KR102414231B1 - Methods for the simultaneous extraction of astaxanthin, its derivatives, and lipids in high yield from cell wall-enhanced Haematococcus pluvialis - Google Patents

Abstract

The present invention relates to a method for simultaneously extracting astaxanthin, astaxanthin derivatives and lipids from Haematococcus fluvialis by a one-pot method, and the method according to the present invention is easily performed by heat and light. There is an effect of extracting decomposed astaxanthin in a high yield, and obtaining a high astaxanthin recovery rate even in a short time and low-speed rotational crushing process. Therefore, the present invention is expected to be usefully used as a method for extracting astaxanthin, a high value-added substance exhibiting antioxidant activity and anti-inflammatory activity, in an energy-efficient, cost-effective, simpler and safer way.

Description

Methods for the simultaneous extraction of astaxanthin, its derivatives, and lipids in high yield from cell wall-enhanced Haematococcus pluvialis }

The present invention relates to a method of simultaneously extracting astaxanthin, astaxanthin derivatives and lipids in Haematococcus pluvialis in a single reaction vessel (one-pot) method, and the like.

Haematococcus pluvialis ( Haematococcus pluvialis ) is a microalgae that lives in freshwater and has the ability to accumulate astaxanthin, a carotenoid, in 3-5% of dry weight. Haematococcus pluvialis produces spore structures to combat various stresses such as nutrient depletion, exposure to strong light, increased salinity, and temperatures that are too low or too high. In this process, secondary metabolites such as lipids and carotenoids are accumulated to resist stress, and among them, astaxanthin (3,3'-dihydroxy-β,β'- carotene-4,4'-dione) is attracting attention as a functional substance with excellent antioxidant effect.

Most of astaxanthin present in Haematococcus cells exists in a form bound to a fatty acid ester, palmitic acid (16:0), oleic acid (18:1), or It exists in mono- or diester-bonded form with linoleic acid (18:2). This conformation helps the highly polar astaxanthin molecules to exist in non-polar lipid droplets within the cell. Astaxanthin is present in Haematococcus spores as 70% monoesters, 25% diesters, and only 5% free form.

Astaxanthin is an antioxidant functional substance that removes harmful free radicals. Compared to beta-carotene, it has one more hydroxyl group (-OH) and one ketone group (=O) at both terminal groups. It has significantly higher antioxidant activity. Astaxanthin has antioxidant activity that is 500 times higher than vitamin E, a representative antioxidant, and 20 times higher than beta-carotene. Due to this high antioxidant activity, astaxanthin is widely used as pharmaceuticals, food additives, and feed additives for animals and fry, and its demand and application range is expected to rapidly expand.

Due to the increase in demand and the expansion of the range of use as described above, the development of a technology for extracting astaxanthin in a high yield is also being actively conducted. In order to accumulate astaxanthin at a high concentration, Haematococcus fluvialis is cultured under stress conditions (high temperature, high salt concentration, low nitrogen concentration, light removal). There have been technical difficulties in effectively extracting astaxanthin derivatives and lipids. In addition, astaxanthin is very vulnerable to heat and light and has a property of being easily decomposed, so when the astaxanthin is decomposed by a strong physical method as above, astaxanthin is also decomposed and the yield is very reduced.

In order to extract free astaxanthin, astaxanthin derivatives, and lipids in the reinforced cell wall to date, various pretreatment processes (mechanical grinding, chemical treatment using strong acids and strong bases, use of ionic liquids, high temperature and high pressure) that usually destroy the cell wall treatment, enzymatic degradation, dormant release cell septum rupture, switchable hydrophilic solvent, etc.) have been used, but there is a problem in that the reinforced cell wall is not completely destroyed and a lot of energy is consumed in the pretreatment process. In addition, some chemicals are very expensive and have to be treated for a long time, and there has been a problem in that astaxanthin is deactivated during the pretreatment process. Furthermore, various extraction methods (Soxhlet extraction, supercritical carbon dioxide extraction, organic solvent batch extraction, ultrasonic extraction, etc.) have been proposed after the pretreatment process, but when the pretreatment process-extraction process is linked, the yield of astaxanthin is 8 to 23 mg There is also a slightly lower problem with /g .

Accordingly, the present inventors have developed Hematococcus fluvialis at low temperature and in a short time through a one-pot method in order to overcome the disadvantages of the existing two-step pretreatment-extraction process. The present invention was completed by developing a method for destroying the cell wall of Fluvialis and extracting astaxanthin at the same time.

Cheng, X.; Qi, Z. B.; Burdyny, T.; Kong, T.; Sinton, D. Low pressure supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis demonstrated on a microfluidic chip. Bioresour. Technol. 2018, 250, 481-485.

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a method for simultaneously extracting astaxanthin from Haematococcus pluvialis .

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention as described above, the present invention provides a simultaneous extraction method of astaxanthin from Haematococcus pluvialis comprising the following steps:

(a) suspending Haematococcus fluvialis in a solvent selected from the group consisting of ethyl acetate, ethyl lactate and isoamyl acetate;

(b) extracting astaxanthin while crushing Haematococcus fluvialis suspended in the solvent; and

(c) filtering the extract obtained from step (b).

In one embodiment of the present invention, the Haematococcus fluvialis may be a strain NIES-144, but is not limited thereto.

In another embodiment of the present invention, the astaxanthin may be free astaxanthin, but is not limited thereto.

In another embodiment of the present invention, the method may be characterized in that free astaxanthin, astaxanthin derivative and lipid are simultaneously extracted, but is not limited thereto.

In another embodiment of the present invention, in the method, the destruction of the Haematococcus fluvialis cell wall and the extraction of astaxanthin may be performed at the same time.

In another embodiment of the present invention, the method may be carried out in a one-pot process, but is not limited thereto.

In one embodiment of the present invention, the method may be carried out at 60 ℃ or less, but is not limited thereto.

In another embodiment of the present invention, the mass ratio of the solvent to Hematococcus fluvialis in step (a) may be 1:5 to 1:20, but is not limited thereto.

In another embodiment of the present invention, step (b) may be performed at 10 °C to 60 °C, but is not limited thereto.

In another embodiment of the present invention, the step (b) may be performed under an inert gas, but is not limited thereto.

In one embodiment of the present invention, the step (b) may be performed by a ball mill process, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be performed at a rotation speed of 100 to 200 rpm, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be performed for 5 to 30 minutes, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be performed for 15 minutes at a rotation speed of 100 rpm, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be performed for 10 minutes at a rotation speed of 200 rpm, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be repeatedly performed with a ball milling time of 5 to 10 minutes and a pause of 5 to 10 minutes, but is not limited thereto.

In another embodiment of the present invention, the ball mill process may be performed using a ball made of zirconia, but is not limited thereto.

In another embodiment of the present invention, the zirconia ball may have a diameter of 2.5 to 10 mm, but is not limited thereto.

In addition, the present invention provides a method for simultaneous extraction of astaxanthin from Haematococcus fluvialis, which may further include the following steps before step (a):

(a-1) growing Haematococcus fluvialis to an aplanospore stage; and

(a-2) Harvesting and freeze-drying Hematococcus fluvialis in the immobilization stage.

In addition, the present invention provides a method for simultaneous extraction of astaxanthin from Haematococcus fluvialis, which further comprises the step of removing the solvent from the filtrate obtained in step (c).

Using the extraction method according to the present invention, astaxanthin, astaxanthin derivatives and lipids can be simultaneously extracted from Haematococcus pluvialis , Since the extraction is made at the same time as the cell wall breaks through within time, there is an effect of minimizing the decomposition reaction of the extracted astaxanthin to achieve a high yield. Therefore, it is expected that the present invention can be usefully used to produce a large amount of astaxanthin having antioxidant and anticancer activity.

1 is a diagram schematically illustrating the cell wall destruction and simultaneous extraction of astaxanthin from Haematococcus fluvialis by a single reaction vessel method.
2 is a digital camera image of the ball mill. The left (a) image is dry grinding, and the right (b) image is wet grinding (i, state immediately after ball milling; ii, cake on the wall of the bowl) Whether a layer is formed; iii, whether a cake is coated on the surface of the zirconia balls).
Figures 3a to 3c are a road comparing the single reaction vessel method and the two-step method, Figure 3a is a diagram comparing the extraction yield (%) and recovery (mg / g) of astaxanthin under the condition at 200 rpm, 3B is an image taken with a digital camera (top) and a scanning electron microscope (middle and bottom), respectively, of Hematococcus fluvialis cyst cells from the left before ball milling, after performing the two-step method, and after performing the single reaction vessel method, respectively 3c is an HPLC chromatogram, where (a) is an astaxanthin standard dissolved in DMSO at a concentration of 1000 ppm, (b) is acetone at 65 ° C. for 24 hours without destruction of the cell wall of Haematococcus fluvialis. In the case of Soxhlet extraction, (c) is the case of extraction by the two-step method, (d) and (e) are the case of extraction using the single reaction vessel method, respectively, before (d) and after (e) saponification.
4 is a diagram showing the effect of ball milling speed and time on cell wall disruption, residual red content of solid residues, and total astaxanthin recovery in liquid extracts.
5 is a diagram showing the effect of the solvent on the residual red content of the solid residue and the total astaxanthin recovery rate (a, lyophilized Hematococcus fluvialis cyst cells before extraction; b to e, cells collected after extraction Digital camera images of fragments; b, ethanol; c, acetone; d, isopropyl alcohol; e, hexane; f, ethyl acetate).
6 is a graph showing the astaxanthin extraction yield ( %) and a graph comparing the recovery rate (mg/g).

In the one-pot, simultaneous cell wall destruction and extraction process of the present invention, Haematococcus fluvialis and an acetate-based organic solvent are introduced into a ball mill chamber, and low rotation of 100 to 200 rpm When the ball mill process was performed for a short time of 10 to 30 minutes at the speed, it was found that the reinforced cell wall is effectively destroyed and at the same time, free astaxanthin, astaxanthin derivatives and lipids present in the cell wall are effectively dissolved and released. The present invention was completed. Astaxanthin obtained by the above method showed a very high yield of 32 mg/g.

In an embodiment of the present invention, a method of efficiently extracting astaxanthin and the like was discovered by performing grinding and extraction of dried Haematococcus fluvialis by a single process using a one-pot method. (See Example 1).

In addition, in one embodiment of the present invention, as a result of comparative analysis of the single reaction vessel method and the two-step method according to the present invention, the simultaneous extraction method according to the single reaction vessel method of the present invention has a higher astaxanthin recovery rate and lipid extraction yield It was confirmed that it has (see Example 2).

In another embodiment of the present invention, by comparing the extraction efficiency according to the method, rotation speed and process time of the ball mill process, it shows a higher astaxanthin recovery rate and lipid extraction yield despite a low rotation speed and a short process time shown (see Example 3).

In another embodiment of the present invention, as a result of comparing the extraction yield and astaxanthin recovery rate of lyophilized Hematococcus fluvialis in a single reaction vessel method according to the present invention by using different solvents, ethyl acetate solvent is used. It was confirmed that astaxanthin can be obtained in the maximum yield as in the case of using ethanol or acetone solvent (see Example 4).

In another embodiment of the present invention, wet Haematococcus fluvialis was pulverized and extracted by the single reaction vessel method according to the present invention in order to save additional energy required for freeze-drying, and ethyl acetate, ethyl lactate as solvents Alternatively, it was confirmed that astaxanthin can be recovered in the maximum yield when isoamyl acetate is used (see Example 5).

In addition, in another embodiment of the present invention, it was confirmed that when the extraction was performed using an acetate-based solvent of ethyl acetate, ethyl lactate or isoamyl acetate, better antioxidant activity and anti-radical activity were exhibited as compared to the case of extraction with acetone or ethanol. (See Example 6).

Summarizing the above results, the simultaneous extraction method by the single reaction vessel method can simultaneously extract astaxanthin, astaxanthin derivatives and lipids from Haematococcus fluvialis in high yield, especially ethyl In the case of extraction using acetate, ethyl lactate, and isoamyl acetate as a solvent, it is proved that astaxanthin can be obtained with a more improved recovery rate than when ethanol or acetone is used as a solvent.

Accordingly, the present invention can provide a method for simultaneously extracting astaxanthin from Haematococcus pluvialis comprising the following steps:

(a) suspending the dried Haematococcus fluvialis in a solvent selected from the group consisting of ethyl acetate, ethyl lactate and isoamyl acetate;

(b) extracting astaxanthin while crushing Haematococcus fluvialis suspended in the solvent; and

(c) filtering the extract obtained from step (b).

As another aspect of the present invention, the present invention may provide a method for simultaneously extracting astaxanthin from Haematococcus fluvialis, which may further include the following steps before step (a):

(a-1) growing Haematococcus fluvialis to an aplanospore stage; and

(a-2) Harvesting and freeze-drying Hematococcus fluvialis in the immobilization stage.

As another aspect of the present invention, the present invention provides a method for simultaneously extracting astaxanthin from Haematococcus fluvialis, further comprising removing the solvent from the filtrate obtained in step (c). can

In the present invention, the Haematococcus fluvialis of the present invention may be a strain of NIES-144, but is not limited thereto.

In addition, in the present invention, astaxanthin according to the present invention may be free (free) astaxanthin, but is not limited thereto.

In addition, the method according to the present invention can extract free astaxanthin, astaxanthin derivative and lipid at the same time.

As used herein, the term "astaxanthin derivative" refers to an astaxanthin monoester or astaxanthin diester containing a fatty acid cluster having 16 to 18 carbon atoms.

As used herein, the term "lipid" is used as a generic term for fatty acids and fat-soluble vitamins including neutral lipids, phospholipids, and glycolipids.

In the present invention, an organic solvent such as ethanol, acetone, ethyl acetate, ethyl lactate or isoamyl acetate may be used as a solvent for extracting astaxanthin and the like from the dried Haematococcus fluvialis, preferably may use an acetate-based solvent. Astaxanthin can also be extracted with a solvent such as ethanol or acetone, but since the solvent is a polar solvent such as water, the two solvents are mixed to remove the ethanol or acetone, and additional effort and cost are required. have. On the other hand, the acetate-based solvent has high solubility in astaxanthin, astaxanthin derivatives and lipids, and at the same time does not mix with water to form a layer. There is this. In addition, when an acetate-based solvent is used, a recovery rate of about 30 mg/g or more, which is the maximum astaxanthin yield, can be achieved, which is equivalent to or improved compared to the recovery rate when a solvent such as ethanol or acetone is used. Therefore, in one embodiment of the present invention, an acetate-based solvent was used.

As the acetate-based solvent, ethyl acetate, ethyl lactate, or isoamyl acetate may be used, but is not limited thereto.

In the present invention, in the method according to the present invention, destruction of the Haematococcus fluvialis cell wall and extraction of astaxanthin can be performed simultaneously.

In addition, in the present invention, the method may be performed in a single reaction vessel (one-pot) process. More specifically, compared to the two-step method of performing the extraction process after the crushing process or the pretreatment process, the method according to the present invention provides high yield even at high efficiency and low cost because the crushing and extraction processes are simultaneously performed in a single reaction vessel. It has the advantage of being able to extract Tarxanthin and the like.

In the present invention, all steps of the extraction method according to the present invention may be performed at 60 ° C. or less, thereby minimizing the decomposition of free astaxanthin and astaxanthin derivatives.

Further, in the present invention, when the dried H. fluvialis is suspended in a solvent, the mass ratio may be 1:5 to 1:20 (mass of dried H. fluvialis: mass of solvent). For example, after suspending 5 g to 20 g of a solvent with respect to 1 g of a dry hematococcus sample, the crushing and extraction steps may be performed by a ball mill process or the like.

In addition, in the present invention, the crushing and simultaneous extraction step of step (b) may be performed at 10 ℃ to 60 ℃. Accordingly, it is possible to prevent decomposition of astaxanthin and the like by frictional heat during the crushing and extraction process.

In addition, in the present invention, the crushing and simultaneous extraction of step (b) may be performed under an inert gas such as argon or nitrogen to prevent degradation of astaxanthin.

In the present invention, the step (b) may be performed by a ball mill process, but is not limited thereto.

As used herein, the term "ball mill" is a kind of grinder used to pulverize and mix a sample, and a mechanism that operates according to the principle of impact and friction in which balls fall from the top of the chamber or a mechanical device. A ball mill consists of a hollow cylindrical shell rotating about an axis, the axis of which may be horizontal or at a small angle to the horizontal. The grinding medium is a ball, and may be made of chrome steel, stainless steel, ceramic or rubber. The ball mill process according to the present invention may be performed using a ball made of zirconia, but is not limited thereto. In addition, the ball preferably has a diameter of 2.5 to 10 mm, but is not limited thereto.

As used herein, the term “zirconia” refers to zirconium oxide and most typically refers to various stoichiometry for ZrO ₂ , and may also be known as zirconium oxide or zirconium dioxide. The zirconia may contain up to 30% by weight of other chemical moieties, such as yttrium oxide (Y ₂ O ₃ ) and organic matter.

In the present invention, the rotation speed of the ball mill may be performed at a low speed of 100 to 200 rpm, in order to minimize the generation of frictional heat. In addition, the ball mill process time may be performed for a short time of 5 minutes to 30 minutes, and when the ball mill process is performed for 5 minutes or more, ball milling of 5 minutes to 10 minutes and a time of 5 minutes to 10 minutes The stop can be repeated to allow the ball milling chamber to cool. For example, the ball mill process may be performed for 15 minutes at a rotation speed of 100 rpm, and may be performed by repeating 5 minutes of ball milling and a pause of 5 minutes three times. As another example, the ball mill process may be performed for 10 minutes at a rotation speed of 200 rpm, and may be performed by repeating 5 minutes of ball milling and a pause of 5 minutes twice.

In addition, in the present invention, Haematococcus fluvialis is grown to the aplanospore stage using an outdoor microalgal culture system under autotrophic conditions, and then collected and directly extracted astaxanthin and lipids in a wet state. Alternatively, astaxanthin and lipids can be extracted after freeze-drying. In the case of growth to the antifreeze stage, a large amount of astaxanthin is accumulated, and the recovery rate can be increased.

Therefore, when astaxanthin is extracted from Haematococcus fluvialis by the method according to the present invention, astaxanthin can be obtained with a recovery rate of 80% or more. In addition, depending on the process time, astaxanthin can be recovered at a recovery rate of 70% or more, or 80% or more, or 90% or more, or 99% or more (30 mg or more of astaxanthin is recovered per 1 g of Haematococcus fluvialis sample). can be obtained. This recovery rate is remarkably high compared to the previously reported method for extracting astaxanthin and the like, which directly proves the superiority of the method according to the present invention.

The terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor may properly define the concept of the term in order to best describe his invention. Based on the principle that there is, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Experimental materials and methods

1. Haematococcus pluvialis ( Haematococcus pluvialis )

The strain used in the present invention was purchased from the National Institute for Environmental Studies in Japan as a H. fluvialis NIES-144 wild-type strain. Haematococcus fluvialis cells were sufficiently grown until a large amount of astaxanthin accumulated in the aplanospore stage using an outdoor microalgal culture system.

2. Solvents and Reagents

High purity argon (Ar) gas (≥99.999%) was purchased from JC Gas (Korea) and used. Dichloromethane (DCM; purity, ≥99.8%), acetone (purity, ≥99.9%), methanol (purity, ≥99.8%), hexane (purity, ≥95.0%) and ethyl acetate (EA; purity, ≥99.8%) ) was purchased from Daejeong Hwaeum (Korea). Ethyl alcohol (purity, ≥99.99%), ethyl lactate, isoamyl acetate and isopropyl alcohol (IPA; purity, ≥99.9%) were obtained from Buddick & Jackson (USA). Purchased. Deionized water was purchased from Samchun Pure Chemical Industries (Korea). potassium persulfate (purity, ≥95%) and sodium hydroxide (purity, ≥97%); astaxanthin standard (purity, ≥97% from Haematococcus fluvialis); 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; purity, >98%); 1,1-diphenyl-2-picryl hydrazyl (DPPH; purity, >95%); gallic acid (purity, >97%); Folin-Ciocalteu phenol reagent (concentration: 2N) and Trolox (6-hydroxy-2,5,7,8-tetramethyl chromine-2-carboxylic acid; purity, >97%) was purchased from Sigma-Aldrich (USA).

3. Thermogravimetric analysis

Moisture and mineral content of dried Haematococcus fluvialis was measured by performing thermogravimetric analysis (TGA) in a temperature range of 30 to 800 °C using a Q50 TGA instrument (TA Instruments, USA). The heating rate was set at 8° C./min, and the gas flow rate was fixed at 60 mL/min.

4. Chromatographic analysis

After extraction with different organic solvents, the concentration of astaxanthin was quantified twice using a reverse-phase HPLC method. Alliance HPLC (model e2695; Water, USA) equipped with a Modal 87E UV-vis detector was used for quantification of astaxanthin. As the column, a YMC-C30 reversed-phase carotenoid column (250 mm length × 4.6 mm inner diameter × 5 μm particle size; YMC Co., Japan) was used. The temperature of the column was maintained at 30 °C during the analysis. The diluted sample was passed through a 0.45 μm PTFE syringe filter and then injected into the sample vial. The composition of the mobile phase was adjusted with solvent A (acetone/water in 84:16 ratio, v/v) and solvent B (acetone/water in 97:3 ratio, v/v). Separation of free astaxanthin from ester derivatives and other carotenoids was achieved by programming the mobile phase elution as follows: isocratic elution with solvent A for the first 10 min followed by solvent B for 100 min. gradient elution from 0% to 100% with solvent A, followed by isocratic elution again with solvent A for 10 min. The flow rate of the mobile phase was set to 1 mL/min, and the sample injection volume was set to 10 μL using an autosampler. Absorbance was recorded at 480 nm using a UV-vis detector. A standard calibration curve with a regression coefficient of R ² =0.9997 was plotted by varying concentrations in the range of 80-656 μg/L using astaxanthin standards in DCM. The free astaxanthin content of the extract was calculated using the retention time for the astaxanthin standard, and other peaks in HPLC chromatograms with ester derivatives of astaxanthin and other types of carotenoids were used in the relevant literature. and designated.

5. Analysis of changes in coloration and morphology

Morphological changes before and after extraction of Hematococcus fluvialis cyst cells were observed using a field emission scanning electron microscope (FESEM; JSM7500F; JEOL, Japan). The extraction efficiency was compared by estimating the residual pigment of the solid residue after extraction and quantifying the color change of the solid residue by calculating RGB pixel values by digital camera image processing using MATLAB software (2012b). Residual pigment was estimated by measuring normalized red content using RGB values.

6. Analysis of antiradical activity of extracts

Because astaxanthin has double bonds and terminal phenolic groups, it acts as an anti-radical by electron or hydrogen atom transfer for radical scavenging. We used two control antioxidants (gallic acid and trolox) and two assays (DPPH ^● and ABTS ^{● +} ) to test the antioxidant activity of crude extracts without further saponication and purification. did The phenol content of the extract was determined using the Pauline-Siocalto reagent. The antioxidant activity of astaxanthin extract was compared with standard free astaxanthin purchased from Sigma-Aldrich.

7. DPPH ^● Scavenging activity assay of (2,2-diphenyl-1-picrylhydrazyl)

The antioxidant activity of the extract was determined by analyzing its scavenging activity against DPPH ^● and comparing it with two types of standard antioxidant solutions (trolox and standard free astaxanthin) at a concentration of 1 mM. Briefly, a homogeneous 0.15 mM ^DPPH stock solution was prepared in methanol and set aside for 20 min. 100 μL of each crude extract obtained using different organic solvents was mixed with 100 μL DPPH ^● solution separately. Next, the mixture was incubated in the dark at 25 °C for 30 min. The absorbance of the mixture was read at 517 nm using a microplate spectrophotometer (Power wave X 340; Biotech, Korea). The radical scavenging activity was determined by DPPH ^● discoloration percentage by Equation 1 below.

[Formula 1]

In Equation 1, absorbance DPPH, absorbance S, and absorbance SC represent the absorbance of DPPH-methanol solution, DPPH-methanol solution and mixture of extract and pure extract, respectively. Measurement of inhibitory concentration for 50% elimination of DPPH ^● IC ₅₀ values were calculated using linear interpolation. A graph of the radical scavenging activity of each extract was plotted by the concentration change of the extract in the range of 40-700 μg/mL.

In addition, a standard curve of Trolox was drawn by varying the concentration in the range of 4-100 μM, and the ^DPPH value of the extract was expressed in μmol as Trolox equivalent per 1 g of extract. The higher the DPPH ^● value, the higher the antioxidant activity, and the lower the DPPH ^● value, the lower the antioxidant activity.

8. Statistical analysis of significance

The reproducibility of the experiments performed in the present invention was confirmed by performing three extractions for each ball milling duration in each solvent and averaging the values within ±5% standard deviation. The anti-radical activity test of each extract obtained in different solvents was performed three times, and average values with standard deviation were shown.

Example 1: Simultaneous crushing and extraction process in a single reaction vessel (one-pot)

Harvested Hematococcus fluvialis cyst cells were freeze-dried at -55°C using a freeze dryer (model FDB-5503; Operon, Korea). A schematic diagram of the simultaneous grinding and extraction process by the single reaction vessel method is shown in FIG. 1 .

First, in order to prevent astaxanthin from being decomposed in the dried Haematococcus fluvialis, the dried sample was stored at -80°C until use. 1 g dried Hematococcus fluvialis sample, 70 g zirconia balls and 10 experimentally required solvents in a vessel of a planetary-type micro ball mill device (PULVERISETTE 7 premium line; FRITSCH, Germany) g was put in. To avoid potential contamination that may occur during the single reaction vessel grinding and extraction process, zirconia balls with a diameter of 5 mm were used as grinding media with good ductility and abrasion resistance. After adding Hematococcus fluvialis, zirconia balls and a solvent, the ball mill chamber was purged with high-purity argon (Ar) gas to maintain an inert gas environment during cell destruction and astaxanthin extraction. For each extraction solvent, cell disruption was performed at various time intervals (5-30 min) and rotational speed (100-200 rpm). The ball mill unit was coupled with a forced air cooling jacket to keep the internal temperature below 25 °C during rotation. In addition, to remove heat from the ball milling chamber, the chamber was allowed to cool through a short ball milling time (5 minutes) and a pause equal to this time. After the experimentally desired ball milling time had elapsed, the chamber was opened and the mixture was collected in a beaker. The grinding media (zirconia balls) and inner dome were washed with 100 mL of the same solvent used for extraction to allow complete recovery of the extract. Then, the mixture was filtered using a pre-weighed filter paper, and the solid residue remaining on the filter paper was dried in a conventional oven at a temperature of 80° C. for 12 hours.

Example 2: Comparison with two-step grinding and extraction process

2.1. Calculation of extraction method, yield and recovery rate

In order to compare the wet grinding process and the dry grinding process, a conventional dry grinding process was performed by introducing Haematococcus fluvialis to the grinding media in the same ratio without adding a solvent. Specifically, the Hematococcus fluvialis sample pretreated by ball milling without adding a solvent was extracted using acetone at 65° C. for 12 hours by the Soxhlet method.

After the simultaneous process of cell wall disruption and extraction in a single reaction vessel, the mass change of the solid sample was calculated using a high-accuracy digital mass balance (Model X124 Explorer series; OHAUS, USA). The overall extraction yield of the extract present in the dried Haematococcus fluvialis was calculated using Equation 2 below.

[Equation 2]

The amount of astaxanthin in the extract was calculated from Equation 3 below using high performance liquid chromatography (HPLC).

[Equation 3]

In Equation 3, astaxanthin (mg) is the total mass of astaxanthin in the extract, and the concentration of astaxanthin (ppm) is the concentration of astaxanthin measured in the extraction solution using HPLC, and the extraction mass ( g) is the total mass of the extraction solution.

In addition, the recovery rate of astaxanthin was quantified using Equation 4 below.

[Equation 4]

2.2. Comparison of astaxanthin recovery rate and total extraction amount

The main difference between the single reaction vessel method according to the present invention and the two-step extraction strategy used in most existing studies is that the approach is different in rupturing the cells and extracting the solute. In a two-step method, dried biomass is initially milled and subsequent extraction is performed using Soxhlet, supercritical fluid or other extraction methods. In contrast, wet grinding and extraction were performed simultaneously in the single reaction vessel method. During grinding and extraction by the single reaction vessel method, the close contact between the solvent and the ruptured cells can extract high yields of astaxanthin even under short processing time and room temperature conditions, unlike that obtained using dry grinding and subsequent extraction. could Therefore, the one-step approach as in the present invention can be viewed as a very energy-efficient, fast, simple and safe method for recovering astaxanthin in high yield from dried H. fluviris.

During dry ball milling of red cysts of Haematococcus pluvialis, a typical cell debris cake was formed, resulting in the formation of a coating of cell debris layer on the walls of the chamber and on the zirconia balls. . As shown in the left photo of FIG. 2 , after pulverizing Haematococcus fluvialis dried at 200 rpm for 10 minutes, extensive cake formation and coating layers were observed (see left (a) ii and iii of FIG. 2 ). In principle, mechanical disruption by ball milling typically induces a strong shear force on the cell wall and ultimately peels off the cell wall of the cyst cell. However, caking and coating phenomena caused by preferential aggregation of H. fluvialis fragments may lead to cell wall destruction and a leveling effect on astaxanthin recovery. That is, once the cake layer has been created, additional grinding to rupture the cyst cells trapped under the cake bed becomes less effective. Therefore, the leveling effect can cause a significant increase in the pretreatment time and a decrease in the grinding efficiency. Thus, to achieve complete cell wall disruption requires very energy intensive continuous ball milling for extended periods of time, which significantly increases the overall process cost. In addition, caking and coating may frequently be interrupted by pretreatment processes to remove a sticky layer of cell debris from the grinding media and the dome of the ball mill.

In the dry grinding of Haematococcus fluvialis, a thick and sticky slurry is produced by residues of cyst cells, eluted lipids and disrupted cell debris (see Fig. 2(a)). The slurry forms a cake layer on the walls of the ball mill chamber and on the ball surface. In addition, the continuous impact of the balls causes compression of the cake layer, which may lead to loss of bioactivity of the eluted astaxanthin. That is, the energy obtained by the caking mixture by continuously striking the balls at high rotational speed may result in the decomposition of astaxanthin by excitation of unsaturated double bonds. Therefore, the process is energy intensive, inefficient, expensive and on a practical scale due to the prolonged grinding time, the inevitable frequent interruption of the pretreatment process, the need for additional techniques for recovery of the cell debris layer and the degradation of astaxanthin associated with dry grinding. Not attractive to implement. In addition, free astaxanthin and its ester derivatives eluted with the use of large amounts of solvents (eg organic solvents, supercritical carbon dioxide and dimethyl ether) and extended extraction times (1-24 hours) in subsequent extraction steps is needed to recover

The recovery of astaxanthin by a single reaction vessel, simultaneous cell wall disruption and astaxanthin extraction approach according to the present invention has many advantages over the two-step method: first, the cyst cell wall destroyed by shear force during wet ball milling is adequately It can be directly dissolved in the solvent to suppress the formation of cakes and coating layers on the chamber surface and the ball surface (see Fig. 2 (b)). Second, unlike the subsequent extraction (eg Soxhlet) in the two-step method, the single reaction vessel method uses a very small amount of solvent, reducing the energy required to recover the dried astaxanthin powder by drying the solvent. Reduce. For example, 250 mL of acetone was used at a siphon rate of 4/h for 12 h for Soxhlet extraction of disrupted cell wall fragments, whereas in the single reaction vessel method, the same amount of dried Haematococcus fluvi It was found that only 100 ml of acetone was sufficient for extraction and thorough washing using Aliss. Third, in the single reaction vessel method, the time required for cell wall destruction and extraction is significantly reduced compared to the time required in the two-step method, thereby reducing the total operating cost. In the two-step method, the total treatment time required for pretreatment and extraction is generally more than 12 hours, but it has been found that only about 30 minutes is sufficient for complete extraction of astaxanthin in the single reaction vessel method. Fourth, in the single reaction vessel method, effective cell wall destruction and extraction can be performed even at very low rotational speed (100-200 rpm) and low temperature (less than 25 ℃) conditions, and thus astaxanthin with high thermal instability is degraded risk can be reduced. Astaxanthin's electron-rich paired double bond structure makes it highly susceptible to physical and chemical degradation when exposed to air, high temperatures, and light. For example, high temperature inhibits the function of astaxanthin because it can be decomposed into first-order reaction kinetics even at very low temperatures (50-60 °C). Finally, the single vessel method can be performed using many low toxic solvents such as ethanol, acetone, n-hexane, isopropyl alcohol and acetate-based solvents, which are typical solvent-based extraction (e.g. Soxhlet) processes. This is because the need for a low-boiling solvent to inhibit the degradation of astaxanthin during the process has been overcome. Therefore, in the recovery of astaxanthin, the single reaction vessel method for simultaneous cell wall disruption and extraction according to the present invention is more energy efficient, more flexible, more cost-effective, simpler and safer than the two-step method.

The results of comparing the astaxanthin recovery rate and the total extraction amount of the two-step and single reaction vessel method are shown in FIGS. 3A and 3B. In the pretreatment of the two-step method, the destruction of the dried Haematococcus fluvialis cell wall was carried out at 200 rpm for 10 minutes, and the pretreated sample was extracted by the Soxhlet method using acetone solvent for 12 hours. The two-step method resulted in a recovery of astaxanthin of 21.0 mg/g from the dried H. fluvialis, and the yield of the liquid extract was 32.0 wt%. In the single vessel method, dried H. fluvialis, zirconia balls and acetone having a H. fluvialis to solvent ratio of 1:10 w/w are introduced together into the vessel of the extractor, at 200 rpm and 10 It was carried out under the condition of minutes. The single reaction vessel method has higher astaxanthin than 27 mg/g (>82% recovery) from dried Haematococcus fluvialis compared to the two-step method under the same ball milling time and rotation speed as the two-step method It showed recovery and better lipid extraction yield of 42.0 wt%. In the case of using a high rotation speed of 300 rpm in the single reaction vessel method, a high lipid extraction yield of 45.9 wt% could be obtained even with a very short ball milling time of 6 minutes. That is, the above results prove that the time-lapse efficiency for cell wall destruction and extraction is significantly higher in the single reaction vessel method than in the two-step method.

The red color of the cell debris collected after extraction is shown in the upper part of FIG. 3B . After performing the two-step method, the collected cell fragments still contained pink pigment, confirming that the red-orange pigment was not completely extracted. In contrast, the color of the cell debris collected by the single reaction vessel method was pale green, confirming that the red-orange pigment including astaxanthin was efficiently extracted except for some residual chlorophyll with low solubility in acetone. . In addition, as shown in the scanning electron microscope (SEM) image of the solid residue (bottom of Fig. 3b), the single reaction vessel method confirmed a higher degree of cyst cell destruction compared to the two-step method, which This suggests that there is a correlation between high extraction yield and high degree of cell wall destruction.

The high extraction rates and astaxanthin recovery observed in the single vessel method are due to the effective contact of the solvent with the extractable material and the agitation generated by ball milling. The reason that cell wall destruction occurred efficiently in the single reaction vessel method according to the present invention is considered to be because the organizational properties of the cell wall were changed by immersion in a solvent in a solid matrix.

2.3. Component analysis by HPLC

As shown in FIG. 3c , the HPLC chromatogram of a solution obtained by Soxhlet extraction of uncrushed Hematococcus fluvialis showed very low extraction yields of <11 wt% and <1 mg/g, respectively, and total astaxanthin recovery. (See Fig. 3c (b)). In particular, the HPLC chromatograms in (c) and (d) of FIG. 3c confirmed that the number and intensity of peaks appearing in the chromatogram extracted by the single reaction vessel method were higher than those of the two-step method, and the extraction of the single reaction vessel strategy It was demonstrated that the yield is quite high. Therefore, the single reaction vessel method according to the present invention enables cell wall disruption and extraction at the same time and solves the problems of caking and coating occurring during mechanical pretreatment of dried H. fluvialis. In addition, almost completely free astaxanthin and its fatty acid monoester or diester derivatives do not require prolonged application of high temperature, high pressure and high rotational speed for cell wall disruption and extraction, and are produced under mild conditions (short time, low temperature, atmospheric pressure and low rotational speed).

Example 3: Comparison of extraction efficiency according to speed and process time of ball mill

Table 1 below shows the effects of process parameters such as time and ball milling speed on the extraction yield by breaking the cell wall of a single reaction vessel and extraction of dried Haematococcus fluvialis. Ethanol was used as the solvent, and the microalgae to solvent ratio was 1/10 w/w.

Item time (minutes) Extraction yield (wt%) ^a Single reaction vessel extraction Step 2 Extraction ^b 100 rpm 200 rpm 200 rpm One 5 33.6 ± 1.6 36.9 ± 1.8 17.5 ± 1.0 2 10 38.3 ± 1.9 42.4 ± 2.1 23.3 ± 1.3 3 15 42.7 ± 2.1 44.5 ± 2.3 27.6 ± 1.5 4 20 43.8 ± 2.2 45.6 ± 2.3 32.7 ± 1.6 5 25 44.9 ± 2.3 46.8 ± 2.4 34.1 ± 1.8 6 30 45.5 ± 2.3 46.9 ± 2.4 36.5 ± 2.0

a, grams of extract per gram of dry sample;

b, Extraction yield by 200 rpm ball milling and Soxhlet extraction in acetone for 12 h.

As shown in Table 1, the extraction yield at a low rotation speed of 100 rpm increased from 33.6 to 45.5 wt% as the time increased from 5 to 30 minutes. Conversely, when the rotation speed was increased to 200 rpm, the extraction yield reached 42.4 wt% in only 10 minutes, and increased to 46.9 wt% as the time was further increased to 30 minutes. Compared with the previously reported method, the single reaction vessel method according to the present invention resulted in an unprecedented high yield of more than 47 wt%, which is a total extract of red Haematococcus fluvialis ( lipid, pigment, moisture, etc.) content was very close. This indicates that almost complete lipid extraction can be achieved using the single reaction vessel method. Ester derivatives of astaxanthin mainly consist of astaxanthin monoesters with C ₁₆ -C ₁₈ fatty acid clusters near the cytoplasmic inner nucleus of H. fluvialis cyst cells. In the Haematococcus fluvialis strain extract, the main fatty acid was oleic acid (C18: 1) monoester, and the peak pattern of the extract in the HPLC chromatogram was in good agreement with that reported in the previous study (see Fig. 3c). The high extraction yields under mild conditions (200 rpm, 25 °C and 1 atm) and short treatment time (30 minutes) suggest that the single reaction vessel method according to the present invention is very effective for the collection of lipids rich in unsaturated fatty acids. Thus, the method according to the present invention can be used to produce biodiesel from extracted lipids and to produce other supplements.

As the liquid extraction yield increased, the residual pigment continued to decrease in the solid residue (see Fig. 4). In the antispore stage of Haematococcus pluvialis, astaxanthin accounted for a major fraction (>80%) of total carotenoids, and concomitantly pigments include β-carotene (approximately 1%) and canthaxanthin (approximately 5.1%). this was included Thus, the red appearance of Haematococcus fluvialis is largely due to the presence of astaxanthin and its esterified derivatives in free form. Astaxanthin derivatives are C ₁₆ -C ₁₈ fatty acid monoesters and diesters, which are very slightly acidic or neutral in nature. Therefore, the presence of these fatty acid monoethers and diesters may not change the red intensity of astaxanthin. Therefore, the change of the solid residue from dark red to colorless or light gray after extraction indicates the destruction of the cell wall and the degree of extraction of the red pigment, and it is possible to directly predict the extraction efficiency through the degree of red reduction of the cell debris calculated using Metlab. can As shown in FIG. 4 , the residual red content in the solid residue had a close correlation with the experimentally determined astaxanthin recovery rate. During the first 5 min of cell wall disruption and extraction by the single reaction vessel method, a significant decrease in the red content of the solid residue was observed, indicating that the cyst cells were initially effectively disrupted. A further increase in treatment time gradually decreased the red content after 30 minutes of extraction. A rotation speed of 200 rpm was found to be more effective in reducing the normalized red content by more than 80% in just 10 minutes compared to 100 rpm. The decrease in red intensity indicates that a total astaxanthin recovery of 21 mg/g from dried H. fluvialis was achieved with a very short time of 5 min ball milling at 200 rpm, which is H. fluvialis. This corresponds to about 65% of astaxanthin present in Ali's cyst. When the time was extended to 10 minutes at 200 rpm, approximately 82% of astaxanthin was recovered. When the wet ball milling time was further increased to 30 minutes, a very high astaxanthin recovery of >31.6 mg/g (about 99%) was achieved. It was found that astaxanthin recovery at a very low rotation speed of 100 rpm was consistently lower than that at 200 rpm in all time points investigated. These results indicate that a longer treatment time or higher shear force is required for a very rigid cell wall for complete destruction of the cyst.

Example 4: Comparison of extraction efficiency and recovery rate of astaxanthin according to solvent

In order to find out the extraction yield and recovery rate of astaxanthin according to the type of solvent, a comparative experiment was conducted using the single reaction vessel method as in Example 1. Each solvent was added to the dried Haematococcus fluvialis, and grinding and extraction were performed at 200 rpm for 30 minutes. The extraction yield and recovery rate of astaxanthin are shown in Table 2 and FIG. 5 below.

menstruum Extraction yield (wt%) ^a Astaxanthin Recovery Rate
(mg/g) ethanol 46.9 ± 2.7 30.1 ± 1.5 acetone 44.8 ± 2.3 30.6 ± 1.5 isopropyl alcohol 43.5 ± 2.1 24.5 ± 1.2 hexane 39.5 ± 1.9 16.6 ± 0.8 ethyl acetate 44.7 ± 2.3 29.9 ± 1.5

a, number of grams of extract based on dry sample

As a result, as shown in Table 2 and FIG. 5, it was confirmed that the highest extraction yield and astaxanthin recovery rate were obtained when ethanol, acetone and ethyl acetate solvents were used.

Example 5: Simultaneous One-pot Grinding and Extraction Process from Wet Haematococcus Fluvialis

Ethyl acetate does not mix with water, so it has the advantage of reducing time and cost when extracting astaxanthin from it. Accordingly, the present inventors confirmed whether the extraction yield and astaxanthin recovery rate were maintained at the maximum when other acetate-based solvents were used as compared to ethanol or acetone.

On the other hand, in Examples 1 to 4, freeze-drying was performed to remove water from the Hematococcus fluvialis harvested in the culture reactor, and then the dried Hematococcus fluvialis was used to grind and extract a single reaction vessel at the same time. The process was investigated. Haematococcus fluvialis harvested in the culture reactor contains about 75-80 wt% of water inside the cells, so additional energy is required when the freeze-drying method is used to dry the water. Therefore, in order to save energy in the entire process, in Example 4, 2 g of a wet Hematococcus fluvialis sample having a water content of 77 wt%, 70 g of zirconia balls and experimentally required 10 g of solvent was added. In addition, 5 g of water was additionally introduced into the reaction vessel to facilitate liquid-liquid separation. In this case, the solvent is ethyl acetate, ethyl lactate, and isoamyl acetate, which are relatively harmless to the human body, environmentally friendly solvent, and liquid-liquid separation is possible in consideration of human harm and environmental toxicity. was used. For each extraction solvent, cell disruption was performed at a process time of 20 minutes and a ball mill speed of 200 rpm. The ball mill unit was coupled with a forced air cooling jacket to keep the internal temperature below 25 °C during rotation. In addition, to remove heat from the ball milling chamber, the chamber was allowed to cool through a short ball milling time (5 minutes) and a pause equal to this time. After the experimentally desired ball milling time had elapsed, the chamber was opened and the mixture was collected in a beaker. The grinding media (zirconia balls) and inner dome were washed with 100 mL of the same solvent used for extraction to allow complete recovery of the extract. Then, the mixture was filtered using a pre-weighed filter paper, and the solid residue remaining on the filter paper was dried in a conventional oven at a temperature of 80° C. for 12 hours. The extraction level and astaxanthin recovery according to each solvent are shown in Table 3 below.

menstruum Extraction yield (wt%) ^a Astaxanthin Recovery Rate
(mg/g) ethyl acetate 42.8 ± 2.5 31.1 ± 1.2 ethyl lactate 44.2 ± 2.7 30.1 ± 1.5 isoamyl acetate 46.2 ± 2.9 30.5 ± 1.1 ethanol 37.1 ± 2.6 25.2 ± 1.2 acetone 38.5 ± 2.8 24.7 ± 1.5

a, number of grams of extract based on wet sample

As shown in Table 3 and FIG. 6, when astaxanthin and lipids are recovered from wet Hematococcus fluvialis in a single reaction vessel using an acetate-based solvent, astaxant is better than ethanol and acetone as solvents. Tin recovery and extraction yield were higher. This is thought to be because when an acetate-based solvent is used, a phase separation with water is formed, and astaxanthin and its derivatives, which are fat-soluble, can be more effectively recovered.

Example 6: Evaluation of antioxidant activity and anti-radical activity

The antioxidant activity and anti-radical activity of astaxanthin obtained in Example 5 was evaluated according to the above-described experimental method. The results are shown in Table 4 below.

Antioxidants in
auxiliary solvent DPPH radical scavenging activity IC ₅₀
(μg/mL) ^a Trolox equivalent
(Trolox equivalents;
μmol of TE/g of extract) ^b ethyl acetate 230 188 ethyl lactate 225 187 isomyl acetate 221 183 acetone 325 151 ethanol 393 52

a, the concentration of antioxidant required to remove 50% of the DPPH present in the mixture;

b, Trolox equivalent per gram of extract.

As shown in Table 4, when an acetate-based solvent of ethyl acetate, ethyl lactate and isoamyl acetate was used, the IC ₅₀ value was as low as 221-230, and in the case of Trolox equivalent, it was 183-188, resulting in ethanol and acetone. was higher than that of the solvent. On the other hand, the extract extracted using acetone and ethanol had a higher IC ₅₀ value of 325-393, and the equivalent of Trolox showed as low as 52-151. These results suggest that the antioxidant activity and anti-radical activity of the extract prepared by using the acetate-based solvent is higher.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (19)

(a) suspending Haematococcus fluvialis in a solvent selected from the group consisting of ethyl acetate, ethyl lactate and isoamyl acetate;
(b) extracting astaxanthin while crushing Haematococcus fluvialis suspended in the solvent; and
(c) as a simultaneous extraction method of astaxanthin from Haematococcus pluvialis , comprising the step of filtering the extract obtained from step (b), the method comprising a single reaction vessel ( One-pot) process, characterized in that the destruction of the Haematococcus fluvialis cell wall and the extraction of astaxanthin are performed at the same time, the simultaneous extraction method.
According to claim 1,
The Hematococcus fluvialis is characterized in that the NIES-144 strain, simultaneous extraction method.
According to claim 1,
The astaxanthin is free (free) astaxanthin, characterized in that the simultaneous extraction method.
According to claim 1,
The method is characterized in that the simultaneous extraction of free astaxanthin, astaxanthin derivative and lipids, a simultaneous extraction method.
delete delete According to claim 1,
Simultaneous extraction method, characterized in that the mass ratio of the solvent to Haematococcus fluvialis in step (a) is 1:5 to 1:20.
According to claim 1,
The step (b) is characterized in that carried out at 10 ℃ to 60 ℃, the simultaneous extraction method.
According to claim 1,
The step (b) is characterized in that it is carried out under an inert gas, the simultaneous extraction method.
According to claim 1,
The step (b) is characterized in that performed by a ball mill (ball mill) process, simultaneous extraction method.
11. The method of claim 10,
The ball mill process is characterized in that performed at a rotation speed of 100 to 200 rpm, simultaneous extraction method.
11. The method of claim 10,
The ball mill process is characterized in that performed for 5 to 30 minutes, the simultaneous extraction method.
11. The method of claim 10,
The ball mill process is characterized in that it is performed for 15 minutes at a rotation speed of 100 rpm, simultaneous extraction method.
11. The method of claim 10,
The ball mill process is characterized in that it is performed for 10 minutes at a rotation speed of 200 rpm, simultaneous extraction method.
11. The method of claim 10,
The ball mill process is characterized in that the ball milling time of 5 to 10 minutes and a pause of 5 to 10 minutes are repeatedly performed, the simultaneous extraction method.
11. The method of claim 10,
The ball mill process is a simultaneous extraction method, characterized in that performed using a ball (ball) of zirconia (zirconia) material.
17. The method of claim 16,
The zirconia balls have a diameter of 2.5 to 10 mm, characterized in that the simultaneous extraction method.
According to claim 1,
Simultaneous extraction method, characterized in that the method further comprises the following steps before step (a):
(a-1) growing Haematococcus fluvialis to an aplanospore stage; and
(a-2) Harvesting and freeze-drying Hematococcus fluvialis in the immobilization stage.
According to claim 1,
Simultaneous extraction method, characterized in that the method further comprises the step of removing the solvent from the filtrate obtained in step (c).

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