CN1791667A - Red pigment originated from Hahella chejuensis, having algalcidal effect - Google Patents
Red pigment originated from Hahella chejuensis, having algalcidal effect Download PDFInfo
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- CN1791667A CN1791667A CNA2004800124600A CN200480012460A CN1791667A CN 1791667 A CN1791667 A CN 1791667A CN A2004800124600 A CNA2004800124600 A CN A2004800124600A CN 200480012460 A CN200480012460 A CN 200480012460A CN 1791667 A CN1791667 A CN 1791667A
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- 241000045411 Hahella chejuensis Species 0.000 title claims abstract description 31
- 230000000694 effects Effects 0.000 title claims description 60
- 238000000034 method Methods 0.000 claims abstract description 31
- 241000206757 Heterosigma akashiwo Species 0.000 claims abstract description 17
- 241000913578 Gymnodinium impudicum Species 0.000 claims abstract description 15
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 241000195493 Cryptophyta Species 0.000 claims description 110
- 239000000049 pigment Substances 0.000 claims description 75
- 244000005700 microbiome Species 0.000 claims description 33
- 230000001580 bacterial effect Effects 0.000 claims description 30
- 241001300810 Cochlodinium Species 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 241000046129 Hahella Species 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000010521 absorption reaction Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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Abstract
The present invention relates to a Hahella chejuensis-derived red pigment having an algicidal effect, and more particularly, to a method for producing a red pigment (RP10356) by culturing Hahella chejuensis 96CJ10356 strain (KCTC 2396). Moreover, the invention relates to a red pigment (RP10356) having an algicidal effect, prepared by this method, an algicidal formulation containing this red pigment as an active ingredient, a method for removing red tide organisms, which is characterized by the use of an organic solvent extract of the culture broth or the use of the red pigment. The inventive red pigment (RP10356) has an excellent algicidal effect against red tide-causing species, such as Cochlodinium polykrikoides, Gyrodinium impudicum, and Heterosigma akashiwo, and thus, is useful as an active ingredient of algicidal formulations for removing red tide organisms.
Description
Technical field
The present invention relates to be derived from the red pigment of Hahella chejuensis, this pigment has molten algae effect.Especially, the present invention relates to a kind of method that obtains red pigment by cultivation Hahella chejuensis 96CJ10356 bacterial strain (KCTC2396).The invention still further relates to the red pigment (RP10356) that obtains by aforesaid method, contain the molten algae preparation of this red pigment, and utilize the meat soup of the described bacterial strain of cultivation or the method that red pigment is removed microorganisms of red tide as activeconstituents with molten algae effect.
Background technology
After the 1980s, taken place harmful and deleterious red tide, the central area that red tide takes place is the algae pollution zone at Korea S beach, and be that water industry brings significant damage the red tide every year that is caused by microalgae.Especially, the cochlodinium sp (Cochlodinium polykrikoides) of generation afterwards in annual August causes significant damage (Park et al., J.Korean Fish.Soc., 31:767-73,1998) for Korea S's seashore water industry.For this reason, people are to the death of the microalgae that causes red tide with suppress growth and launched research (Yoshinaga et al., Japan Fish Sci., 61:780-6,1995).
In Marine ecosystems, micropopulation is to the generation and the termination of red tide play an important role (Furuka et al., Marine Pollution Bull., 23:189-93,1992).The quantity of molten phycomycete reaches highest level at red tide latter stage, and this red tide depends on the host microorganism (Yoshinaga etal., Japan Fish Sci., 61:780-6,1995) in the red tide generation marine site.
Known bacterium with molten algae effect comprises alternately Zymomonas mobilis (Alteromonas), Cytophaga (Cytophaga), Flavobacterium (Flavobaterium), pseudoalteromonas (Pseudoalteromonas), Sporospira and vibrios (Vibrio).It is reported that such bacterium is got involved in (Lovejoy et al., Appl.Environ.Microbiol., 64:2806-13,1993) in the disappearing of red tide directly or indirectly.Molten phycomycete can kill or dissolve multiple algae, and every kind in these bacteriums all has the specific molten algae effect of algal kind.For example, molten phycomycete Cytophaga (Cytophoga sp.) can act on Diatomacae (Bacillariophyceae), Raphydophyceae and Dinophyceae (Dinophyceae) (Imai et al., Mar.Biol., 116:527-32,1993) respectively.
Yet, the research of the molten algae mechanism of molten phycomycete is not also finished, it is said cytolysis (the Imai et al. that subduing fast of microorganisms of red tide given the credit to microorganisms of red tide, Mar.Biol., 116:527-32,1993), microorganism growth is suppressed and death (the Imi et al. that causes, Fisheries Science, 61:628-36,1995) and by suppress microorganism from monogony to amphigenetic crossover process, as chain Alexander algae (Sawayama et al., Nippon Suisan Gakkaishi, 59:291-4,1993).
The molten algae mechanism of molten phycomycete can be divided into two classes: (1) a kind of mechanism is that molten phycomycete causes microorganisms of red tide to dissolve (Imai et al. by contacting with the microorganisms of red tide surface, Mar.Biol., 116:527-32,1993), (2) another kind of mechanism is the molten algae material of the external secretion of molten phycomycete and induce microorganisms of red tide that dissolving takes place or growth is suppressed.The mechanism of action of most of molten phycomycetes belongs to second kind, report that in addition molten algae mechanism is by (Kawano et al. due to the microbiotic, J.Mar.Biotechnol., 5:225-9,1997), by (Sawayama et al., Nippon Suisan Gakkaishi, 59:291-4 due to the low-molecular-weight oligopeptides, 1993), by (Baker and Herson, Appl.Environ.Microbiol., 35:791-6 due to the protein, 1978) (Mitsutani et al. and due to the thermally labile ECM, Nippon Suisan Gakkaishi, 58:2159-69,1992).Microalgae is occurring in several days reducing with the postvaccinal several hrs of most of molten phycomycetes.The difference in reaction times is directly related with the effect of molten algae material, and the reaction times is different and different with the difference of molten algae material output and inductor kind.
Therefore, the throw oneself into molten algae material that utilizes molten algae microorganisms of people suppresses to cause the growth of the microalgae of red tide, thereby reduces the research of red tide to the harm of marine products industry.Especially cause the nineteen eighty-two cochlodinium sp of red tide for the first time, cause red tide continuously from nineteen ninety.After nineteen ninety-five, from Korea S's south coast to southeast seashore the highdensity extensive red tide of 10000 cells/ml having taken place, thus marine products has already been brought serious harm.For this reason, carried out the prevention red tide and made the minimized research of harm.Yet, do not see so far and utilize the report of pigment as molten algae material.
Pigment can be divided into natural pigment and synthetic dyestuff, and pigment is used in the multiple use, comprises purposes such as food, makeup, medicine and fodder additives.Compare with obtain pigment from animal and plant, obtain natural pigment from microorganism, as can obtaining a large amount of natural pigments by fermentation, and pigment property is stable.The color of the pigment that obtains from microorganism is different and different according to cultural method and substratum, even same bacterial strain, shade of color is different (Carels et al., Can.J.Microbiol. with the different of medium component, culture temperature and pH value etc., 23:1360-72,1977).Known pigment is a secondary metabolite, obtain as the typical regulation mechanism as shown in the fermentation of microbiotic and toxin by secondary metabolite, their output is subjected to influence (the Wong et al. of carbon source, nitrogenous source, phosphoric acid and trace element largely, Myclogia, 73:649-54,1981).
Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396=IMSNU 11157), this marine microorganism that separation obtains from the settling that Korea S's Cheju island seashore is collected is a kind of Gram-negative sports type bacillus.This bacillus produces a large amount of exocellular polysaccharide and red pigment (Lee et al., Int.J.Systematic and Evolutionary Microbiology, 51:661-6,2001).
But the molten algae that is derived from the pigment of the Hahella microorganism belonging to genus that comprises Hahella chejuensis 96CJ10356 bacterial strain acts on not clear.
Summary of the invention
The inventor has carried out extensive studies to find a kind of new microbe with better molten algae effect, thereby, the red pigment (RP10356) that discovery obtains from aforesaid Hahella chejuensis 96CJ10356 bacterial strain has good molten algae effect, has finished the present invention thus.
Therefore, a main purpose of the present invention is to provide a kind of production to have the method for the red pigment of molten algae effect, the method is characterized in that by the Hahella microorganism belonging to genus is cultivated.
Another object of the present invention is to provide a kind of red pigment (RP10356) with molten algae effect, and a kind of molten algae preparation that contains this red pigment as activeconstituents.
To achieve these goals, scheme of the present invention is to provide a kind of production to have the method for the pigment material of molten algae effect, and it comprises: cultivate the Hahella microorganism belonging to genus; With an organic solvent separating also from culture broth, purifying has the pigment material of molten algae effect.
In the method for the present invention, the Hahella microorganism belonging to genus is Hahella chejuensis preferably, more preferably Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Another scheme of the present invention is to provide a kind of red pigment with molten algae effect that is obtained by aforesaid method, and (sherwood oil: acetone: the Rf value methyl alcohol=5: 3: 2) is 0.98, and maximum absorption is 539nm at silica gel tlc for it.The present invention also provides a kind of molten algae preparation that red pigment is an activeconstituents that contains.
Red pigment among the present invention shows has molten algae effect to cochlodinium sp, Gyrodinium impudicum or Heterosigma akashiwo (Heterosigma akashiwo).
Further scheme of the present invention has been to provide a kind of method of eliminating microorganisms of red tide, the method is characterized in that to have used red pigment, the culture broth of Hahella microorganism belonging to genus or the extractive with organic solvent of culture broth.
In the present invention, utilize the culture broth of organic solvent extraction Hahella chejuensis 96CJ10356 bacterial strain, obtain pure red pigment (RP10356) thereby separate.For obtain to have the red pigment (RP10356) of molten algae effect from Hahella chejuensis96CJ10356 bacterial strain, microorganism must be cultivated in the suitable culture base, and substratum contains the inorganic salt and aged (aged) seawater of nutritive salt, trace.
The spendable nutritional medium of the present invention comprises: 5% glucose as carbon source, 0.5% peptone as nitrogenous source, and 0.083g/L KH
2PO
4, 0.067g/L K
2HPO
4, 5g/L MgSO
4And 1g/L CaCl
2The inorganic salt of trace preferably include 0.005g/L FeCl
26H
2O, 0.001g/LMnCl
24H
2O, 0.001g/L Na
2MoO
4, 0.001g/L ZnCl
2Deng.
The red pigment RP10356 that the thick pigment that extraction obtains from microorganism culturing meat soup and this thick pigment of purifying obtain is detected the molten algae effect that has cochlodinium sp, Gyrodinium impudicum, Heterosigma akashiwo, chain Alexander algae and Procentrum micans.These algae all are the microalgaes that causes red tide.This detected result shows that thick pigment and red pigment RP10356 have good molten algae effect to cochlodinium sp, Gyrodiniumimpudicum and Heterosigma akashiwo.Red pigment RP10356 of the present invention is a kind of fat-soluble red pigment, and the maximum absorption wavelength in the ethanol is 486nm and 539nm.
Description of drawings
Fig. 1 illustrates the process of extracting and separating red pigment from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Fig. 2 shows the molten algae effect to cochlodinium sp of the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Fig. 3 shows the molten algae effect to Gyrodinium impudicum of the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Fig. 4 shows the molten algae effect to Heterosigma akashiwo of the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Fig. 5 shows the molten algae effect to cochlodinium sp of the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396).
Fig. 6 shows the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) and causes the metamorphosis of test bacterium, A: cochlodinium sp owing to producing molten algae effect; B:Gyrodinium impudicum; C: Heterosigma akashiwo; D: chain Alexander algae and E:Procentrummicans.
Fig. 7 shows in the culture of Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396), the situation of pH, cell and RP10356 output.
Fig. 8 shows the thick pigment that obtains from Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) and from the thin-layer chromatography result of the isolated pigment RP10356 of thick pigment, 1:2-propanol extraction thing (thick pigment); 2:RP10356.
Fig. 9 shows from the UV of the RP10356 of Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) acquisition and absorbs.
Figure 10 shows 4 components that obtain by silica gel chromatography from the thick pigment of Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) acquisition, 1:2-propanol extraction thing (thick pigment); 2: component 1; 3: component 2; 4: component 3; 5: component 4; Solvent: sherwood oil: acetone: methyl alcohol=5: 3: 2.
Figure 11 shows from the result of the efficient liquid phase chromatographic analysis of the RP10356 of Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) acquisition.
Figure 12 shows from the high performance liquid chromatography result of the pigment RP10356 of Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396) acquisition, and wherein stratographic analysis detects at 300-550nm by the DAD detector.
Detailed Description Of The Invention and preferred forms
The present invention will be described in detail by following embodiment.For those of ordinary skills is conspicuous, and these embodiment only are used to illustrate, and the present invention also not only is confined to embodiment.
The extraction of embodiment 1:RP10356 with separate
The bacterial strain that this embodiment adopts is Hahella chejuensis 96CJ10356 bacterial strain (KCTC 2396=IMSNU 11157, from the settling that Korea S's Cheju island seashore is collected, separate and obtain (Lee et al., Int.J.Systematic and Evolutionary Microbiology, 51:661-666,2001)).Inoculation this bacterial strain (following table 1) on the STN solid medium was cultivated 48 hours at 25 ℃, stored under 4 ℃ then, and per two weeks are changed fresh culture, carry out inferior the cultivation.
Table 1:STN substratum
| Composition | Concentration |
| Sucrose | 20g/L |
| Tryptone | 10g/L |
| NaCl | 10g/L |
| MgSO 4 | 5g/L |
| CaCl 2 | 1g/L |
| KH 2PO 4 | 0.083g/L |
| K 2HPO 4 | 0.067g/L |
| FeCl 3 | 0.005g/ |
| MnC | |
| l2 | 0.001g/L |
| Na 2MoO 4 | 0.001g/L |
| ZnCl 2 | 0.001g/L |
| Distilled water | 1,000ml |
| pH | 7.0 |
Preparation is during RP10356 from the 96CJ10356 bacterial strain, and this inoculation to basic medium SZoBell medium (table 2), was cultivated 3 days under 25 ℃ and 120rpm then.From culture broth, isolate pigment RP10356 at last.
Table 2:SZoBell medium
| Composition | Concentration |
| Glucose | 20g/L |
| Peptone | 5g/L |
| Yeast extract | 1g/L |
| FePO 4 | 0.01g/L |
| The aged seawater | 750ml |
| Distilled water | 250ml |
| pH | 7.2 |
Utilize method shown in Figure 1, from the culture broth of 96CJ10356 bacterial strain, extract red pigment.For the polysaccharide in the 96CJ10356 strain culturing meat soup that will produce a large amount of exocellular polysaccharides is removed, and isolate pigment, the 2-Virahol that in 1 liter of strain culturing meat soup, adds 2 times of volumes, (BRANSON 8210 to use Ultrasonic Cell Disruptor then, USA) handled 1 hour, placed 24 hours down at 4 ℃ again, thus the polysaccharide in the removal culture broth.12, under the 000g, with centrifugal 30 minutes of the culture broth of having removed polysaccharide, (Whatmann, Φ 47mm USA) filtered, and isolate the thick pigment of removing cell to use the GF/F filter then.
In order from the 2-Virahol pigment extract (thick pigment) of having removed polysaccharide and cell, to isolate pigment RP10356,2-Virahol pigment extract is carried out concentrating under reduced pressure under 45 ℃, remove the 2-Virahol.Then, add to the sherwood oil of 1 volume in the water layer and jolting 1 hour, isolate petroleum ether layer by separatory funnel, thereby remove water soluble component and a large amount of salt, and from the residue water layer, extract fat-soluble component.Use the anhydrous sodium sulfate drying petroleum ether layer, remove moisture wherein, (No 5, and Watmann USA) filters, and 40 ℃ of following concentrating under reduced pressure filtrates sherwood oil steamed and remove (Fig. 1) by filter paper then.
Utilize silica gel chromatography from thick pigment extract, to isolate pigment with molten algae performance.Thick pigment extract is added to silica gel 60 (0.040-0.063mm; Merk, Germany) in, (separate thick pigment among the Φ 2.5 * 30cm), eluting solvent is sherwood oil and acetone (7: 3) at the glass column of having filled equal resin.The Rf value of 4 components that obtain is respectively 0.98 (light yellow), 0.96 (dark red), 0.88 (bright red) and 0.59 (blueness).Wherein, the Rf value is that 0.88 pigment component is main pigment, and this pigment component is called as " RP10356 ", and detects its molten algae effect.
The molten algae effect of embodiment 2:RP10356
The algae that present embodiment adopted is shown in Table 3.This slightly algae be that the red tide research group of BuKung university (BKU) water industry system and Korea S's ocean research and Institute of Development Studies (KORDI) provides.Other little algae is directly isolated from the marine site that red tide took place.
Table 3
| Classification | The science title | Medium | Bacterial strain | The source |
| Dinophyceae (Dinophyceae) | Cochlodinium sp (Cochlodinium polykrikoides) | f/2 | - | BKU |
| Gyrodinium impudicum | f/2 | KG03 | KORDI | |
| Procentrum micans | f/2 | - | KORDI | |
| Pin born of the same parents algae (Raphidophyceae) | Chain Alexander algae (Alexandrium catenella) | f/2 | - | KORDI |
| Heterosigma akashiwo (Heterosigma akashiwo) | f/2 | - | KORDI |
The f/2 medium of employing table 4 is cultivated little algae.The algae incubator (Je-il Science, Korea) in, in 22.5 ℃ and 150 μ Ein/m
2Carry out incubation under the light intensity of/s, incubator is regulated to 16 hours/bright/dark circulation of 8 hours.For little algae culture, adopt the cell of logarithmic phase to detect molten algae effect.(F-2000, Hitachi Japan) detect the growth of little algae, or are fixed in the 2%Rugol solution, use inverted microscope (Axon-1000, Zeiss, Germany) counting then with luminoscope.
Table 4
1.f/2 medium
| Composition | Concentration |
| NaNO 3 | 0.075g |
| NaH 2PO 4·H 2O | 0.005g |
| Na 2SiO 3·9H 2O | 0.030g |
| F/2 trace-metal solution | 1ml |
| The f/2 vitamin solution | 1ml |
| Aged seawater | 1,000ml |
2.f/2 trace element solution
| Composition | Concentration (g/L) |
| MnCl 2·4H 2O | 0.18 |
| ZnSO 4·7H 2O | 0.022 |
| CuSO 4·5H 2O | 0.01 |
| Na 2MoO 4·2H 2O | 0.007 |
| CoCl 2·6H 2O | 0.01 |
| FeCl 2·6H 2O | 3.15 |
| Na 2-EDTA·2HxO | 4.35 |
3.f/2 vitamin solution
| Composition | Concentration |
| Vitamin B12 | 1μg |
| Biotin | 1μg |
| VITMAIN B1-HCl | 200mg |
| Distilled water | 1,000ml |
(1) the molten algae effect of thick pigment
Will be from embodiment 1 isolated thick pigment and RP10356, with concentration is that 0,0.1,1.0,10,50 and 100 μ g/ml add to and contain 1 * 10 of little algae (cochlodinium sp, Gyrodinium impudicum, Heterosigma akashiwo, chain Alexander algae and Procentrum micans) of causing red tide
3In little algae culture broth of cell/ml.Cultivated 30 minutes, 1 hour, 2 hours and after 24 hours, each little algae after will handling respectively is fixed in the 2%Rugol solution, with inverted microscope (Zeiss, Axon100, Germany) the not dissolved cell count of statistics is calculated cell count dissolved in the cell of initial inoculation then.Molten algae effect is calculated according to following formula:
Molten algae effect (%)=[(initiator cell number-survival cells/(initiator cell number)] * 100
Mensuration is dissolved in the molten algae effect of the thick pigment in the ethanol, and the result shows when the concentration of the thick pigment in joining cochlodinium sp is 0.1 μ l/ml that the molten algae effect after 30 minutes and 24 hours is respectively 3.8% and 72.1%.When the concentration that adds was 1.0 μ l/ml, the molten algae effect after 30 minutes and 24 hours was respectively 63.8% and 98.7%.When the concentration that adds during for the highest 100 μ l/ml, the molten algae in the time of 30 minutes act as 91.3%.As can be seen, thick pigment is greater than the concentration of 0.1 μ l/ml the time, and its molten algae effect to the cochlodinium sp that causes maximum red tide harm is maximum, especially when lower concentration, has fabulous initial molten algae effect (Fig. 2).
When the concentration of the thick pigment in adding to Gyrodinium impudicum was 0.1 μ l/ml, the molten algae effect after 30 minutes and 24 hours was respectively 1.4% and 40.8%.When concentration was 1.0 μ l/ml, the molten algae effect after 30 minutes and 24 hours was respectively 28.7% and 93.0%.When highest point reason concentration 100 μ l/ml, the molten algae in the time of 30 minutes act as 60.6%, shows that the molten algae effect of comparison cochlodinium sp (Fig. 3) is lower slightly.
When the concentration of the thick pigment in adding to Heterosigma akashiwo was 0.1 μ l/ml, the molten algae effect after 30 minutes and 24 hours was respectively 6.5% and 78.8%.When concentration was 1.0 μ l/ml, the molten algae effect after 30 minutes and 24 hours was respectively 23.8% and 96.3%.For highest point reason concentration 100 μ l/ml the time, the molten algae after 30 minutes act as 46.4% (Fig. 4).
In addition, thick pigment has significant molten algae effect to chain Alexander algae and Procentrum micans.As seen, the thick pigment that extracts from the culture broth of 96CJ10356 strain has different molten algae effects to each the little algae that causes red tide, and the strongest to the molten algae effect of cochlodinium sp.
(2) the molten algae effect of RP10356
Detect isolated as mentioned above 4 kinds of components and to causing Korea S every year the molten algae effect of the cochlodinium sp of red tide harm takes place, the molten algae effect to this algae in molten algae test of thick pigment is the strongest.The method of the molten algae effect test of 4 kinds of components is identical with the method for thick pigment.Compare with the molten algae effect of thick pigment, have only RP10356 to demonstrate molten algae effect in 4 kinds of components.Molten algae effect when comparing 30 minutes, when 1 μ l/ml concentration, the molten algae of thick pigment act as 63.8%, and RP10356 has presented the molten algae effect 83.5% of obvious enhanced.Concentration with RP10356 of 50% molten algae effect is 0.65 μ l/ml in effect in the time of 30 minutes, and concentration is 0.17 μ l/ml (Fig. 5 and table 5) in the time of 24 hours.
Table 5
| Time (hr) | 0.5hr | 1hr | 2hr | 24hr |
| Contrast | -2.37 | 0.79 | -0.40 | 9.49 |
| Ethanol (10 -3％) | -4.76 | 0.79 | 2.38 | 9.92 |
| 0.1μl/ml RP10356 | 13.39 | 45.61 | 43.93 | 69.46 |
| 1.0μl/ml RP10356 | 83.46 | 96.54 | 99.23 | 99.62 |
| 10μl/ml RP10356 | 91.36 | 95.47 | 95.88 | 96.71 |
| 50μl/ml RP10356 | 94.91 | 95.27 | 95.64 | 96.00 |
| 100μl/ml RP10356 | 94.47 | 94.47 | 95.32 | 94.89 |
(3) morphologic observation of little algae
Observe the metamorphosis of the cell after thick pigment and RP10356 processing with inverted microscope.The metamorphosis such as growth-inhibiting, cell expansion, cytolysis and the degeneration of first sheath of the cell of observation after RP10356 handles.
When the thick pigment that is 1.0 μ l/ml with concentration, is derived from the 96CJ10356 strain adds in little algae, can observe cell in cochlodinium sp, Gyrodinium impudicum and the Heterosigma akashiwo and expand and dissolve along with time lengthening, show the molten algae effect (Fig. 6 A, 6B and 6C) of thick pigment.Degrade but in chain Alexander algae, only observe part first sheath, show of the molten algae effect lower (Fig. 6 D) of thick pigment this algae.The form of Procentrum micans has also shown minimum variation (Fig. 6 E).
When handling little algae with pigment RP10356, in cochlodinium sp, observe same metamorphosis as shown in Figure 6A, show that molten algae material is the RP10356 in the thick pigment.
The preparation of embodiment 3:RP10356
Carry out the mass production of molten algae material RP10356 with Hahella chejuensis 96CJ10356 bacterial strain, with the 96CJ10356 strain be inoculated into final volume be 3 liters contain 5wt% glucose, 0.1wt% peptone, 0.42g/L KH
2PO
4, 0.34g/L K
2HPO
4, 0.5g/L MgSO
4, 2.0g/L CaCl
2, 0.001g/L CoCl
26H
2O, 0.001g/L MnCl
3, 0.001g/L ZnSO
4, 0.001g/LNaMoO
4, in 750ml distilled water and the aged seawater.At 25 ℃, under the condition of 200rpm and ventilation speed 1.5vvm, in 5 liters every batch culture system, cultivate the material of being inoculated.
Along with the prolongation of incubation time, the output of polysaccharide increases, and when cultivating 81 hours, polysaccharide yield reaches maximum.Until cultivating 48 hours, cell yield is all increasing always, but when having cultivated 74 hours, the cell growth reduces.Detect the concentration of the RP10356 produced, the 1ml culture broth is added in the cold ethanol of 2ml, handled 30 minutes,, under the 000g centrifugal 15 minutes, remove cell then 8 with the cytoclasis instrument.Optical density (A with the spectrophotometer measurement supernatant liquor
539).The result shows that the concentration of the RP10356 of final acquisition is 209mg/L (Fig. 7).
The characteristic of embodiment 4:RP10356
(1) TLC (thin-layer chromatography) analyzes
Purifying 2-propanol extraction thing (thick pigment) obtains RP10356.The thick pigment of 1 μ g is dissolved in the 1ml ethanol.With 5 μ l solution points at 60F
254SOn the TLC plate (Merck), this plate is expanded to the height (Fig. 8) of 10cm in the mixed solution of sherwood oil-acetone-methyl alcohol (5: 3: 2).The Rf value of 4 kinds of components that obtain is respectively 0.98 (light yellow), 0.96 (dark red), 0.88 (bright red) and 0.59 (blueness).
(2) utilize the UV spectrophotometer to carry out absorption spectroanalysis
The optical density of measure R P10356.(UV-VIS2401PC, Shimadzu Japan) measure the maximum absorption (Fig. 9) of the methanol solution of 1 μ g/ml RP10356 in the 300-700nm wavelength region to utilize spectrophotometer.Measuring result shows that RP10356 presents maximum absorption at the wavelength place of 486nm and 539nm, and main peak is the peak at 539nm place.
(3) carry out the large-scale purification of pigment by silica gel chromatography
For extensive the separation and purifying pigment from the thick pigment of the 2-propanol extraction of culture broth, with silica gel chromatography (silica gel 60,0.040-0.063mm, Merck, Germany) the thick pigment of purifying, eluting solvent are the mixture (Figure 10) of acetone and methyl alcohol (9: 1).Utilize silica gel chromatography that thick pigment is separated and purifying, obtain 4 kinds of components, be respectively light yellow, dark red, bright red and blue, with coming to the same thing of TLC according to elution order.In 4 kinds of components, isolated the RP10356 of large red.
(4) HPLC analyzes
With HP 1050 systems (Hewlett Packard, USA) pigment of analysis purifying.In analysis, use silicagel column (YMC-Pack SIL, 250 * 4.6mm, Japan), detector be DAD-UV (HewlettPackard, USA), the detection wavelength is 539nm, 486nm and 300-800nm, and per minute flows out 1.0ml acetone-methyl alcohol (9: 1) (Figure 11 and 12).The HPLC analytical results shows that under these conditions the retention time of RP10356 is 3.52 minutes, even separation obtains unimodal in the wavelength region of 300-800nm.
Industrial applicibility
As mentioned above, the invention provides the red pigment with algae-lysing that is derived from Hahella chejuensis. This red pigment (RP10356) has good algae-lysing to causing the algae of red tide such as cochlodinium sp, Gyrodinium impudicum and Heterosigma akashiwo. Therefore, can be as the active component of the molten algae preparation of removing microorganisms of red tide.
Claims (15)
1. method for preparing pigment material with the effect of molten algae, it comprises step:
(a) cultivate the Hahella microorganism belonging to genus; With
(b) separating also from culture broth with organic solvent, purifying has the pigment material of molten algae effect.
2. preparation according to claim 1 has the method for the pigment material of molten algae effect, and wherein said Hahella microorganism belonging to genus is Hahella chejuensis.
3. preparation according to claim 2 has the method for the pigment material of molten algae effect, and wherein said Hahella chejuensis is 96CJ10356 bacterial strain (KCTC 2396).
4. one kind has the red pigment that molten algae acts on, and it is obtained by method any among the claim 1-3, and (sherwood oil: acetone: the Rf value methyl alcohol=5: 3: 2) is 0.98 to this pigment, and maximum absorption is at about 539nm at silica gel tlc.
5. red pigment according to claim 4, wherein red pigment has molten algae effect to cochlodinium sp, Gyrodinium impudicum or Heterosigma akashiwo.
6. red pigment according to claim 4, wherein red pigment is RP10356.
7. molten algae preparation that contains the described red pigment of claim 4 as activeconstituents.
8. molten algae preparation according to claim 7, wherein said red pigment has molten algae effect to cochlodinium sp, Gyrodinium impudicum or Heterosigma akashiwo.
9. a method of removing microorganisms of red tide is characterized in that, this method has been utilized the culture broth of Hahella microorganism belonging to genus or the extractive with organic solvent of this culture broth.
10. the method for removal microorganisms of red tide according to claim 9, wherein said Hahella microorganism belonging to genus is Hahella chejuensis.
11. the method for removal microorganisms of red tide according to claim 9, wherein said Hahellachejuensis is 96CJ10356 bacterial strain (KCTC 2396).
12. the method for removal microorganisms of red tide according to claim 9, wherein said solvent are the 2-Virahol.
13. the method for removal microorganisms of red tide according to claim 9, wherein said red tide are caused by cochlodinium sp, Gyrodinium impudicum or Heterosigma akashiwo.
14. a method of removing microorganisms of red tide is characterized in that this method has been used the red pigment in the claim 4.
15. the method for removal microorganisms of red tide according to claim 14, wherein said red tide are caused by cochlodinium sp, Gyrodinium impudicum or Heterosigma akashiwo.
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| KR10-2003-0029526 | 2003-05-09 | ||
| KR10-2003-0029526A KR100495271B1 (en) | 2003-05-09 | 2003-05-09 | The algicidal effect of red pigment produced by Hahella chejuensis 96CJ10356 strain and a method for production of red pigment having algalcidal effect therefrom |
| KR1020030029526 | 2003-05-09 | ||
| PCT/KR2004/001072 WO2004099391A1 (en) | 2003-05-09 | 2004-05-10 | Red pigment originated from hahella chejuensis, having algalcidal effect |
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| JP (1) | JP4469840B2 (en) |
| KR (1) | KR100495271B1 (en) |
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| KR100758997B1 (en) * | 2005-11-30 | 2007-09-17 | 인하대학교 산학협력단 | Method to control dinoflagellate of Heterosigma sp. by microorganism of Pseudomonas sp. |
| KR100661175B1 (en) | 2005-12-20 | 2006-12-22 | 한국해양연구원 | Algicidal agent containing prodigiosin and prodigiosin biosynthetic gene cluster |
| KR100770204B1 (en) | 2005-12-21 | 2007-10-26 | 한국해양연구원 | Use of Exopolysaccharide Produced from Hahella Chejuensis Mutant m10356 |
| KR100770205B1 (en) | 2007-07-24 | 2007-10-26 | 한국해양연구원 | Drug Delivery Carrier Using Exopolysaccharide Produced from Hahella Chejuensis Mutant m10356 and Method Thereof |
| CN104232524B (en) * | 2014-08-29 | 2017-05-31 | 厦门大学 | Algistatic activity material prodigiosin and preparation method thereof |
| KR102127466B1 (en) | 2019-02-13 | 2020-06-29 | 충남대학교산학협력단 | Algicidal Composition for Controlling Freshwater Algae |
| KR102135037B1 (en) * | 2019-05-28 | 2020-07-20 | 한국생명공학연구원 | Erythrobacter sp. 3-20A1M strain producing pyrrole-2-carboxylic acid and having algicidal activity and uses thereof |
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