JP2000060532A - Production of astaxanthin-containing hematococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To industrially produce the subject algae rich in astaxanthin by culturing algae Hematococcus by two-stage culture method so as to diminish the adverse effect of predators or parasites in an outdoor culture pond. SOLUTION: This algae is produced by the following procedure: algae Hematococcus is proliferated in a closed-type culture unit and then cultured in an outdoor culture pond to produce and accumulate astaxanthin in the Hematococcus; the culture operation is completed before predators or parasites for the Hematococcus contaminate the culture pond and is proliferated therein; preferably, a C-medium for freshwater algae is placed in a tank-type culture unit and adjusted to pH 7-8 with acetic acid (salt) and then subjected to high- pressure steam sterilization; subsequently, Hematococcus is inoculated into the medium and subjected to submerged culture at 25-28 deg.C while keeping the medium at pH 7-8. It is recommended that, in the outdoor culture pond, the Hematococcus is transferred into the sterilized pond 10-30 cm deep free from any nitrogen source and cultured at the initial Hematococcus concentration of 5-20 gDCW/m2.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hematococcus containing 3% or more of astaxanthin which is useful as a pigment for foodstuffs, cosmetics, pharmaceuticals, health foods, and for deep-fried foodstuffs such as seafood and egg yolk. The present invention relates to an industrial manufacturing method of.

[0002]

2. Description of the Related Art Astaxanthin is a kind of carotenoid pigment that exhibits a red color and is widely distributed in nature. For example, fish such as red sea bream and salmon and trout, or crustaceans, etc. accumulate astaxanthin in their epidermis, muscles or outer shells, so that the epidermis or meat exhibits a beautiful red or pink color, but these organisms Astaxanthin cannot be biosynthesized by itself.

[0003] Therefore, astaxanthin is derived from a natural product and is useful as a pigment for foodstuffs, and when aquaculture these fish and shellfish, astaxanthin is usually added to the feed for coloring, so-called frying. (JP-A-54
-70995, JP-A-7-67546). It is also used as a feed for poultry for the purpose of improving the color of chicken eggs (Patent No. 2561198), and more recently, the strong antioxidative effect of astaxanthin has been noticed, and it is used as cosmetics, pharmaceuticals, and health foods. Is also under consideration (JP-A-63
-83017, JP-A-2-49091).

The astaxanthin source used for these includes chemically synthesized products, as well as krill / Ami shrimp and Phaffia yeast containing astaxanthin. In the market, natural products are more favored from the viewpoint of safety, and methods for extracting and purifying pigments from krill and culturing methods for Phaffia yeast are being actively researched (JP-A-6-
No. 200179, JP-A-8-508885).

However, these organisms have a low astaxanthin content and have problems in extraction and purification. Currently, the most chemically synthesized products are used, but from the viewpoint of safety, astaxanthin derived from natural products is used. There is a strong demand for cheaper use.

[0006] The algae Haematococcus has attracted particular attention in recent years as a source of astaxanthin derived from natural products because it has a significantly higher astaxanthin content than the above-mentioned organisms. However, it has long been known that hematococcus produces and accumulates astaxanthin (TW Goodwin,
et. al., Biochem. J., 57, p376 (1954)), but despite the various studies that have been conducted since then, large-scale culture technology has not yet been established. The reason is that Haematococcus is a relatively weak algae and is difficult to culture.

[0007] In order to produce and accumulate a large amount of astaxanthin in Haematococcus, it is important to irradiate a large amount of intense light, and for photosynthetic culture of algae, sunlight is the cheapest and most powerful light source. Therefore, normally, an outdoor pond type culture device under sunlight (hereinafter referred to as an outdoor culture pond) is used.

[0008] However, when hematococcus is cultured in an outdoor culture pond, it is very difficult to prevent external contamination of the culture pond with animals or parasite-causing microorganisms that feed on hematococcus. Conventionally, algae that have been successfully produced commercially in outdoor culture ponds have been limited to chlorella that grows fast, or spirulina or donariella that can prevent contamination by culturing under conditions of alkali or high salt concentration.

When hematococcus is cultivated in an inexpensive outdoor culture pond, zooplankton such as ciliates and rotifers and fungi contaminate the hematococcus after a few days, so that hematococcus is cultivated. It was impossible.

In order to prevent predation or contamination of parasites, various closed culture devices such as tubulars and culture methods have been devised (Japanese Patent Publication No. 2-501189, JP-A-5-68).
No. 585). However, all of them are still in the research stage because they have problems such as complicated apparatus, high manufacturing cost, and inability to sufficiently prevent contamination.

[0011]

The problem to be solved by the present invention is to provide an industrial method for producing highly astaxanthin-containing hematococcus which has reduced the effects of predation or parasites in an outdoor culture pond. is there.

[0012]

Means for Solving the Problems As a result of earnest research, the present inventors have found that when hematococcus is cultured in an outdoor culture pond, an animal that feeds on hematococcus (hereinafter referred to as a predator) or a parasitic microorganism ( Due to the contamination of (parasitic microorganisms), the amount of algal cells begins to decrease 4 to 8 days after the start of cultivation, and the concentration of hematococcus algal cells at the start of cultivation in an outdoor culture pond (hereinafter referred to as the initial algae concentration) Examine the relationship of astaxanthin production rate, that there is an initial algal concentration suitable for efficiently producing and accumulating astaxanthin,

[0013] Hematococcus is grown in a closed type culture device, inoculated into an outdoor culture pond after sterilization, and astaxanthin is produced and accumulated in the hematococcus to reduce algae due to contamination with predators and parasitic microorganisms. Previously, it was found that a Haematococcus alga having a high astaxanthin content can be produced, and the present invention has been completed.

That is, according to the present invention, (a) an alga Haematococcus is grown in a closed type culture device, and then astaxanthin is produced and accumulated in the hematococcus in an outdoor culture pond, and an organism that feeds or parasites the hematococcus is cultured. A method of producing astaxanthin-containing hematococcus by a two-step culture method, which comprises culturing before being contaminated in a pond and growing.

(B) The initial concentration of hematococcus in the outdoor culture pond for producing and accumulating astaxanthin is 5 to 20 g DC.
W / m ² and a method for producing astaxanthin-containing hematococcus according to (a),

(C) The closed culture device is a device that does not artificially irradiate the culture solution with light, (b)
Or a method for producing an astaxanthin-containing hematococcus according to (b),

(D) The astaxanthin-containing hematto according to (a) or (b), characterized in that the culture solution in the outdoor culture pond does not substantially contain a nitrogen source as a growth nutrient for hematococcus. And a method for producing coccus.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The hematococcus used in the present invention is a unicellular alga belonging to the genus Hematococcus of the family Chlamydomonas of the order Chloridomonaceae of the alga Chlorophyta, which need not be limited to a specific alga strain and can be stored in universities and research institutions. Algae strain,
Alternatively, algae strains collected and purely separated from lakes, rivers, puddle, seaside, etc. around the world can be used.

As an example of the former, Haematococcus pluvialis is NIES144 of the National Institute for Environmental Studies, UTEX2 of the University of Texas Algae Conservation Facility.
505, Haematococcus lacustris (H. lacustri
s) is ATCC3040 from American Type Culture Collection
2, 30453, IAM C-392, Institute of Applied Microbiology, University of Tokyo,
C-393, C-394, C-339, UTEX 16, 294, H. capensis (UTEX LB102)
3, H. droebakens
is), UTEX 55, and Haematococcus zimbabwiensis (H. zimbabwiensis) are UTEX LB1758.

As the latter, for example, when rainwater accumulated in the depressions of a tombstone or a rock has a red color, it is collected and smeared on a plate agar medium of a freshwater algae medium to give Haematococcus. Can be separated.

Under suitable conditions, Haematococcus becomes teardrop-shaped zoospore cells having two isometric flagella and proliferates by cell division. When various stresses such as nitrogen deficiency, strong light irradiation, and high salt concentration are applied to this zoospore,
It is known that growth stops and the morphology changes, resulting in spherical cyst cells without flagella (MR Droop, Arch.Mi.
krobiol., 21, p267 (1955)). With the formation of cysts, astaxanthin is often produced and accumulated in the protoplasm in many cases. In addition, this intracellular structure may be called hematochrome.

When hematococcus is cultivated in an outdoor culture pond, as shown in the reference example, contamination of predators and parasitic microorganisms always occurs. After sterilizing the culture pond with hypochlorite, etc., start culturing after 8 days, or if the pond was not sterilized after culturing, after 4 days. Parasitic microorganisms are observed and the algae concentration decreases.

Examples of predators include ciliates and rotifers, as well as amoeba and chironomid larvae (ascaris), which feed on both zoospores and cyst cells of Haematococcus. On the other hand, examples of the parasitic microorganisms include kitrid which belongs to the fungus Aspergillus, which parasites and kills cyst cells of Haematococcus specifically.

For the purpose of preventing such predation or contamination of parasites, in the two-step culture of the present invention, hematococcus is first grown in a closed (closed) culture device free from the contamination of predation or parasites. The device used here may be one that can prevent contamination of predators or parasitic microorganisms, and examples thereof include tank-type, tubular-type, and air-dome-type culture devices, but are not limited to these. .

The tank-type culture device capable of high-pressure steam sterilization is
Since Haematococcus can be cultivated purely, it is suitable for this purpose. Acetic acid or acetate in a medium for freshwater algae is 1 to 100 mmol / l, preferably 5 to 30 mm
ol / l is added, the pH is adjusted to 6-9, preferably 7-8, and autoclaved. The medium for freshwater algae contains nitrogen, phosphorus, potassium, magnesium, which are necessary for the growth of algae.
Contains inorganic salts of iron and other trace metals and vitamins such as thiamine. For example, VT medium, C medium, MBM medium, MDM
Examples include culture media (algal research method, edited by Mitsuo Chihara and Kazutoshi Nishizawa, Kyoritsu Shuppan, 1979).

Among them, the C medium contains Tris hydrochloride,
It is preferable because the pH can be easily adjusted. Hematococcus is inoculated into this and cultured at 20 to 32 ° C, preferably 25 to 28 ° C with aeration and stirring. When the growth starts, acetic acid is consumed and the pH rises, and the growth is inhibited as it is. Therefore, it is preferable to add acetic acid, hydrochloric acid or the like to maintain the pH at 6 to 9, preferably 7 to 8.

This culture can be carried out while irradiating with light, and in that case, carbon dioxide can be used instead of acetic acid as a carbon source. However, the growth is faster when acetic acid is used. In the present invention, in the following outdoor culture pond culture, in order to accumulate astaxanthin in hematococcus using inexpensive sunlight, light irradiation in a closed type culture device that requires expensive equipment and operating cost is not always do not need.

By culturing in a closed-type culturing apparatus, clean Haematococcus alga bodies composed of green, brown or red zoospores or cysts free from contamination by predators and parasitic microorganisms can be obtained. It is then transferred to an outdoor culture pond for rapid production and accumulation of astaxanthin.

The outdoor culture pond comprises a concrete or plastic circular or raceway type pond, a device for stirring the culture liquid, and a device for supplying carbon dioxide to the culture liquid, such as chlorella, spirulina, It is also possible to use those generally used for culturing Donariella,
Its surface is open to the atmosphere and sunlight, and it is necessary that the outdoor culture pond is sealed with glass or the like in order to prevent invasion of predators and parasitic microorganisms into the outdoor culture pond. Absent.

It is preferable that the outdoor culture pond is sterilized before the hematococcus cultured in the closed type culture device is transferred to the outdoor culture pond. The sterilization of the outdoor culture pond may be any method as long as it kills predators and parasitic microorganisms, but the chemical solution sterilization with hypochlorite or ozone is simple and suitable for the present invention.

Specifically, after washing the outdoor culture pond, it is sufficient to dissolve hypochlorite or ozone in the medium and fill the outdoor culture pond, and at the same time, the medium is sterilized. After sterilization, residual chlorine and ozone disappear from the medium by irradiation with sunlight and stirring, so that the culture can be started as it is.

The sterilization condition is represented by the CT value which is the product of the added concentration (ppm) and the time (minute). In the present invention,
For sodium hypochlorite and calcium hypochlorite C
T value is 5 to 500, and in case of ozone, CT value is 0.5 to
Sterilize to 10. When these sterilization operations are performed,
Hematococcus can be cultured in an outdoor culture pond for up to 7 days. When the sterilization operation is not performed, the culture can be performed for up to 3 days.

Originally, it is preferable to perform it while confirming that there are no contaminants during the culture in the outdoor culture pond.
According to the present invention, the predation or parasitism of Haematococcus is contaminated in the culture pond, and that the culture is completed before the proliferation, before the hematococcus cultured in the closed type culture device is transferred to the outdoor culture pond, the outdoor Up to 7 if the culture pond is sterilized
If the outdoor culture pond is not sterilized for one day, it means culturing for a maximum of 3 days.

The presence or absence of contamination is checked by a microscope, but when a predator or a parasitic microorganism is found in one drop of the culture solution, the alga concentration often starts to decrease. These days are important because cleaning and sterilizing the pond also becomes difficult.

The medium in the outdoor culture pond is obtained by removing some or all of the inorganic salts contained in the medium for freshwater algae, and at least does not substantially contain a nitrogen source as a growth nutrient source for hematococcus. Use one. It may be groundwater, river water, agricultural water or drinking water itself. By using such a nutrient-deficient medium, hematococcus zoospores stop growing and become cysts, and astaxanthin is produced and accumulated in hematococcus.

This phenomenon can also be promoted by increasing the salt concentration by adding 0.3 to 0.4% sodium chloride or the like (MR Droop, Arch. Mikrobio.
L., 20, 391 (1954)). These media are sterilized by the above-mentioned chemical solution sterilization, ultraviolet sterilization, heat sterilization or the like, and then placed in an outdoor culture pond.

Next, an alga of Haematococcus algae, which was cultivated in a closed-type culture apparatus, was inoculated into an outdoor culture pond, and the initial algae concentration at this time had a great influence on the productivity of astaxanthin and the content of astaxanthin in Hematococcus. To do.
At this time, the astaxanthin generation rate and the astaxanthin content are closely related to the light amount.

The amount of light is expressed by photon flux density (E),
The amount of light in the area will of course vary depending on the location, weather, etc.
The amount of light in a place suitable for culturing Tococcus is 25 to 100E.
/ M ²・ Day and yearly average of 50E / m²-It is about a day. First shot
Per algae concentration and light quantity, and astaxanthin culture area
Production rate and astaxanthin content of Haematococcus
The relationship with is specifically shown in Examples. Initial algae concentration is 5
gDCW / m²Below, the area was hit at any amount of light
It was found that the astaxanthin productivity was low.

In the present invention, gDCW is an abbreviation for dry cell weight, and is JIS K 0101 (industrial water test method), JIS K
Suspended substances in water (S
The culture solution was filtered with GS-25 (pore size of about 1 μm, filter paper treated with an organic binder (acrylic resin) of ultrafine borosilicate glass fiber) manufactured by Advantech Toyo Co., Ltd., which is widely used for measuring S). After that, the filter paper 1
After being dried at 05 ° C. for 6 hours to a constant weight, the dry cell weight obtained by measuring the weight is expressed in g.

Hematococcus is characterized by an astaxanthin content of up to 5%, but the light intensity is 25%.
When the E / m ² · day is weak, the initial algae concentration is 20 gDC
When it exceeds W / m ² , the astaxanthin content in hematococcus does not reach 3% within 3 days, and when the initial algal concentration exceeds 30 g DCW / m ² , the astaxanthin content falls within 7 days. It does not reach 3%, and astaxanthin cannot be sufficiently produced and accumulated.

Therefore, in order to produce Haematococcus containing 3% or more of astaxanthin, the initial algae concentration in the outdoor culture pond is 5 when the outdoor culture pond is sterilized and the culture period is within 7 days. ~30gDCW / m ^2, without sterilization an outdoor pond, in the case within three days 5~20gDCW /
Must be m ² .

In the present invention, it is preferable that the initial concentration of Haematococcus in the outdoor culture pond is 5 to 20 g DCW / m ² . The liquid depth of the outdoor culture pond can be changed arbitrarily,
From the effective use of sunlight, preferably 5 cm ~
It is 40 cm, more preferably 10 cm to 30 cm.

For example, when the liquid depth in the outdoor culture pond is 10 cm, the initial concentration of hematococcus in the outdoor culture pond is 5 to 5.
20 g DCW / m ² means that the hematococcus concentration of the culture solution is 50 mg / l to 200 mg / l, and 25 mg / l to 100 mg / l when the liquid depth is 20 cm.
means l.

Culturing in the outdoor culture pond is carried out for 3 to 7 days with appropriate stirring. The pH of the medium increases in the daytime by consumption of carbon dioxide by photosynthesis, and decreases in the nighttime by exhaustion of carbon dioxide by respiration. During the daytime, the concentration of carbon dioxide decreases and the rate-determining step of photosynthesis is reached. Therefore, carbon dioxide is added from the outside to maintain the pH at 6 to 9, preferably 7 to 8.

During this period, the zoospores of Haematococcus stop growing and become cysts, and the number of cells does not increase. However, astaxanthin is produced and accumulated by photosynthesis, the cells become large and the apparent alga concentration increases. By the two-step culture method of the present invention, a Haematococcus alga containing a high concentration of 3% or more astaxanthin can be efficiently produced.

[0046]

EXAMPLES The present invention will be described below with reference to Examples and Comparative Examples, but the present invention is not limited to these.

Reference Examples 1 to 5 Reduction of algae concentration due to contamination (sterilized culture pond) Haematococcus pluvialis NIES144
Also, Haematococcus sp. DY-1 purely separated by the present inventor was cultured in a tank-type culture device to obtain green zoospore algal cells.
An outdoor circular culture pond (1.2 m ² ) filled with drinking water to a depth of 10 cm was sterilized with a chemical solution under the conditions shown in the table, and 20 g DCW / m ² (200 mg / l) of algal cells were added to the sterilized solution. The resulting mixture was inoculated as described above and cultivated with stirring at 12 rpm while controlling the pH to 7.5 with carbon dioxide. Every day, one drop of the culture solution was dropped on a slide glass and examined under a microscope, and the alga concentration was measured. The results are shown in Table 1. In all the cultures, contamination was observed after the 8th day, and the concentration of hematococcus also started to decrease after the 8th day.

[0048]

[Table 1]

(Reference Examples 6 to 12) Reduction of algae concentration due to contamination (culture pond without sterilization) An outdoor circular culture pond (1.2 m ² ) or a raceway type culture pond (5 m ² ) was simply washed. Fill the drinking water to a depth of 10 cm without chemical sterilization, and refer to Reference Example 1 to
20 g DCW / m ² (200 mg /
l), and the culture was carried out with stirring while controlling the pH to 7.5 with carbon dioxide. Specimens and algal concentrations were measured daily. The results are shown in Table 2. In all of the culture examples, contamination was observed after the 4th day, and the hematococcus concentration also started to decrease after the 5th day.

[0050]

[Table 2]

(Example 1) To a 5 L tank-type culture device equipped with a paddle type impeller, 2.8 L of a medium prepared by adding sodium acetate to 10 mmol / l to C medium which had been concentrated 2 times was added, and high-pressure steam sterilization was performed. did. 200 ml of a culture solution of H. pluvialis NIES144 cultivated in a flask was inoculated into this, 25 ° C, stirring speed 50 rpm, aeration amount 300
Cultured at ml / min. The medium pH was controlled at 7.5 by adding 1 M acetic acid. After culturing for 10 days, a sterile culture solution containing green zoospores and having an alga concentration of 600 mg / l was obtained.

Next, 17 L of C medium, which had been double concentrated, was added to a 25 L acrylic cylindrical closed culture tank, and sterilized with sodium hypochlorite (CT value = 120). Inoculate this with 3 L of the above culture solution, and illuminate with a sunlight lamp at 25 ° C. and an air flow rate of 2 L / min (light amount = 4 E / L / day).
Cultured. The medium pH is 7.5 by the addition of carbon dioxide.
Controlled to. After culturing for 5 days, a culture solution containing green zoospores and having an alga concentration of 600 mg / L was obtained. Nitrate nitrogen contained in the original medium was not detected at all from this culture, and no contamination of predators or parasitic microorganisms was observed.

The culture broth thus obtained was washed with drinking water for 1, 2,
It was diluted 3, 6, 12 and 24 times, and inoculated to 1.2 m ² outdoor circular culture pond sterilized with sodium hypochlorite (CT value = 60) so that the liquid depth would be 10 cm, and the pH was The culture was performed while controlling the temperature to 7.5 by adding carbon dioxide. The temperature during the culture period is 25 to 32 ° C., and the light amount is 26 E / average on average.
It was adjusted with an agricultural light-shielding sheet so that m ² · day and 48 E / m ² · day.

The green zoospores rapidly cystified and produced and accumulated astaxanthin. The astaxanthin production rate and the astaxanthin content on the 3rd and 7th days of the culture are shown in Table 3 (light amount average 26 E / m ² · day) and Table 4 (light amount average 48 E / m ² · day). No contamination of predators or parasitic microorganisms was observed until the 7th day of culture.

[0055]

[Table 3]

[0056]

[Table 4]

Example 2 To a 50 L tank type culture device equipped with a paddle type impeller, 27 L of a medium prepared by adding sodium acetate to 10 mmol / l to C medium which had been concentrated twice was added and sterilized under high pressure steam. Example 1
Cultured in a 5L tank type culture device in the same manner as in
3 L of a culture solution of H. sp. DY-1 that had become mg / l was inoculated and cultured at 25 ° C, a stirring speed of 40 rpm, and an aeration rate of 3 L / min. The medium pH was controlled at 7.5 by adding 1 M acetic acid. Cultured for 8 days, alga concentration of green zoospores 6
A sterile culture of 00 mg / l was obtained. Nitrate nitrogen contained in the original medium was not detected at all from this culture solution.

A washed 1.2 m ² outdoor circular culture basin was filled with 100 L of drinking water, and 20 L of the above culture solution was inoculated into this (liquid depth 10 cm), and the pH was adjusted to 7.5 by adding carbon dioxide. The culture was performed for 3 days while controlling. The temperature during the culture period was 25 to 32 ° C., and the average daily light intensity was 46 E / m ² · day. After 3 days, the Haematococcus alga bodies consisting of red cysts were harvested, concentrated by centrifugation, and then freeze-dried to obtain 41 g of alga bodies with an astaxanthin content of 3.2%. Microscopic examination was performed at the time of harvest, but no contamination with predators or parasitic microorganisms was observed.

(Comparative Example 1) To a 5 L tank-type culture device equipped with a paddle type impeller, 2.8 L of a medium prepared by adding sodium acetate to 10 mmol / l to C medium which had been concentrated 2 times was added, and autoclaved. did. 200 ml of a culture solution of H. pluvialis NIES144 cultivated in a flask was inoculated into this, and the temperature was 25 ° C, the stirring speed was 50 rpm, and the aeration rate was 300 m.
Cultured at 1 / min. The medium pH was controlled at 7.5 by the addition of 1M acetic acid. After culturing for 10 days, a sterile culture solution containing green zoospores and having an alga concentration of 600 mg / l was obtained. At this time, all of the nitrate nitrogen contained in the original medium was consumed and was not detected.

To an outdoor 0.3 m ² acrylic square-shaped culture basin, 27 L of C medium concentrated twice was added and sterilized with sodium hypochlorite (CT value = 60). This was inoculated with 3 L of the above culture solution (10 cm deep) and cultured. The medium pH was controlled at 7.5 by adding carbon dioxide.
Cultured for 10 days, brown algae concentration 840
A culture solution of mg / l was obtained. The temperature during the culture period is 25 to 3
At 0 ° C., the average daily light intensity was 36 E / m ² · day. Nitrate nitrogen contained in the original medium was not detected at all from this culture solution, and 1-3 microscopic ciliata showing green by feeding Haematococcus were observed under a microscope. It was

A washed 1.2 m ² outdoor circular culture basin was filled with 90 L of drinking water, and 30 L of the above-mentioned culture solution was inoculated into the culture tank (depth 10 cm), and the pH was adjusted to 7.5 by adding carbon dioxide. Cultured under control. Temperature during culture is 2
The temperature was 5 to 32 ° C., and the average daily light intensity was 41 E / m ² · day.
Two days later, when the culture solution was sampled, the alga concentration of hematococcus consisting of reddish brown cysts was 31 g DCW /
m ² (310 mg / l), the astaxanthin content of the algal cells was 1.9%. However, from this day, ciliates and rotifers proliferated rapidly to supplement Haematococcus, and when he was microscopically examined on the 3rd day, hematococcus was not seen at all.

[0062]

EFFECTS OF THE INVENTION According to the method for producing hematococcus containing astaxanthin of the present invention, it is possible to prevent contamination by obtaining clean Haematococcus alga in a closed culture device, and transfer it to an outdoor culture pond to produce astaxanthin. By accumulating, 3% or more high astaxanthin-containing Haematococcus can be produced inexpensively and efficiently.

Claims (4)

[Claims] 1. An alga Haematococcus is grown in a closed-type culture device, and then astaxanthin is produced and accumulated in the hematococcus in an outdoor culture pond, and an organism that feeds or parasitizes the hematococcus is contaminated and proliferated in the culture pond. A method for producing astaxanthin-containing hematococcus by a two-step culture method, which comprises culturing before completion. 2. The initial concentration of hematococcus in an outdoor culture pond for producing and accumulating astaxanthin is 5 to 20 g DCW /
The method for producing astaxanthin-containing hematococcus according to claim 1, wherein the method is m ² . 3. The method for producing astaxanthin-containing hematococcus according to claim 1, wherein the closed culture device is a device that does not artificially irradiate the culture solution with light. 4. The production of astaxanthin-containing hematococcus according to claim 1 or 2, wherein the culture solution in the outdoor culture pond contains substantially no nitrogen source as a growth nutrient source for hematococcus. Method.