CN114774497B - Method for preparing lipopeptide antibiotic iturin A by using capsicum stalks - Google Patents
Method for preparing lipopeptide antibiotic iturin A by using capsicum stalks Download PDFInfo
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- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 20
- 239000001390 capsicum minimum Substances 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 17
- 108010028921 Lipopeptides Proteins 0.000 title claims abstract description 14
- 230000003115 biocidal effect Effects 0.000 title claims abstract description 10
- 240000008574 Capsicum frutescens Species 0.000 title claims description 16
- 108010082754 iturin A Proteins 0.000 title claims description 16
- 239000010902 straw Substances 0.000 claims abstract description 62
- 239000000287 crude extract Substances 0.000 claims abstract description 40
- 238000006731 degradation reaction Methods 0.000 claims abstract description 40
- 239000007857 degradation product Substances 0.000 claims abstract description 39
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 37
- 230000015556 catabolic process Effects 0.000 claims abstract description 37
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 34
- 239000011347 resin Substances 0.000 claims abstract description 23
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- 230000014759 maintenance of location Effects 0.000 claims abstract description 16
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 14
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 11
- 238000004440 column chromatography Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 239000007788 liquid Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 239000006185 dispersion Substances 0.000 claims description 17
- 238000013375 chromatographic separation Methods 0.000 claims description 11
- RDUGMXONDQDIRN-UHFFFAOYSA-N iso-beta-AA-C14-Iturin A Natural products CC(C)CCCCCCCCC1CC(=O)NC(CC(=O)N)C(=O)NC(Cc2ccc(O)cc2)C(=O)NC(CC(=O)N)C(=O)NC(CCC(=O)N)C(=O)N3CCCC3C(=O)NC(CC(=O)N)C(=O)NC(CO)C(=O)N1 RDUGMXONDQDIRN-UHFFFAOYSA-N 0.000 claims description 11
- UZIRWCBDDZAYTQ-UHFFFAOYSA-N iturin A3 Natural products O=C1NC(CC(N)=O)C(=O)NC(CCC(N)=O)C(=O)N2CCCC2C(=O)NC(CC(N)=O)C(=O)NC(CO)C(=O)NC(CCCCCCCCC(C)CC)CC(=O)NC(CC(N)=O)C(=O)NC1CC1=CC=C(O)C=C1 UZIRWCBDDZAYTQ-UHFFFAOYSA-N 0.000 claims description 11
- 239000000945 filler Substances 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 239000003463 adsorbent Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 11
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- 241000208293 Capsicum Species 0.000 abstract 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
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- 238000012360 testing method Methods 0.000 description 7
- 230000000843 anti-fungal effect Effects 0.000 description 6
- 238000009777 vacuum freeze-drying Methods 0.000 description 6
- 229940121375 antifungal agent Drugs 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 241000223602 Alternaria alternata Species 0.000 description 4
- 240000004160 Capsicum annuum Species 0.000 description 4
- 239000006002 Pepper Substances 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000722363 Piper Species 0.000 description 3
- 235000016761 Piper aduncum Nutrition 0.000 description 3
- 235000017804 Piper guineense Nutrition 0.000 description 3
- 235000008184 Piper nigrum Nutrition 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000010517 secondary reaction Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 235000002567 Capsicum annuum Nutrition 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001511 capsicum annuum Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108010002015 fengycin Proteins 0.000 description 1
- CUOJDWBMJMRDHN-VIHUIGFUSA-N fengycin Chemical compound C([C@@H]1C(=O)N[C@H](C(=O)OC2=CC=C(C=C2)C[C@@H](C(N[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCC(N)=O)C(=O)N1)[C@@H](C)O)=O)NC(=O)[C@@H](CCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)C[C@H](O)CCCCCCCCCCCCC)[C@@H](C)CC)C1=CC=C(O)C=C1 CUOJDWBMJMRDHN-VIHUIGFUSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for preparing lipopeptid antibiotic iturinA by using capsicum stalks, belonging to the technical field of compound preparation. According to the invention, the capsicum straw is mixed with a degradation reagent, and degradation treatment is carried out under the microwave condition to obtain a straw degradation product; inoculating bacillus amyloliquefaciens into the straw degradation product for fermentation to obtain a fermentation crude extract; separating the fermented crude extract sequentially by macroporous adsorption resin column chromatography and liquid chromatography, and collecting components with retention time of 14.8min and 22.0min to obtain lipopeptide antibiotics C14-iturinA and C15-iturinA. The method provided by the invention has the advantages of low cost and large-scale production potential.
Description
Technical Field
The invention relates to the technical field of compound preparation, in particular to a method for preparing lipopeptid antibiotics iturin A by using capsicum straws.
Background
The lipopeptides antibiotics mainly comprise three families of iturin, surfaction and fengycin, wherein the iturin family is a cyclic lipopeptides containing 7 amino acids and has strong antifungal effect; the iturin A in the iturin family can also release electrolyte and polymer aggregate, increase the conductivity and permeability of a biomass membrane, influence the surface tension of a cell membrane and inhibit the formation of pathogenic bacteria spores. At present, no method for preparing the lipopeptide antibiotic iturin A on a large scale exists, but the existing laboratory preparation method generally needs to adopt a eutrophic culture medium, such as an LB culture medium or an NB culture medium, and the like, so that the cost is high, and the large-scale production is not facilitated.
Disclosure of Invention
The invention aims to provide a method for preparing lipopeptid antibiotic iturin A by using capsicum stalks, which has the advantages of low cost and large scale production potential.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for preparing lipopeptid antibiotics iturin A by using capsicum stalks, which comprises the following steps:
mixing capsicum stalks with a degradation reagent, and carrying out degradation treatment under a microwave condition to obtain stalk degradation products;
inoculating bacillus amyloliquefaciens into the straw degradation product for fermentation to obtain a fermentation crude extract;
separating the fermented crude extract by macroporous adsorption resin column chromatography and liquid chromatography in sequence, and collecting components with retention time of 14.8min and 22.0min to obtain lipopeptide antibiotics C14-iturin A and C15-iturin A;
the conditions for the liquid chromatographic separation include:
the model of the chromatographic column is SunFire, and the filler is silica gel; the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m; the column temperature is 25+/-5 ℃;
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system was 0.6mL/min.
Preferably, the degradation agent comprises water or sulfuric acid, and the mass fraction of the sulfuric acid is 0.1-0.5 wt%.
Preferably, the dosage ratio of the capsicum stalks to the degradation agent is 1g: (10-30) mL.
Preferably, the degradation treatment conditions include: the temperature is 120-180 ℃, the microwave power is 600-800W, and the time is 10-40 min.
Preferably, the bacillus amyloliquefaciens is bacillus amyloliquefaciens CAS02.
Preferably, the temperature of the fermentation is 25-37 ℃ and the time is 48-112 h; the fermentation is carried out under the shaking condition, and the rotating speed of the shaking is 150-250 rpm.
Preferably, the macroporous adsorption resin in the macroporous adsorption resin column is XAD-16 macroporous adsorption resin.
Preferably, the macroporous adsorption resin column chromatographic separation method comprises the following steps: mixing the fermented crude extract with water to obtain a fermented crude extract aqueous dispersion; loading the fermented crude extract aqueous dispersion liquid onto a macroporous adsorption resin column, eluting by using water, a methanol aqueous solution and methanol as eluent in sequence, and collecting eluent obtained after eluting by using the methanol.
Preferably the volume fraction of the aqueous methanol solution is 30 to 50%.
The invention provides a method for preparing lipopeptid antibiotics iturin A by using capsicum stalks, which comprises the following steps: mixing capsicum stalks with a degradation reagent, and carrying out degradation treatment under a microwave condition to obtain stalk degradation products; inoculating bacillus amyloliquefaciens into the straw degradation product for fermentation to obtain a fermentation crude extract; separating the fermented crude extract by macroporous adsorption resin column chromatography and liquid chromatography in sequence, and collecting components with retention time of 14.8min and 22.0min to obtain lipopeptide antibiotics C14-iturin A and C15-iturin A; the conditions for the liquid chromatographic separation include: the model of the chromatographic column is SunFire, and the filler is silica gel; the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m; the column temperature is 25+/-5 ℃; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system was 0.6mL/min. According to the invention, the capsicum stalks are efficiently degraded under the microwave condition, the stalk degradation products are fermented by utilizing bacillus amyloliquefaciens to obtain a fermented crude extract, and then the fermented crude extract is further separated and purified by macroporous adsorption resin column chromatographic separation and liquid chromatographic separation to obtain the high-purity lipopeptide antibiotic iturin A. The invention utilizes the low-cost pepper straw fermentation to obtain the high-value antifungal compound iturin A, has the advantages of low cost and large scale production potential, can solve the problem of straw waste treatment, and can also improve the added value of crop planting.
Drawings
FIG. 1 is a graph showing the growth curve of Bacillus amyloliquefaciens CAS02 in capsicum annuum straw degradation products;
FIG. 2 is a graph showing the growth curve of Bacillus amyloliquefaciens CAS02 in tomato straw degradation products;
FIG. 3 is a growth curve of Bacillus amyloliquefaciens CAS02 in cucumber straw degradation products;
FIG. 4 is a graph showing the results of antibacterial test of different fermented crude extracts against Alternaria alternata;
FIG. 5 is a graph showing the results of the antibacterial test of the compound having a retention time of 14.8min in example 1;
FIG. 6 is a graph of the compound having a retention time of 22.0min and a graph of the antibacterial test result in example 1.
Biological preservation information
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAS02 has a preservation date of 2018, 3 and 26 days, a preservation place of China general microbiological culture Collection center, a specific address of China national academy of sciences of China, including North Chen West Lu No.1 and No. 3 of the Korean area of Beijing, and a preservation number of CGMCC No.15514.
Detailed Description
The invention provides a method for preparing lipopeptid antibiotics iturin A by using capsicum stalks, which comprises the following steps:
mixing capsicum stalks with a degradation reagent, and carrying out degradation treatment under a microwave condition to obtain stalk degradation products;
inoculating bacillus amyloliquefaciens into the straw degradation product for fermentation to obtain a fermentation crude extract;
separating the fermented crude extract by macroporous adsorption resin column chromatography and liquid chromatography in sequence, and collecting components with retention time of 14.8min and 22.0min to obtain lipopeptide antibiotics C14-iturin A and C15-iturin A;
the conditions for the liquid chromatographic separation include:
the model of the chromatographic column is SunFire, and the filler is silica gel; the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m; the column temperature is 25+/-5 ℃;
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system was 0.6mL/min.
According to the invention, the capsicum straw is mixed with the degrading agent, and degradation treatment is carried out under the microwave condition, so that the straw degradation product is obtained. In the invention, the chilli straw is preferably dried, ground and sieved sequentially before use; the screening is preferably to screen the ground powder through a 20-mesh screen, and mix the screened straw powder with the degradation reagent.
In the present invention, the degradation agent preferably includes water or sulfuric acid, and the mass fraction of the sulfuric acid is preferably 0.1 to 0.5wt%, more preferably 0.15 to 0.25wt%, and further preferably 2wt%; the invention preferably adopts the degradation reagent of the type, which is beneficial to ensuring better degradation effect on the chilli straw. In the invention, the dosage ratio of the chilli straw to the degradation agent is preferably 1g: (10-30) mL, more preferably 1g: (10-20) mL, more preferably 1g:15mL. In the present invention, the degradation treatment conditions include: the microwave heating temperature is preferably 120-180 ℃, more preferably 120-150 ℃; the microwave power is preferably 600-800W, more preferably 600W; the time is preferably 10 to 40 minutes, more preferably 20 minutes.
After the degradation treatment, the invention preferably cools the obtained product system to 55-65 ℃, and carries out centrifugal separation for 5-10 min under the condition of 4000-8000 rpm, and the obtained supernatant is used as straw degradation liquid; and drying the straw degradation liquid to obtain a straw degradation product. In the present invention, the drying mode is preferably vacuum freeze drying; the invention preferably adopts vacuum freeze drying, which is beneficial to avoiding secondary reaction of degradation products. In the invention, the obtained straw degradation product is preferably stored in a refrigerator at the temperature of minus 20 ℃.
After the straw degradation product is obtained, bacillus amyloliquefaciens is inoculated into the straw degradation product for fermentation, and a fermentation crude extract is obtained. The invention preferably disperses the straw degradation products in water to obtain straw degradation product aqueous dispersion liquid; inoculating bacillus amyloliquefaciens into the straw degradation product water dispersion liquid for fermentation to obtain a fermentation crude extract. In the present invention, the concentration of the aqueous dispersion of the straw degradation product is preferably 1 to 10g/L, more preferably 5g/L. In the invention, the bacillus amyloliquefaciens is preferably bacillus amyloliquefaciens CAS02, and the preservation number of the bacillus amyloliquefaciens CAS02 is CGMCC NO.15514. According to the invention, bacillus amyloliquefaciens bacterial liquid is preferably inoculated into the straw degradation product water dispersion liquid, and the OD value of the bacillus amyloliquefaciens bacterial liquid is preferably 0.1-0.5, more preferably 0.3; the preparation method of the bacillus amyloliquefaciens bacterial liquid preferably comprises the following steps: culturing bacillus amyloliquefaciens in an LB culture medium to obtain bacillus amyloliquefaciens bacterial liquid; the time of the culture is preferably 12 to 36 hours, more preferably 24 hours. In the present invention, it is preferable that the culture medium is diluted with LB medium until the OD value of the resulting Bacillus amyloliquefaciens bacterial liquid is 0.3.
In the invention, the volume ratio of the straw degradation product aqueous dispersion to the bacillus amyloliquefaciens bacterial liquid is preferably 1000: (1 to 10), more preferably 1000:1. in the present invention, the temperature of the fermentation is preferably 25 to 37 ℃, more preferably 28 to 36 ℃, still more preferably 28 ℃; the time is preferably 48 to 112 hours, more preferably 112 hours; the fermentation is preferably carried out under shaking conditions, the rotational speed of the shaking is preferably 150 to 250rpm, more preferably 160 to 180rpm, still more preferably 175rpm. The invention preferably ferments the straw degradation products based on bacillus amyloliquefaciens CAS02, can effectively utilize the degradation products and generate lipopeptid antibiotics iturin A; the fermentation is preferably carried out under the conditions, which is beneficial to improving the yield of the lipopeptid antibiotic iturin A.
After the fermentation, the invention preferably carries out centrifugal separation on the obtained fermentation product system for 5-10 min under the condition of 4000-8000 rpm, and takes supernatant fluid for drying to obtain a fermentation crude extract; the drying mode is preferably vacuum freeze drying. The invention preferably adopts vacuum freeze drying, which is beneficial to avoiding secondary reaction of the fermented crude extract and reducing activity.
After the fermentation crude extract is obtained, the fermentation crude extract is sequentially subjected to macroporous adsorption resin column chromatographic separation and liquid chromatographic separation, and the components with retention time of 14.8min and 22.0min are collected to obtain lipopeptid antibiotics C14-iturin A and C15-iturin A. In the invention, the macroporous adsorption resin in the macroporous adsorption resin column is preferably XAD-16 macroporous adsorption resin; the column body of the macroporous adsorption resin column is preferably a glass column, the length of the glass column is preferably 800mm, and the inner diameter of the glass column is preferably 40mm. Preferably, the fermentation crude extract is mixed with water to obtain an aqueous dispersion of the fermentation crude extract; the aqueous dispersion of the fermented crude extract was loaded onto a glass column wet packed with XAD-16 macroporous adsorbent resin, and then eluted with an eluent. In the present invention, the concentration of the aqueous dispersion of the crude fermentation extract is preferably 0.01 to 0.2g/mL, more preferably 0.1g/mL. In the present invention, the elution includes water elution, aqueous methanol elution, and methanol elution which are sequentially performed; the volume of the detergent used for each elution is independently preferably 500-700 mL, more preferably 600mL; the volume fraction of the aqueous methanol solution used in the elution of the aqueous methanol solution is preferably 30 to 50%, more preferably 35 to 45%, and even more preferably 40%. The invention preferably collects the eluent obtained after methanol elution, and carries out liquid chromatographic separation after concentration; the concentration is preferably performed by using a rotary evaporator.
In the present invention, the conditions for the liquid chromatography separation include:
the model of the chromatographic column is SunFire, and the filler is silica gel; the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m; the column temperature is 25+/-5 ℃;
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system was 0.6mL/min.
In the present invention, the apparatus used for the liquid chromatography separation is preferably an Agilent 1290 system equipped with a SunFire chromatographic column (C18, 250×4.6mm,5 μm, waters, USA); the sample volume for the liquid chromatography is preferably 10. Mu.L.
After liquid chromatography separation, the invention collects the components with retention time of 14.8min and 22.0min to obtain lipopeptides antibiotics C14-iturin A and C15-iturin A, wherein the component with retention time of 14.8min is C14-iturin A, and the component with retention time of 22.0min is C15-iturin A.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Drying pepper straw, grinding into powder, sieving with a 20-mesh sieve, taking 2g of straw powder below the sieve, adding 30mL of water, placing the obtained material into a microwave reactor (CEM iPrep), and carrying out degradation treatment for 20min under the conditions that the microwave heating temperature is 120 ℃ and the microwave power is 600W; when the obtained product system is cooled to 60 ℃, carrying out centrifugal separation for 5min under the condition of 5000rpm, and taking the obtained supernatant as straw degradation liquid; vacuum freeze drying the straw degradation liquid to obtain straw degradation products, and storing the straw degradation products in a refrigerator at the temperature of minus 20 ℃;
(2) Culturing bacillus amyloliquefaciens CAS02 (with the preservation number of CGMCC No. 15514) in an LB culture medium for 24 hours, and diluting the bacillus amyloliquefaciens CAS02 with the preservation number of CGMCC No.15514 to an OD value of 0.3 by using the LB culture medium to obtain bacillus amyloliquefaciens bacterial liquid; dispersing 5g of the straw degradation product in 1L of water, inoculating 1mL of the bacillus amyloliquefaciens bacterial liquid into the obtained straw degradation product aqueous dispersion, and carrying out shake fermentation for 112h at 28 ℃ under the condition of 175rpm to obtain a fermentation product system; centrifuging at 5000rpm for 5min, and vacuum freeze drying the supernatant to obtain fermented crude extract;
(3) 5g of the crude fermentation extract was dissolved in 50mL of water, then loaded onto a glass column (800 mm. Times.40 mm, ID) wet packed with XAD-16 macroporous adsorbent resin, continuously eluted with 600mL of water, 600mL of 40% aqueous methanol solution and 600mL of 100% methanol, respectively, the eluate obtained by eluting with 100% methanol was concentrated by a rotary evaporator, and the remainder was recorded as methanol eluted phase; the methanol eluted phase was subjected to liquid chromatography column separation and purification using an Agilent 1290 system equipped with a SunFire column (C18, 250×4.6mm,5 μm, waters, USA) under chromatographic conditions including: the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system is 0.6mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and the components with the retention time of 14.8min and 22.0min are collected to obtain two kinds of iturin A lipoid peptide antibiotics, namely C14-iturin A and C15-iturin A, respectively, and the purity is more than 95%.
Examples 2 to 6
Lipopeptides antibiotic iturin A was prepared as in example 1, except for the degradation reagents and degradation temperatures, the specific conditions being set forth in Table 1.
Comparative examples 1 to 12
Lipopeptides antibiotic iturin a was prepared as in example 1, except for the type of straw, degradation agent and degradation temperature, the specific conditions being listed in table 1.
Table 1 straw types, degradation agents, and degradation temperatures in examples 1 to 6 and comparative examples 1 to 12
Characterization and performance testing
1. In the step (2) of the examples and the comparative examples, after the bacillus amyloliquefaciens bacterial liquid is inoculated into the straw degradation product aqueous dispersion, the OD600 value of the bacillus amyloliquefaciens CAS02 under different shake culture time conditions is tested, and the growth curves of the bacillus amyloliquefaciens in different straw degradation products are obtained. FIGS. 1 to 3 are graphs showing the growth of Bacillus amyloliquefaciens CAS02 in pepper, tomato or cucumber straw degradation products, respectively, LJ-pepper straw, FQ-tomato straw, HG-cucumber straw; the H-degradation reagent is water; the S-degrading reagent is 0.2wt% sulfuric acid; 120. 150, 180-degradation treatment temperature. As can be seen from the graph, the Bacillus amyloliquefaciens CAS02 has different growth conditions under different types of straws and different degradation conditions, wherein the strain growth conditions at the degradation treatment temperature of 120 ℃ and 150 ℃ are better than the strain growth conditions at the treatment temperature of 180 ℃.
2. The crude extracts obtained in step (2) of examples and comparative examples were dissolved in sterile water, respectively, to obtain crude extract solutions with a concentration of 100mg/mL, filtered through a 0.22 μm filter membrane, and the obtained filtrates were added to a petri dish, and the antagonistic effect of the crude extracts on Alternaria alternata was observed (the filtrate addition amount at each well was 40. Mu.L) with Alternaria alternata as a target. FIG. 4 is a graph showing the results of antifungal experiments of different fermented crude extracts against Alternaria alternata, wherein LHC-capsicum straw water-degradable fermented crude extract, LSC-capsicum straw 0.2wt% sulfuric acid-degradable fermented crude extract, FHC-tomato straw water-degradable fermented crude extract, FSC-tomato straw 0.2wt% sulfuric acid-degradable fermented crude extract, HHC-cucumber straw water-degradable fermented crude extract, LSC-cucumber straw 0.2wt% sulfuric acid-degradable fermented crude extract, and 120, 150, 180-degradation treatment temperatures. As can be seen from fig. 4, under the condition of fermenting the degradation products of the capsicum stalks at 120 ℃, the fermented crude extract has obvious antibacterial effect on the red star germs.
3. The C14-iturin A and C15-iturin A standards (standard purchase: sigma-Aldrich Co., st.Louis, MO, USA; standard dissolved in methanol at 1mg/mL on-line) were subjected to liquid chromatography and compared with the compound collected in example 1, and the results showed that the retention times were consistent, demonstrating that the compounds collected in example 1 were C14-iturin A and C15-iturin A. The compounds collected in example 1 were subjected to liquid chromatography-mass spectrometry analysis and antifungal testing was performed as described above with red star germs as targets. FIG. 5 is a graph of the compound with a retention time of 14.8min and the graph of the antifungal test results in example 1, and FIG. 6 is a graph of the compound with a retention time of 22.0min and the graph of the antifungal test results in example 1, showing that the compounds with retention times of 14.8min and 22.0min have anti-eubacterial activity, and the lipopeptidic antibiotics C14-iturin A and C15-iturin A are verified by mass spectrometry detection.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A method for preparing lipopeptid antibiotic iturin A by using capsicum stalks comprises the following steps:
sequentially drying, grinding and screening the chilli straw, mixing the screened straw powder with a degradation reagent, and carrying out degradation treatment under the microwave condition to obtain a straw degradation product; the degradation reagent is water; the dosage ratio of the straw to the degradation reagent is 1g: (10-30) mL; the degradation treatment conditions include: the microwave heating temperature is 120-150 ℃, the microwave power is 600-800W, and the time is 10-40 min;
dispersing the straw degradation products in water to obtain straw degradation product aqueous dispersion liquid; inoculating bacillus amyloliquefaciens into the straw degradation product water dispersion liquid for fermentation to obtain a fermentation crude extract; the concentration of the straw degradation product aqueous dispersion is 1-10 g/L, the fermentation temperature is 25-28 ℃, and the fermentation time is 48-112 h; the bacillus amyloliquefaciens is bacillus amyloliquefaciens CAS02, and the preservation number is CGMCC NO. 15514;
separating the fermented crude extract by macroporous adsorption resin column chromatography and liquid chromatography in sequence, and collecting components with retention time of 14.8min and 22.0min to obtain lipopeptide antibiotics C14-iturin A and C15-iturin A;
the conditions for the liquid chromatographic separation include:
the model of the chromatographic column is SunFire, and the filler is silica gel; the specification of the chromatographic column is as follows: the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m; the column temperature is 25+/-5 ℃;
the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an acetic acid aqueous solution with the volume fraction of 0.1%, the mobile phase B is acetonitrile, and the volume ratio of the mobile phase A to the mobile phase B is 60:40; the flow rate of the mobile phase system was 0.6mL/min.
2. The method according to claim 1, wherein the fermentation is carried out under shaking conditions, and the shaking speed is 150-250 rpm.
3. The method of claim 1, wherein the macroporous adsorbent resin in the macroporous adsorbent resin column is XAD-16 macroporous adsorbent resin.
4. A method according to claim 1 or 3, wherein the method of macroporous adsorbent resin column chromatographic separation comprises: mixing the fermented crude extract with water to obtain a fermented crude extract aqueous dispersion; loading the fermented crude extract aqueous dispersion liquid onto a macroporous adsorption resin column, eluting by using water, a methanol aqueous solution and methanol as eluent in sequence, and collecting eluent obtained after eluting by using the methanol.
5. The method according to claim 4, wherein the volume fraction of the aqueous methanol solution is 30-50%.
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| CN112725381A (en) * | 2020-12-29 | 2021-04-30 | 山东省农业科学院农产品研究所 | Method for extracting antifungal active substance produced by antagonistic bacillus amyloliquefaciens |
| CN114369539A (en) * | 2022-01-11 | 2022-04-19 | 吉林大学 | Preparation method of culture medium taking agricultural wastes as raw materials, culture medium and application |
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| CN112725381A (en) * | 2020-12-29 | 2021-04-30 | 山东省农业科学院农产品研究所 | Method for extracting antifungal active substance produced by antagonistic bacillus amyloliquefaciens |
| CN114369539A (en) * | 2022-01-11 | 2022-04-19 | 吉林大学 | Preparation method of culture medium taking agricultural wastes as raw materials, culture medium and application |
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| Title |
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| "Genomic insights on fighting bacterial wilt by a novel Bacillus amyloliquefaciens strain Cas02";Depeng Chu et al.;《Microbial Biotechnology》;第15卷;第1152-1167页 * |
| "Production of antifungal iturins from vegetable straw: A combined chemical-bacterial process";Yuan Yuan et al.;《Bioresource Technology》;第378卷;第1-9页 * |
| "微波辅助稀酸降解秸秆的工艺研究";朱李妍 等;《粮食与饲料工业》(第9期);第56-59、67页 * |
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