CN101444228B - Ocean actinomycete fermentation extract, composition thereof, and application in biofouling resistance - Google Patents
Ocean actinomycete fermentation extract, composition thereof, and application in biofouling resistance Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 20
- 230000004151 fermentation Effects 0.000 title claims abstract description 20
- 239000000203 mixture Substances 0.000 title claims description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241001042912 Salinispora pacifica Species 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 claims abstract description 5
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- 235000002639 sodium chloride Nutrition 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 3
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- 238000004587 chromatography analysis Methods 0.000 abstract 1
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- 230000001954 sterilising effect Effects 0.000 description 3
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- 241000195493 Cryptophyta Species 0.000 description 1
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- 241000191984 Staphylococcus haemolyticus Species 0.000 description 1
- 241001234514 Vibrio halioticoli Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to an application of ocean actinomycete (Salinispora pacifica) SCSIO 00013 fermentation extract in biofouling resistnace and is characterized in that the ocean actinomycete SCSIO 00013 stain is inoculated to a Gause 1 cultural medium so as to be fermented; the gained mycelium is separated from fermentation solution and is then extracted by organic solvent; coarse extract is gained by concentrating and combining the extract; the coarse extract is suspended on water and extracted by chloroform or ethyl acetate; chromatography of the extract is carried out by a normal-pressure silica gel column; a solution system such as chloroform-acetone and the like is used as eluent so as to carry out gradient elution; thin layer chromatography analysis of fraction is carried out; chloroform-acetone with the volume ratio of 4:1 is used as solvent to extend the system; the fraction with the Rf value Rf ranging from 0.20 to 0.75 is collected and is then combined; subsequently, the pigment is removed, thus gaining target extract. The fermentation extract can be used for halobios fouling resistance; the coating consisting of the fermentation extract and film-forming material contains no heavy metal, has no harm on the environment and can be suitable for the cultivation of marine pearl oyster.
Description
Technical field
The present invention relates to a kind of ocean actinomycete fermentation extract and composition thereof and the application aspect anti-biofouling, related in particular to extract that obtains by marine actinomycete (Salinispora pacifica) SCSIO 00013 fermentation and the composition that contains this extract and this extract in the application aspect the anti-marine biofouling.
Underwater facility in the marine aquaculture, as net cage, hull, fishing net; buoy; drainpipe etc.; it is halobiontic stained that Chang Rongyi is subjected to coming from marine bacteria, marine fungi, marine algae, ocean protozoa and mollusk etc.; these fouling organisms are the final fouling organism group that forms on the facility under water also; thus the energy consumption and the weight of underwater facility increased, reduced efficient and speed, also can blocking pipeline; reduce the conveying of nutriment and oxygen, even cause the death of sea farming product.Biological corrosion and biodeterioration almost are simultaneous, but also exist the relation of mutually promoting, be the speed that biodeterioration can quicken biological corrosion, biological corrosion can impel the further gathering of fouling organism again, with the marine aquaculture is example, biodeterioration causes net cage, the obstruction of pearl shell cage etc., the aquatic products fish of culturing, shrimp, anoxic of shellfish etc. or scarce nutriment and death, fouling organism is attached to the weight that has increased hull on the hull in addition, strengthen resistance, reduced the dynamic efficiency of hull, increased oil consumption, the corrosion of hull is further accelerated, thereby shortened the service life of hull greatly.Because the considerable damage effect of marine biofouling, the direct economic loss that causes every year just reach tens dollars.
Present stage mainly is artificial or mechanical physical removal method and the chemical scavenging method that adopts chemicals to the Disposal Measures of marime fouling.But physical removal method efficient is too low, and the used chemicals toxicity of chemical scavenging method is too big again, certainly will influence the marine environment of periphery.
The objective of the invention is to develop material nontoxic and the efficient anti-biofouling of energy by marine actinomycete, another order provides its composition, and further purpose is to develop the application of this material aspect anti-marine biofouling.
We pass through marine actinomycete (Salinispora pacifica) SCSIO 00013 bacterial classification Gause I medium culture, mycelium that obtains and zymotic fluid with organic solvent extraction after, again through the silicagel column gradient elution, the mixture that obtains is nontoxic and have a characteristic of anti-marine biofouling, with coat the underwater facility surface after filmogen mixes, can prevent marine organisms stained to the underwater facility surface effectively, thereby realize purpose of the present invention.
Ocean actinomycete fermentation extract of the present invention is characterized in that being obtained by the fermentation of following step by marine actinomycete (Salinispora pacifica) SCSIO00013:
(1) with marine actinomycete SCSIO 00013 bacterial classification inoculation to the shaking in the bottle of Gause I medium arranged, temperature is 28 ℃ on shaking table, 40 rev/mins of speed, cultivate after 3 days, be transferred to again in the seed bottle, through in the Gause I medium, 28 ℃ of temperature, 40 rev/mins of speed were cultivated after 7 days, were transferred to the shaking in the bottle of Gause I medium, 28 ℃ of temperature, 40 rev/mins of speed were cultivated 10 days, will obtain mycelium then and separate with zymotic fluid, described Gause I medium, pH 7.0～7.2, every premium on currency soluble-containing starch 10～30g, K
2HPO
40.5～1g, KNO
30.5～1.0g, MgSO
47H
2O 0.5～1.0g, sea salt 1.5%～3%;
(2) mycelium and the zymotic fluid of step (1) are used solvent extraction respectively, concentrate and obtain extract 1 and extract 2, then two extracts are merged, obtain crude extract, described mycelium solvent extraction is to destroy cell wall with ultrasonic, extract with ethyl acetate, described zymotic fluid solvent extraction is to extract with ethyl acetate again;
(3) crude extract that step (2) is obtained is suspended in water, uses ethyl acetate extraction, concentrates to obtain acetic acid ethyl ester extract;
(4) acetic acid ethyl ester extract that step (3) is obtained carries out the normal pressure silica gel column chromatography, with the chloroform-methanol solvent system is eluant, eluent, carry out wash-out according to 10: 0 to 0: 10 gradient of volume ratio, cut carries out thin layer chromatography analysis, with chloroform-acetone of 4: 1 of volume ratio is the solvent development system, collects Rf value R
f0.20～0.75 cut merges and removes pigment, obtains product.
The described separation of step (1) can be adopted for example centrifugation etc. of conventional method.
Described marine actinomycete (Salinispora pacifica) SCSIO 00013 is that (known bacterial strain sees reference document: Jensen P R, Mafnas C.Environmental microbiology, 2006,8:1881-1888), the applicant guarantees to provide this bacterial classification in from the applying date 20 years.This bacterial strain separates from the north, the South Sea (116 ° of 19.945E in September, 2006,20 ° 36.012 ' N), the husky depositional environment of the grey that the depth of water is 657 meters, it is Halophiles, can adopt Gause I culture fluid or other to cultivate with the salt culture fluid, belong to rayfungi, its 16Sr rna gene sequence is compared with the marine actinomycete S.pacifica that delivered in 2005, similitude reaches more than 99%, and network analysis itself and S.pacifica have nearest affiliation.So this dientification of bacteria is the bacterial strain SCSIO 00013 of S.pacifica.
The preparation method of ocean actinomycete fermentation extract of the present invention is with above-mentioned.
The composition that contains ocean actinomycete fermentation extract of the present invention is characterized in that being made up of ocean actinomycete fermentation extract of the present invention and filmogen.Described filmogen is a synthetic resin, natural resin etc.With mixture and filmogen such as the synthetic resin of being produced by marine actinomycete of the present invention, mixing such as natural resin make it film forming, are applied on the surface of underwater facility again.
Found through experiments ocean actinomycete fermentation extract of the present invention and have the excellent antibiotic activity, anti-kentrogon adheres to active concentration IC
50Be 25.61 μ g/mL, prove to can be used for anti-marine biofouling; This extract is to the LC of penguin pearl shell
50＞1000 μ g/mL think that this extract is nontoxic to penguin pearl shell seedling, can be used for anti-biofouling in the pearl breedinmg.
The present invention obtains nontoxic from marine actinomycete by simple extracting method, the extract of anti-marine biofouling efficiently, the coating that is made by this mixture does not contain heavy metal, environmental sound can be applied to the anti-biofouling in the underwater facilities such as the breed of marine pearl oyster and hull.
Following examples are to further specify of the present invention, but the invention is not restricted to following examples.
Embodiment 1:
A 500mL through sterilization shakes in the bottle with marine actinomycete (Salinispora pacifica) SCSIO 00013 bacterial classification inoculation to 2, and the medium that shakes in the bottle is that (pH 7.0～7.2, every premium on currency soluble-containing starch 10～30g, K for the Gause I medium
2HPO
40.5～1g, KNO
30.5～1.0g, MgSO
47H
2O 0.5～1.0g, sea salt 1.5%～3%,), temperature is 28 ℃ on shaking table, and 40 rev/mins of speed are cultivated after 3 days, be transferred to respectively in 10 seed bottles, through in the Gause I medium, 28 ℃ of temperature, 40 rev/mins of speed, cultivate after 7 days, the actinomycetes of 20 seed bottles are transferred to shaking in the bottle of 500mL, shake the Gause I medium that bottle is contained 150mL, condition of culture is 28 ℃ of temperature, 40 rev/mins of speed, cultivated 10 days, treat the actinomycetes bulk fermentation after, with super-magnum centrifuge centrifugation mycelium and zymotic fluid.Zymotic fluid with isopyknic ethyl acetate extraction three times, is merged extract, obtain extract 1.Handle mycelium with ultrasonic echography, use isopyknic ethyl acetate extraction three times again, merge extract, obtain extract 2.With extract 1 and extract 2 concentrating under reduced pressure, obtain pitchy material 3.2g respectively.
Above-mentioned pitchy material is suspended in the 100mL redistilled water, uses equal volume of ethyl acetate then, obtain acetic acid ethyl ester extract.Acetic acid ethyl ester extract is crossed particle diameter 40～60 μ m, and the silicagel column of 64g is a solvent system with the chloroform-methanol, carried out gradient elution from 10: 0 to 0: 10,2 droplets/second of flow velocitys, cut carries out thin layer chromatography analysis, with 4: 1 chloroform-acetone of volume ratio is the solvent development system, collects Rf value R
f0.20～0.75 cut merges and with Sephadex LH-20 gel column depigmentation, obtains 0.55g extract of the present invention.
Embodiment 2: embodiment 1 extract is antibiotic to adhere to activity test with the biological larva of fouling resistance
Adopt the agar-agar diffusion process to measure antibacterial activity.Place the aseptic scraps of paper (5mm i.d.) that are soaked with 50 μ g target extracts on the agar-agar plate of coating bacterium, the inhibition zone size unit is represented with mm.The scraps of paper that are soaked with same volume DMSO are done blank, are added with rifampin (3-[[(4-methyl isophthalic acid-piperazinyl) imino group] methyl]-rifamycin) the scraps of paper do positive control.After at room temperature cultivating 24 hours then, measure the inhibition zone size.The anti-bacterial result sees Table 1.
The antibacterial activity of table 1 target extract and rifampin (size unit of inhibition zone L, mm; Every content, 50 μ g/disc)
The size of inhibition zone L is by vernier caliper measurement in the table 1, the inhibition zone of irregularly shaped size average (L=R-r).Show as can be known that by table 1 antibacterial activity of extract is stronger, larva is adhered to induce bacterium Staphylococcushaemolyticus especially, Pseudoalteromonas sp., Vibrio halioticoli and Rhodovuium sp. have stronger inhibitory action.
Embodiment 3: the biological larva of embodiment 1 extract fouling resistance adheres to activity test
The anti-kentrogon of adopting 24 orifice plates to measure embodiment 1 extract respectively adheres to activity.Add the culture fluid that 1mL contains the kentrogon of 10～15 maturations in each plate hole, embodiment 1 extract is dissolved among the DMSO, is diluted to different concentration with the sterilization seawater then.3 of each concentration are parallel, and aseptic seawater is done blank.Culture plate placed under the room temperature cultivated 24 hours, the larva number that statistics is adhered under anatomical lens carries out statistical analysis with the SPSS program.Statistics sees Table 2:
The adhesive rate (%) of table 2 embodiment 1 extract kentrogon under variable concentrations
As can be seen from Table 2: right with the DMSO contrast ratio, it is 60% that the adhesive rate of the larva of 4 concentration of embodiment 1 extract has only 0.1 μ g/mL, does not have notable difference, and other 3 concentration all have notable difference.
The anti-larva that adheres to inhibiting rate=(blank-adhesive rate)/blank * 100% computerized compound according to larva adheres to activity.
Obtain to draw a conclusion by analysis: the anti-larva of (1) embodiment 1 extract adheres to active concentration IC
50Be 25.61 μ g/Ml; (2) the target extract has the activity that anti-kentrogon adheres to.
Embodiment 4: the deadly test of 1 pair of pearl shell seedling of embodiment
Adopt vial to measure embodiment 1 extract respectively to cultivating the deadly test of 7 days pearl shell seedling.Add the penguin pearl shell seedling culture fluid that 2mL contains 10～15 maturations in each small beaker, embodiment 1 extract is dissolved among the DMSO, is diluted to different concentration with the sterilization seawater then.3 of each concentration are parallel, and aseptic seawater is done blank.Culture plate placed under the room temperature cultivated 24 hours, the dead seedling number of statistics is used LD under magnifying glass
50Program is carried out statistical analysis.Statistics sees Table 3:
Embodiment 1 target extract under table 3 variable concentrations is to cultivating the lethality rate (%) of 7 days penguin pearl shell
Calculate lethality rate and the lethal concentration of embodiment 1 extract according to corrected mortality=(control group survival rate-processed group survival rate)/control group survival rate * 100% to the penguin pearl shell.
Obtain to draw a conclusion by analysis: the target extract is to the LC of penguin pearl shell
50＞1000 μ g/mL think that compound is nontoxic to penguin pearl shell seedling.
Embodiment 5: the preparation of compositions that contains extract of the present invention that is used for the pearl breedinmg anti-biofouling
Adopt the technology of existing preparation coating, in coating, add the anti-biofouling activity extract that the present invention relates to.For example, activity extract of the present invention is mixed synthetic resin, in natural resin or other filmogen, preparation makes it film forming, is applied on the boxes and baskets or other underwater facility that use in the pearl breedinmg process again.
Claims (4)
1. ocean actinomycete fermentation extract is characterized in that being obtained by following step fermentation by marine actinomycete (Salinispora pacifica) SCSIO00013:
(1) with marine actinomycete SCSIO 00013 bacterial classification inoculation to the shaking in the bottle of Gause I medium arranged, temperature is 28 ℃ on shaking table, 40 rev/mins of speed, cultivate after 3 days, be transferred to again in the seed bottle, through in the Gause I medium, 28 ℃ of temperature, 40 rev/mins of speed were cultivated after 7 days, were transferred to the shaking in the bottle of Gause I medium, 28 ℃ of temperature, 40 rev/mins of speed were cultivated 10 days, will obtain mycelium then and separate with zymotic fluid, described Gause I medium, pH 7.0～7.2, every premium on currency soluble-containing starch 10～30g, K
2HPO
40.5～1g, KNO
30.5～1.0g, MgSO
47H
2O 0.5～1.0g, sea salt 1.5%～3%;
(2) mycelium and the zymotic fluid of step (1) are used solvent extraction respectively, concentrate and obtain extract 1 and extract 2, then two extracts are merged, obtain crude extract, described mycelium solvent extraction is to destroy cell wall with ultrasonic, extract with ethyl acetate, described zymotic fluid solvent extraction is to extract with ethyl acetate again;
(3) crude extract that step (2) is obtained is suspended in water, uses ethyl acetate extraction, concentrates to obtain acetic acid ethyl ester extract;
(4) acetic acid ethyl ester extract that step (3) is obtained carries out the normal pressure silica gel column chromatography, with the chloroform-methanol solvent system is eluant, eluent, carry out wash-out according to 10: 0 to 0: 10 gradient of volume ratio, cut carries out thin layer chromatography analysis, with chloroform-acetone of 4: 1 of volume ratio is the solvent development system, collects Rf value R
f0.20～0.75 cut merges and removes pigment, obtains product.
2. a composition that contains the described extract of claim 1 is characterized in that being made up of described ocean actinomycete fermentation extract of claim 1 and filmogen.
3. the described ocean actinomycete fermentation extract of claim 1 is in the application of anti-marine biofouling.
4. the application of the described ocean actinomycete fermentation extract of claim 3 is the application of anti-marine biofouling in pearl breedinmg.
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| CN2008102204277A CN101444228B (en) | 2008-12-25 | 2008-12-25 | Ocean actinomycete fermentation extract, composition thereof, and application in biofouling resistance |
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| CN107164267B (en) * | 2017-06-05 | 2020-03-17 | 天津大学 | Marine actinomycete U-19-3 with broad-spectrum antibacterial activity and application thereof |
| CN110983816B (en) * | 2019-11-07 | 2021-05-18 | 浙江理工大学 | Application of actinomycin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1023008C (en) * | 1987-05-18 | 1993-12-08 | 阿托化学公司 | Process for preparing halogenated imides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1023008C (en) * | 1987-05-18 | 1993-12-08 | 阿托化学公司 | Process for preparing halogenated imides |
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