CN114917258A - Method for extracting ciliate killing substance from nannochloropsis - Google Patents
Method for extracting ciliate killing substance from nannochloropsis Download PDFInfo
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- CN114917258A CN114917258A CN202210688134.1A CN202210688134A CN114917258A CN 114917258 A CN114917258 A CN 114917258A CN 202210688134 A CN202210688134 A CN 202210688134A CN 114917258 A CN114917258 A CN 114917258A
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- nannochloropsis
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 241000224474 Nannochloropsis Species 0.000 title claims abstract description 23
- 239000000126 substance Substances 0.000 title claims abstract description 16
- 241000223782 Ciliophora Species 0.000 title claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 8
- 241000159660 Nannochloropsis oculata Species 0.000 claims abstract description 5
- 244000005700 microbiome Species 0.000 claims abstract description 3
- 239000002994 raw material Substances 0.000 claims abstract 2
- 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
- 239000012071 phase Substances 0.000 claims description 7
- 239000006228 supernatant Substances 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
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 5
- 241000195493 Cryptophyta Species 0.000 claims description 4
- 238000005286 illumination 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
- 238000005119 centrifugation Methods 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
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 239000002609 medium Substances 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
- 238000012258 culturing Methods 0.000 claims description 2
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims 1
- 239000000575 pesticide Substances 0.000 abstract description 5
- 231100000252 nontoxic Toxicity 0.000 abstract description 4
- 230000003000 nontoxic effect Effects 0.000 abstract description 4
- 241001300629 Nannochloropsis oceanica Species 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000005416 organic matter Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 8
- 241001465977 Coccoidea Species 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000002110 toxicologic effect Effects 0.000 description 4
- 231100000027 toxicology Toxicity 0.000 description 4
- 244000144974 aquaculture Species 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 235000019687 Lamb Nutrition 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
- 238000001704 evaporation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 239000003960 organic solvent Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000131066 Coccinella Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 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
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 229960000355 copper sulfate Drugs 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
- 238000012136 culture method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229960001781 ferrous sulfate Drugs 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- -1 formaldehyde, pyrethrins Chemical class 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
- 229940107698 malachite green Drugs 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
- 239000000463 material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 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
- 229940070846 pyrethrins Drugs 0.000 description 1
- 239000002728 pyrethroid Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 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
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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
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- 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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Abstract
The invention relates to a method for extracting ciliate killing substances from nannochloropsis, belonging to the technical field of organic matter extraction. The present invention uses nannochloropsis (Nannochloropsis oceanica) LAMB204 as raw material, and separates by liquid chromatography after breaking. The nannochloropsis LAMB204 is disclosed in the patent with application number 202110639406.4, the preservation number is CGMCC NO.20713, the preservation date is 2020, 10 and 23 days, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the Beijing city, Chaoyang district, North Chen Xilu No. 1, and the post code is 100101. The invention provides a method for separating and purifying ciliate-killing substances in nannochloropsis oculata LAMB204, which can effectively separate and extract the substances through liquid chromatography. Provides a new method for extracting nontoxic and harmless biological pesticide.
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
Various diseases occur frequently while the aquaculture industry is developed in a blowout manner. In recent years, in the cultivation of various economic animals and plants (fishes, crustaceans, algae, sea cucumbers, etc.), diseases caused by ciliates have been increasing in both the occurrence range and the degree of damage. When ciliate diseases occur, drugs prepared by taking copper sulfate, ferrous sulfate, zinc sulfate, formaldehyde, pyrethrins and the like as main components are mostly adopted in the aquatic product market for treatment, and even malachite green and other forbidden drugs are abused. The drugs provided above can cause serious consequences such as water pollution, drug residue, resistance reduction of aquaculture animals, carcinogenesis and the like, so that the aquaculture species are degraded and the diseases are serious, and finally, producers suffer huge economic losses. Aiming at the phenomena, how to extract nontoxic and harmless biological pesticide from plants is a problem which needs to be solved urgently at present.
Disclosure of Invention
Aiming at the problem that no toxic and harmless biological pesticide is lacked in the prior art, the invention provides a method for extracting ciliate killing substances from nannochloropsis so as to solve the problem. The invention separates and purifies the effective substances in the nannochloropsis LAMB204, can be used for killing ciliates, and provides a new method for nontoxic and harmless biological pesticides.
The technical scheme of the invention is as follows:
a method for extracting ciliate-killing substance from Nannochloropsis sp (Lamb 204) comprises crushing Lamb, 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 2020, 10 and 23 days, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the Beijing city, Chaoyang district, North Chen Xilu No. 1, and the post code is 100101.
The method for crushing nannochloropsis LAMB204 comprises the following steps: culturing the nannochloropsis LAMB204 to plateau stage, and centrifuging the algae solution at plateau stage at 8000r/min for 10 min. The supernatant after centrifugation was filtered to ensure that the supernatant was free of algal cells.
The culture medium of Nannochloropsis oculata LAMB204 is F/2 seawater culture medium. The F/2 seawater culture medium comprises the following substances: every 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 0.07mg of O, vitamin B 1 0.1mg, vitamin B 12 0.5 μ g and biotin 0.5 μ g.
The culture method of nannochloropsis LAMB204 comprises the following steps: the culture temperature is 23 +/-1 ℃, and the illumination intensity is 70 mu molphostons/s/m 2 irradation (12:12), salinity of 30 ‰, and pH of 7.8.
The high performance liquid chromatography method comprises the following steps:
the instrument comprises: waters1525 liquid chromatography system with 2998PDA detector and 2707 autosampler
A chromatographic column: agilent Zorbax SB-C184.6x250mm, 5 um; column temperature 30 deg.C
Ultraviolet wavelength: 190nm-400nm full wavelength scanning
Sample injection amount: 10 μ L
Mobile phase: a-methanol; b-0.1% trifluoroacetic acid in water
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 preparative liquid chromatography method was as follows:
the instrument comprises: waters2545 series, 2789 UV/Vis Detector and 2767 fraction collector
A chromatographic column: waters XTerra prep RP c187.8x150mm, 10um
Ultraviolet wavelength: 200nm
Sample introduction amount: 100uL
Mobile phase: a-methanol; b-0.5% aqueous acetic acid 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 marine cocculi.
The beneficial effects of the invention are as follows:
the invention provides a method for separating and purifying substances capable of killing ciliates in nannochloropsis oculata LAMB204, which can effectively separate and extract the substances through liquid chromatography and provides a new method for extracting nontoxic and harmless biological pesticides.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is an HPLC chromatogram of a sample to be separated in example 1 of the present invention.
FIG. 2 is a prepared liquid phase spectrum of a sample to be separated in example 1 of the present invention.
FIG. 3 is a graph showing the population growth of the toxicological test flagellates of example 2 of the present invention.
FIG. 4 is a graph showing the morphological changes of flagellates in toxicological experiments in example 2 of the present invention.
FIG. 5 is a graph showing the population growth of marine coccid in the toxicological test of example 2 of the present invention.
FIG. 6 is a diagram showing the morphological changes of marine coccid in the toxicological test of example 2 of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for extracting ciliate killing substance from nannochloropsis sp comprises the following steps:
(1) nannochloropsis LAMB204(Nannochloropsis oceanica) was prepared and cultured in F/2 seawater medium containing, per 1L of seawater: 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 μ g and biotin 0.5 μ g. The culture temperature is 23 +/-1 ℃, and the illumination intensity is 70 mu molphostons/s/m 2 irradiation (12:12), salinity of 30 per mill, and pH of 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 the supernatant was free of algal cells. And (3) carrying out liquid chromatography analysis on the filtered supernatant, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: waters1525 liquid chromatography System with 2998PDA Detector and 2707 autosampler
A chromatographic column: agilent Zorbax SB-C184.6x250mm, 5 um; column temperature 30 deg.C
Ultraviolet wavelength: 190nm-400nm full wavelength scanning
Sample injection amount: 10 μ L
Mobile phase: a-methanol; b-0.1% trifluoroacetic acid aqueous 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 results of the detection are shown in FIG. 1.
(3) And (3) separating and purifying the supernatant through a prepared liquid phase according to the detection result of the step (2), wherein the conditions of the prepared liquid phase are as follows:
the instrument comprises: waters2545 series, 2789 UV/Vis Detector and 2767 fraction collector
A chromatographic column: waters XTerra prep RP c187.8x150mm, 10um
Ultraviolet wavelength: 200nm
Sample introduction amount: 100uL
Mobile phase: a-methanol; b-0.5% aqueous acetic acid 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 results of the detection are shown in FIG. 2. The material was collected at RT 15.03 min. And (3) completely evaporating the mobile phase in the preparation of the active substance by using a rotary evaporator, and blowing nitrogen for 5min after the evaporation is finished so as to ensure that the organic solvent is completely evaporated and avoid the influence of the organic solvent on the experimental result.
Example 2
To be sent toExample 1 to active substances isolated from nannochloropsis, 2ml of F/2 seawater medium was added and after solubilization, toxicity tests were performed on flagellates and marine coccinella. The specific experimental steps are as follows: the experiment was carried out in a 24-well plate by adding 20 ml of the liquid prepared in example 1 to each well and 20 flagellates and coccinedia oceanica to 20 ml of the liquid prepared in example 1, respectively, at a culture temperature of 23. + -. 1 ℃ and a light intensity of 100. mu. m m -2 .s -1 The illumination period is 12: 12. To obtain the density, population growth rate and generation time of flagellates and marine coccid, they were counted every 12 hours on a Research invested System Microcope. The flagellates and marine coccid in each well were counted three times and then averaged. Population growth of flagellates and marine coccinellids is measured in terms of time and density. The results are shown in detail in FIGS. 3 to 6.
As can be seen from the graphs of fig. 3 and 5, in the solution prepared in example 1, flagellates were completely killed within 3 hours and marine coccyx were completely killed within 2.5 hours. In FIG. 4, A → F is the morphogram of the apoptosis process of flagellate, and FIG. 6 is the morphogram of the apoptosis process of marine coccyx. As can be seen from fig. 4 and 6, in the solution prepared in example 1, flagellates and marine coccinellids were effectively killed. Therefore, the nannochloropsis oculata extract prepared by the invention can be proved to be capable of effectively killing ciliates.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. A method for extracting substance for killing ciliates from nannochloropsis is characterized in that nannochloropsis (Nannochloropsis sorbifolia) LAMB204 is used as raw material, and separation is carried out by using prepared liquid phase after crushing; the nannochloropsis oculata LAMB204 has a preservation number of CGMCC NO.20713, a preservation date of 10 and 23 days in 2020, a preservation unit of common microorganism center of China Committee for culture Collection of microorganisms, a preservation address of No. 3 of Xilu No. 1 of Beijing Kogyo area of Chaoyang, and a zip code of 100101.
2. The method of claim 1, wherein the nannochloropsis LAMB204 is disrupted by: culturing nannochloropsis LAMB204 to plateau stage, and centrifuging the plateau stage algae solution at 8000r/min for 10 min; the supernatant after centrifugation was filtered.
3. The method of claim 2, wherein the culture medium of nannochloropsis LAMB204 is F/2 seawater culture medium.
4. The method of claim 3, wherein the F/2 seawater medium comprises the following: every 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. mu.g, biotin 0.5. mu.g.
5. The method of claim 1, wherein the nannochloropsis LAMB204 is cultured by: the culture temperature is 23 +/-1 ℃, and the illumination intensity is 70 mu molphostons/s/m 2 The day and night irradiation ratio is 12:12, the salinity is 30 per mill, and the pH is 7.8.
6. The method of claim 1, wherein the preparative liquid chromatography method is as follows:
the instrument comprises the following steps: a Waters2545 series, equipped with 2789 uv-vis detector and 2767 fraction collector;
a chromatographic column: waters XTerra prep RP c187.8x150mm, 10 um;
ultraviolet wavelength: 200 nm;
sample introduction amount: 100 uL;
mobile phase: a-methanol; b-0.5% aqueous acetic acid;
elution gradient:
7. a ciliate-killing substance extracted by the method of claim 1.
8. A method of detecting the substance of claim 7, wherein the high performance liquid chromatography method comprises:
the instrument comprises: a Waters1525 liquid chromatography system with 2998PDA detector and 2707 autosampler;
a chromatographic column: agilent Zorbax SB-C184.6x250mm, 5 um; the column temperature is 30 ℃;
ultraviolet wavelength: scanning at 190nm-400nm full wavelength;
sample introduction amount: 10 mu L of the solution;
mobile phase: a-methanol; b-0.1% trifluoroacetic acid in water;
elution gradient:
9. the method of claim 1, wherein the ciliates are flagellates and marine filarial worms.
Priority Applications (1)
| 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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| US20100112649A1 (en) * | 2008-06-04 | 2010-05-06 | Willson Bryan Dennis | Compositions, methods and uses for growth of microorganisms and production of their products |
| 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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Patent Citations (6)
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| US20100112649A1 (en) * | 2008-06-04 | 2010-05-06 | Willson Bryan Dennis | Compositions, methods and uses for growth of microorganisms and production of their products |
| 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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