CN108117787A - A kind of method that antifouling activity substance is extracted from microalgae - Google Patents
A kind of method that antifouling activity substance is extracted from microalgae Download PDFInfo
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- CN108117787A CN108117787A CN201711128342.1A CN201711128342A CN108117787A CN 108117787 A CN108117787 A CN 108117787A CN 201711128342 A CN201711128342 A CN 201711128342A CN 108117787 A CN108117787 A CN 108117787A
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- activity substance
- antifouling activity
- microalgae
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/14—Paints containing biocides, e.g. fungicides, insecticides or pesticides
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- Engineering & Computer Science (AREA)
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- Separation Using Semi-Permeable Membranes (AREA)
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Abstract
The present invention provides a kind of method that antifouling activity substance is extracted from microalgae, including freeze thawing broken wall, resuspension, salt fractionation, aqueous two-phase extraction, ultrafiltration purification and product configuration.The method of the present invention preparation process is simple, favorable reproducibility, manufacturing cost are cheap, and the recovery rate for extracting antifouling activity substance is high.The finished product being finally prepared has excellent hydrophobicity performance coated on rear fast filming in substrate, the protective film of formation.The activity of phosphoprotein phosphatase can effectively be inhibited, make protein that Hyperphosphorylationof occur, cause cell rupture, the biology such as seaweed, planktonic organism and shellfish is effectively prevented and adheres in substrate.
Description
Technical field
The present invention relates to bioactive substances to extract field, is specifically a kind of side that antifouling activity substance is extracted from microalgae
Method.
Background technology
The aggravation of water body eutrophication degree and the expansion of scope cause freshwater microalgae algal bloom increasingly universal, seriously
Water body and ambient enviroment are destroyed, to eliminate microalgae pollution, annual substantial amounts of microalgae is salvaged up, at the same time salvages
The recycling of microalgae has become microalgae emphasis research topic.
There is more than 2000 kinds of fouling organism in ocean, majority lives in the offshore sea waters such as seashore and bay.The children of fouling organism
Worm and spore can float travelling, develop to after certain phase that just attachment is settled down, be stained Offshore Units, such as hull, float
Mark, bridge pier, harbour, net cage and netting gear etc., main harm include increase ship resistance, obstruction pipeline, accelerate metal erosion,
Make instrument and rotating machine failure, the influence to acoustic instrument and influence to aquatic products industry etc., as the damage caused by biodeterioration
Mistake is difficult to estimate.Marine anti-pollution technology is for marine organisms to be prevented to be stained the attachment of Offshore Units, are started in ship pollution prevention
The research of coating, and it is antifouling for Major Technology with poison release type, it is anti-by releasable copper, tin, mercury, lead etc. in coating
Dirty agent forms the concentration of poison layer for having toxic action to sea-plant spore and marine animal larva in surrounding materials, so as to
Reach anti-fouling effect.However, such antifouling paint is in use, part toxic agent (such as organotin, DDT) can cause seawater water
Body pollution triggers biomutation, jeopardizes marine food chain, has to marine ecology balance and human health potentially hazardous.
The content of the invention
It is an object of the invention to provide a kind of preparation method is simple, favorable reproducibility, manufacturing cost is cheap, recovery rate is high
A kind of method that antifouling activity substance is extracted from microalgae, hydrophobicity is good after the antifouling activity substance extracted forms a film, to dirt
Larva and the spore harmful effects for damaging biology are good and can natural degradation.
The problem of present invention in background technology for mentioning, the technical solution taken is：One kind is extracted antifouling from microalgae
The method of active material, including freeze thawing broken wall, resuspension, salt fractionation, aqueous two-phase extraction, ultrafiltration purification and product configuration.Production
Product be formulated as in obtained after ultrafiltration purification 10 parts of antifouling activity substances add in 5 ~ 10 parts of glycerine, 0.001 ~ 0.002 part of EDTA,
0.0005 ~ 0.001 part of DTT and 0.3 ~ 0.5 part of resorcinol and glycidol ether are uniformly mixed and obtain finished product.The method of the present invention
Preparation process is simple, favorable reproducibility, manufacturing cost are cheap, and the recovery rate for extracting antifouling activity substance is high.Finally it is prepared
Finished product is coated on rear fast filming, electrostatic attraction, hydrogen bond and Van der Waals force between the protective film and hydrone of formation in substrate
Smaller, protective film shows stronger hydrophobicity.Protect film surface can low, the water contact angle of protective film（WCA）For 170 ° ~
185 °, roll angle（SA）For 0.4 ° ~ 5 °, there is excellent hydrophobicity performance.The activity of phosphoprotein phosphatase can effectively be inhibited, make albumen
Hyperphosphorylationof occurs for matter, causes cell rupture, and it is attached in substrate to be effectively prevented the biology such as seaweed, planktonic organism and shellfish
It；Resorcinol and glycidol ether have coupling with glycerine, EDTA and DTT, can play that adjust antifouling activity substance steady
It qualitatively acts on, can reconcile the activity and anisotropy of each component, increase the uniformity and compactness of protective film, make protective film
After long-term use will not peeling, come off, extend the service life of substrate, reduce cost.
Freeze thawing broken wall is：Add in ultra-pure water in microalgae mud, the mass volume ratio of microalgae mud and ultra-pure water is 1g：0.6~
2.0ml is freezed at a temperature of -25 ~ -15 DEG C, is thawed at a temperature of 15 ~ 25 DEG C, multigelation number 1 ~ 3 time, 2 after freeze thawing
8000 ~ 12000r/min centrifuges 6 ~ 15min at a temperature of ~ 6 DEG C, preserves supernatant.Its condition of the method for freeze thawing broken wall is milder,
Protein denaturation is not easily caused, it is increased using the ice pellets and the salinity of cell liquid formed into the cell under freezing conditions and is drawn
The swelling risen is so that clasmatosis albumen dissolves out.Above-mentioned freeze thawing operation is the most preferably change of freeze thawing temperature or number of freezing and thawing
Change can generate harmful effect to antifouling activity species activity or recovery rate.
Resuspension is：In the precipitation obtained after freeze thawing broken wall add in 3 ~ 6 times of volumes Tris-HCl buffer solutions, repeat from
The heart 2 ~ 4 times merges supernatant；The Tris-HCl pH of buffer is 7.5 ~ 8.0.In the precipitation obtained after freeze thawing broken wall there are still
The antifouling activity substance of a part is operated using above-mentioned resuspension, can improve the recovery rate of antifouling activity substance, reduces loss.
Salt fractionation is：The supernatant obtained after the supernatant and resuspension that will be obtained after freeze thawing broken wall merges, and adds in sulphur
Sour ammonium makes ammonium sulfate saturation degree in solution reach 15 ~ 25%, stirs, and 4000 ~ 6000r/min centrifuges 3 ~ 8min at a temperature of 2 ~ 6 DEG C,
Precipitation is taken, in precipitation plus ultrapure water dissolution adds in ammonium sulfate and ammonium sulfate saturation degree in solution is made to reach 30 ~ 40%, stirs, 2 ~ 6
4000 ~ 6000r/min centrifuges 3 ~ 8min at a temperature of DEG C, takes supernatant.Ammonium sulfate molecule can rapid damage antifouling activity substance week
The hydrated sheath enclosed reduces its solubility in water, purifies antifouling activity substance.
Aqueous two-phase extraction is：PEG4000 and potassium phosphate KHP, regulation system are added in salt fractionation obtains supernatant
PH is that 5.5 ~ 6.5, PEG4000 concentration is that 12 ~ 20%, KHP concentration is 10 ~ 15%, and 400 ~ 800r/min stirrings are transferred to liquid separation
It is layered in funnel, removes phase, obtain antifouling activity substance solution.When system pH is 5.5 ~ 6.5, antifouling activity material molecule table
The charge of face institute band is neutralized (or considerably less), and the influence of net charge at this time can be ignored, and influences antifouling activity substance point
Match somebody with somebody mainly its surface nature, antifouling activity material collection in polyethylene glycol phase and other impurities are then assigned to inorganic salts phase
In.
Ultrafiltration purification is：The antifouling activity substance solution that aqueous two-phase extraction obtains is transferred in ultra-filtration centrifuge tube, 2 ~ 6 DEG C
At a temperature of 8000 ~ 12000r/min centrifuge 8 ~ 15min, obtain antifouling activity substance；The ultra-filtration centrifuge tube specification for 20 ~
40KDa。
Compared with prior art, the advantage of the invention is that：The method of the present invention preparation process is simple, favorable reproducibility, preparation
Of low cost, the recovery rate for extracting antifouling activity substance is high.Fast filming after the finished product being finally prepared is coated in substrate,
The protective film of formation has excellent hydrophobicity performance.The activity of phosphoprotein phosphatase can effectively be inhibited, make protein that excessive phosphorus occur
Acidifying, causes cell rupture, is effectively prevented the biology such as seaweed, planktonic organism and shellfish and adheres in substrate；Resorcinol and
Glycidol ether has coupling with glycerine, EDTA and DTT, can play the role of adjusting antifouling activity material stability, can
To reconcile the activity and anisotropy of each component, increase the uniformity and compactness of protective film, make protective film in long-term use
After will not peeling, come off, extend the service life of substrate, reduce cost.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of method that antifouling activity substance is extracted from microalgae, comprises the following steps：
1）Freeze thawing broken wall：Add in ultra-pure water in microalgae mud, the mass volume ratio of microalgae mud and ultra-pure water is 1g：1ml, in -20
Freeze at a temperature of DEG C, thaw at a temperature of 20 DEG C, multigelation number 2 times, after freeze thawing at a temperature of 4 DEG C 10000r/min from
Heart 10min preserves supernatant；
2）Resuspension：The Tris-HCl buffer solutions of 5 times of volumes of addition in the precipitation obtained after freeze thawing broken wall, repeated centrifugation 3 times,
Merge supernatant；Tris-HCl pH of buffer is 7.8；
3）Salt fractionation：The supernatant obtained after the supernatant and resuspension that will be obtained after freeze thawing broken wall merges, and adds in ammonium sulfate,
Ammonium sulfate saturation degree in solution is made to reach 20%, is stirred, 5000r/min centrifugations 5min, takes precipitation at a temperature of 4 DEG C, adds in precipitation
Ultrapure water dissolution, which adds in ammonium sulfate, makes ammonium sulfate saturation degree in solution reach 35%, stirs, and 5000r/min is centrifuged at a temperature of 4 DEG C
5min takes supernatant；
4）Aqueous two-phase extraction：PEG4000 and potassium phosphate KHP is added in salt fractionation obtains supernatant（KH2PO4：K2HPO4=
1:1）, regulation system pH is that 6.0, PEG4000 concentration is that 16%, KHP concentration is 12%, 600r/min stirrings, is transferred to liquid separation leakage
It is layered in bucket, removes phase, obtain antifouling activity substance solution；
5）Ultrafiltration purification：The antifouling activity substance solution that aqueous two-phase extraction obtains is transferred in ultra-filtration centrifuge tube, 4 DEG C of temperature
Lower 10000r/min centrifuges 10min, obtains crude antifouling activity substance；The ultra-filtration centrifuge tube specification is 30KDa；
6）Product configuration：In the 10 parts of antifouling activity substances obtained after ultrafiltration purification add in 8 parts of glycerine, 0.001 part of EDTA,
0.0005 part of DTT and 0.4 part of resorcinol and glycidol ether are uniformly mixed and obtain finished product.
Embodiment 2：
A kind of method that antifouling activity substance is extracted from microalgae, comprises the following steps：
1）Freeze thawing broken wall：Add in ultra-pure water in microalgae mud, the mass volume ratio of microalgae mud and ultra-pure water is 1g：1.5ml, in-
It freezes at a temperature of 25 DEG C, thaws at a temperature of 20 DEG C, multigelation number 2 times, 10000r/min at a temperature of 4 DEG C after freeze thawing
10min is centrifuged, preserves supernatant；
2）Resuspension：The Tris-HCl buffer solutions of 6 times of volumes of addition in the precipitation obtained after freeze thawing broken wall, repeated centrifugation 3 times,
Merge supernatant；Tris-HCl pH of buffer is 7.8；
3）Salt fractionation：The supernatant obtained after the supernatant and resuspension that will be obtained after freeze thawing broken wall merges, and adds in ammonium sulfate,
Ammonium sulfate saturation degree in solution is made to reach 22%, is stirred, 5000r/min centrifugations 5min, takes precipitation at a temperature of 4 DEG C, adds in precipitation
Ultrapure water dissolution, which adds in ammonium sulfate, makes ammonium sulfate saturation degree in solution reach 38%, stirs, and 5000r/min is centrifuged at a temperature of 4 DEG C
5min takes supernatant；
4）Aqueous two-phase extraction：PEG4000 and potassium phosphate KHP is added in salt fractionation obtains supernatant（KH2PO4：K2HPO4=
1:1）, regulation system pH is that 5.8, PEG4000 concentration is that 15%, KHP concentration is 12%, 600r/min stirrings, is transferred to liquid separation leakage
It is layered in bucket, removes phase, obtain antifouling activity substance solution；
5）Ultrafiltration purification：The antifouling activity substance solution that aqueous two-phase extraction obtains is transferred in ultra-filtration centrifuge tube, 4 DEG C of temperature
Lower 10000r/min centrifuges 10min, obtains crude antifouling activity substance；The ultra-filtration centrifuge tube specification is 30KDa；
6）Product configuration：In the 10 parts of antifouling activity substances obtained after ultrafiltration purification add in 7 parts of glycerine, 0.002 part of EDTA,
0.0005 part of DTT and 0.3 part of resorcinol and glycidol ether are uniformly mixed and obtain finished product.
Embodiment 3：
A kind of method that antifouling activity substance is extracted from microalgae, Optimization Steps are：
1）Freeze thawing broken wall：Add in ultra-pure water in microalgae mud, the mass volume ratio of microalgae mud and ultra-pure water is 1g：1ml, in -20
Freeze at a temperature of DEG C, thaw at a temperature of 20 DEG C, multigelation number 2 times, after freeze thawing at a temperature of 4 DEG C 10000r/min from
Heart 10min preserves supernatant；
2）Resuspension：The Tris-HCl buffer solutions of 5 times of volumes of addition in the precipitation obtained after freeze thawing broken wall, repeated centrifugation 3 times,
Merge supernatant；Tris-HCl pH of buffer is 7.8；
3）Salt fractionation：The supernatant obtained after the supernatant and resuspension that will be obtained after freeze thawing broken wall merges, and adds in ammonium sulfate,
Ammonium sulfate saturation degree in solution is made to reach 20%, is stirred, 5000r/min centrifugations 5min, takes precipitation at a temperature of 4 DEG C, adds in precipitation
Ultrapure water dissolution, which adds in ammonium sulfate, makes ammonium sulfate saturation degree in solution reach 35%, stirs, and 5000r/min is centrifuged at a temperature of 4 DEG C
5min takes supernatant；
4）Aqueous two-phase extraction：PEG4000, potassium phosphate KHP are added in salt fractionation obtains supernatant（KH2PO4：K2HPO4=
1:1）With sebacic acid diisopropyl ester, regulation system pH is that 6.0, PEG4000 concentration is that 16%, KHP concentration is 12%, sebacic acid two
Isopropyl ester concentration stirs for 0.1%, 600r/min, is transferred in separatory funnel and is layered, remove phase, it is molten to obtain antifouling activity substance
Liquid.Sebacic acid diisopropyl ester, which is dissolved in PEG4000, can increase selectivity of the two phase aqueous extraction system to antifouling activity substance, and
The surface tension of phase up and down can be increased, reduce the loss of antifouling activity substance in extraction process；
5）Ultrafiltration purification：The antifouling activity substance solution that aqueous two-phase extraction obtains is transferred in ultra-filtration centrifuge tube, 4 DEG C of temperature
Lower 10000r/min centrifuges 10min, obtains crude antifouling activity substance；The ultra-filtration centrifuge tube specification is 30KDa；
6）Product configuration：In the 10 parts of antifouling activity substances obtained after ultrafiltration purification add in 8 parts of glycerine, 0.001 part of EDTA,
0.0005 part of DTT and 0.4 part of resorcinol and glycidol ether are uniformly mixed and obtain finished product.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of method that antifouling activity substance is extracted from microalgae, including freeze thawing broken wall, resuspension, salt fractionation, aqueous two-phase
Extraction, ultrafiltration purification and product configuration, it is characterised in that：The product configuration is that 10 parts obtained after ultrafiltration purification are antifouling
5 ~ 10 parts of glycerine, 0.001 ~ 0.002 part of EDTA, 0.0005 ~ 0.001 part of DTT and 0.3 ~ 0.5 part of isophthalic two are added in active material
Phenol and glycidol ether are uniformly mixed and obtain finished product.
2. a kind of method that antifouling activity substance is extracted from microalgae according to claim 1, it is characterised in that：Described
Freeze thawing broken wall is：Add in ultra-pure water in microalgae mud, the mass volume ratio of microalgae mud and ultra-pure water is 1g：0.6 ~ 2.0ml, in-
It freezes at a temperature of 25 ~ -15 DEG C, thaws at a temperature of 15 ~ 25 DEG C, multigelation number 1 ~ 3 time, 2 ~ 6 DEG C of temperature after freeze thawing
Lower 8000 ~ 12000r/min centrifuges 6 ~ 15min, preserves supernatant and precipitation.
3. a kind of method that antifouling activity substance is extracted from microalgae according to claim 1, it is characterised in that：Described
Resuspension is：The Tris-HCl buffer solutions of 3 ~ 6 times of volumes of addition in the precipitation obtained after freeze thawing broken wall, repeated centrifugation 2 ~ 4 times,
Merge supernatant；The Tris-HCl pH of buffer is 7.5 ~ 8.0.
4. a kind of method that antifouling activity substance is extracted from microalgae according to claim 1, it is characterised in that：Described
Salt fractionation is：The supernatant obtained after the supernatant and resuspension that will be obtained after freeze thawing broken wall merges, and adds in ammonium sulfate, makes molten
Ammonium sulfate saturation degree reaches 15 ~ 25% in liquid, stirs, and 4000 ~ 6000r/min centrifuges 3 ~ 8min at a temperature of 2 ~ 6 DEG C, takes precipitation,
In precipitation plus ultrapure water dissolution adds in ammonium sulfate and ammonium sulfate saturation degree in solution is made to reach 30 ~ 40%, stirs, at a temperature of 2 ~ 6 DEG C
4000 ~ 6000r/min centrifuges 3 ~ 8min, takes supernatant.
5. a kind of method that antifouling activity substance is extracted from microalgae according to claim 1, it is characterised in that：It is described
Aqueous two-phase extraction be：PEG4000 and potassium phosphate KHP is added in salt fractionation obtains supernatant, regulation system pH is 5.5
~ 6.5, PEG4000 concentration are that 12 ~ 20%, KHP concentration is 10 ~ 15%, and 400 ~ 800r/min stirrings are transferred in separatory funnel and divide
Layer, removes phase, obtains antifouling activity substance solution.
6. a kind of method that antifouling activity substance is extracted from microalgae according to claim 1, it is characterised in that：Described
Ultrafiltration purification is：The antifouling activity substance solution that aqueous two-phase extraction obtains is transferred in ultra-filtration centrifuge tube, at a temperature of 2 ~ 6 DEG C
8000 ~ 12000r/min centrifuges 8 ~ 15min, obtains antifouling activity substance；The ultra-filtration centrifuge tube specification is 20 ~ 40KDa.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711128342.1A CN108117787A (en) | 2017-11-15 | 2017-11-15 | A kind of method that antifouling activity substance is extracted from microalgae |
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| CN201711128342.1A CN108117787A (en) | 2017-11-15 | 2017-11-15 | A kind of method that antifouling activity substance is extracted from microalgae |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016198950A2 (en) * | 2015-06-11 | 2016-12-15 | Biomimetx, S.A. | Antifouling composition and process for production thereof |
| CN107298710A (en) * | 2017-07-06 | 2017-10-27 | 浦江县欧立生物技术有限公司 | A kind of extracting method of spirulina phycocyanin |
-
2017
- 2017-11-15 CN CN201711128342.1A patent/CN108117787A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016198950A2 (en) * | 2015-06-11 | 2016-12-15 | Biomimetx, S.A. | Antifouling composition and process for production thereof |
| CN107298710A (en) * | 2017-07-06 | 2017-10-27 | 浦江县欧立生物技术有限公司 | A kind of extracting method of spirulina phycocyanin |
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Application publication date: 20180605 |