KR100543088B1 - The manufacturing method of high-enriched Astaxanthin - Google Patents

Abstract

The present invention relates to a highly concentrated astaxanthin pigment oil extraction method and a method for producing free asanthine.

Specifically, astaxanthin is cultured by a known method using a photobioreactor of Hematococcus algae (Haematococcus species, Phytochemistry, 20, 2561, (1981)), an alga that forms astaxanthin as a natural pigment. Hematococcus algae containing up to 5% was obtained, dried and pulverized and pressurized to about 2000-2500 bar to destroy the cell walls. Hematococcus algae was continuously circulated through the solvent to extract astaxanthin. It is characterized by extracting astaxanthin pigment oil of high concentration (5-15%) through filtration, concentration, separation and purification.

The present invention also provides astaxanthin in the form of free astaxanthin concentrate containing 5-15% astaxanthin pigment oil or hematococcus algae containing 1-5% hydrolysis in basic solution. It is characterized by obtaining.

Hematococcus, Astaxanthin

Description

Extraction method of astaxanthin pigment oil of high concentration and the manufacturing method of free astaxanthin {The manufacturing method of high-enriched Astaxanthin}             

1 shows a soxhlet device with an ultrasonic wave generating terminal, used in an embodiment of the invention.

The present invention relates to a highly concentrated astaxanthin pigment oil extraction method and a method for producing free asanthine.

Specifically, astaxanthin is cultured by a known method using a photobioreactor of Hematococcus algae (Haematococcus species, Phytochemistry, 20, 2561, (1981)), an alga that forms astaxanthin as a natural pigment. Hematococcus algae containing up to 5% was obtained, dried and pulverized and pressurized to about 2000-2500 bar to destroy the cell walls. Hematococcus algae was continuously circulated through the solvent to extract astaxanthin. The present invention relates to a method for extracting high concentration (5-15%) astaxanthin pigment oil through filtration concentration, separation, and purification.

The present invention also provides astaxanthin in the form of free astaxanthin concentrate containing 5-15% astaxanthin pigment oil or hematococcus algae containing 1-5% hydrolysis in basic solution. It is about how to get it.

In the present invention as described above, astaxanthin-containing materials, in addition to hematococcus algae, Phaffia rhodozyma, shrimp, crab, lobster or salmon may be used in the same principle and method.

Astaxanthin is a natural carotenoid that is extracted from hematococcus algae and has an antioxidant effect of 550 to 1000 times more than tocopherol (vitamin E) .It removes harmful oxygen from the body, which is the source of various diseases, It is the next generation antioxidant. Astaxanthin is not only effective in preventing aging due to antioxidant effects, but also in enhancing immunity, inhibiting damage to the eyes, skin and central nervous system by ultraviolet rays, treating Helicobacter infection, preventing cardiovascular disease and diabetes, preventing dementia and cancer, and improving vision. It is known to have excellent effects.

Antioxidants are substances that neutralize the action of free radicals.The free radical damage from oxygen in living organisms that use oxygen and the free radicals of lipids, proteins, nucleic acids, and carbohydrates caused by oxygen (single oxygen, ozone, etc.) A substance that blocks, suppresses or delays damages.

More than 95% of the oxygen we drink is mostly used to generate energy in the body, and oxygen itself is converted into water. However, about 2-5% of them are converted to harmful toxic oxygen, which is very reactive and active, which is free radicals. One of the fundamental causes of aging is the increased free radicals in the body that oxidize the cells, which are continually attacked by oxidants throughout their lives. Environmental factors mainly affect humans, such as air pollution, tobacco smoke, exposure to chemicals, and ultraviolet rays. Oxidants react mainly with proteins, nucleic acids, and fats to alter the molecular structure of these substances, causing cell aging, arteriosclerosis, anemia, presbyopia, and carcinogens. Much of human health depends on the elimination of free radicals and how much they reduce damage to tissues, cells and DNA by oxidation, and antioxidants play an important role in preventing disease.

Beta carotene and vitamin E are known as representative antioxidants, but according to US FDA data, astaxanthin is about 550-1000 times more effective than vitamin E and about 10 times more effective than beta carotene. It is very useful to be applied in various fields such as (removing live oxygen, preventing cell damage), food field (food additives, health supplements), cosmetics (anti-aging, whitening, sun protection), feed (fish, poultry feed) It can be called a substance.

Conventionally, it has been used as a method of extracting astaxanthin pigment having the above-mentioned advantages, and cultured yeast strain Papifa rhodozyma (Phytochemistry) by treating with an appropriate method, dissolving with organic solvent, and then separating and concentrating. And astaxanthin, which are synthesized by an organic chemical method, and separated and purified to obtain astaxanthin pigment (August). However, most of the methods of extracting astaxanthin by culturing yeast strains are known to be possible to produce only in the laboratory due to low extraction yield, high destruction rate of pigment at the time of extraction, and high production cost. There is no useful mass production, and the production of astaxanthin according to the method of synthesizing and purifying by organic chemical method is actually made, but as the safety of the isomer obtained as a by-product is not secured, There is a disadvantage that you can not use.

In addition, there is a method of obtaining astaxanthin extract having a content of about 5-10% by using a supercritical fluid after culturing hematococcus algae, using a supercritical fluid, but the extraction cost is very high and the price is very economical. It is a low extraction method. In addition, there is an extraction method using microwave, which is a method of culturing hematococcus algae, suspending it in water, treating with microwaves, extracting with a solvent, and concentrating by centrifugation. After irradiating microwaves by connecting about twelve Teflon tubes of about 8 mm, the astaxanthin pigment is extracted using a solvent. When the small Teflon tube is used, mass production is difficult and foreign substances such as proteins are adsorbed inside the tube. Due to the difficulty in washing, mass production is not easy, and there are many problems in the method of suspending hematococcus algae in water.

On the other hand, astaxanthin present in the hematococcus is composed of free astaxanthin, which accounts for about 5% of the total carotenoid content by ester bonding with protein, 70% monoester astaxanthin and 10% diester astaxanthin. As a method of obtaining a high-concentration high-purity product containing more than 98% of free astaxanthin, it is generally obtained by removing protein using cholesterol esterase, which is very expensive. It is used only for analysis.

An object of the present invention is to solve the above problems and to provide a method that can supply the astaxanthin to the market at a low price by increasing the productivity by extracting a high concentration of astaxanthin pigment oil with a low cost, high efficiency and simple apparatus and extraction conditions There is. Therefore, the ultimate purpose is to maximize the utility of astaxanthin by expanding its utilization and application range in the industry including pharmaceuticals.

Another object of the present invention is a high-purity free high astaxanthin content of a large amount of free form of astaxanthin compared to enzymatic hydrolysis by hydrolyzing the astaxanthin ester in hematococcus algae using an organic solvent in a basic state. There is a new way to get astaxanthin.

According to the present invention having the above object, high concentration of Asta is obtained under high efficiency extraction conditions while irradiating ultrasonic waves using a natural solvent such as alcohol obtained after drying and pulverizing hematococcus algae containing astaxanthin. Provided is a method for extracting astaxanthin pigment oil, characterized by extracting xanthine.

The present invention also provides a method of obtaining 20-98% or more pure free astaxanthin in a very economically free state compared to enzymatic hydrolysis by hydrolyzing the astaxanthin ester in the Hematococcus algae in a basic solution without using an enzyme. .

In the present invention, in order to obtain a high-efficiency extract in large quantities, the cell wall is crushed with a pressurizing device, and the extraction efficiency is increased by reducing the extraction time and the extraction time as well as the productivity by reducing the production cost by irradiating ultrasonic waves using a conventional extraction device. A new extraction method for extracting natural pigment astaxanthin in large quantities is disclosed. Also disclosed is a method of obtaining high purity free astaxanthin in a general basic solution without using an enzyme.

The present invention for achieving the first object described above,

Drying and pulverizing a material selected from the group consisting of hematococcus algae including astaxanthin, Phaffia rhodozyma, shrimp, crab, lobster and salmon;

b) pressing the ground material to break the cell wall;

c) extracting astaxanthin oil by separating the cell wall material into the natural solvent in a natural solvent and applying ultrasonic wave of rated output 200-2000 watts and 16-25 kHz, and concentrating under reduced pressure; high concentration of astaxanthin pigment oil through It is characterized in that the extraction.

At this time, conventionally known between the step b) of pressing the ground material to destroy the cell wall, and c) extracting astaxanthin oil by putting in a natural solvent and applying ultrasonic wave of rated output 200-2000 watts and 16-25 kHz. The method may further include removing impurities other than astaxanthin using the enzyme treatment method (ie, adding a cholesterol esterase solution). In addition, the pressure in the pressurization step is preferably 2,000-2,500 bar, and the extraction solvent used is selected from the group consisting of edible alcohol, methanol, acetone, nucleic acid, petroleum ether, chloroform and ethyl acetate which can be obtained in nature. It may be any one or a mixture thereof. Ultrasound is also preferably treated for 10-100 minutes continuously or intermittently.

The present invention for achieving the second object described above,

a) drying and pulverizing a material selected from the group consisting of hematococcus algae, papia rodjima, shrimp, crab, lobster and salmon, including astaxanthin;

b) pressing the ground material to break the cell wall;

c) hydrolyzing the material in which the cell wall is destroyed in a basic solution state without using an enzyme; it is characterized by producing a high concentration of free astaxanthin.

At this time, between the step b) of breaking the cell wall by pressing the ground material and the step c) of hydrolysis in a basic solution state, the material whose cell wall is destroyed is placed in a natural solvent, and the rated output is 200-2000 watts and 16-25 kHz. The method may further include extracting astaxanthin oil by applying an ultrasonic wave of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, formic acid, citric acid, and the like after hydrolyzing the cell wall-broken material in a basic solution without using an enzyme. The step of neutralizing the hydrogen ion concentration of 2-6 as one or a mixture thereof selected from the group consisting of lactic acid is followed.

In addition, the pressure in the pressurization step is preferably 2,000-2,500 bar, and the extraction solvent used is selected from the group consisting of edible alcohol, methanol, acetone, nucleic acid, petroleum ether, chloroform and ethyl acetate which can be obtained in nature. It is characterized in that any one or a mixture thereof. In addition, when the sonication process is performed, it is preferable to process for 10-100 minutes continuously or intermittently. Further, the basic solution to be used is any one selected from the group consisting of alcohol, methanol, acetone, nucleic acid, petroleum ether, chloroform and ethyl acetate, or a mixture thereof, ② a small amount of water, It is preferable to use a solution prepared by mixing any one selected from the group consisting of ammonium hydroxide, sodium carbonate, sodium hydrogen carbonate, ammonium carbonate and magnesium carbonate so as to have a hydrogen ion concentration of 8-12.

Regardless of the materials used, the principle and method are the same. When the present invention is described as using a hematococcus algae as a material, hematoma having an astaxanthin content of about 1-5% obtained by culturing by a known method is used. By drying and pulverizing the Caucasus algae, pressurizing it to about 2000-2500 bar, and then breaking the cell wall, extracting, filtering, separating and concentrating by using low-priced edible alcohol or organic solvent and ultrasonic wave, which are obtained in nature, With a simple device and low extraction cost, high concentrations of hematococcus concentrates with astaxanthin content of 5-15% can be obtained. In addition, hematococcus algae containing 5-15% astaxanthin or hematococcus algae having 1-5% astaxanthin content up to the cell wall destruction step was obtained by using a basic solution by chemical method. Astaxanthin will be able to obtain astaxanthin having a purity of 20-98% or more.

As described above, the present invention maximizes the efficiency of extraction by completely destroying the cell wall by pressing and drying at about 2000-2500 bar after drying and pulverizing the hematococcus algae having an astaxanthin content of 1-5%, which is obtained using an optical incubator. It is characterized by. In addition, the ultrasonic treatment method used in the present invention is characterized in that 10-600 minutes or continuous treatment using the rated output 200-2000 watts and ultrasonic vibration of 16-25 kHz continuously, which can be applied to the existing extraction device have. In addition, the extraction solvent is characterized in that any one of edible alcohols and organic solvents obtainable in nature, or a solvent in which these are selectively mixed.

In addition, since astaxanthin and carotenoids are sensitive to light and heat, it is found that the content measurement and extraction concentration in the process of the present invention is preferably performed in a cool dark place without natural light.

Hereinafter, an example of implementing the present invention will be described.

Example 1 Cell Wall Destruction of Hematococcus

Light culture, drying, grinding and pressing of 2000-2500 bar of haematococcus algae to crush the cell walls were performed by Applicant's Swedish processing company Bioprocess, which provides about 1-5% of astaxanthin. Instead of being provided.

2. Example 2 Preparation and Assay of Astaxanthin Standard Solution

Sigma's 98% synthetic astaxanthin reagent (A9335 Astaxanthin: 3,3'-Dihydroxy-β, β-carotene-4,4'-dione MDL number: MFCD00672621, 472 to determine the content of astaxanthin in Hematococcus algae) -61-7 minimum 98%, CAS Number: 472-61-7) was used as the standard reagent.

First take 30 mg of astaxanthin standard in a 1 L volumetric flask and record the correct mass. Acetone is added and sonicated for 5 minutes to completely dissolve. After stabilization for 1 hour, acetone is added to adjust the volumetric flask and used as a standard solvent. Prepare a standard solution by accurately taking 20 ml of standard solvent into a 100 ml volumetric flask with a graduated pipette and calibrating with acetone.

Measure the absorbance of the standard solution at 474 nm from the UV spectrometer and compare it with the following equation to determine the accuracy of the experiment and the accuracy of the equation. A calibration curve is drawn up to determine the content of astaxanthin. The result is shown below.

Astaxanthin (g / ml or mg / L) = Absorbance x 10,000

                                           2100

Astaxanthin Concentration in Standard Solution Astaxanthin Standard (/ 1L) (x20ml) (/ 100ml) 30.2 mg 5.9192 g / ml

(Graph 1) UV spectrum of astaxanthin standard solution

Standard solution concentration Absorbance Astaxanthin Concentration content (%) 30.2 mg (/ 1 L) (x20 ml) (/ 100 ml) 1.2448 5.9276 µg / ml 100 (± 0.15)

3. Example 3 Experiment of Determination of Astaxanthin Content in Hematococcus Algae

The following method is used to determine the content of astaxanthin in Hematococcus algae. Take approximately 10 mg of hematococcus algae, place in a centrifuge tube and record the correct value. Place glass beads in two or three tubes. 100 μl of distilled water is added to the tube and allowed to stand for 60 minutes by spinning slowly until the sample is completely wet.

The tube is stirred vigorously for 10 seconds. After standing, 5 ml of acetone is added to the tube, followed by vigorous stirring. Using a centrifuge to deposit the insoluble material for 5 minutes at 3500 ~ 4000rpm and transfer to a volumetric flask using a pipette. 5 ml of acetone was added to the tube, followed by strong stirring, followed by centrifugation to separate the supernatant solution, which was transferred to a volumetric flask. Repeat this method 7-8 times until the layer of acetone is no color.

Adjust the volumetric flask with acetone. The flask is capped and mixed to a uniform concentration. The solution is measured for absorbance at 474 nm with acetone blanked from an ultraviolet spectrometer. The formula given is used to determine the content of astaxanthin. The result is shown below.

Astaxanthin Content (%) =

Astaxanthin (g / ml or mg / L) x 100

 sample (g or mg) / volume of volumetric flask (ml or L)

(Graph 2) UV spectrum of carotenoids in hematococcus

Hematococcus bird Total concentration Absorbance Astaxanthin Concentration content (%) 31.3mg (/ 250ml) 125.2 μg / ml 1.0660 5.0762 µg / ml 4.05 28.6mg (/ 250ml) 114.4 µg / ml 0.9452 4.5010 µg / ml 3.93

4. Example 4 Experiment of Determination of Astaxanthin Content in Hematococcal Algae by HPLC Method

In order to analyze carotenoids in hematococcus algae, algae are extracted with a solvent and analyzed by HPLC. Take 10 mg of hematococcus algae into a tube. Add 3 ml of acetone, add 30 µl of distilled water and 3 ml of nucleic acid, and stir vigorously for 30 seconds. After centrifugation at 3500 rpm for 30 seconds, the nucleic acid layer is transferred to a 10 ml tube. After drying under reduced pressure under nitrogen injection, 50 μl of a mixture of chloroform and methanol (1: 1) was added thereto, followed by HPLC analysis.

Astaxanthin in Hematococcus is ester-bound with protein, so to measure the content of astaxanthin in carotenoids, cholesterol cholesterol ester, which can break the ester bond with protein, is measured using HPLC. do.

1.5 ml of acetone is added to the hematococcus and extracted, followed by 1 ml of Tris hydrochloric acid buffer (pH 7.0). After standing at 37 ° C for 2 minutes, dilute the cholesterol esterase solution ( Pseudomonas flourescens Sigma C-9281, 10,000 units per gram) to prepare 100µl 20 unit / ml, and add 10µl of this solution at 45 ° C. React for a minute.

0.5 g of Na ₂ SO ₄ (H ₂ O) is added and 1 ml of petroleum ether is added. After vigorous stirring for 30 seconds, centrifugation (3000-3500 rpm) for 3 minutes is carried to transfer the layer of petroleum ether to a tube containing 0.5 g of Na ₂ SO ₄ (anhydrous). The extraction is repeated using petroleum ether. Centrifuge it and transfer it to a clean tube. During this process, Na ₂ SO ₄ is rinsed with petroleum ether to transfer all carotenoids. After drying under reduced pressure, petroleum ether was removed, dissolved in a mixed solution of chloroform and methanol (1: 1), filtered through a micro filter, and analyzed using HPLC.

HPLC analysis conditions are as follows.

HPLC (Waters 2695 Separations Module, 2487 Dual Absorbance Detector) analysis conditions for the mobile phase using the gradient method shown in the table below, the flow rate is 1.2 ml / min, the column temperature is fixed at 25 ℃ and the injection amount is 20 μl. The column used Waters Symmetry C18 (5m, 3.9Ⅹ150nm) and the absorbance at 474nm was used. The result is shown below.

HPLC gradient protocol: Time (min) Flow rate ml / min % Methanol % Water % Ethyl Acetaete Condition 0 1.2 82 8 10 Injection 20 1.2 29 One 70 Linear gradient 22 1.2 29 One 70 Equilibration 24 1.2 82 8 10 Linear gradient 30 1.2 82 8 10 Equilibration

(Graph 3) HPLC spectrum of astaxanthin standard solution

(Graph 4) HPLC spectrum of hematococcus algae

5. Example 5 Hematococcus Extraction Method with Astaxanthin 5-15%

200 g of the sample of Example 1 (2.5% astaxanthin content) was placed in a filter barrel, and placed in a soxhlet apparatus (see FIG. 1) equipped with an ultrasonic wave generating terminal, and about 2 L of ethanol for alcohol was extracted for 24 hours. After extraction, the resultant was filtered with a vacuum filter, reduced pressure was used to evaporate ethanol, and dried under reduced pressure to obtain 48.05 g (yield 96.1%) of hematococcus extract having a content of about 10% astaxanthin. The content of astaxanthin was measured using a UV spectrophotometer.

In order to measure the astaxanthin content of the prepared astaxanthin concentrate, 30 mg of astaxanthin concentrate was measured using an electronic balance in a 50 ml beaker and the exact mass was recorded. 10 ml of acetone is added to the beaker to dissolve and the solution is transferred to a 250 ml volumetric flask. Repeat this process several times until the beaker is completely red until no color is visible, and then scale the volumetric flask with acetone. The solution is measured for absorbance at 474 nm with acetone blanked from an ultraviolet spectrometer. Using the formulas (1) and (2) presented above, the content of astaxanthin is determined. The result is shown below.

(Graph 5) UV spectrum of concentrate using ethanol solvent

Extraction solvent Extract status Extraction amount Solution preparation Total concentration Absorbance Astaxanthin Concentration content (%) Acetone Oil phase 31% 42.5 mg (/ 250 ml) (x20 ml) (/ 100 ml) 34.0 μg / ml 0.6115 2.91 µg / ml 8.56% ethanol Oil phase 34% 19.0mg (/ 250ml) 76.0 µg / ml 1.1022 5.25 µg / ml 6.91%

6. Example 6 Preparation Method of Priasanthin by Hydrolysis by Basic Solution of Hematococcus Algae

In general, to measure the content of astaxanthin in algae in the hematococcus is measured by using a cholesterol esterase that can break the ester bond of astaxanthin and protein, followed by HPLC. However, cholesterol esterases are not suitable for mass production due to their high cost.

Instead of expensive esterases, chemical methods were used to achieve the same results as using cholesterol esterases, and to provide a way for mass production. The ester bond was terminated with a basic alcohol solution, neutralized with an acidic aqueous solution, and confirmed by HPLC.

A basic alcohol solution was prepared by dissolving 0.072 g of sodium hydroxide in 100 ml of methanol and 0.1 ml of distilled water. 0.1 g of hematococcus alga was added to the prepared basic methanol solution, and the mixture was stirred under reflux at 50 ° C. After 4-5 hours, cool to room temperature and neutralize with diluted acidic solution until neutral using litmus species or pH meter.

0.5 g of Na ₂ SO ₄ (H ₂ O) is added to the reaction and 1 ml of petroleum ether is added. After vigorous stirring for 30 seconds, centrifugation (3500 rpm) for 3 minutes is carried to transfer a layer of petroleum ether to a tube containing 0.5 g of Na ₂ SO ₄ (anhydrous). The extraction is repeated using petroleum ether. Centrifuge it and transfer to a clean tube. During this process, Na ₂ SO ₄ is rinsed with petroleum ether to transfer all carotenoids. Drying under reduced pressure to remove petroleum ether, dissolved in a 4: 1 mixed solvent of ethyl acetate and methanol, filtered using a micro filter, and analyzed using HPLC. HPLC results showed the same results as those measured with esterase, which is shown in Figure 7.

An alcoholic basic solution was prepared by dissolving 5 g of sodium hydroxide in 100 g of methanol and 0.1 g of distilled water. 10 g of hematococcus alga was added to the prepared methanol basic solution, and the mixture was stirred at reflux at 50 ° C. After 2-3 hours, the mixture is cooled to room temperature and neutralized with a diluted acidic solution using a litmus species or pH meter until neutral. Measured after pretreatment in the same manner as the above method for analysis by HPLC. As a result of HPLC, the same result was obtained as shown in Fig. 8.

(Graph 6) Astaxanthin HPLC spectrum in hematococcus algae (cholesterol esterase method)

(Graph 7) Preparation of priasanthin by hydrolysis with basic solution of hematococcus alga HPLC spectrum

(Graph 8) Preparation of priasanthin by hydrolysis with basic solution of hematococcus alga HPLC spectrum

According to the present invention as described above, astaxanthin during the extraction process is pressurized to 2000-2500bar to completely destroy the cell wall, treated using ultrasonic vibration of rated output 200-2000 watts and 16-25kHz, obtained in nature as extraction solvent By using edible edible alcohols and organic solvents, it is possible to mass produce high concentrations of astaxanthin at low price by extracting high concentrations of astaxanthin with low cost, high efficiency and simple device and extraction conditions, thereby increasing productivity. It can have a great ripple effect when used as additives, cosmetics, feed, photosensitizers or pigments for electronic materials.

Claims (12)

delete delete delete delete delete a) drying and pulverizing a material selected from the group consisting of hematococcus algae, papia rodjima, shrimp, crab, lobster and salmon, including astaxanthin; b) pressing the ground material to break the cell wall; c) hydrolyzing the material in which the cell wall is destroyed in a basic solution state without using an enzyme; Method for producing highly concentrated free astaxanthin. The method of claim 6, Between step b) of pressing the ground material to break the cell wall and c) hydrolyzing in basic solution state, The method comprising the steps of extracting astaxanthin oil by putting the material in which the cell wall is destroyed in a natural solvent and applying an ultrasonic power of 200-2000 watts and 16-25 kHz at a rated output. Method for producing highly concentrated free astaxanthin. The method according to claim 6 or 7, The pressure in the pressurization step is characterized in that 2,000-2,500bar Method for producing highly concentrated free astaxanthin. The method of claim 7, wherein The extraction solvent used is any one selected from the group consisting of edible alcohols, methanol, acetone, nucleic acids, petroleum ether, chloroform and ethyl acetate which can be obtained in nature, or a mixture thereof. Method for producing highly concentrated free astaxanthin. The method according to claim 6 or 7, The basic solution used is Any one selected from the group consisting of alcohol, methanol, acetone, nucleic acid, petroleum ether, chloroform and ethyl acetate or mixtures thereof; With a small amount of water, Sodium hydroxide, calcium hydroxide, calcium hydroxide, ammonium hydroxide, sodium carbonate, sodium hydrogen carbonate, ammonium carbonate and magnesium carbonate any one or a mixture thereof selected from the group consisting of a solution prepared by mixing so that the hydrogen ion concentration is 8-12 By Method for producing highly concentrated free astaxanthin. The method according to claim 6 or 7, During the ultrasonic treatment step characterized in that the ultrasonic treatment for 10-600 minutes continuously or intermittently Method for producing highly concentrated free astaxanthin. The method according to claim 6 or 7, After the step of hydrolyzing the material whose cell wall is destroyed in a basic solution without using an enzyme, Neutralizing the hydrogen ion concentration to 2-6 as any one or a mixture thereof selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, formic acid, citric acid and lactic acid Method for producing highly concentrated free astaxanthin.

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