorizing with TS-3 decolorizing resin column at an outlet of the eluate with antibacterial peptide flow out, loading the resin at a flow rate of 15L/hr to obtain decolorized solution; concentrating the decolorized solution at 50 deg.C, concentrating under reduced pressure to obtain 8000ml concentrated solution, adjusting pH of the concentrated solution to 2.8, adding 8% sodium sulfate as precipitant, stirring, filtering, collecting precipitate, dissolving with 3200ml 70% ethanol-water, filtering to obtain filtrate, concentrating the filtrate at 50 deg.C under reduced pressure to obtain antibacterial peptide extract with HPLC content of 76.50% and yield of 73.94%.
Experimental example 9 measurement of antibacterial Activity
The antibacterial activity was measured by the Minimum (MIC) method as follows:
1) sample group: respectively adding samples to be detected into a 96-well plate, and respectively adding 105 bacterial suspensions/ml into corresponding holes, wherein the total volume is 200 mu l/hole.
2) Detection of bacteria control group: and respectively adding solvents with the same volume as the sample to be detected in the sample group into a 96-well plate, and respectively adding 105 bacterial suspensions per ml into corresponding holes, wherein the total volume is 200 mu l per hole.
3) Medium blank control group: 200. mu.l/well of assay medium.
4) And (3) bacterial culture: incubate at 37 ℃ for 24h, and read the OD per well using a 1420Victor2 multi-standard counter at 600 nm.
5) And (3) fungus culture: incubate at 26 ℃ for 48h, and read the OD per well using a 1420Victor2 multi-standard counter at 600 nm.
6) The OD was determined 90 for each antibiotic substance in triplicate. The results are shown in Table 4
TABLE 4 determination of antibacterial Activity
Claims (5)
1. An antimicrobial peptide compound a, characterized by: the antibacterial peptide compound A is shown in a formula (I), the antibacterial peptide compound A is derived from Brevibacillus laterosporus (Brevibacillus laterosporus) with the preservation number of CGMCC No.16337, the molecular weight is 1556Da, the sequence of the antibacterial peptide compound A is Leu-Val-Dhb-Orn-Val-Val-Val-Lys-Val-Leu-Lys-Tyr-Leu-Thr-ol,
formula (I).
2. An antibacterial peptide extract, which is characterized in that the antibacterial peptide compound A of claim 1 is taken as a main component, other components are analogues of the antibacterial peptide compound A, and the molecular weight is between 1542-1602Da, and the preparation method comprises the following steps:
1) taking a fermentation liquid of Brevibacillus laterosporus with the preservation number of CGMCC No.16337, wherein the formula of the fermentation medium is as follows: 20-50g of glucose, 5-20g of glycerol, 2-5g of inorganic ammonium salt, 0.05-0.5g of dipotassium hydrogen phosphate, 3-10g of yeast powder or corn steep liquor powder, 5-15g of industrial peptone, 5-10g of sodium chloride, 1-3g of calcium chloride, 1-4g of valine and 1000ml of water, wherein the pH value is 6.0-7.0; fermenting for 12-25 hr, adjusting pH to 2.5-3.5, adding filter aid, and filtering to obtain antibacterial peptide filtrate;
2) adjusting the pH of the antibacterial peptide filtrate to 5.5-6.5, adsorbing the filtrate by macroporous resin, and then resolving by a resolving agent to obtain an antibacterial peptide resolving solution;
3) decolorizing the analytic solution by a decolorizing resin column to obtain a decolorized solution;
4) concentrating the decolorized solution to small volume, adjusting pH of the solution to 2.5-3.5, adding precipitant, filtering, collecting precipitate, dissolving with solvent, and filtering to obtain solution;
5) concentrating the dissolved solution to obtain antibacterial peptide extract;
wherein the macroporous resin in the step 2) is any one of XR925CS, XR925J, D312 and HD-2 resin; wherein the resolving agent in the step 2) is 70-80% ethanol or 70-80% acetone aqueous solution; wherein the decolorizing resin in the step 3) is any one of XRTS-3, TS-4, D315 and D304; wherein the precipitator in the step 4) is any one of calcium chloride, sodium sulfate, ammonium sulfate and magnesium sulfate, and the amount of the precipitator is 5-15% of the volume of the solution by mass-volume ratio; wherein the solvent in the step 4) is 70-80% ethanol, 70-80% acetone or 70-80% methanol aqueous solution.
3. A process for preparing the antibacterial peptide compound a according to claim 1, which comprises the steps of:
1) taking a fermentation broth of Brevibacillus laterosporus CGMCC No.16337, wherein the formula of the fermentation medium is as follows: 20-50g of glucose, 5-20g of glycerol, 2-5g of inorganic ammonium salt, 0.05-0.5g of dipotassium hydrogen phosphate, 3-10g of yeast powder or corn steep liquor powder, 5-15g of industrial peptone, 5-10g of sodium chloride, 1-3g of calcium chloride, 1-4g of valine, 1000ml of water and pH 6.0-7.0; fermenting for 12-25 hr, adjusting pH to 2.5-3.5, adding filter aid, and filtering to obtain antibacterial peptide filtrate;
2) adjusting the pH of the antibacterial peptide filtrate to 5.5-6.5, adsorbing the filtrate by macroporous resin, and then resolving by a resolving agent to obtain an antibacterial peptide resolving solution;
3) decolorizing the analytic solution by a decolorizing resin column to obtain a decolorized solution;
4) concentrating the decolorized solution to small volume, adjusting pH of the solution to 2.5-3.5, adding precipitant, filtering, collecting precipitate, dissolving with solvent, and filtering to obtain solution;
5) concentrating the dissolved solution to obtain an antibacterial peptide extract, performing ODS preparative chromatography, performing gradient elution with 25-35% of 0.5-1 ‰ trifluoroacetic acid-acetonitrile aqueous solution, performing liquid phase detection, collecting chromatographic peak eluate with peak time of 12.5min, concentrating, and drying to obtain antibacterial peptide compound A;
wherein the macroporous resin in the step 2) is any one of XR925CS, XR925J, D312 and HD-2 resin; wherein the resolving agent in the step 2) is 70-80% ethanol or 70-80% acetone aqueous solution; wherein the decolorizing resin in the step 3) is any one of XRTS-3, TS-4, D315 and D304; wherein the precipitator in the step 4) is any one of calcium chloride, sodium sulfate, ammonium sulfate and magnesium sulfate, and the amount of the precipitator is 5-15% of the volume of the solution by mass-volume ratio; wherein the solvent in the step 4) is 70-80% ethanol, 70-80% acetone or 70-80% methanol aqueous solution.
4. Use of the antimicrobial peptide compound a of claim 1 or the antimicrobial peptide extract of claim 2 for the manufacture of a medicament for the prevention and/or treatment of bacterial and/or fungal infections.
5. Use of the antimicrobial peptide compound a of claim 1 or the antimicrobial peptide extract of claim 2 for the preparation of a product with bacteriostatic and/or bactericidal properties.
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| 侧孢短芽孢杆菌B8发酵液中抑菌物质理化性质研究;赵秀香等;《河南农业科学》;20111231;第40卷(第6期);第90-93页 * |
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