CN101235075B - Method for extracting phycocyanin from phormidium tenue - Google Patents
Method for extracting phycocyanin from phormidium tenue Download PDFInfo
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- CN101235075B CN101235075B CN2008100467687A CN200810046768A CN101235075B CN 101235075 B CN101235075 B CN 101235075B CN 2008100467687 A CN2008100467687 A CN 2008100467687A CN 200810046768 A CN200810046768 A CN 200810046768A CN 101235075 B CN101235075 B CN 101235075B
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- phormidium tenue
- phormidium
- tenue
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Abstract
The invention discloses a method for extracting phycocyanin from phormidium tenue, which comprises preparing sodium nitrate, dibasic potassium phosphate, magnesium sulfate, calcium chloride, citrate, ammonium iron citrate, disodium editate, sodium carbonate and micro element solution into phormidium tenue culture medium, arranging phormidium tenue into the culture medium to cultivate and prepare phormidium tenue powder, extracting and purifying phormidium tenue phycocyanin from the phormidium tenue powder. The invention has simple process, easy operation, high reliability, high yield and high quality, and the equipments are simple equipment and suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method of from Phormidium tenue, extracting Phycocyanins, C-, belong to biological technical field.
Background technology
Phormidium tenue is a kind of filamentous cyanobacteria that is distributed widely in the desert habitat, and it belongs to Cyanophyta, Cyanophyceae, hormogon[ium order, Oscillariaceae, Phormidium.At present, the utilization of Phormidium tenue generally only rested on carry out on the artificial algae skinning, to other application and developments of Phormidium tenue still blank out almost.Why Phormidium tenue is used to form artificial algae skinning is carried out desert treatment, and main is because it is rich in Phycocyanins, C-.Phycobiliprotein is a class pigment conjugated protein that is present in some algae (mainly being red algae and blue-green algae) phycobilisome.Phycobiliprotein has the intensive fluorescence, sends out fluorescent red-orange, itself then takes on a red color, purple or blueness etc., so be coloured polypeptide.Can be divided into four classes to phycobiliprotein by spectral response curve: phycoerythrin, Phycocyanins, C-, phycoerythrocyanin (pec) and allophycocyanin.Phycocyanins, C-is a class blue pigment albumen wherein.Phycocyanins, C-also has very high application and development and is worth: can be used as the pigment of pure natural, be used for industry such as food, makeup and dyestuff except that can be used for photosynthesis of plant research; The SILVER REAGENT Phycocyanins, C-can be made into fluorescent reagent, is applied in medical clinic applications and the research fields such as immunology and biotechnology; Also can be made into medicine and be used for health care.The cyanine that uses in industries such as food at present mainly still is the chemosynthesis pigment, and natural blue pigment uses seldom, domestic Phycocyanins, C-is to extract to obtain from spirulina and kelp mostly, their Phycocyanins, C-content is mostly lower than the Phormidium tenue, in addition, the cost of cultivating kelp wants high more than the cost of cultivating Phormidium tenue.
Summary of the invention
The object of the invention just provides a kind of method of extracting Phycocyanins, C-from Phormidium tenue.
For achieving the above object, the invention provides a kind of method of extracting Phycocyanins, C-from Phormidium tenue may further comprise the steps:
A, the preparation of nutrient solution: add SODIUMNITRATE 0.8～2.5g by every premium on currency, dipotassium hydrogen phosphate 0.02～0.05g, sal epsom 0.06～0.09g, calcium chloride 0.02～0.05g, citric acid 0.004～0.008g, ferric ammonium citrate 0.004～0.008g, disodium EDTA 0.001～0.003g, yellow soda ash 0.01～0.04g, trace element solution 0.5～2ml, wherein trace element solution is by adding boric acid 270～300mg in every 100ml distilled water, tetrahydrate manganese chloride 170～200mg, Zinc Sulphate Heptahydrate 18～25mg, Sodium Molybdate Dihydrate 15～30mg, cupric sulfate pentahydrate 6～10mg stirs evenly after the dissolving fully and makes nutrient solution;
The cultivation of B, Phormidium tenue: at first be that one-level is cultivated, when inserting in the nutrient solution aerated culture, regulate air-flow at 1.5～3.5L.min by the Phormidium tenue kind
-1, light intensity is controlled at 65～85 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, after cultivating 5～10 days, is transferred to secondary and cultivates; Next is that secondary is cultivated, and the culture of one-level being cultivated gained is linked into aerated culture in the new nutrient solution, regulates air-flow at 2.5～5L.min
-1, aseptically process, light intensity are controlled at 75～95 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates and is transferred to three grades of cultivations after 5～10 days; The 3rd is three grades of cultivations, and the culture of secondary being cultivated gained is linked into aerated culture in the new nutrient solution, regulates air-flow at 3～6L.min
-1, aseptically process, light intensity are controlled at 70～100 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates after 5～10 days as the inoculation provenance that continues to cultivate; The 4th is to continue to cultivate, and at first is that the partial circulating cultivation pool is cultivated, and with the algae kind access partial circulating cultivation pool of gained after three grades of cultivations, controlled temperature utilizes natural light to carry out illumination at 22～35 ℃, and the control intensity of illumination is at 120～180 μ E.m
-2.s
-1, stir substratum; Next is that the systemic circulation cultivation pool is cultivated, and the Phormidium tenue of cultivating in the partial circulating cultivation pool after 4～7 days is transferred in the systemic circulation cultivation pool, and the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool; Controlled temperature utilizes natural light to carry out illumination at 22～35 ℃, and the control intensity of illumination is at 120～180 μ E.m
-2.s
-1The Phormidium tenue filtration of cultivating after 10～20 days is received to such an extent that the Phormidium tenue algae is starched;
When above-mentioned Phormidium tenue is inoculated, in the ratio access of every liter of nutrient solution 0.1～0.5g weight in wet base.
The preparation of C, Phormidium tenue powder: algae is starched through centrifugal acquisition Phormidium tenue algae mud, then Phormidium tenue algae mud is obtained the Phormidium tenue powder after concentrated, dehydration, drying, pulverizing;
The extracting of D, Phormidium tenue Phycocyanins, C-: the 0.05～0.1mol.L that in the Phormidium tenue powder, adds 7～10 times of weight
-1The pH value is 6.0～8.0 phosphate buffer solution, fully stir evenly be placed on-10～-20 ℃ down freezing, wait to freeze the back and take out, place 20～30 ℃ melt and dissolved down, treat its fully melt and dissolved after, again that it is freezing, so multigelation is 3～5 times, and then centrifugal 10～15min under 3000～6000rpm, collect supernatant liquor, be the Phycocyanins, C-crude extract;
E, purifying: under condition of ice bath, in the crude extract of Phycocyanins, C-, add saturation ratio and be 50～70% ammonium sulfate lucifuge, the 20～40min that saltouts, at 3～5 ℃ of following centrifugal 15～25min of 3000～6000rpm, the reject supernatant liquor is with 0.05 long-pending～0.1mol.L of monoploid then
-1, the pH value is 6.0～8.0 phosphoric acid buffer dissolution precipitation thing, and is centrifugal behind lucifuge dialysis 20～30h, gets supernatant liquor with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 the good DEAE-52 ion exchange column of phosphoric acid buffer balance, and with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 phosphoric acid buffer and 0.1～0.3mol.L
-1The NaCl eluant solution, with collecting blue color component, lyophilize gets the Phycocyanins, C-of purifying.
The present invention has the following advantages and distinguished effect: easy to implement the method, easy to operate, economy is quick, safe and reliable, output is high, good product quality, and equipment used simply is applicable to commercial scale production.
Embodiment
The concrete implementation step of extracting the Phormidium tenue Phycocyanins, C-is as follows:
The preparation of A, nutrient solution
Add SODIUMNITRATE 1.5g, dipotassium hydrogen phosphate 0.04g, sal epsom 0.075g, calcium chloride 0.036g, citric acid 0.006g, ferric ammonium citrate 0.006g, disodium EDTA 0.001g, yellow soda ash 0.02g, trace element solution 1ml by every premium on currency.Wherein trace element solution stirs evenly after the dissolving fully by adding boric acid 286mg, tetrahydrate manganese chloride 181mg, Zinc Sulphate Heptahydrate 22.2mg, Sodium Molybdate Dihydrate 25.2mg, cupric sulfate pentahydrate 7.9mg in every 100ml distilled water.
The cultivation of B, Phormidium tenue
(1) one-level is cultivated: the Phormidium tenue kind is inserted the triangular flask that nutrient solution is housed, and (in 150～200ml), static cultivation or aerated culture during aerated culture, are regulated the air-flow size at 2.5L.min
-1, air-flow carries out aseptically process through cotton ball, and light intensity is controlled at 70 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.Cultivate and be transferred to the secondary cultivation after 7～10 days.
(2) secondary is cultivated: the culture that one-level is cultivated gained is linked into the culturing bottle that nutrient solution is housed, and (in 500～1000ml), aerated culture is regulated the air-flow size at 3L.min
-1, aseptically process, light intensity are controlled at 80 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.After cultivating 7-10 days, be transferred to three grades of cultivations.
(3) three grades of cultivations: the culture that secondary is cultivated gained is linked into the culturing bottle that nutrient solution is housed, and (in 5000～20000ml), aerated culture is regulated the air-flow size at 5L.min
-1, aseptically process, light intensity are controlled at 90 μ E.m
-2.s
-1, temperature is controlled at 30 ℃.After cultivating 7～10 days, just can be used as the inoculation provenance that continues cultivation.
(4) continue to cultivate
1. partial circulating cultivation pool (1～2m
3) cultivate: adopts the algae kind of gained after three grades of cultivations, access partial circulating cultivation pool.Adopt glass temperature canopy controlled temperature at 30 ℃, utilize natural light to carry out illumination, adopt shading screen control intensity of illumination at 120～180 μ E.m
-2.s
-1, impeller or submersible pump stir substratum.
2. systemic circulation cultivation pool (40～60m
3) cultivate: the Phormidium tenue of cultivating in the partial circulating cultivation pool 4～7 days is transferred in the systemic circulation cultivation pool, the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool, and other culture condition are identical with the culture condition of partial circulating cultivation pool.
The preparation of C, Phormidium tenue powder
The Phormidium tenue of cultivating in the systemic circulation cultivation pool after 15～20 days is extracted into filtration unit with pump---on the vibration inclined-plane sieve, behind vibration inclined-plane sieve, receive Phormidium tenue algae slurry, then that the algae slurry is centrifugal through whizzer, obtain Phormidium tenue algae mud.With Phormidium tenue algae mud concentrate, dehydration, dry, obtain the Phormidium tenue powder after pulverizing.
The extracting of D, Phormidium tenue Phycocyanins, C-
0.05～the 0.1mol.L that in the Phormidium tenue powder, adds 7～10 times of weight
-1, the pH value is the phosphate buffer solution of 6.0-8.0, fully stirs evenly.Place then-10～-20 ℃ down freezing, wait to freeze the back take out place 20～30 ℃ melt and dissolved down, treat its melt and dissolved fully after, again that it is freezing.So multigelation is 3～5 times, and most Phycocyanins, C-s are extracted out from the fake valoniopsis pachynema cell, then the centrifugal 10～15min of 3000～6000rpm.Collect supernatant liquor, be the Phycocyanins, C-crude extract.
E, purifying
Under condition of ice bath, in the crude extract of Phycocyanins, C-, add saturation ratio and be 50～70% ammonium sulfate lucifuge, the 20～40min that saltouts, at 3～5 ℃ of centrifugal 15～25min of 3000～6000rpm down, the reject supernatant liquor is with 0.05 long-pending～0.1mol.L of monoploid then
-1, the pH value is 6.0～8.0 phosphoric acid buffer dissolution precipitation thing, and is centrifugal behind lucifuge dialysis 20～30h, gets supernatant liquor with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 the good DEAE-52 ion exchange column of phosphoric acid buffer balance, and with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 phosphoric acid buffer and 0.1～0.3mol.L
-1The NaCl eluant solution, collect blue color component with automatic Fraction Collector, lyophilize gets the Phycocyanins, C-of purifying.
Claims (2)
1. a method of extracting Phycocyanins, C-from Phormidium tenue is characterized in that comprising the steps:
A, the preparation of nutrient solution: add SODIUMNITRATE 0.8～2.5g by every premium on currency, dipotassium hydrogen phosphate 0.02～0.05g, sal epsom 0.06～0.09g, calcium chloride 0.02～0.05g, citric acid 0.004～0.008g, ferric ammonium citrate 0.004～0.008g, disodium EDTA 0.001～0.003g, yellow soda ash 0.01～0.04g, trace element solution 0.5～2ml, wherein trace element solution is by adding boric acid 270～300mg in every 100ml distilled water, tetrahydrate manganese chloride 170～200mg, Zinc Sulphate Heptahydrate 18～25mg, Sodium Molybdate Dihydrate 15～30mg, cupric sulfate pentahydrate 6～10mg stirs evenly after the dissolving fully and makes nutrient solution;
The cultivation of B, Phormidium tenue: at first be that one-level is cultivated, when inserting in the nutrient solution static cultivation or aerated culture, regulate air-flow at 1.5～3.5L.min by the Phormidium tenue kind
-1, light intensity is controlled at 65～85 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, after cultivating 5～10 days, is transferred to secondary and cultivates; Next is that secondary is cultivated, and the culture of one-level being cultivated gained is linked into aerated culture in the new nutrient solution, regulates air-flow at 2.5～5L.min
-1, aseptically process, light intensity are controlled at 75～95 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates and is transferred to three grades of cultivations after 5～10 days; The 3rd is three grades of cultivations, and the culture of secondary being cultivated gained is linked into aerated culture in the new nutrient solution, regulates air-flow at 3～6L.min
-1, aseptically process, light intensity are controlled at 70～100 μ E.m
-2.s
-1, temperature is controlled at 22～35 ℃, cultivates after 5～10 days as the inoculation provenance that continues to cultivate; The 4th is to continue to cultivate, and at first is that the partial circulating cultivation pool is cultivated, and with the algae kind access partial circulating cultivation pool of gained after three grades of cultivations, controlled temperature utilizes natural light to carry out illumination at 22～35 ℃, and the control intensity of illumination is at 120～180 μ E.m
-2.s
-1, stir substratum; Next is that the systemic circulation cultivation pool is cultivated, the Phormidium tenue of cultivating in the partial circulating cultivation pool after 4～7 days is transferred in the systemic circulation cultivation pool, with cultivate in the systemic circulation cultivation pool Phormidium tenue after 10～20 days filter receive Phormidium tenue algae slurry;
The preparation of C, Phormidium tenue powder: algae is starched through centrifugal acquisition Phormidium tenue algae mud, Phormidium tenue algae mud is obtained the Phormidium tenue powder after concentrated, dehydration, drying, pulverizing;
The extracting of D, Phormidium tenue Phycocyanins, C-: the 0.05～0.1mol.L that in the Phormidium tenue powder, adds 7～10 times of weight
-1The pH value is 6.0～8.0 phosphate buffer solution, fully stir evenly be placed on-10～-20 ℃ down freezing, wait to freeze the back and take out, place 20～30 ℃ melt and dissolved down, treat its fully melt and dissolved after, again that it is freezing, so multigelation is 3～5 times, and then centrifugal 10～15min under 3000～6000rpm, collect supernatant liquor, be the Phycocyanins, C-crude extract;
E, purifying: under condition of ice bath, in the crude extract of Phycocyanins, C-, add saturation ratio and be 50～70% ammonium sulfate lucifuge, the 20～40min that saltouts, at 3～5 ℃ of following centrifugal 15～25min of 3000～6000rpm, the reject supernatant liquor is with 0.05 long-pending～0.1mol.L of monoploid then
-1, the pH value is 6.0～8.0 phosphoric acid buffer dissolution precipitation thing, and is centrifugal behind lucifuge dialysis 20～30h, gets supernatant liquor with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 the good DEAE-52 ion exchange column of phosphoric acid buffer balance, and with 0.05～0.1mol.L
-1, the pH value is 6.0～8.0 phosphoric acid buffer and 0.1～0.3mol.L
-1The NaCl eluant solution, with collecting blue color component, lyophilize gets the Phycocyanins, C-of purifying.
2. according to the described method of extracting Phycocyanins, C-from Phormidium tenue of claim 1, it is characterized in that: the volume ratio of nutrient solution is 1: 20～25 in partial circulating cultivation pool and the systemic circulation cultivation pool.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI626892B (en) * | 2015-12-18 | 2018-06-21 | 旭海生物科技有限公司 | Acrochaetium extract and extraction method thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103408657B (en) * | 2013-08-27 | 2015-08-05 | 福清市新大泽螺旋藻有限公司 | Phycocyanins, C-production technique and products thereof |
| CN104130964A (en) * | 2014-07-31 | 2014-11-05 | 青岛农业大学 | Seawater spirulina culture solution |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130028A (en) * | 1995-02-27 | 1996-09-04 | 陈志龙 | Method for preparing phycocyan |
| US5807536A (en) * | 1996-04-09 | 1998-09-15 | The Regents Of The University Of California | Anatomical imaging with phycocyanins |
| CN1344723A (en) * | 2001-09-03 | 2002-04-17 | 中国科学院海洋研究所 | High-purity biliprotein separating process |
| JP2003342489A (en) * | 2002-05-28 | 2003-12-03 | Dainippon Ink & Chem Inc | Method of extracting phycocyanin from blue-green algae |
-
2008
- 2008-01-25 CN CN2008100467687A patent/CN101235075B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130028A (en) * | 1995-02-27 | 1996-09-04 | 陈志龙 | Method for preparing phycocyan |
| US5807536A (en) * | 1996-04-09 | 1998-09-15 | The Regents Of The University Of California | Anatomical imaging with phycocyanins |
| CN1344723A (en) * | 2001-09-03 | 2002-04-17 | 中国科学院海洋研究所 | High-purity biliprotein separating process |
| JP2003342489A (en) * | 2002-05-28 | 2003-12-03 | Dainippon Ink & Chem Inc | Method of extracting phycocyanin from blue-green algae |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI626892B (en) * | 2015-12-18 | 2018-06-21 | 旭海生物科技有限公司 | Acrochaetium extract and extraction method thereof |
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