CN114917258B - Method for extracting ciliate killing substance from nannochloropsis - Google Patents
Method for extracting ciliate killing substance from nannochloropsis Download PDFInfo
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- CN114917258B CN114917258B CN202210688134.1A CN202210688134A CN114917258B CN 114917258 B CN114917258 B CN 114917258B CN 202210688134 A CN202210688134 A CN 202210688134A CN 114917258 B CN114917258 B CN 114917258B
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- 241000224474 Nannochloropsis Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000000126 substance Substances 0.000 title claims abstract description 16
- 241000223782 Ciliophora Species 0.000 title claims abstract description 13
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 8
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 239000013535 sea water Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000012071 phase Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- FDJOLVPMNUYSCM-UVKKECPRSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2,7, Chemical compound [Co+3].N#[C-].C1([C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)[N-]\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O FDJOLVPMNUYSCM-UVKKECPRSA-L 0.000 claims description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 238000012136 culture method Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 231100000252 nontoxic Toxicity 0.000 abstract description 5
- 230000003000 nontoxic effect Effects 0.000 abstract description 5
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000005416 organic matter Substances 0.000 abstract description 2
- 239000000575 pesticide Substances 0.000 abstract description 2
- 241000509521 Nannochloropsis sp. Species 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000004660 morphological change Effects 0.000 description 4
- 230000002110 toxicologic effect Effects 0.000 description 4
- 231100000027 toxicology Toxicity 0.000 description 4
- 230000000853 biopesticidal effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000003495 flagella Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000199914 Dinophyceae Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- VQXSOUPNOZTNAI-UHFFFAOYSA-N Pyrethrin I Natural products CC(=CC1CC1C(=O)OC2CC(=O)C(=C2C)CC=C/C=C)C VQXSOUPNOZTNAI-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- HYJYGLGUBUDSLJ-UHFFFAOYSA-N pyrethrin Natural products CCC(=O)OC1CC(=C)C2CC3OC3(C)C2C2OC(=O)C(=C)C12 HYJYGLGUBUDSLJ-UHFFFAOYSA-N 0.000 description 1
- VJFUPGQZSXIULQ-XIGJTORUSA-N pyrethrin II Chemical compound CC1(C)[C@H](/C=C(\C)C(=O)OC)[C@H]1C(=O)O[C@@H]1C(C)=C(C\C=C/C=C)C(=O)C1 VJFUPGQZSXIULQ-XIGJTORUSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Alternative & Traditional Medicine (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for extracting ciliate killing substances from nannochloropsis, belonging to the technical field of organic matter extraction. The invention takes nannochloropsis sp. The nannochloropsis LAMB204 is disclosed in the patent with application number 202110639406.4, the preservation number is CGMCC NO.20713, the preservation date is 10 month and 23 days in 2020, the preservation unit is China general microbiological culture Collection center, the preservation address is North Chen West road No. 1, 3 in the Korean region of Beijing city, and the post code is 100101. The invention provides a method for separating and purifying ciliate-killing substances in nannochloropsis LAMB204, which can effectively separate and extract the substances by liquid chromatography. Provides a new method for extracting nontoxic and harmless biological pesticides.
Description
Technical Field
The invention belongs to the technical field of organic matter extraction, and particularly relates to a method for extracting ciliate killing substances from nannochloropsis.
Background
While the well-blown type aquaculture industry develops, various diseases are abused. In recent years, in the cultivation of various economic animals and plants (fish, crustacean, algae, sea cucumber, etc.), diseases caused by ciliates are in increasing trend in occurrence range and hazard degree. When ciliate diseases occur, medicines prepared by taking copper sulfate, ferrous sulfate, zinc sulfate, formaldehyde, pyrethrin and the like as main components are mostly adopted in the aquatic market for treatment, and even forbidden medicines such as malachite green and the like are abused. The medicines provided by the method can cause serious consequences such as water pollution, medicine residue, resistance reduction of aquaculture animals, cancerogenesis and the like, so that the cultured varieties are degraded and the diseases are serious, and the final producer suffers from huge economic loss. Aiming at the phenomena, how to extract nontoxic and harmless biopesticide from plants is a problem which needs to be solved at present.
Disclosure of Invention
Aiming at the problem of lack of nontoxic and harmless biological pesticides in the prior art, the invention provides a method for extracting ciliate killing substances from nannochloropsis so as to solve the problem. The method for separating and purifying the effective substances in the nannochloropsis LAMB204 can be used for killing ciliates and provides a novel method for nontoxic and harmless biopesticide.
The technical scheme of the invention is as follows:
a method for extracting ciliate killing substance from nannochloropsis comprises pulverizing nannochloropsis (nannochloropsis) LAMB204 as raw material, and separating by liquid chromatography. The nannochloropsis LAMB204 is disclosed in the patent with application number 202110639406.4, the preservation number is CGMCC NO.20713, the preservation date is 10 month and 23 days in 2020, the preservation unit is China general microbiological culture Collection center, the preservation address is North Chen West road No. 1, 3 in the Korean region of Beijing city, and the post code is 100101.
The crushing method of the nannochloropsis LAMB204 comprises the following steps: culturing nannochloropsis LAMB204 to a platform stage, and centrifuging the alga solution in the platform stage for 10min at 8000 r/min. The supernatant after centrifugation was filtered to ensure that no algal cells were present in the supernatant.
The culture medium of the nannochloropsis LAMB204 is F/2 seawater culture medium. The F/2 seawater culture medium comprises the following substances: each 1L of seawater comprises: naNO 3 75mg、NaH 2 PO 4 ·H 2 O 5mg、Na 2 SiO 3 ·9H 2 O 20mg、Na 2 EDTA 4.36mg、FeCl 3 ·6H 2 O 3.16mg、CuSO 4 ·5H 2 O 0.01mg、ZnSO 4 ·7H 2 O 0.023mg、CoCl 2 ·6H 2 O 0.012mg、MnCl 2 ·4H 2 O 0.18mg、Na 2 MoO 4 ·2H 2 O0.07 mg, vitamin B 1 0.1mg, vitamin B 12 0.5 μg, biotin 0.5 μg.
The culture method of the nannochloropsis LAMB204 comprises the following steps: the culture temperature is 23+/-1 ℃, and the illumination intensity is 70 mu mol photons/s/m 2 irradication (12:12), salinity 30%, ph=7.8.
The high performance liquid chromatography method is as follows:
instrument: waters1525 liquid chromatography system with 2998PDA detector and 2707 autosampler
Chromatographic column: agilent Zorbax SB-C18.6X250 mm,5um; column temperature of 30 DEG C
Ultraviolet wavelength: full wavelength scan of 190nm-400nm
Sample injection amount: 10 mu L
Mobile phase: a-methanol; b-0.1% aqueous trifluoroacetic acid solution
Elution gradient:
| time (min) | Flow rate (mL/min) | A％ | B％ |
| 0 | 1 | 5 | 95 |
| 30 | 1 | 65 | 35 |
| 35 | 1 | 5 | 95 |
| 40 | 1 | 5 | 95 |
The preparation method of the liquid chromatograph comprises the following steps:
instrument: waters2545 series, UV-visible detector 2789 and fraction collector 2767
Chromatographic column: waters XTerra prep RP C18 A.sub.18.8x150mm, 10um
Ultraviolet wavelength: 200nm
Sample injection amount: 100uL
Mobile phase: a-methanol; b-0.5% acetic acid aqueous solution
Elution gradient:
| time (min) | Flow rate (mL/min) | A％ | B％ |
| 0 | 3 | 0 | 100 |
| 12 | 3 | 6 | 94 |
| 13 | 3 | 50 | 50 |
| 15 | 3 | 50 | 50 |
| 16 | 3 | 0 | 100 |
| 18 | 3 | 0 | 100 |
The ciliates are flagellates and sea tail worms.
The beneficial effects of the invention are as follows:
the invention provides a method for separating and purifying ciliate-killing substances in nannochloropsis LAMB204, which can effectively separate and extract the substances by a liquid chromatography method and provides a novel method for extracting nontoxic and harmless biopesticide.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is an HPLC chromatogram of a sample to be separated in example 1 of the present invention.
FIG. 2 is a preparation liquid-phase spectrum of a sample to be separated in example 1 of the present invention.
FIG. 3 is a graph showing population growth of the dinoflagellates of example 2 toxicological experiments of the present invention.
FIG. 4 is a graph showing the morphological changes of the toxicological test dinoflagellates of example 2 of the present invention.
FIG. 5 is a graph of population growth of the marine tail worms of example 2 toxicological experiments of the present invention.
FIG. 6 is a graph showing morphological changes of the marine tail worm according to the toxicological test of example 2 of the present invention.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
A method for extracting ciliate killing substance from nannochloropsis comprises the following specific steps:
(1) Preparation of nannochloropsis LAMB204 (nannochlopsisoceica) cultured in F/2 seawater medium, each 1L of seawater comprising: naNO 3 75mg、NaH 2 PO 4 ·H 2 O 5mg、Na 2 SiO 3 ·9H 2 O 20mg、Na 2 EDTA 4.36mg、FeCl 3 ·6H 2 O 3.16mg、CuSO 4 ·5H 2 O 0.01mg、ZnSO 4 ·7H 2 O 0.023mg、CoCl 2 ·6H 2 O 0.012mg、MnCl 2 ·4H 2 O 0.18mg、Na 2 MoO 4 ·2H 2 O0.07 mg, vitamin B 1 0.1mg, vitamin B 12 0.5 μg, biotin 0.5 μg. The culture temperature is 23+/-1 ℃, and the illumination intensity is 70 mu mol photons/s/m 2 irradication (12:12), salinity 30%, ph=7.8.
(2) The algae liquid cultured to the plateau stage is centrifuged for 10min at 8000 r/min. The supernatant after centrifugation was filtered to ensure that no algal cells were present in the supernatant. And (3) carrying out liquid chromatography on the filtered supernatant, wherein the chromatographic conditions are as follows:
instrument: waters1525 liquid chromatography system with 2998PDA detector and 2707 autosampler
Chromatographic column: agilent Zorbax SB-C18.6X250 mm,5um; column temperature of 30 DEG C
Ultraviolet wavelength: full wavelength scan of 190nm-400nm
Sample injection amount: 10 mu L
Mobile phase: a-methanol; b-0.1% aqueous trifluoroacetic acid solution
Elution gradient:
| time (min) | Flow rate (mL/min) | A％ | B％ |
| 0 | 1 | 5 | 95 |
| 30 | 1 | 65 | 35 |
| 35 | 1 | 5 | 95 |
| 40 | 1 | 5 | 95 |
The detection results are shown in FIG. 1.
(3) Separating and purifying the supernatant according to the detection result of the step (2) through a preparation liquid phase, wherein the preparation liquid phase is prepared under the following conditions:
instrument: waters2545 series, UV-visible detector 2789 and fraction collector 2767
Chromatographic column: waters XTerra prep RP C18 A.sub.18.8x150mm, 10um
Ultraviolet wavelength: 200nm
Sample injection amount: 100uL
Mobile phase: a-methanol; b-0.5% acetic acid aqueous solution
Elution gradient:
| time (min) | Flow rate (mL/min) | A％ | B％ |
| 0 | 3 | 0 | 100 |
| 12 | 3 | 6 | 94 |
| 13 | 3 | 50 | 50 |
| 15 | 3 | 50 | 50 |
| 16 | 3 | 0 | 100 |
| 18 | 3 | 0 | 100 |
The detection results are shown in FIG. 2. Collect material at rt=15.03 min. And (3) completely evaporating the mobile phase during preparation of the active substances by using a rotary evaporator, and blowing nitrogen for 5min after evaporation is finished so as to ensure complete evaporation of the organic solvent and avoid influence of the organic solvent on experimental results.
Example 2
To the active substance isolated from nannochloropsis in example 1, 2ml of F/2 seawater medium was added, and after dissolution, toxicity test was performed on flagella and sea tail worm. The specific experimental steps are as follows: experiments were carried out in 24-well plates, 20 ml of the liquid prepared in example 1 was added to each well, 20 flagellates and sea tail worms were added to each 20 ml of the liquid prepared in example 1, the culture temperature was 23.+ -. 1 ℃ and the illumination intensity was 100. Mu. m m -2 .s -1 The illumination period is 12:12. To obtain density, population growth rate and generation time of the flagellates and marine tail worms, counts were taken at Research Inverted System Microscope every 12 hours. The flagella and sea tail worms in each well were counted three times and then averaged. Population growth of flagellates and marine tail worms is measured in terms of time and density. Detailed resultsSee fig. 3-6.
As can be seen from the graphs of fig. 3 and 5, in the solution prepared in example 1, the flagella can be completely killed in 3 hours, and the sea tail worms can be completely killed in 2.5 hours. In fig. 4, a→f is a morphological change map of the process of apoptosis of the flagellate, and fig. 6 is a morphological change map of the process of apoptosis of the marine tail worm. As can be seen from fig. 4 and 6, in the solution prepared in example 1, the flagellates and the marine tail worms can be effectively killed. Therefore, the nannochloropsis extract prepared by the invention can prove that ciliates can be effectively killed.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. A detection method for extracting ciliate killing substances from nannochloropsis is characterized by using nannochloropsis @ to perform detectionNannochloropsisoceanica) LAMB204 is used as a raw material, the nannochloropsis LAMB204 is cultivated to a platform stage, and the alga solution in the platform stage is centrifuged for 10min at 8000 r/min; filtering the centrifuged supernatant, performing high performance liquid chromatography on the filtered supernatant, separating and purifying the supernatant by preparing a liquid phase according to an analysis result, and collecting a substance at the RT=15.03 min;
the preservation number of the nannochloropsis LAMB204 is CGMCC NO.20713, the preservation date is 10 months and 23 days in 2020, the preservation unit is the China general microbiological culture Collection center, the preservation address is North Chen Xiyu No. 1, 3 in the Korean region of Beijing, and the postal code is 100101; the culture medium of the nannochloropsis LAMB204 is an F/2 seawater culture medium; the F/2 seawater culture medium comprises the following substances:each 1L of seawater comprises: naNO 3 75mg、NaH 2 PO 4 ·H 2 O 5mg、Na 2 SiO 3 ·9H 2 O 20mg、Na 2 EDTA 4.36mg、FeCl 3 ·6H 2 O 3.16mg、CuSO 4 ·5H 2 O 0.01mg、ZnSO 4 ·7H 2 O 0.023mg、CoCl 2 ·6H 2 O 0.012mg、MnCl 2 ·4H 2 O 0.18mg、Na 2 MoO 4 ·2H 2 O0.07 mg, vitamin B 1 0.1mg, vitamin B 12 0.5 μg, biotin 0.5 μg; the culture method of the nannochloropsis LAMB204 comprises the following steps: the culture temperature is 23+/-1 ℃, and the illumination intensity is 70 mu mol photons/s/m 2 The irradiation day and night ratio is 12:12, the salinity is 30 per mill, and the pH=7.8;
the high performance liquid chromatography method comprises the following steps:
instrument: waters1525 liquid chromatography system, equipped with 2998PDA detector and 2707 autosampler;
chromatographic column: agilent Zorbax SB-C18.6X250 mm,5um; column temperature is 30 ℃;
ultraviolet wavelength: scanning at a full wavelength of 190nm-400 nm;
sample injection amount: 10. Mu.L;
mobile phase: a-methanol; b-0.1% trifluoroacetic acid aqueous solution;
elution gradient:
；
The preparation liquid chromatography method comprises the following steps:
instrument: waters2545 series, 2789 uv-visible detector and 2767 fraction collector;
chromatographic column: waters XTerra prep RP C18 7.8x150mm,10um;
ultraviolet wavelength: 200nm;
sample injection amount: 100uL;
mobile phase: a-methanol; b-0.5% acetic acid aqueous solution;
elution gradient:
。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210688134.1A CN114917258B (en) | 2022-06-17 | 2022-06-17 | Method for extracting ciliate killing substance from nannochloropsis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210688134.1A CN114917258B (en) | 2022-06-17 | 2022-06-17 | Method for extracting ciliate killing substance from nannochloropsis |
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| Publication Number | Publication Date |
|---|---|
| CN114917258A CN114917258A (en) | 2022-08-19 |
| CN114917258B true CN114917258B (en) | 2023-12-19 |
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| CN103592283A (en) * | 2013-11-21 | 2014-02-19 | 中国科学院青岛生物能源与过程研究所 | Method for quickly detecting microalgae energy-generating process |
| CN109390036A (en) * | 2018-10-31 | 2019-02-26 | 湘潭大学 | A method of excavating selection microalgae grease anabolism marker |
| CN109839453A (en) * | 2017-11-29 | 2019-06-04 | 中国科学院大连化学物理研究所 | A kind of content assaying method of microalgae carotenoid composition |
| CN113388523A (en) * | 2021-06-08 | 2021-09-14 | 日照职业技术学院 | Marine nannochloropsis oculata LAMB204 resisting disease ciliates and application thereof |
| WO2022119062A1 (en) * | 2020-12-01 | 2022-06-09 | 한국에너지기술연구원 | Solid acid catalyst for producing biodiesel, solid base catalyst for producing biodiesel, methods for preparing same, and method for producing biodiesel using these catalysts |
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| WO2009149260A1 (en) * | 2008-06-04 | 2009-12-10 | Solix Biofuels, Inc. | Compositions, methods and uses for growth of microorganisms and production of their products |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592283A (en) * | 2013-11-21 | 2014-02-19 | 中国科学院青岛生物能源与过程研究所 | Method for quickly detecting microalgae energy-generating process |
| CN109839453A (en) * | 2017-11-29 | 2019-06-04 | 中国科学院大连化学物理研究所 | A kind of content assaying method of microalgae carotenoid composition |
| CN109390036A (en) * | 2018-10-31 | 2019-02-26 | 湘潭大学 | A method of excavating selection microalgae grease anabolism marker |
| WO2022119062A1 (en) * | 2020-12-01 | 2022-06-09 | 한국에너지기술연구원 | Solid acid catalyst for producing biodiesel, solid base catalyst for producing biodiesel, methods for preparing same, and method for producing biodiesel using these catalysts |
| CN113388523A (en) * | 2021-06-08 | 2021-09-14 | 日照职业技术学院 | Marine nannochloropsis oculata LAMB204 resisting disease ciliates and application thereof |
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