KR101477204B1 - Methods for the simultaneous production of astaxanthin and lipid from Haematococcus pluvialis - Google Patents

Abstract

The present invention relates to a method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis, wherein the microalgae, Hematococcus pluvialis, is chemically treated with an aqueous surfactant solution to destroy the cell wall, A polar organic solvent or a mixed polar organic solvent is put into a batch extractor, and ultrasonic waves are applied while stirring to extract a ketocarotenoid and a lipid as a raw material of a biofuel into an organic solvent. A nonpolar organic solvent and ultrapure water The ketocarotenoid, which is a functional active substance, and the lipid, which is a raw material of the biofuel, are separated from each other through layer separation, and a ketocarotenoid-enriched polar organic solvent and an aqueous solution layer are supplied to the nonpolar polymer adsorption column By producing highly concentrated astaxanthin, the high-performance active substance Asta It relates to a process for simultaneous production of lipids (lipid) used as a raw material for tin and biofuels.

Description

Methods for simultaneous production of astaxanthin and lipid from Haematococcus pluvialis (Haematococcus pluvialis)

The present invention relates to a method for simultaneously producing astaxanthin and lipid from hematococcus pluvialis, and more particularly, to a method for producing hematococcus pluvialis, which is a microalgae, through chemical treatment and organic solvent- And a method for simultaneously producing astaxanthin and lipid, which are raw materials of biofuel, from a separated liquid after phase separation by a centrifugal separator.

Natural astaxanthin extracted from crustaceans such as shrimp or lobster or algae such as Haematococcus is rich in color and has a flavor effect through enhancement of color such as salmon and 500 times more than tocopherol (vitamin E) Ketocarotenoid with antioxidant function is known to have effects such as anticancer action, activation of immune function and vitamin A precursor through removal of active oxygen radical in human body.

In addition, microalgae, including Haematococcus, contain lipids that are converted to biofuel feedstocks.

The natural astaxanthin (3,3'-dihydroxy-β, β'-carotene-4,4'-dione) can be produced by using an organic solvent or supercritical carbon dioxide to produce microalgae such as Bioresource Technology 99 (2008) 5556 -5560) or a yeast strain, J Zhejiang Univ. Sci. B, 2008 9 (1): 51-59), and the color of the crystal is dark violet-brown. It has a melting temperature of 224 ° C and a molecular weight of 596.86 g / mol (C ₄₀ H ₅₂ O ₄ ). It is insoluble in aqueous solution but soluble in most organic solvents (dichloromethane, chloroform, acetone, dimethylsulfoxide, methanol, ethanol and ethylacetate) .

Astaxanthin, a red ketocarotenoid, is a kind of carotenoid pigment with a chemical structure such as β-carotene, which is capable of eliminating harmful free radicals that cause aging and cancer. Is an antioxidant functional substance.

Astaxanthin has a much higher antioxidant activity than conventional antioxidants because of its unique molecular structure, which has one hydroxyl group (-OH) and one ketone group (═O) at both ends in comparison with beta-carotene. (Fisheries Sci., 62: 134, 1996, Crit. Rev. Biotechnol., 11: 297,1991), which is 500 times more potent than Jane's vitamin E and 20 times more potent than beta-carotene.

Due to these antioxidant functions, astaxanthin is widely used as a feed additive for pharmaceuticals, food additives, and animal and poultry, and its demand and application range is expected to increase rapidly.

Conventionally commercialized astaxanthin is chemically synthesized (Pure Appl. Chem., 74: 1369-1382, 2002) and is produced from crustaceans and microorganisms.

The chemically synthesized astaxanthin is mostly free astaxanthin, which is inactivated in vivo, has low stability against light or temperature, and is restricted for use in some countries in Europe.

Various natural biological materials of astaxanthin have also been reported, including crustaceans and their extracts, green algae Haematococcus fluvialis, and red yeast Xanthophyllomyces dendrorhous. Particularly, since the content of astaxanthin is relatively low and the content of moisture, ash and chitin is high in conventional crustacean diets, the green algae Haematococcus pluvialis and the red yeast xanthophylla maescedendroos (Xanthophyllomyces dendrorhous) has recently become an important ingredient in astaxanthin for industrial production. Among them, Haematococcus pluvialis has a high content of astaxanthin (6-8%) and is a natural product that produces 3S, 3'S isomeric form of astaxanthin in high bioavailability (Trends Biotechnol., 21: 210-216 2003; Chemosphere, 54: 413-417 2004; J. Chromatogr., 1076: 189-192 2005; Biotechnol. Lett., 19: 443-446 1997; Patent No. 10-2009-0131991).

The Haematococcus pluvialis is a unicellular green algae, one of the photosynthetic microorganisms, which accumulates a large amount of astaxanthin in the cells in the dormant phase.

Therefore, in order to extract astaxanthin from Hematococcus pluvialis, it is necessary to break the cell wall and free the pigment inside, so that it takes a long time for extraction and it is difficult to extract.

For this reason, various methods have been developed for effectively recovering astaxanthin accumulated in the cells, but most methods using many kinds of organic solvents are dominant. In addition, the use of organic solvents and freeze-drying And the like, and it is difficult to commercialize it due to an increase in the unit price due to expensive processing cost. That is, an efficient method of obtaining a high purity astaxanthin pigment extract has not reached the commercial level (Korean Patent No. 10-2010-0030327).

As a mechanical treatment method of the conventional method, the cultured and collected strains are frozen in liquid nitrogen, pulverized using an impact mill (high speed impact mill or jet mills), and the cell wall of the strain is disrupted, A method of breaking down the cell wall using a mechanical method such as a method of extracting taenzen (US Pat. Nos. 4871551 and 6022701) and a French pressure or homogenizers and then extracting it with a solvent have.

In addition, a method of treating microwaves and extracting them using ethanol, methanol, acetone, and a mixed solvent thereof (Korean Patent No. 2000-0072136) is known, but a method of extracting astaxanthin and biofuels from microalgae Methods for simultaneous production and optimization of solvent mixing ratios to achieve this are needed.

As an example of a biological treatment method, a method using a cell wall degrading enzyme derived from Trichoderma reesei ATCC 13631 (Phaffia yeast) is known (Korean Patent No. 1995-0005983).

It is also known that a water-immiscible organic solvent containing an antioxidant and / or an oil is used to prepare a powdery preparation in which the active ingredient is finely divided (Korean Patent No. 1999-0072792).

However, when carotenoids are extracted by destroying the cell wall using various methods as described above, there are disadvantages such as low pigment extraction rate and high pigment destruction rate, and labor cost, high processing cost, There is a problem that excessive organic solvent may cause cost and environmental pollution.

On the other hand, microalgae are widely used not only for the production of astaxanthin as described above but also for the production of raw materials for biofuels. However, there is still a method for simultaneously producing raw materials of astaxanthin and biofuels from Hematococcus pluvialis Is not being studied.

It is an object of the present invention to solve the above-mentioned problems, and an object of the present invention is to provide a method for treating microalgae, Haematococcus pluvialis, which comprises a chemical treatment step, an organic solvent-supporting organic solvent extraction step, A step of producing lipid from the separated liquid, and a step of producing astaxanthin by the non-polar polymer adsorbent treatment of the separated liquid in order, thereby providing a method for simultaneously producing astaxanthin and lipid while reducing the amount of organic solvent used There is.

According to an aspect of the present invention, there is provided a method of treating a hematocookus pluvialis cell, comprising the steps of: (a) treating a cell wall of Hematococcus pluvialis with sodium dodecyl sulfate;

An ultrasound-assisted organic solvent extraction step of adding a polar organic solvent to a hematocookus pluvialis with cell walls destroyed and obtaining an extract containing keto-carotenoids and lipids by supersonic wave extraction;

Separating the upper nonpolar organic solvent layer in which the lipid is dissolved and the lower polar organic solvent layer into which the ketocarotenoid is concentrated by injecting a nonpolar organic solvent into the extract;

A lipid production step of evaporating the phase-separated non-polar organic solvent layer to obtain a lipid;

And a step of producing astaxanthin by supplying a phase-separated polar organic solvent layer to an adsorption column filled with a nonpolar polymer adsorbent to produce astaxanthin, and a method for simultaneous production of astaxanthin and lipid from hematococcus pluvialis ≪ / RTI >

In a preferred embodiment of the present invention, the keto-carotenoid recovery step may include recovering the keto-carotenoid contained in the supernatant discharged from the chemical treatment step by liquid-liquid extraction with a nonpolar solvent.

The present invention is a preferred embodiment, wherein the chemical treatment step comprises: impregnating and stirring the hematocookus pluvialis with a solution of sodium dodecyl sulfate aqueous solution to chemically destroy the cell wall;

Centrifuging the mixture of Hematococcus pluvialis and sodium dodecylsodium to obtain wet Hematococcus pluvialis with supernatant and cell walls destroyed;

And then washing the dodecyl sulfate remaining in the wet Hematococcus pluvialis with ultra pure water to remove the dodecyl sulfate.

In a preferred embodiment of the present invention, an aqueous solution having a sodium dodecyl sulfate concentration of 1.0 wt% may be added in an amount of 6 to 9 mL per 1 g of hematococcus pluvialis.

In the present invention, the extraction of the supersonic wave organic solvent includes a step of introducing a polar organic solvent into a wet hematocookus pluvialis which has undergone a chemical treatment step into an extraction reactor having an ultrasonic generator inserted therein, injecting ultrasonic waves for a predetermined period of time, An ultrasound assisted extraction step of extracting the carotenoid and lipid into an organic solvent;

Then, centrifugation was carried out on Hematococcus pluvialis, an organic solvent extract mixed with the extracted ketocarotenoid and lipid, followed by centrifugation to separate the hematocookus pluvialis wetted with organic solvent into the supernatant (ketocarotenoid + lipid + organic solvent) Step.

In the liquid-liquid extraction step of the present invention, the non-polar organic solvent is mixed with the supernatant liquid centrifuged through the supersonic wave-assisted organic solvent extraction step, and then the ultrapure water is added to the upper nonpolar organic solvent layer, The keto carotenoid may be phase separated into a mixture of concentrated lower organic solvent and ultrapure water.

According to a preferred embodiment of the present invention, the step of producing asbestanthine comprises: an evaporation-drying step of evaporating and drying a mixture of an aqueous solution of ultrapure water and a keto-carotenoid to obtain a crude keto-carotenoid;

A dissolution step in which a non-polar organic solvent is added to a Crude keto carotenoid powder to dissolve it;

Adsorption step of passing the solution in which the crude keto carotenoid is dissolved to an adsorption tower filled with a nonpolar polymer adsorbent to adsorb astaxanthin and to discharge a component having a polarity lower than that of astaxanthin to a discharge liquid;

A desorption step of desorbing astaxanthin by supplying a non-polar solvent to the non-polar polymer adsorbent adsorbed by astaxanthin which is discharged from the discharged liquid;

Then, the effluent discharged from the adsorption tower is evaporated and dried to obtain astaxanthin powder.

The present invention is a preferred embodiment, wherein the keto carotenoid recovery step comprises filtering the supernatant separated in the centrifugation step of the chemical treatment step;

Adding a mixed solution of ethyl acetate and methanol, which are non-polar organic solvents, to the filtered supernatant liquid to extract liquid-liquid;

And then the ketocarotenoid and the lipid can be recovered by evaporating and drying the upper ethyl acetate layer in which the ketocarotenoid and the lipid are dissolved.

In a preferred embodiment of the present invention, 50 to 132 ml of a non-polar organic solvent having a volume ratio of ethyl acetate: methanol of 1.7: 1 can be added per 1.0 g of Hematococcus pluvialis.

In a preferred embodiment of the present invention, the polar organic solvent is a monopolar organic solvent or a mixed polar organic solvent in which the monopolar organic solvent is mixed, wherein the monopolar organic solvent may be selected from methanol, ethanol, ethyl acetate, and acetone.

The mixed polar organic solvent may be used in combination of acetone and methanol, acetone and ethanol, acetone and ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol.

In the present invention, the acetone and methanol may be ultrasonically extracted by adding 35 to 45 ml per 1 g of hematococcus pluvialis after mixing in a molar ratio of acetone in the range of 0.3 to 0.9.

In the present invention, the acetone and ethanol may be ultrasonically extracted by adding 35 to 45 ml per 1 g of hematocookus pluvialis after mixing in a range of 0.5 to 0.95 mole ratio of acetone.

In the present invention, the acetone and the ethyl acetate can be ultrasonically extracted by adding 35 to 45 ml per 1 g of hematococcus pluvialis after mixing in the range of 0.2 to 0.95 mole ratio of acetone.

According to a preferred embodiment of the present invention, the ethyl acetate and methanol may be ultrasonically extracted by adding 35 to 45 ml per 1 g of hematocookus pluvialis after mixing in a range of 0.2 to 0.8 mole ratio of ethyl acetate.

In a preferred embodiment of the present invention, the ethyl acetate and ethanol may be mixed in a range of the mole ratio of ethyl acetate to the range of 0.35 to 0.9.

In a preferred embodiment of the present invention, the non-polar organic solvent used in the liquid-liquid extraction step may be hexane or petromium ether.

In a preferred embodiment of the present invention, when hexane is used as the non-polar organic solvent, when the mixed polar organic solvent used in the liquid-liquid extraction step is acetone: methanol having a volume ratio of 3: 1, hexane and ultrapure water 0.31 to 1.53: 0.4 to 1.54.

As described above, the present invention relates to a method for producing a microalgae, which comprises the steps of: chemical treatment using hematocookus pluvialis, sodium dodecylsulfate, organic solvent extraction treatment using ultrasonic waves, liquid-liquid extraction treatment with a nonpolar organic solvent, The advantage of being able to simultaneously produce astaxanthin and lipid, which is a raw material for biofuels, by sequentially passing the adsorbent treatment step,

In addition, the extraction efficiency of ketocarotenoid, which is a functional active substance, and lipid, which is a raw material of biofuels, is enhanced by introducing an organic solvent extraction treatment step for supersonic waves,

In addition, the non-polar organic solvent layer obtained in the liquid-liquid extraction step with the nonpolar organic solvent is evaporated to produce lipids of 10.0 to 11.41 g per 100 g of the final hematocookus pluvialis,

Also, by treating the polar organic solvent layer obtained in the liquid-liquid extraction step with the nonpolar organic solvent with a nonpolar polymer adsorbent, it is possible to obtain an aqueous solution of 2.77 to 3.9 g of astaxanthin having a concentration ratio of 3S, 3'S-astaxanthin per 100g of final hematococcus pluvialis of 80% The advantage of being able to produce xanthine,

In addition, it is a useful invention having advantages of solubility enhancement effect to astaxanthin by using a polar mixed solvent and supersonic wave assisted extraction method to reduce the amount of solvent used.

1 is an exemplary view showing a production process according to an embodiment of the present invention,
FIG. 2 is a graph showing the (3S, 3'S) -astaxanthin extracting result in Hematococcus pluvialis through a single organic solvent and supersonic wave assisted extraction according to Example 1,
FIG. 3 is a graph showing the extraction yield of (3S, 3'S) -astaxanthin according to the mixing ratio of ethyl acetate, methanol, ethyl acetate, acetone, ethyl acetate and ethanol according to Example 4,
4 is a graph showing the extraction yield of (3S, 3'S) -astaxanthin according to the mixing ratio of acetone, methanol, acetone and ethanol, acetone and ethyl acetate according to Example 5,
FIG. 5 is a graph showing the extraction yield of keto-carotenoid according to the number of times of supersonic wave extraction in the optimum mixing polarity organic solvent mixing ratio according to Example 6. FIG.
FIG. 6 is a graph showing an ultrasonic-assisted organic solvent extraction amount of microalgae treated with SDS (sodium dodecylsulfate) aqueous solution at an optimum mixed polar organic solvent mixing ratio according to Example 7. FIG.
FIG. 7 is a graph showing the results of high-speed chromatography analysis of upper and lower layer samples of liquid-liquid extraction using 3S, 3'S-astaxanthin, supersonic wave organic solvent extract, and nonpolar organic solvent according to Example 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

1 shows a process flow of a method for simultaneous production of astaxanthin and biofuel raw material lipid from Hematococcus pluvialis according to one embodiment of the present invention.

As shown, the simultaneous production of astaxanthin and lipid, which is a raw material of biofuel, from hematococcus pluvialis, a microalgae, is mainly performed by adding sodium dodecylsulfate to hematococcus sp. A chemical treatment step (S100) of destroying the cell wall of ruby alice;

An ultrasound-assisted organic solvent extraction step (S200) for obtaining an extract containing ketocarotenoid and lipid by ultrasound-assisted extraction of an organic solvent into a hematocookus pluvialis with a cell wall destroyed;

Liquid extraction step (S300) of phase-separating the nonpolar organic solvent layer in which the lipid is dissolved and the lower polar organic solvent layer in which the ketocarotenoid is concentrated, by injecting a nonpolar organic solvent into the extract;

A lipid production step (S400) of evaporating a phase-separated nonpolar organic solvent layer to obtain a lipid as a raw material of the biofuel;

And an asan xanthine production step (S500) of supplying a phase-separated polar organic solvent layer to an adsorption tower filled with a non-polar polymer adsorbent to obtain an isocyanane.

In the present invention, the mixture of the supernatant solution in which the sodium dodecylsulfate, a surfactant, is centrifuged in the chemical treatment step (S100) is subjected to liquid-liquid extraction with ethyl acetate and methanol, which are nonpolar organic solvents, And a keto carotenoid recovery step (S600) for removing sodium dodecyl sulfate as an alcohol layer and separating the ketocarotenoid into an ethyl acetate layer.

At this time, the amount of ketocarotenoid isolated is insufficient in the final yield of the present invention because the amount of the ketocarotenoid is small and the purity is low as compared with the ketocarotenoid obtained in the step S400.

The polar organic solvent used as the monopolar organic solvent or the mixed polar organic solvent in which the supersonic wave supporting organic solvent is extracted (S200) is methanol or ethanol or ethyl acetate (acetyl acetate) or acetone.

In addition, the supersonic wave supporting treatment may be performed by adding a chemically treated hematocookus pluvialis into a batch reactor together with a monopolar organic solvent or a mixed polar organic solvent to prepare an ultrasonic generator (probe) having a rated output of 500 watt and 20 kHz according to an embodiment ) To extract ketocarotenoids and lipids by ultrasonic extraction.

The step of producing astaxanthin (S500) comprises: evaporating and drying a phase-separated polar organic solvent layer to dissolve the powdered ketocarotenoid in a nonpolar mixed organic solvent, and then adding the non-polar polymeric adsorbent to the adsorption tower The nonpolar mixed organic solvent used in this step is a mixed solvent of hexane and ethyl acetate.

More specifically, the chemical treatment step S100, the supersonic wave-assisted organic solvent extraction step S200, the liquid-liquid extraction step S300, the lipid production step S400, the astaxanthin production step S500 and the keto- The step S600 will be described.

The chemical treatment step (S100) is performed by first mixing microalgae Haematococcus pluvialis with an aqueous solution of sodium dodecylsulfate (SDS) Followed by impregnation and agitation to chemically destroy the cell wall (S101). At this time, an aqueous solution having a sodium dodecylsulfate concentration of 1.0 wt% is added in an amount of 6 to 9 mL per 1 g of hematococcus pluvialis. In the range of such a range, the cell wall is destroyed with a minimum amount of sodium dodecyl sulfate.

Centrifuging step (S102) of centrifuging a mixture of hematocookus pluvialis and sodium dodecylsulfate impregnated and agitated to obtain wet hematocococcus pluvialis with supernatant and cell walls sufficiently destroyed .

And a cleaning step (S103) for cleaning with dewatered water to remove sodium dodecyl sulfate remaining in the wet Hematococcus pluvialis.

The centrifugation step and the washing step in which the mixture of the ultrapure water and the hematocookus pluvialis are centrifuged again and separated into the supernatant in which the wet hematococcus pluvialis and the sodium dodecyl sulfate are dissolved is repeated. Washing is usually carried out 3 to 5 times until no bubbles are visually observed.

The extraction step (S200) of supersonic wave supporting organic solvent may be performed by adding a monopolar organic solvent or a mixed polar organic solvent mixed with wet monocotyledonous hematocococcus pluvialis through a chemical treatment step (S100) The keratocarotenoids present in the cytoplasm of the hematocococcus pluvialis cell wall, which has been destroyed by the ultrasonic wave for a certain period of time (20 to 120 minutes), are placed in an extraction reactor equipped with a wave generator, (Step S201).

The monopolar organic solvent is preferably one of methanol, ethanol, ethyl acetate (ethyl acetate), and acetone.

The mixed polar organic solvent is preferably used in combination of acetone and methanol, acetone and ethanol, acetone and ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol.

In this case, the molar fraction of acetone and methanol for acetone and methanol is in the range of 0.3 to 0.9, the mole fraction of acetone and acetone is 0.5 to 0.95 for acetone and ethanol, the mole fraction of acetone and acetone is in the range of 0.2 to 0.95, Ethyl acetate and ethanol are mixed in a molar ratio range of 0.35 to 0.9. Ethyl acetate and ethanol are mixed with 35 ~ 45 ml per 1 g of hematococcus pluvialis. At such mixing ratio, the extraction amount of (3S, 3'S) -astaxanthin is at least 0.6 wt% or more.

Hematococcus pluvialis and extracts of keto-carotenoids and lipids extracted with organic solvents were centrifuged to remove the hematocococcus pluvialis and the supernatant (ketocarotenoid + lipid extracted with organic solvent) (Step S202).

Also, the number of times to repeat the centrifugation step (S202) and the supersonic wave extraction step (S201) until the naked eye becomes almost white is performed three to four times.

In the liquid-liquid extraction step (S300), the nonpolar organic solvent is mixed with the supernatant liquid centrifuged through the supersonic wave-assisted organic solvent extraction step (S200), and ultrapure water is added to perform phase separation. The liquid-liquid extraction step is phase-separated into a mixture of a lipid-concentrated upper nonpolar organic solvent layer and a lower polar organic solvent concentrated ketocarotenoid and ultrapure water.

The nonpolar organic solvent used herein is hexane or petroleum ether.

When hexane is used as the nonpolar organic solvent, hexane and ultrapure water are mixed in a volume ratio of 0.31 to 1.53: 0.4 to 1.54 when the mixed polar organic solvent used in the previous liquid-liquid extraction step has a volume ratio of acetone: methanol of 3: . The reason for this is that the phase separation efficiency is good during the liquid-liquid extraction when the numerical value ratio is used.

The lipid production step (S400) has an evaporation / drying step (S401) for evaporating the organic solvent in the mixture layer of the lower polar organic solvent and the ultrapure water in which the ketocarotenoid is concentrated after the liquid-liquid extraction step (S300).

After evaporation and drying, it will obtain the lipid powder which is the raw material of the biofuel.

The step of producing astaxanthin (S500) includes an evaporation drying step (S501) of evaporating and drying a mixture of the ultrapure water solution and the ketocarotenoid to obtain crude ketocarotenoid.

Thereafter, the crude ketocarotenoid powder obtained after evaporation and drying has a dissolution step (S502) in which hexane and ethyl acetate mixed solvent, which is a nonpolar solvent, are added to dissolve the crude ketocarotenoid powder.

Thereafter, the solution in which the crude keto-carotenoid is dissolved is passed through an adsorption column filled with a polystyrene-divinylbenzene-based amberlite-based nonpolar polymer adsorbent to adsorb astaxanthin, and an adsorbing step of discharging components with lower polarity than astaxanthin to the discharged liquid (S503).

And a desorption step (S504) for desorbing astaxanthin by supplying a nonpolar mixed solvent of hexane and ethyl acetate to the nonpolar polymer adsorbent adsorbed on astaxanthin which is discharged therefrom.

And thereafter evaporating and drying the discharged solution discharged from the adsorption tower through the desorption step (S505). After this evaporation and drying step, astaxanthin powder is obtained.

On the other hand, the discharged liquid of the component having a lower polarity than the astaxanthin discharged from the adsorption tower through the adsorption step (S503) is obtained as a raw material of the biofuel through the evaporation drying step (S506).

Meanwhile, the keto carotenoid recovery step (S600) has a step (S601) of filtering the supernatant separated in the centrifugal separation step (S102) of the chemical treatment step (S100).

Thereafter, a step (S602) of adding a mixed solution of ethyl acetate and methanol to the filtered supernatant liquid to extract liquid-liquid is carried out.

Thereafter, the lower aqueous solution / alcohol mixture layer in which sodium dodecylsulfate is dissolved is discarded and the upper ethyl acetate layer in which the ketocarotenoid is dissolved is evaporated and dried (S603). After evaporation drying, the ketocarotenoid is recovered.

Hereinafter, preferred embodiments of the present invention will be described.

The analytical methods of Examples and Comparative Examples are as follows: violaxanthin, neoxanthin, (3S, 3'S) -astaxanthin, lutein, zeaxanthin, echinenone, canthaxanthin, beta carotene, chlorophyll a, chlorophyll b Were analyzed by high performance liquid chromatography with C ₁₈ ODS (octadecylsilane, reverse phase, 5 μm, 250 mm × 4.6 mm) columns. A gradient method was used in which the mobile phase A and the mobile phase B were mixed according to time and supplied.

The mobile phase A is acetonitrile: methanol: ultrapure water = 78: 10: 12 (volume percentage) and mobile phase B is acetonitrile: methanol: ultrapure water: dichloromethane = 45.5: 28: 4.5: 22 (volume percentage).

In the gradient method, 100% for mobile phase A and 0% for mobile phase B, 10% for mobile phase A, 10% for mobile phase A, 90% for mobile phase B and 0% The mobile phase A is 10% and the mobile phase B is 90%. In 40-45 minutes, the mobile phase A is changed from 10% to 0%, the mobile phase B is changed from 90% to 100% B is 100%. In 50-55 minutes, mobile phase A is 0% to 100%, mobile phase B is 100% to 0%, 55-70 minutes mobile phase A is 100% and mobile phase B is 0% And the flow rate was 1 ml / min at all times.

20 μl of the sample was injected and analyzed with a visible light detector at a wavelength of 455 nm. (DHI, 0.841 mg / l), echinenone (DHI, 0.786 mg / l), neaxanthin (DHI, 0.745 mg / l), violaxanthin (DHI, 0.955 mg / l), chlorophyll a (Sigma), chlorophyll b (Sigma), beta-carotene (Sigma) and lutein (Sigma) were used as standard samples.

The concentration of the standard sample was concentrated, and 20 μl of the solution having the concentration of each component of 0 to 12 mg / l was injected into the high-speed liquid grommatography, and the area of the peak obtained was plotted as a concentration, and the coefficient of linear function obtained by linear regression To determine the concentration of each component contained in the extract.

(Example 1) Ultrasonic assisted extraction experiment and result using a single organic solvent

0.2 g of microalgae Haematococcus pluvialis was dissolved in dichloromethane (DCM), acetone, methanol (MeOH), chloroform, ethyl acetate (EtOAc, ethylacetate) (EtOH, ethanol), and hexane were added to each extractor, ultrasonicated for 60 minutes at a temperature of 4 ° C, and the mixture was stirred for 60 minutes while the ultrasonic injection was stopped. Thus, lipid and keto-carotenoid (ketocarotenoid) component into an organic solvent.

Extract 1 ml of the filtrate is completely evaporated in a nitrogen atmosphere, and the sample dissolved in 1 ml of methanol is analyzed by high-speed chromatography (HPLC).

Typically, the weight extracted from 100 g of microalgae from the (3S, 3'S) -astaxanthin concentration is shown in FIG.

The weight of (3S, 3'S) -astaxanthin extracted per 100g of microalgae was 0.772g, 0.59g, 0.521g, 0.479g, 0.474g, 0.339g, 0.034g in the order of DCM, Acetone, MeOH, EtOAc, (3S, 3'S) -astaxanthin in the order of DCM, Acetone, MeOH, EtOAc, chloroform, EtOH and hexane in the order of 64.22%, 61.37%, 58.19% 59.92%, 62.33%, 45.03%, and 13.2%.

(Example 2) Ultrasonic assisted extraction using a mixed organic solvent of dichloromethane and methanol

A microalgae Haematococcus pluvialis was added to four extractors containing 20 ml of a solution of methanol (MeOH) and dichloromethane (DCM) in a volume ratio of 1: 5.7, 1: 3, 1: (100 ml of an organic solvent per 1.0 g of microalgae) and extracted at 4 캜 for 70 minutes while applying ultrasonic waves thereto. 1 ml of the extract was filtered through a 0.5 μm PTFE membrane and then completely evaporated in a nitrogen atmosphere. The sample was dissolved in 1 ml of methanol and analyzed by high-speed chromatography.

The maximum extraction of (3S, 3'S) -astaxanthin was 0.902 wt% (or (3S, 3'S) -astaxanthin 0.902 g per 100 g of microalgae) in DCM: MeOH = 3: 1 The area ratio of the (3S, 3'S) -astaxanthin component in the obtained extract was 63.92%.

(Example 3) Ultrasonic assisted extraction using mixed organic solvent of chloroform and ethanol

0.2 g of microalgae Haematococcus pluvialis was added to four extractors containing 20 ml of a solution having a volume ratio of chloroform and ethanol of 1: 1, 1: 3, 1: 1 and 3: 1, And extracted with ultrasonic waves at 4 캜 for 70 minutes. 1 ml of the extract was filtered through a 0.5 μm PTFE membrane and then completely evaporated in a nitrogen atmosphere. The sample was dissolved in 1 ml of methanol and analyzed by high-speed chromatography.

The maximum extraction of (3S, 3'S) -astaxanthin was 0.682wt% (or (3S, 3'S) -astaxanthin 0.682g per 100g of microalgae) in chloroform: EtOH = 7: The area ratio of the (3S, 3'S) -astaxanthin component in the obtained extract was 62.91%.

(Example 4) Ultrasonic assisted extraction using a mixed organic solvent of ethyl acetate (ethyl acetate), acetone, ethyl acetate, methanol, ethyl acetate and ethanol

To five extractors containing 20 ml of a mixed solvent of ethyl acetate and acetone in a volume ratio of 1: 9, 1: 3, 1: 1, 3: 1 and 9: 1, 0.2 g of Haematococcus pluvialis Were added to five extractors containing 20 ml of a solvent containing ethyl acetate and methanol in a volume ratio of 1: 3, 1: 1, 7: 3, 4: 1, and 9: 1, respectively, to obtain Haematococcus pluvialis. 0.2 g of Haematococcus plusvialis was added to each of four extractors containing 20 ml of a solvent of ethyl acetate and ethanol in a volume ratio of 9: 1, 3: 1, 1: 1 and 1: And extracted at 4 캜 for 70 minutes.

FIG. 3 shows the weight of (3S, 3'S) -astaxanthin extracted per 100 g of Haematococcus pluvialis under the condition that the mixing ratio of ethyl acetate to methanol, ethyl acetate to ethanol, and ethyl acetate to acetone were different.

Typically, in a mixed solvent of ethyl acetate and methanol, the extraction amount of (3S, 3'S) -astaxanthin is maximized to 0.805 wt% when the volume ratio is 4: 1, and when the volume ratio of ethyl acetate to acetone is 1: (3S, 3'S) -astaxanthin was found to be the largest at 0.677 wt% in the mixed solvent of ethyl acetate and ethanol at the volume ratio of 3: 1 and 0.74 wt% (3S, 3'S) -astaxanthin area ratio of 59.44%, 61.08%, and 56.33% was obtained from the HPLC analysis results.

(Example 5) Ultrasonic assisted extraction using a mixed organic solvent of acetone, ethyl acetate, acetone, methanol, acetone, and ethanol

2. Add 0.2 g of microalgae Haematococcus pluvialis to an extractor containing 20 ml of a solvent having a volume ratio of acetone to methanol of 1: 3, 1: 1, 3: 1 and 9: 1, 0.2 g of Haematococcus pluvialis was added to four extractors containing 20 ml of a solvent having a volume ratio of ethanols of 9: 1, 3: 1, 1: 1 and 1: 3, (Haematococcus plusvialis) was added to each of 5 extractors containing 20 ml of a solvent having a volume ratio of 9: 1, 3: 1, 1: 1, 1: 3, 1: 9, sonicated, (3S, 3'S) -astaxanthin was determined according to the volume ratio of the mixed solvent.

Fig. 4 shows the weight of (3S, 3'S) -astaxanthin extracted per 100 g of Haematococcus pluvialis under different conditions of mixing ratio of acetone and methanol, acetone and ethanol, acetone and ethyl acetate. The maximum extraction amount of (3S, 3'S) -astaxanthin was 0.796 wt% when the volume ratio of acetone to methanol was 3: 1, 0.687 wt% when the volume ratio of acetone to ethanol was 3: 1, (3S, 3'S) -astaxanthin were 60.86%, 58.65% and 61.08%, respectively.

(Example 6) Extraction amount of ketocarotenoid according to the number of extraction in mixed polar organic solvent and supersonic wave assisted extraction

Case ① 100 ml of a mixed solvent of 2.5 g of Haematococcus pluvialis and 3: 1 by volume of acetone: methanol (Acetone: MeOH) (40 ml of organic solvent per 1.0 g of microalgae)

Case (2) A mixture of 2.5 g of Haematococcus pluvialis and 100 ml of a mixed solvent of acetone: ethanol (EtOH) in a volume ratio of 3: 1 (40 ml of organic solvent per 1.0 g of microalgae)

case (3) 100 ml of a mixed solvent (2.5 ml of Haematococcus pluvialis and 3: 1 volume ratio of acetone: Acetone: EtOAc) (40 ml of organic solvent per 1.0 g of microalgae)

case ④ A mixture of 2.5 g of Haematococcus pluvialis and 100 ml of a mixed solvent of 4: 1 by volume of ethyl acetate: methanol (EtOAc: MeOH) (40 ml of organic solvent per 1.0 g of microalgae)

case ⑤ 2.5 ml of Haematococcus pluvialis and 100 ml of a mixed solvent of 3: 1 by volume of ethyl acetate: ethanol (EtOAc: EtOH) (40 ml of organic solvent per 1.0 g of microalgae) were added to five different extractors And the mixture was subjected to primary extraction for 70 minutes while applying ultrasonic waves at 4 ° C, followed by centrifugation to obtain a first supernatant. After the centrifugal separation, the secondary algae were subjected to the second extraction. In the same manner as the first extraction, 100 ml of a mixed solvent of acetone: methanol having a volume ratio of 3: 1, acetone: ethanol in the volume ratio of 3: , 100 ml of a mixed solvent of acetone: ethyl acetate in a volume ratio of 3: 1, 100 ml of a mixed solvent of ethyl acetate and methanol in a volume ratio of 4: 1, a mixed solvent of ethyl acetate: ethanol in a volume ratio of 3: 1 Were added to each well and subjected to secondary extraction with ultrasonic waves at 4 ° C for 70 minutes. Subsequently, a second supernatant obtained by centrifugation was obtained.

After the centrifugation, the residue of the microalgae was subjected to a third extraction. In the same manner as the second extraction, 100 ml of a mixed solvent of acetone: methanol having a volume ratio of 3: 1 and an acetone: ethanol volume ratio of 3 , 100 ml of a mixed solvent of acetone: ethyl acetate in a volume ratio of 3: 1, 100 ml of a mixed solvent of ethyl acetate and methanol in a volume ratio of 4: 1, a mixed solvent of ethyl acetate: ethanol in a volume ratio of 3: 1 Were added to each well and subjected to tertiary extraction with ultrasonic waves at 4 ° C for 70 minutes. Followed by centrifugation to obtain a tertiary supernatant. case (1) to (5) was completely evaporated in a nitrogen atmosphere and dissolved in 2.5 ml of methanol to prepare analytical samples (25-fold dilution). 100 μl of the secondary extracts of cases (1) to (5) The solvent was completely evaporated and dissolved in 0.5 ml of methanol to make analytical sample (5-fold dilution). 100 μl of the third extract of cases ① to ⑤ was completely evaporated in a nitrogen atmosphere and dissolved in 100 μl of methanol. Samples were prepared and analyzed by high-speed chromatography. 5 shows the amount and total amount of (3S, 3'S) -astaxanthin per 100 g of the microalgae obtained in the first, second and third extraction of the cases (1) to (5) (Neo), lutein (Lut), canthaxanthin (Can), echinenone (Ech), beta-carotene (b-Car), chlorophyll , and chlorophyl b. The total amount of (3S, 3'S) -astaxanthin, neoxanthin (Neo), lutein (Lut), canthaxanthin (Can), echinenone (Ech), and beta-carotene (Acetone: EtOAc) volume ratio of 3: 1 in case of volume ratio of 3: 1, case ③ Case 3: volume ratio of acetone: ethanol (Acetone: EtOH) 0.89 g in case of 3: 1 case ④ 0.972 g in case of volume ratio of ethyl acetate: methanol (EtOAc: MeOH) of 4: 1, case ⑤ 0.892 in case of volume ratio of ethyl acetate: ethanol (EtOAc: EtOH) g, and the content of (3S, 3'S) -astaxanthin was 88.5 ~ 91.7% (see Table 1).

Extraction of keto-carotenoid according to extraction frequency in mixed polar organic solvent and supersonic wave extraction

(Example 7) Ultrasonic-assisted organic solvent extraction of micro-algae treated with aqueous sodium dodecylsulfate (SDS) solution

According to the method detailed in Fig. 1 of the present invention, a recovery experiment of astaxanthin, which is a lipid as a raw material of a biofuel and a highly functional active substance, from a microalga, Haematococcus pluvialis, Respectively.

2.5 g of Hematococcus pluvialis with a moisture content of 5.23% and an aqueous solution having a sodium dodecyl sulfate concentration of 1.0 wt% were placed in a reactor and mixed and stirred at 20 ° C for 60 minutes (S101) Lt; RTI ID = 0.0 > chemically < / RTI > At this time, 8 ml of SDS aqueous solution is added per 1 g of Hematococcus pluvialis. The supernatant obtained by primary centrifugation (S102) of a mixture of Hematococcus pluvialis and SDS is stored in a separate container, and the chemically treated Hematococcus pluvialis is transferred to an extractor.

In order to remove SDS remaining in the chemically treated microalgae, ultrapure water is added to the extractor to wash the hematocookus pluvialis (S103), and 16 ml of ultrapure water per 1.0 g of hematocookus pluvialis is added. The supernatant obtained by secondary centrifugation of the mixture is combined with the primary centrifugation supernatant. The micro-algae are cleaned with ultrapure water until the bubbles can not be seen with the naked eye, usually 2-3 times.

The centrifuged supernatant contained SDS components and ketocarotenoids and lipids excreted in aqueous solution from the cytoplasm of Hematococcus pluvialis during the chemical treatment. The SDS component and keto The carotenoid is separated and filtered (S601). For this purpose, Per 1.0 ml A solvent having a volume ratio of 1.7: 1 ethyl acetate: methanol was added to the extraction reactor in an amount of 50-132 ml per 1.0 g of hematococcus pluvialis. After thoroughly stirring, the mixture was stabilized until separated into an upper organic solvent layer and a lower aqueous solution layer .

At this time, the lower aqueous solution layer in which SDS is dissolved is discarded and the lower organic solvent layer is evaporated to dry (S603) to obtain ketocarotenoid and lipid.

Case (1) A mixed solvent of 2.5 g of chemically treated hematocookus pluvialis and aceton: methanol (Acetone: MeOH) in a volume ratio of 3: 1 was added to 40 ml per gram of hematococcus pluvialis,

Case 2) A mixed solvent of 2.5 g of chemically treated hematocookus pluvialis and a 3: 1 volume ratio of acetone: ethanol (Acetone: EtOH) was added to 40 ml per gram of hematococcus pluvialis,

Case ③ A mixed solvent of 2.5 g of chemically treated hematocookus pluvialis and acetone: Acetone: EtOAc in a volume ratio of 3: 1 was added to 40 ml of 1 g of hematocookus pluvialis,

Case ④ A mixture of 2.5 g of chemically treated Hematococcus pluvialis and 40 ml of a mixed solvent of 4: 1 by volume of ethyl acetate: methanol (EtOAc: MeOH) was added to 1 g of hematocookus pluvialis,

case ⑤ A mixed solvent of 2.5 g of chemically treated hematocookus pluvialis and 3: 1 volume ratio of ethyl acetate: ethanol (Acetone: EtOH) was added to 5 different extractors in an amount of 40 ml per 1 g of hematococcus pluvialis, (Step S201) for 70 minutes while maintaining the temperature at 4 占 폚. Thereafter, the supernatant solution is centrifuged (S202) by supersonic wave support and the organic solvent-extracted mixture is centrifuged (S300). The supernatant solution is stored in a cylindrical liquid-liquid extractor (S300). The hematocookus pluvialis residues after centrifugation are subjected to a secondary extraction and a tertiary extraction by introducing a polar organic solvent having the same conditions as the case (1) to the case (5), and the supernatant obtained by centrifugation is added to a cylindrical liquid-liquid extractor .

case (1) to (5) were completely evaporated in a nitrogen atmosphere and dissolved in 2.5 ml of methanol to prepare analytical samples (25-fold dilution). 100 μl of the secondary extracts of cases (1) to (5) After completely evaporating the solvent and dissolving in 0.5 ml of methanol, samples for analysis (5-fold dilution) were prepared and 100 μl of the third extracts of cases ① to ⑤ were completely evaporated in a nitrogen atmosphere and dissolved in 100 μl of methanol. Samples were prepared and analyzed by high-speed chromatography. 6 and Table 2 show the liquid-liquid extraction step Total extraction yield of 3S, 3'S-astaxanthin obtained from supersonic wave support organic solvent extraction step is shown.

Ultrasonic-assisted organic solvent extraction conditions and results of SDS (sodiumdodecylsulfate) aqueous solution-treated microalgae

Case (1) per 100 g of hematococcus pluvialis case (1) Case of ① case of 1: 3 volume ratio of acetone: methanol (Acetone: MeOH), case ② volume ratio of acetone: ethanol (Acetone: EtOH) is 3 : 1.07 g in case 1, case ③ 1.088 g in case of 3: 1 volume ratio of acetone: Acetone: EtOAc case ④ 1.062 g in case of volume ratio of ethyl acetate: methanol (EtOAc: MeOH) case ⑤ The volume ratio of ethyl acetate: ethanol (EtOAc: EtOH) was 1.066 g in the case of 3: 1, which was not chemically treated with SDS and compared with the cases ① to ⑤ of the mixed organic solvent supersonic wave- Increased by 9.3 ~ 21.0%. The extraction yield of 3S, 3'S-astaxanthin is 4.5 ~ 8.4% of the total extraction amount in the SDS aqueous solution treatment and 77.4 ~ 88.0% of the total extraction amount in the first extraction.

In order to separate the ketocarotenoid and the lipid, hexane and ultrapure water were added to the first extraction supernatant obtained through the ultrasound support extraction step (S201) and the centrifugal separation step (S202) of the cases (1) and (2) The extraction step S300 is performed to phase separate the lipid into the upper hexane layer and the ketocarotenoid into the lower polar organic solvent / aqueous layer.

In case of the supernatant of case ③, case ④, case ⑤ that has been passed through supersonic wave extraction step (S201) and centrifugation step (S202) containing ethyl acetate, the organic solvent is removed by evaporation, The mixture is dissolved in a mixed organic solvent and hexane and ultrapure water are added thereto to carry out a liquid-liquid extraction step (S300) to convert the lipid to the upper hexane layer and the ketocarotenoid to the lower polar organic solvent / Phase separation.

As shown in Table 3, 130 ml of the first extraction supernatant was put into a liquid-liquid extractor, and 50 ml of hexane and 20 ml of ultrapure water were added thereto. The ratio of acetone: methanol: hexane: ultrapure water was 3: 1: 1.53: 0.4 v / v / v), case ⓑ The first extraction supernatant (130 ml) was put into a liquid-liquid extractor, and 10 ml of hexane and 50 ml of ultrapure water were added thereto. The ratio of acetone: methanol: hexane: ultrapure water was 3: 1: 0.31: 1.54 (v / v / v / v) was prepared by adding 30 ml of hexane and 30 ml of ultrapure water to a mixture of acetone: methanol: hexane: ultrapure water of 3: 1: 0.92: ) And lipid (ketocarotenoid) were separated from each other.

After the liquid-liquid extraction step (S300), the hexane of the upper nonpolar organic solvent layer was evaporated to dryness (S401) to obtain 10.0 to 11.41 g of lipid as a raw material of the biofuel per 100 g of microalgae. 2.77 to 3.9 g per 100 g of the microalgae Haematococcus pluvialis was obtained from the crude extract of 3S, 3'S-astaxanthin crude extract obtained by evaporation drying (S501).

The liquid-liquid extraction step (S300) using the above nonpolar organic solvent has the effect of increasing the concentration ratio of 3S, 3'S-astaxanthin in the stock solution from 56% to 58% to 80.0%. FIG. 7 is a high-speed chromatogram analysis of upper and lower layer samples of liquid-liquid extraction using 3S, 3'S-astaxanthin, supersonic wave organic solvent extract, and nonpolar organic solvent.

Conditions and Results of Liquid - Liquid Extraction of Organic Solvent Extract Supporting Ultrasonic Wave of Microalgae Using Nonpolar Organic Solvent

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined in the appended claims and their equivalents. Of course, such modifications are within the scope of the claims.

Description of the Related Art
(S100): chemical treatment step
(S200): extraction step of supersonic wave supporting organic solvent
(S300): liquid-liquid extraction step
(S400): stage of lipid production
(S500): Step of producing astaxanthin
(S600): keto carotenoid recovery step

Claims (18)

A chemical treatment step of injecting sodium dodecyl sulfate to destroy the cell wall of Hematococcus pluvialis;
An ultrasound-assisted organic solvent extraction step of adding a polar organic solvent to a hematocookus pluvialis with cell walls destroyed and obtaining an extract containing keto-carotenoids and lipids by supersonic wave extraction;
Separating the upper nonpolar organic solvent layer in which the lipid is dissolved and the lower polar organic solvent layer into which the ketocarotenoid is concentrated by injecting a nonpolar organic solvent into the extract;
A lipid production step of evaporating the phase-separated non-polar organic solvent layer to obtain a lipid;
And a step of producing astaxanthin by supplying a phase-separated polar organic solvent layer to an adsorption column filled with a nonpolar polymer adsorbent to produce astaxanthin, and a method for simultaneous production of astaxanthin and lipid from hematococcus pluvialis .
The method according to claim 1,
Further comprising the step of recovering keto carotenoid and lipid contained in the supernatant discharged from the chemical treatment step by liquid-liquid extraction with a nonpolar solvent and recovering keto carotenoid from the hematocookus pluvialis. Simultaneous production of lipids.
The method according to claim 1,
The chemical treatment step includes: impregnating and stirring the hematocookus pluvialis with a dodecyl sulfate aqueous solution to chemically destroy the cell wall;
Centrifuging the mixture of Hematococcus pluvialis and sodium dodecylsodium to obtain wet Hematococcus pluvialis with supernatant and cell walls destroyed;
And then washing the dodecyl sulfate dodecyl sulfate remaining in the wet Hematococcus pluvialis with ultrapure water and removing the dodecylsodium dodecylsulfate. The method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis.
The method according to claim 1 or 3,
A method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis, wherein an aqueous solution having a sodium dodecylsulfate concentration of 1.0 wt% is added in an amount of 6 to 9 mL per 1 g of hematococcus pluvialis.
The method according to claim 1,
In the step of extracting supersonic wave supporting organic solvent, a polar organic solvent is introduced into a wet hematocookus pluvialis which has undergone a chemical treatment step into an extraction reactor having an ultrasonic generator inserted therein, and ultrasonic waves are injected for a predetermined period of time to remove keto carotenoids and lipids into organic solvents Extracting ultrasonic support;
Then, centrifugation was carried out on Hematococcus pluvialis, an organic solvent extract mixed with the extracted ketocarotenoid and lipid, followed by centrifugation to separate the hematocookus pluvialis wetted with organic solvent into the supernatant (ketocarotenoid + lipid + organic solvent) A method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis.
The method according to claim 1,
In the liquid-liquid extraction step, the nonpolar organic solvent is mixed with the supernatant separated by centrifugation through the supersonic wave-assisted organic solvent extraction step, and then the ultrapure water is added to the upper nonpolar organic solvent layer and the lower nonpolar organic solvent layer in which the keto carotenoid is concentrated. Wherein the phase is phase separated by a mixture of a solvent and ultrapure water. 2. The method of producing simultaneously astaxanthin and lipid from Hematococcus pluvialis.
The method according to claim 1,
The step of producing astaxanthin comprises: an evaporation-drying step of evaporating and drying a mixture of an aqueous solution of ultrapure water and a keto-carotenoid to obtain a crude keto-carotenoid;
A dissolution step in which a non-polar organic solvent is added to a Crude keto carotenoid powder to dissolve it;
Adsorption step of passing the solution in which the crude keto carotenoid is dissolved to an adsorption tower filled with a nonpolar polymer adsorbent to adsorb astaxanthin and to discharge a component having a polarity lower than that of astaxanthin to a discharge liquid;
A desorption step of desorbing astaxanthin by supplying a non-polar solvent to the non-polar polymer adsorbent adsorbed by astaxanthin which is discharged from the discharged liquid;
And a step of evaporating and drying the effluent discharged from the adsorption tower to obtain astaxanthin powder. The method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis.
The method of claim 2,
Wherein the keto carotenoid recovery step comprises filtering the supernatant separated in the centrifugation step of the chemical treatment step;
Adding a mixed solution of ethyl acetate and methanol, which are non-polar organic solvents, to the filtered supernatant liquid to extract liquid-liquid;
And then evaporating the upper ethyl acetate layer in which the tocarotenoid and lipid are dissolved to recover the ketocarotenoid and the lipid. The method for simultaneous production of astaxanthin and lipid from Hematococcus pluvialis.
The method of claim 8,
Per 1.0 ml of the supernatant A method for simultaneous production of astaxanthin and lipid from hematocookus pluvialis, which comprises adding 50 to 132 ml per 1.0 g of non-polar organic solvent having a volume ratio of ethyl acetate: methanol of 1.7: 1 to hematococcus pluvialis .
The method according to claim 1 or 5,
Wherein the polar organic solvent is a monopolar organic solvent or a mixed polar organic solvent in which the monopolar organic solvent is used, and the monopolar organic solvent is one selected from methanol, ethanol, ethyl acetate, and acetone. A method of simultaneous production of taraxin and lipid.
The method of claim 10,
Wherein the mixed polar organic solvent is selected from the group consisting of acetone, methanol, acetone and ethanol, acetone, ethyl acetate, ethyl acetate, methanol, ethyl acetate and ethanol. Simultaneous production method.
The method of claim 11,
The acetone and methanol are mixed with acetone in a molar ratio ranging from 0.3 to 0.9, and 35 to 45 ml per 1 g of hematococcus pluvialis are added for ultrasonic extraction. Simultaneous production of astaxanthin and lipid from hematococcus pluvialis Way.
The method of claim 11,
Wherein acetone and ethanol are mixed with acetone in a molar ratio ranging from 0.5 to 0.95, and then 35 to 45 ml per 1 g of hematocookus pluvialis are added to ultrasonically extract the mixture. As a result of the simultaneous extraction of astaxanthin and lipid from hematococcus pluvialis Production method.
The method of claim 11,
Wherein acetone and ethyl acetate are mixed with acetone in a molar ratio ranging from 0.2 to 0.95 and then 35 to 45 ml per 1 g of hematococcus pluvialis are subjected to ultrasonic extraction to remove astaxanthin and lipid from the hematococcus pluvialis. Simultaneous production method. The method of claim 11,
The mixture of ethyl acetate and methanol is mixed in the range of 0.2-0.8 of the mole fraction of ethyl acetate and then 35-45 ml per 1 g of hematococcus pluvialis is subjected to ultrasonic extraction to remove astaxanthin and lipid .
The method of claim 11,
Wherein the ethyl acetate and ethanol are mixed in a molar ratio of ethyl acetate in the range of 0.35 to 0.9.
The method according to claim 1 or 6,
Wherein the nonpolar organic solvent used in the liquid-liquid extraction step is hexane or a petromellitic ether, wherein the non-polar organic solvent used in the liquid-liquid extraction step is hexane or petromium ether.
18. The method of claim 17,
When hexane was used as the nonpolar organic solvent, the mixed polar organic solvent used in the liquid-liquid extraction step was mixed with acetone: methanol having a volume ratio of 3: 1, and hexane and ultrapure water were mixed in a volume ratio of 0.31 to 1.53: 0.4 to 1.54 ≪ RTI ID = 0.0 > 1. ≪ / RTI > The method of simultaneous production of astaxanthin and lipid from Hematococcus pluvialis.

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