CN104232524B - Algistatic activity material prodigiosin and preparation method thereof - Google Patents
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Abstract
Algistatic activity material prodigiosin and preparation method thereof, is related to pigment.By Hahella sp.KA22 inoculations after purification in being cultivated on solid 2216E flat boards, picking single bacterium colony is inoculated in after cultivating in 2216E fluid nutrient mediums and obtains final product zymotic fluid, is centrifuged, removal supernatant stays thalline, extraction, adds ethyl acetate after being evaporated, be evaporated extract after standing；Extract is dissolved in methyl alcohol, sephadex post separation is splined on, gained is red, yellow color component rotation is evaporated, and is re-dissolved in verifying algistatic activity in DMSO；Active component ethyl acetate is dissolved, loading and silicagel column, wash-out；Yellow, red and pink component, be evaporated, be dissolved in DMSO and verify algistatic activity；Active component is dissolved in ethyl acetate, chromatographic analysis, colour developing determines active component purity；By active component loading and silicagel column elution；Yellow and red component, be evaporated, be dissolved in DMSO and verify algistatic activity.
Description
Technical field
The present invention relates to a kind of pigment with strong algicdal activity, more particularly to algistatic activity material prodigiosin and its
Preparation method.
Background technology
Ocean complex environment has bred abundant microbial diversity, and marine microorganism is by micro- food ring in marine ecology
Played an important role in the material circulation of system and energy flow, and have complicated relation with red tide algae, be worth one
It is mentioned that,
The various microorganisms such as marine bacteria, actinomyces, virus can in specific manner be suppressed by direct or indirect effect
The growth of algae even cracks frustule, therefore molten algae property microorganism is to adjust one of important factor that red tide life disappears, fully profit
With its interaction feature and rule with red tide plankton, the special efficacy microorganism that development and utilization is derived from frequent occurrence marine site can
Can turn into solves the problems, such as Disaster And Prevention Measures of Red Tides this complexity and important means (Wang Xin, Zhou Yanyan, the Zheng Tianling (2010) of the ocean of sternness
The ecological some advanced subjects of marine bacteria and its New research progress microorganisms journal 50:291-297；Zhang H,An X,
Zhou Y,Zhang B,Zhang S,et al.(2013)Effect of Oxidative Stress Induced by
Brevibacterium sp.BS01on a HABCausing Species-Alexandrium tamarense.PLoS ONE
8:e63018；Lee's Yi, Zheng Wei, Zheng Tianling (2013) marine microbial diversity and its Progress in molecular ecology microorganisms
Learn circular 40:655-668).Marine bacteria plays key player in the life of red tide disappears, and affects marine ecosystems material
With the transport and conversion of energy.Up to now there is substantial amounts of algae-lysing bacterium to be separated to obtain, it is main to include false Alteromonas
Bacterium (Pseudoalteromonas), the vibrios (Vibrio) in γ-Proteobacteria, Cytophage (Cellulophaga),
Flavobacterium (Flavobacterium) in CFB monoids, and low G/C content bacillus (Bacillus)
(Yoshinaga I,Kawai T,Ishida Y(1997)Analysis of algicidal ranges of the
bacteria killing the marine dinoflagellate Gymnodinium mikimotoi isolated
from Tanabe Bay,Wakayama Pref.,Japan.Fisheries science:FS 63:94-98；Lovejoy C,
Bowman JP,Hallegraeff GM(1998)Algicidal effects of a novel marine
Pseudoalteromonas isolate(class Proteobacteria,gamma subdivision)on harmful
algal bloom species of the genera Chattonella,Gymnodinium,and
Heterosigma.Applied and Environmental Microbiology 64:2806；Su J,Yang X,Zhou
Y,Zheng T(2011)Marine bacteria antagonistic to the harmful algal bloom
species Alexandrium tamarense(Dinophyceae).Biological Control 56:132-138)。
In recent years, China's red tide occurrence frequency quickly increases, and red tide occurs scale and drastically expands, and marine site occurs to all near
Bank marine site extends, and time span is gradually increased, and poisonous, harmful red tide plankton persistently increases.It is the highest attention of reply country
Environment Change and marine ecology safety problem, in view of the puzzlement that the harmful algal that China takes place frequently is caused to economic development, research is newly
The efficient red-tide control method of type, is one of significant problem urgently to be resolved hurrily in the current field.Due to physics and chemical method
Administer red tide and apply and easily cause the weak points such as secondary pollution in the presence of large area is difficult to, made using biological ecology each other
Current research focus is turned into (yellow the Changjiang river, the end of the year coastal area of southeastern China of Dong Qiaoxiang, Zheng Lei (1999) 1997 is big for administering red tide
The biological typoiogical classification of scale red tide reason and Ecological Characteristics Oceanologia et Limnologia Sinicas 30:581-589；Skerratt JH,
Bowman JP,Hallegraeff G,James S,Nichols PD(2002)Algicidal bacteria associated
with blooms of a toxic dinoflagellate in a temperate Australian
estuary.Marine Ecology-Progress Series 244:1-15；Su JQ,Yang XR,Zheng TL,Tian
Y,Jiao NZ,et al.(2007)Isolation and characterization of a marine algicidal
bacterium against the toxic dinoflagellate Alexandrium tamarense.Harmful
Algae 6:799-810；Yu Zhiming, Cao Xihua (2002) the enforcement period of the ninth five-year plan coastal area of china harmful algal disaster situation and its preventing and treating
Countermeasure safety and environment journal 2:15-17；Li little Cai, Pei Haiyan, Hu Wenrong (2007) algae-lysing bacterium and algicidal substances study into
Exhibition Treatment of Industrial Water).
The algicidal mode of bacterium includes directly killing algae and killing algae indirectly, the former refer to algae-lysing bacterium directly with cells contacting and
Cause the cracking of frustule dead, the latter is that i.e. algae-lysing bacterium discharges chemical substance and splits and kill by way of similar allelopathy
Frustule or with algae competition nutriment mode work.At present, it has been reported that bacterium olution-type adhesive include egg
White matter (Lee S, Kato J, Takiguchi N, Kuroda A, Ikeda T, et al. (2000) Involvement of an
extracellular protease in algicidal activity of the marine bacterium
Pseudoalteromonas sp.strain A28.Applied and Environmental Microbiology 66:
4334), polypeptide (Imamura N, Motoike I, Shimada N, Nishikori M, Morisaki H, et al. (2001)
An efficient screening approach for anti-Microcystis compounds based on
Knowledge of aquatic microbial ecosystem.The Journal of antibiotics 54), amino
Acid (Yoshikawa K, Adachi K, MIYUKI, N, Tamaki S, Harada K, et al. (2000) Beta-
cyanoalanine production by marine bacteria on cyanide-free medium and its
specific inhibitory activity toward cyanobacteria.Applied and Environmental
Microbiology 66:718), biosurfactant (Dakhama A, No ü e J, Lavoie M (1993) Isolation
and identification of antialgal substances produced by Pseudomonas
aeruginosa.Journal of Applied Phycology 5:297-306), pigment (Kim D, Lee JS, Park YK,
Kim JF,Jeong H,et al.(2007)Biosynthesis of antibiotic prodiginines in the
marine bacterium Hahella chejuensis KCTC2396.J Appl Microbiol 102:937-944) etc..
Therefore, exploitation algae-inhibiting agent is turned into for a new approaches of red tide control by screening efficient, single-minded algistatic activity material.
The content of the invention
The first object of the present invention is to provide Hahella sp.KA22.
The second object of the present invention is to provide a kind of algistatic activity material prodigiosin and preparation method thereof.
The Hahella sp.KA22 were preserved in China typical culture collection center on 06 29th, 2014, protected
Tibetan center deposit number is CCTCC NO：M2014297, collection address is：Chinese Wuhan Wuhan Universitys, postcode
430072。
The molecular formula of the algistatic activity material prodigiosin is C20H26ON3, structural formula is：
Relative molecular weight is 323.
The preparation method of the algistatic activity material prodigiosin, comprises the following steps：
1) by Hahella sp.KA22 inoculations after purification on the solid 2216E flat boards that fresh seawater is prepared, in
Cultivated in constant incubator, the single bacterium colony on picking solid 2216E flat boards is inoculated in the 2216E Liquid Cultures of fresh seawater preparation
After being cultivated in base, zymotic fluid is obtained final product；
2) by step 1) gained zymotic fluid centrifugation, removal supernatant leaves thalline, with acidic methanol extraction, by methanol extraction liquid
Rotation is evaporated, and adds the ethyl acetate isometric with methyl alcohol, is evaporated acetic acid ethyl acetate extract rotation after standing, goes removal of impurities
Matter；
3) by step 2) acetic acid ethyl ester extract that obtains is dissolved in 1mL methyl alcohol, is splined on Sephadex LH-20 Portugals and gathers
Sugared gel column carries out initial gross separation, and elution flow mutually takes 100% methyl alcohol, obtains red component A1, yellow color component A2；By red
Component A1 and yellow color component A2 rotations are evaporated, and are dissolved in DMSO, verify algistatic activity；
4) by active component 1mL ethyl acetate dissolving, loading and silicagel column, eluted using eluent gradient concentration；
5) component for collecting different colours obtains yellow color component B1, red component B2, pink component B3, by yellow color component
B1, red component B2 and pink component B3 rotations are evaporated, and are dissolved in DMSO, verify algistatic activity；
6) active component is dissolved in ethyl acetate, using thin-layer chromatography (thin-layer chromatography,
TLC) analyze, using ultraviolet colour developing, iodine vapor colour developing and ammonium sulfate ethanol develop the color, and determine active component purity；
7) active component loading and silicagel column are eluted using eluent gradient concentration；
8) component for collecting different colours obtains yellow color component C1 and red component C2, by yellow color component C1 and red component
C2 rotations are evaporated, and are re-dissolved in DMSO, verify algistatic activity.
In step 1) in, the Hahella sp.KA22 were preserved in Chinese Typical Representative culture on 06 29th, 2014
Collection, collection deposit number is CCTCC NO：M2014297, collection address is：Wuhan, China Wuhan University,
Postcode 430072；The temperature cultivated in be set forth in constant incubator can be 28 DEG C, and the time of culture can be 24h；It is set forth in fresh
The condition cultivated in the 2216E fluid nutrient mediums that seawater is prepared can be in 28~37 DEG C, 180~250rpm cultures 2d.
In step 2) in, the condition of the centrifugation can be in 10,000~12,000g centrifugations 10min；The acidic methanol
Consumption can be by volume 30 times of thalline；The temperature of the standing can be 4 DEG C；The impurity includes a large amount of blue-black color substances
And other impurities.
In step 4) in, the silicagel column can use 170mm × 30mm, 200~300 mesh silicagel columns；The mobile phase ladder
Spending concentration can be：A () n-hexane elutes 2 column volumes；(b) n-hexane: ethyl acetate=8: 1, calculate by volume, wash-out 2
Individual column volume；(c) n-hexane: ethyl acetate=6: 1, elute 2 column volumes；(d) n-hexane: ethyl acetate=5: 1, wash-out 4
Individual column volume；(e) n-hexane: ethyl acetate=3: 1, elute 2 column volumes；F () elutes 2 column volumes with ethyl acetate.
In step 7) in, the silicagel column can use 170mm × 30mm, 500 mesh silicagel columns；The eluent gradient concentration
Can be：A () n-hexane elutes 2 column volumes；(b) n-hexane: ethyl acetate=8: 1, calculate by volume, elute 2 cylinders
Product；(c) n-hexane: ethyl acetate=6: 1, elute 2 column volumes；(d) n-hexane: ethyl acetate=5: 1, elute 4 cylinders
Product；(e) n-hexane: ethyl acetate=3: 1, elute 2 column volumes；F () elutes 2 column volumes with ethyl acetate.
The present invention obtains one plant of Hahella sp.KA22 with conventional method screening, by fermented and cultured, is contained
The zymotic fluid of potent algal control pigment, by zymotic fluid centrifugation, then the thalline is extracted and is then separated by collects thalline
Pigment, obtains the compound with strong algistatic activity.The potent algistatic activity material is a color containing tripyrrole ring structure
Element.The active material can efficiently, frustule is exclusively killed, with the potential for developing into algae-inhibiting agent, in biodegradable algae
Class, improvement red tide aspect have potential application prospect.
Brief description of the drawings
Fig. 1 is the mass-spectrogram of algistatic activity material prodigiosin of the present invention.In Fig. 1, abscissa represents matter lotus
Than ordinate represents the relative abundance at each peak；By mass spectrogram as can be seen that substantially containing a kind of compound in component C2, should
The molecule [M+H] of compound+Cation adduction peak is respectively 324.2, thus it is speculated that the molecular weight of the compound is about 323.
Fig. 2 is the nuclear magnetic resonance map of algistatic activity material prodigiosin of the present invention.In fig. 2, abscissa is represented
Each peaking displacement study, ordinate represents the intensity at each peak；Using nuclear magnetic resonance technique 1H-NMR (600M), 13C-NMR,
DEPT and H-1H COSY spectrograms, thus it is speculated that structural formula of compound, chemical molecular formula is C20H26ON3, molecular weight is that 323.2, m/z is
323.2065。
Fig. 3 is the effect of algae restraint figure of algistatic activity material prodigiosin of the present invention.In figure 3, abscissa representative office
Reason time, ordinate represents algal control rate；After treatment 3d, the prodigiosin algal control rate of 5 μ g/ml can reach more than 80%, i.e. algae
The growth of cell receives serious suppression.
Fig. 4 is the bacterial strain electron microscope of algistatic activity material prodigiosin of the present invention.In fig. 4 it is shown that transmission electron microscope
Lower observation thalline is obvious corynebacteria, and size is (0.5~0.8) μ m (7.0~8) μm, atrichia.
Specific embodiment
Following examples will the present invention is further illustrated with reference to accompanying drawing.
1st, strain, algae kind and culture medium
Strain：Hahella sp.KA22 are from Xiamen University using red from Fujian sky with environmental microorganism research institute
One plant of bacterium with stronger algicdal activity that woods wetland is separate, this bacterium of Preliminary Identification is river Bordetella (Hahella sp.).
Algae kind：Phaeocystis globosa is without bacterial strain, and algae germline is provided by aquatile research institute of Ji'nan University, aseptic through this experiment
The degerming phaeocystis globosa for obtaining of algae technology is without bacterial strain.Algae used medium is f/2 culture mediums.Algae triangular flask disposed within
Middle culture, temperature is 20 ± 1 DEG C, and illumination condition is 12h illumination: 12h dark.
Culture medium：
Screening and culturing medium (2216E)：Peptone (Peptone) 5g, yeast extract (Yeast Extract) 1g, phosphoric acid
High ferro 0.1g, agar powder 10g (solid medium), pH 7.6~7.8, Chen Haishui constant volumes to 1L.
2nd, the preparation method of the algistatic activity material prodigiosin, comprises the following steps：
(1) by Hahella sp.KA22 inoculations after purification on the solid 2216E flat boards that fresh seawater is prepared,
24h is cultivated in 28 DEG C of constant incubators；Single bacterium colony on flat board described in picking is inoculated in the 2216E liquid of fresh seawater preparation
In culture medium, in 28~37 DEG C, zymotic fluid is obtained final product after 180~250rpm cultures 2d；
(2) zymotic fluid in step (1) is centrifuged 10min in 10,000~12,000g；Removal supernatant leaves thalline；Adopt
Extracted with 30 times of acidic methanols of volume, the rotation of methanol extraction liquid is evaporated, added and the isometric ethyl acetate of methyl alcohol, 4 DEG C quiet
Put overnight；Acetic acid ethyl acetate extract rotation is evaporated, a large amount of blue-black color substances and other impurity are removed；
(3) acetic acid ethyl ester extract is dissolved in 1mL methyl alcohol, being splined on Sephadex LH-20 sephadex columns is carried out
Initial gross separation, elution flow mutually takes 100% methyl alcohol, obtains red component A1, yellow color component A2；Two component is rotated and is steamed
It is dry, it is dissolved in DMSO checking algistatic activities；
(4) by active component 1mL ethyl acetate dissolving, loading and silicagel column (170mm × 30mm, 200~300 mesh),
Eluted using eluent gradient concentration, i.e., (a) n-hexane elutes 2 column volumes；(b) n-hexane: ethyl acetate (v/v)=8: 1,
2 column volumes of wash-out；(c) n-hexane: ethyl acetate=6: 1, elute 2 column volumes；(d) n-hexane: ethyl acetate=5: 1,
4 column volumes of wash-out；(e) n-hexane: ethyl acetate=3: 1, elute 2 column volumes；F () elutes 2 cylinders with ethyl acetate
Product；
(5) component for collecting different colours obtains yellow color component B1, red component B2, pink component B3, by three groups
Divide rotation to be evaporated, be dissolved in DMSO, verify algistatic activity；
(6) active component is dissolved in ethyl acetate, using thin-layer chromatography (thin-layer chromatography,
TLC) analyze, using ultraviolet colour developing, iodine vapor colour developing and ammonium sulfate ethanol develop the color, and determine active component purity；
(7) active component loading and silicagel column (170mm × 30mm, 500 mesh) are eluted using eluent gradient, stream used
Dynamic phase is as described in step (4)；
(8) component for collecting different colours obtains yellow color component C1, red component C2, and two component rotations are evaporated, molten
Solution verifies algistatic activity in DMSO.
The structural formula of obtained algistatic activity material prodigiosin is as follows：
The structural formula is after determining relative molecular mass by ESI-OrbiTrap MS, then through nuclear magnetic resonance map solution
Spectrum, relative molecular weight is about 323.
The mass-spectrogram of algistatic activity material prodigiosin of the present invention is referring to Fig. 1.As seen from Figure 1, component C2
In substantially contain a kind of compound, the molecule [M+H] of the compound+Cation adduction peak is respectively 324.2, thus it is speculated that the compound
Molecular weight be about 323.The nuclear magnetic resonance map of algistatic activity material prodigiosin of the present invention is referring to Fig. 2.Can be with by Fig. 2
Find out, using nuclear magnetic resonance technique 1H-NMR (600M), 13C-NMR, DEPT and H-1H COSY spectrograms, thus it is speculated that compound structure
Formula, chemical molecular formula is C20H26ON3, molecular weight is that 323.2, m/z is 323.2065.Algistatic activity material spirit bacterium of the present invention
The effect of algae restraint figure of red pigment is referring to Fig. 3.As seen from Figure 3, after treatment 3d, the prodigiosin algal control rate of 5 μ g/ml can reach
The growth of more than 80%, i.e. frustule receives serious suppression.The bacterial strain Electronic Speculum of algistatic activity material prodigiosin of the present invention
Figure is referring to Fig. 4.As seen from Figure 4, it is obvious corynebacteria thalline to be observed under display transmission electron microscope, and size is (0.5~0.8)
μ m (7.0~8) μm, atrichia.
The present invention can first pass through a Sephadex LH-20 sephadex column chromatography, and elution flow mutually uses 100%
Methyl alcohol, component is collected by different colours；Silicagel column (170 × 30mm, 200~300 mesh) and silicagel column (170 are then carried out successively
× 30mm, 500 mesh) chromatography, using eluant, eluent n-hexane and ethyl acetate different volumes ratio gradient elution；According to different colours
Collect component.
The Hahella sp.KA22 can be screened by the following method：
(1) Fujian sky Mangrove Wetlands (117 ° of 24 ' -117 ° of 30 ' E, 23 ° of 53 ' -23 ° of 56 ' N) deposit, uses sterilized water
After a series of 10 times dilute, each dilution takes 0.1mL and is coated on 2216E culture medium flat plates, and 5d is cultivated at 25 DEG C；
(2) picking difference bacterium colony size, form single bacterium colony line 2216E solid plates, are placed at a temperature of 28~37 DEG C
Culture 3-5d, verifies whether pure culture, repeats the step until obtaining pure culture；
(3) by inoculation after purification in 3mL 2216E fluid nutrient mediums, 28 DEG C of shaking tables, 150rpm concussions are placed in
Culture 3d；
(4) 0.5mL nutrient solutions are taken to be inoculated in the phaeocystis globosa nutrient solution of exponential growth, in 20 ± 1 DEG C, 12h illumination,
12h is dark, 50 μm of ol photons m-2s-12d is cultivated under the conditions of intensity of illumination, 2216E culture mediums are added in algae solution for control,
Be respectively provided with 3 it is parallel；Whether observation phaeocystis globosa cell settles, and sedimentation contains algal control in representing thalline 3d culture supernatants
Active material, so as to filter out algistatic activity bacterial strain.
Claims (9)
1.Hahella sp.KA22, were preserved in China typical culture collection center, collection on 06 29th, 2014
Deposit number is CCTCC NO：M2014297.
2. the preparation method of algistatic activity material prodigiosin, it is characterised in that the molecule of the algistatic activity material prodigiosin
Formula is C20H26ON3, structural formula is：
Relative molecular weight is 323；
The preparation method is comprised the following steps：
1) by Hahella sp.KA22 inoculations after purification on the solid 2216E flat boards that fresh seawater is prepared, in constant temperature
Cultivated in incubator, the single bacterium colony on picking solid 2216E flat boards is inoculated in the 2216E fluid nutrient mediums of fresh seawater preparation
After culture, zymotic fluid is obtained final product；The Hahella sp.KA22, were preserved in Chinese Typical Representative culture on 06 29th, 2014
Collection, collection deposit number is CCTCC NO：M2014297；
2) by step 1) gained zymotic fluid centrifugation, removal supernatant leaves thalline, is extracted with acidic methanol, by the rotation of methanol extraction liquid
It is evaporated, adds the ethyl acetate isometric with methyl alcohol, be evaporated acetic acid ethyl acetate extract rotation after standing, goes the removal of impurity；
3) by step 2) acetic acid ethyl ester extract that obtains is dissolved in 1mL methyl alcohol, is splined on Sephadex LH-20 glucans and coagulates
Glue post carries out initial gross separation, and elution flow mutually takes 100% methyl alcohol, obtains red component A1, yellow color component A2；By red component
A1 and yellow color component A2 rotations are evaporated, and are dissolved in DMSO, verify algistatic activity；
4) by active component 1mL ethyl acetate dissolving, loading and silicagel column, eluted using eluent gradient concentration；
5) collect different colours component obtain yellow color component B1, red component B2, pink component B3, by yellow color component B1,
Red component B2 and pink component B3 rotations are evaporated, and are dissolved in DMSO, verify algistatic activity；
6) active component is dissolved in ethyl acetate, using thin layer chromatography analysis, using ultraviolet colour developing, iodine vapor develops the color and sulfuric acid
Ammonium ethanol develops the color, and determines active component purity；
7) active component loading and silicagel column are eluted using eluent gradient concentration；
8) component for collecting different colours obtains yellow color component C1 and red component C2, and yellow color component C1 and red component C2 is revolved
Turn to be evaporated, be re-dissolved in DMSO, verify algistatic activity.
3. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 1) in, it is described
The temperature cultivated in constant incubator is 28 DEG C, and the time of culture is 24h.
4. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 1) in, it is described
The condition cultivated in the 2216E fluid nutrient mediums that fresh seawater is prepared is in 28~37 DEG C, 180~250rpm cultures 2d.
5. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 2) in, it is described
The condition of centrifugation is that 10min is centrifuged in 10,000~12,000g；The consumption of the acidic methanol can be by volume the 30 of thalline
Times；The temperature of the standing can be 4 DEG C；The impurity includes a large amount of blue-black color substances and other impurity.
6. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 4) in, it is described
Silicagel column uses 170mm × 30mm, 200~300 mesh silicagel columns.
7. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 4) in, it is described
Eluent gradient concentration is：A () n-hexane elutes 2 column volumes；(b) n-hexane: ethyl acetate=8: 1, calculate by volume,
2 column volumes of wash-out；(c) n-hexane: ethyl acetate=6: 1, elute 2 column volumes；(d) n-hexane: ethyl acetate=5: 1,
4 column volumes of wash-out；(e) n-hexane: ethyl acetate=3: 1, elute 2 column volumes；F () elutes 2 cylinders with ethyl acetate
Product.
8. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 7) in, it is described
Silicagel column uses 170mm × 30mm, 500 mesh silicagel columns.
9. the preparation method of algistatic activity material prodigiosin as claimed in claim 2, it is characterised in that in step 7) in, it is described
Eluent gradient concentration is：A () n-hexane elutes 2 column volumes；(b) n-hexane: ethyl acetate=8: 1, calculate by volume,
2 column volumes of wash-out；(c) n-hexane: ethyl acetate=6: 1, elute 2 column volumes；(d) n-hexane: ethyl acetate=5: 1,
4 column volumes of wash-out；(e) n-hexane: ethyl acetate=3: 1, elute 2 column volumes；F () elutes 2 cylinders with ethyl acetate
Product.
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Non-Patent Citations (2)
| Title |
|---|
| Biosynthesis of antibiotic prodiginines in the marine bacterium Hahella chejuensis KCTC 2396;D. Kim等;《Journal of Applied Microbiology》;20071231;第102卷;第937-944页，尤其是摘要、第938页图1、第939页左边栏第2段、第940页图3 * |
| 灵菌红素对有害藻类的除藻活性研究;刘伯雅等;《中国环境科学》;20101231;第30卷(第4期);第477-482页 * |
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