CN116716200A - Bacillus and fermentation method and application thereof - Google Patents
Bacillus and fermentation method and application thereof Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 83
- 230000004151 fermentation Effects 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 19
- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 claims abstract description 83
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 claims abstract description 83
- 241000830536 Tripterygium wilfordii Species 0.000 claims abstract description 25
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- 239000007788 liquid Substances 0.000 claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 claims abstract description 20
- 239000002054 inoculum Substances 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 7
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 235000015278 beef Nutrition 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 238000004458 analytical method Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
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- 238000000227 grinding Methods 0.000 claims description 3
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- 241000894006 Bacteria Species 0.000 abstract description 32
- 238000000605 extraction Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 229930014626 natural product Natural products 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 12
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000011160 research Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
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- 238000010170 biological method Methods 0.000 description 2
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- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- -1 diterpene lactone compound Chemical class 0.000 description 2
- 241001576300 endophytic bacterium Species 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 238000009629 microbiological culture Methods 0.000 description 2
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- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
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- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 206010018364 Glomerulonephritis Diseases 0.000 description 1
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- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000545405 Tripterygium Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 239000004480 active ingredient Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003509 anti-fertility effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KQJSQWZMSAGSHN-JJWQIEBTSA-N celastrol Chemical compound C([C@H]1[C@]2(C)CC[C@@]34C)[C@](C)(C(O)=O)CC[C@]1(C)CC[C@]2(C)C4=CC=C1C3=CC(=O)C(O)=C1C KQJSQWZMSAGSHN-JJWQIEBTSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 230000001351 cycling effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- 229930004069 diterpene Natural products 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
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- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- GLBMIEFQNYULDO-UFZHVSJGSA-M triptosar Chemical compound [Na+].O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](OC(=O)CCC([O-])=O)[C@]21[C@H]3O1 GLBMIEFQNYULDO-UFZHVSJGSA-M 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The application discloses bacillus and a fermentation method and application thereof, and belongs to the field of natural product extraction. The preservation number of the Bacillus sp is CGMCC No.25391. The application also discloses a method for fermenting triptolide by using the bacillus, which comprises the following steps: inoculating the bacillus into a fermentation culture medium according to the inoculum size of 3-7%, adjusting the pH value to 7.4, and carrying out shake flask fermentation culture at 22-34 ℃ for 20-36h to obtain fermentation liquor; separating and measuring triptolide in the fermentation liquid by using a high performance liquid chromatograph. The application screens out endophytic bacteria of the triptolide with high yield through different tissues of the tripterygium wilfordii, and provides a novel raw material and a biological fermentation method for the production of the triptolide.
Description
Technical Field
The application relates to the field of natural product extraction, in particular to bacillus and a fermentation method and application thereof.
Background
The tripterygium Tripterygium wilfordii hook.f. system spear Wei Ke tripterygium plant has a plurality of pharmacological effects of anti-inflammatory, antibacterial, immunoregulation, anti-tumor, anti-fertility and the like, has extremely wide clinical application of the original medicine and various preparations thereof, and can be used for treating difficult and complicated diseases such as rheumatic arthritis, rheumatoid arthritis, traumatic injury, glomerulonephritis, lupus erythematosus, nephrotic syndrome and the like. Tripterygium wilfordii and Tripterygium wilfordii are two herb plants with similar forms in Tripterygium wilfordii, and are native to southeast China, korea and Japan. Triptolide, also known as triptolide, has a molecular formula of C 2 0H 24 O 6 The epoxy diterpene lactone compound is extracted from the root, leaf, flower and fruit of Tripterygium wilfordii, and forms the main active component of Tripterygium wilfordii extract with alkaloid such as triptolide, is insoluble in water, but is soluble in absolute ethyl alcohol, ethyl acetate and the like. Tripterygium wilfordii hook extract has 3 existing forms: PG 490 containing 97% triptolide; PG27 containing (0.36.+ -. 0.04)% triptolide; PG 490-88 is 14-succinyl sodium salt of triptolide, water-soluble substance, and is hydrolyzed into triptolide in blood plasma. Modern scientific research has shown that: triptolide can be used for resisting rheumatoid diseases and plays a vital role in resisting cancers, and a natural active product of hot spot research is currently occurring in a first-stage anticancer clinical research conducted in the United states.
Along with the continuous deep research on TP, people have more and more deep and wide knowledge on pharmacological, pharmaceutical, toxicological and clinical application aspects and the like. The TP has great potential application value and market prospect, has great adverse reaction besides high bioactivity, greatly influences the development and utilization of TP medicaments, and is a key point for solving the problem how to maintain the bioactivity of TP, so that a novel triptolide preparation with high efficiency and low toxicity is developed by utilizing a modern high-tech means.
Triptolide is the main active ingredient of Tripterygium wilfordii, and has antiinflammatory and neuroprotective effects. However, triptolide is extremely low in tripterygium wilfordii, and although many previous people have studied triptolide production, the tripterygium wilfordii has the problems of large demand of tripterygium wilfordii raw materials, low yield and the like. With the continuous exploration and experiment of triptolide production, triptolide can be extracted with high yield by a simpler method, which is a very necessary choice, thus being beneficial to providing resources for medical pharmacy research and solving unnecessary waste in the exploitation of triptolide.
Disclosure of Invention
The application aims to provide bacillus and a fermentation method and application thereof, so as to solve the problems in the prior art, screen out bacillus with high triptolide yield through different tissues of tripterygium wilfordii, and provide a novel raw material and a biological fermentation method for triptolide production.
In order to achieve the above object, the present application provides the following solutions:
the application provides Bacillus sp, the preservation number of which is CGMCC No.25391; the preservation time is 2022, 7 and 25 days; the preservation unit is China general microbiological culture Collection center; the preservation address is the microbiological institute of China academy of sciences of national institute of sciences No. 1, 3, north Chen West Lu, the Korean area of Beijing city.
The application also provides a method for fermenting triptolide by using the bacillus, which comprises the following steps: inoculating the bacillus into a fermentation culture medium according to the inoculum size of 3-7%, adjusting the pH value to 7.4, and carrying out shake flask fermentation culture at 22-34 ℃ for 20-36h to obtain fermentation liquor; and (3) after the fermentation liquor is treated, separating and measuring triptolide in the fermentation liquor by using a high performance liquid chromatograph.
Preferably, the rotation speed of the shake flask fermentation is 120-240r/min.
Preferably, the fermentation medium comprises the following components in parts by weight: 5.0 parts of beef extract, 10.0 parts of peptone, 5.0 parts of NaCl, 0.5 parts of tripterygium wilfordii, 1000 parts of water and pH7.0.
Preferably, the inoculum size is 5%; the temperature of the fermentation culture is 28 ℃, and the fermentation time is 24 hours.
Preferably, the fermentation broth treatment comprises: separating the fermentation liquid into a supernatant A and a precipitate, grinding the precipitate, mixing with the supernatant, centrifuging, collecting a supernatant B, extracting the supernatant B, and collecting an organic phase for analysis by a high performance liquid analysis chromatograph.
The application also provides an application of the bacillus in production of triptolide.
The application discloses the following technical effects:
the application screens out a bacillus from the tripterygium wilfordii root, and the best fermentation condition of the bacillus strain is determined by optimizing the fermentation condition of the bacillus strain: the inoculation amount is 5%, the initial pH7.4, the fermentation temperature is 28 ℃, the shaking speed is 180r/min, the fermentation time is 24 hours, and the amount of triptolide produced after fermentation under the optimal fermentation condition can reach 24.527 mug/mL. Therefore, the bacillus with high triptolide yield is obtained through screening, a novel raw material and a biological method are provided for triptolide production, and the rapid growth of strains can be ensured in a short time due to the extraction of the triptolide by the biological method, so that resources are rapidly and largely provided for medical pharmacy research, and unnecessary triptolide exploitation is avoided.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a triptolide standard curve;
FIG. 2 shows the effect of the time-varying production of triptolide by root 1 endophytic bacteria;
FIG. 3 shows the effect of leaf 1 endophytic bacteria on time-varying production of triptolide;
FIG. 4 shows the effect of leaf 2 endophytic bacteria on time-varying production of triptolide;
FIG. 5 shows the production of triptolide by root 1 endophytic bacteria under different inoculum size conditions;
FIG. 6 shows the production of triptolide by root 1 endophytic bacteria at different fermentation temperatures;
FIG. 7 shows the production of triptolide by root 1 endophytic bacteria under different fermentation time conditions;
FIG. 8 shows the production of triptolide by root 1 endophyte under different initial pH conditions;
FIG. 9 shows the production of triptolide by root 1 endophytic bacteria at different shake flask rotational speeds.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 isolation, identification, and fermentation condition screening of endophytic bacteria of Tripterygium wilfordii
1. Isolation of endophytic bacteria of Tripterygium wilfordii
From Tripterygium wilfordii leaves and roots which normally grow from the Guangxi Lin Shi Zhenhua lawn large rock pipe station to the roadside of the chicken public rock, 7 endophytes, respectively designated as leaf 1, leaf 2, leaf 3, leaf 4, root 1, root 2 and root 3 endophytes, are obtained according to a conventional screening method of endophytes.
2. Identification of endophytic bacteria
(1) Morphological identification:
the formula of the culture medium comprises: 5.0g of beef extract powder; 10.0g of tryptone; 5.0g of NaCl; 17g of agar powder; 1000mL of water; pH7.0.
The purified strain is streaked on LB solid medium and cultured for 12 hours at 37 ℃, so that the size, shape and color of the colony are observed, and gram staining is carried out to observe the morphological characteristics of the colony: the colony is round, light yellow in color, smooth and raised in surface, positive gram staining and short rod under microscope.
(2) Molecular biology identification:
the extraction of the strain DNA is carried out according to a kit, and the extracted DNA is amplified according to a PCR kit, wherein the primer is a universal primer 27F. The PCR amplification procedure is thermal denaturation at 95 ℃ for 5min;94 ℃/40s; cycling for 40 times at 55 ℃/40s,72 ℃/2 min; extending at 72℃for 10min.
Detecting target amplified products by 1% agarose gel electrophoresis, sending the PCR amplified products qualified by electrophoresis to Shanghai biological engineering Limited company for sequence analysis, and carrying out DNA BLAST comparison analysis on sequencing sequence data on the national center of biotechnology information to obtain sequences with similar comparison indexes. Sequencing results were as follows SEQ ID NO:1 is shown as follows:
GGGGCCTATACTTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTATGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGAAGTACGGTCGCAAGACTGAACTCAAAGGAATTGACGGGGGCCCGCACAGCGGTGGAGCATGTGGTTTAATTCGAAGCACGCGAGATCTTACCAGTCTTGACATCCTCTGACATCCTAGAGATAGACGTCCCCTCGGGGCAGAGTGGACAGGTGGTGCATGTTGTCGTCACTCGTGTCTGAAATGTTGGGTTAGTCCCGCAACGAGCGCACCCTTGATCTTAGTGCAGCATCAGTGGGCACTCTAGGTGACTGCGGTGACAACGGAGAAGTGGGATGACTCATCATCATC。
the Bacillus sp is obtained through identification and separation, and the preservation number of the Bacillus sp is CGMCC No.25391; the preservation time is 2022, 7 and 25 days; the preservation unit is China general microbiological culture Collection center; the preservation address is the microbiological institute of China academy of sciences of national institute of sciences No. 1, 3, north Chen West Lu, the Korean area of Beijing city.
3. Screening of endophytic bacterium fermentation conditions
3.1 test reagents
Beef extract peptone medium (NA medium) formula: beef extract 2.5g, peptone 5.0g, naCl 2.5g, agar 8.5g, water 500mL, pH7.0.
Beef extract peptone liquid medium (NB medium) formulation: beef extract 5.0g, peptone 10.0g, naC l5.0g, water 1000mL were mixed with heating, pH7.0.
The formula of the fermentation medium comprises: beef extract 5.0g, peptone 10.0g, naCl5.0 g, tripterygium wilfordii 0.5g, 1000mL of water, pH7.0.
Tripterygium wilfordii standard (500 μg/mL): triptolide 5mg was dissolved in methanol 10mL.
3.2 test methods
3.2.1 drawing of Tripterygium wilfordii A standard curve
The triptolide solution is diluted to 5 mug/mL, 10 mug/mL, 20 mug/mL, 30 mug/mL and 40 mug/mL, and the triptolide standard curve is prepared by a high performance liquid chromatography method by taking a chromatographic condition of C18 column, column temperature 25 ℃, mobile phase methanol: water=45:55, wavelength 218nm and flow rate 0.8 nm.
3.2.2 endophyte screening for triptolide production
3.2.2.1 Strain activation
Two-loop streaks were taken from root 1 endophyte plates on an ultra clean bench with an inoculating loop on NA medium and incubated in a constant temperature incubator at 28℃for 14-16h. Leaf 1, leaf 2, leaf 3, leaf 4, root 2 and root 3 endophytic bacteria operate in the same manner.
3.2.2.2 seed liquid preparation
Inoculating the activated endophytic bacteria into two loops to 40mL of beef extract peptone liquid culture medium in an ultra-clean workbench, marking, placing in a constant temperature incubator at 28 ℃, and shake culturing for 14-16h at 180 r/min.
Before inoculating the endophyte of root 1, shake-flask fermenting, sterilizing articles such as centrifuge tube, pouring proper amount of seed liquid into a cuvette in a sterile operation table, performing colorimetric determination at 620nm wavelength, controlling absorbance of the seed liquid at 0.8A, and stopping fermenting.
3.2.2.3 shaking flask fermentation to produce triptolide
2mL of bacterial seed liquid is absorbed, inoculated into a triangular flask with 40mL of fermentation medium, two parallel strains are made, two blank control groups are placed in a constant temperature incubator at 28 ℃,180r/min and shake culture is carried out for 24h.
Determination of triptolide content in 3.2.2.4 fermentation liquor
Collecting supernatant A and precipitate A from fermentation broth of shake flask at 10000r/min for 15min, freezing and preserving supernatant A, freezing precipitate A at-80deg.C overnight, taking out, grinding on mortar for 5min, mixing with supernatant A according to mark, centrifuging at 10000r/min for 5min, and collecting supernatant B.
B was extracted with 15mL ethyl acetate and the organic phase was collected. After 3 times the organic phases were combined and the ethyl acetate was evaporated on a rotary evaporator to give a paste. The paste was then poured into a small centrifuge tube and placed in an oven for drying at 60 ℃. Dissolving a proper amount of sample in a certain amount of methanol to prepare 0.1mg/mL liquid to be tested, performing ultrasonic disruption in an ultrasonic cell disruption instrument for 15min to completely dissolve, performing organic membrane filtration treatment on the ultrasonic sample, and placing the ultrasonic sample in a special bottle of a high performance liquid chromatograph to be tested.
3.2.3 fermentation Condition optimization
The same experimental methods as 3.2.2.1 and 3.2.2.2.
(1) Inoculum size
Seed solutions are inoculated into 40mL fermentation culture medium with initial pH of 7 according to inoculum sizes of 3%, 4%, 5%, 6% and 7%, and then shake flask fermentation is carried out for 24h under the condition of 30 ℃ and 180 r/min. And measuring the content in a high performance liquid chromatograph.
(2) Initial pH value
The seed solution was inoculated into 40mL fermentation media with pH values of 6.2, 6.8, 7.4, 8.0 and 8.6, which had been prepared, according to an optimal inoculum size of 3.2.3.1, and then shake-flask fermented at 30℃and 180r/min for 24 hours. And measuring the content in a high performance liquid chromatograph.
(3) Shaking bottle rotating speed
The seed solution is respectively inoculated into 40mL fermentation culture medium with pH of 7.4 obtained in the step (2) according to the inoculum size of 5 percent of the step (1), and then is subjected to shaking fermentation for 24 hours in an incubator with shaking rotation speeds of 120, 150, 180, 210 and 240r/min at the temperature of 30 ℃. And measuring the content in a high performance liquid chromatograph.
(4) Culture temperature
The seed solutions were inoculated in an inoculum size of 5% into 40mL of the prepared fermentation medium having a pH of 7.4, and then subjected to shaking flask fermentation at a temperature of 22℃at 25℃at 28℃at 31℃at 34℃at 2.3.3.3 for 24 hours, respectively. And measuring the content in a high performance liquid chromatograph.
(5) Fermentation time:
the seed solution is inoculated into 40mL of prepared fermentation medium with pH of 7.4 according to the inoculation amount of 5%, and then is subjected to shaking flask fermentation for 20h, 24h, 28h, 32h and 36h under the condition of optimal temperature of 30 ℃ and 180r/min obtained by 2.3.3.4. And measuring the content in a high performance liquid chromatograph.
4. Results and analysis
4.1 drawing Tripterine Standard Curve
Preparing a standard curve of triptolide by a high performance liquid chromatography method. As shown in figure 1, a triptolide standard curve is drawn by taking the abscissa of the triptolide concentration as the X axis and the peak area as the ordinate of the Y axis to calculate a regression equation, wherein Y= 17547X-17324 shows that the triptolide has good linear relation at R= 0.9958.
4.2 endophytic bacteria triptolide
4.2.1 triptolide production by 1 endophytic bacterium
The root 1 endophytic bacteria produced changes in triptolide as shown in figure 2.
As can be seen from FIG. 2, the triptolide content of the root 1 bacteria increased with time, reaching a maximum value of 1.021 μg/mL within 24h, and the yield tended to decrease. The HPLC method is used for measuring the content of triptolide in each part of the Jiangxi tripterygium wilfordii [ J ]. Jiangxi traditional Chinese medicine, 2012,43 (006): 52-54 ] shows that the content of triptolide in the tripterygium wilfordii leaf is 10.87 mug/g, and the content of the triptolide in the fermentation liquor is 0.136 mug/mL, so that the root 1 endophyte can convert the fermentation liquor to produce the triptolide.
4.2.2 production of triptolide by leaf 1 endophytic bacteria
The leaf 1 endophytic bacteria produced changes in triptolide as shown in figure 3.
As can be seen from FIG. 3, triptolide in the fermentation broth of the leaf 1 endophyte increased and then decreased with increasing fermentation time, reaching the highest value of 0.992 mug/mg at 24h.
4.2.3 production of triptolide by leaf 2 endophytic bacteria
The leaf 2 endophytic bacteria produced changes in triptolide as shown in figure 4. As can be seen from FIG. 4, the triptolide content produced by the bacteria in leaf 2 increased with time, reaching the maximum value of 0.988. Mu.g/mL at 24h.
Triptolide was not detected by each of the leaf 3, leaf 4, root 2 and root 3 bacteria, and as can be seen from comparison of fig. 2 to 4, triptolide was higher in the fermentation broth of the root 1 endophytic bacteria, and therefore the root 1 endophytic bacteria were selected as target cells.
4.3 fermentation Condition optimization
4.3.1 Effect of different inoculum size on triptolide yield
As shown in FIG. 5, the effect of different inoculum size on the triptolide content of the bacteria in root 1 is obtained by the method 3.2.3, wherein the triptolide content in the fermentation liquid of the bacteria in root 1 is increased along with the increase of the inoculum size, and reaches the maximum value of 20.083 mug/mL when the inoculum size is 5%.
4.3.2 Effect of different fermentation temperatures on triptolide yield
As shown in FIG. 6, the effect of different fermentation temperatures on the triptolide content of the endophyte in root 1 is shown by the 3.2.3 method, the triptolide content in the fermentation liquor of the endophyte in root 1 increases along with the increase of the fermentation temperature, reaches the highest value of 9.866 mug/mL at 28 ℃, and is unfavorable for the fermentation production of triptolide by the endophyte due to the increase of the temperature.
4.3.3 Effect of different fermentation times on Tripterygium wilfordii
As shown in FIG. 7, the effect of different fermentation times on the triptolide content of the root 1 endophytic bacteria is shown by the 3.2.3 method, the maximum triptolide yield of the root 1 endophytic bacteria reaches 16.923 mug/mL under the condition that the fermentation time is 24 hours, and then the triptolide content in the fermentation liquid is irregularly reduced.
4.3.4 Effect of different initial pH values on triptolide yield
According to the method 3.2.3, the influence of different initial pH values on the triptolide content produced by the bacteria in the root 1 is shown in figure 8, the triptolide content in the fermentation liquor of the bacteria in the root 1 is rapidly increased and then rapidly reduced along with the increase of the initial pH value, and the highest 24.527 mug/mL is achieved when the initial pH value of the fermentation liquor is 7.4.
4.3.5 Effect of different shake flask fermentation Rate on triptolide yield
According to the method 3.2.3, the influence of different shaking fermentation speeds on the triptolide content of the root 1 endophytic bacteria is shown in figure 9, the triptolide content in the root 1 endophytic bacteria fermentation liquid is firstly slowly increased and then slowly reduced along with the increase of the speed, and the maximum value of 10.503 mug/mL is reached when the shaking speed is 180 r/min.
In summary, the optimal fermentation conditions were determined as follows: the inoculation amount is 5%, the initial pH7.4, the fermentation temperature is 28 ℃, the shaking speed is 180r/min, and the fermentation time is 24 hours.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (7)
1. A Bacillus sp, characterized in that the Bacillus has a preservation number of CGMCC No.25391.
2. A method of fermenting triptolide using the bacillus of claim 1, comprising the steps of: inoculating the bacillus into a fermentation culture medium according to the inoculum size of 3-7%, adjusting the pH value to 7.4, and carrying out shake flask fermentation culture at 22-34 ℃ for 20-36h to obtain fermentation liquor; and after the fermentation liquor is treated, separating and measuring triptolide in the fermentation liquor by using a high performance liquid chromatograph.
3. The method of claim 2, wherein the rotational speed of the shake flask fermentation is 120-240r/min.
4. The method of claim 2, wherein the fermentation medium comprises the components in parts by weight: 5.0 parts of beef extract, 10.0 parts of peptone, 5.0 parts of NaCl, 0.5 parts of tripterygium wilfordii, 1000 parts of water and pH7.0.
5. The method of claim 2, wherein the inoculum size is 5%; the temperature of the fermentation culture is 28 ℃, and the fermentation time is 24 hours.
6. The method of claim 2, wherein the fermentation broth treatment comprises: separating the fermentation liquid into a supernatant A and a precipitate, grinding the precipitate, mixing with the supernatant, centrifuging, collecting a supernatant B, extracting the supernatant B, and collecting an organic phase for analysis by a high performance liquid analysis chromatograph.
7. The use of bacillus according to claim 1 for the production of triptolide.
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