JPH078268A - Method for culturing marine microalgae and method for producing docosahexaenoic acid using the same - Google Patents

Abstract

(57) [Summary] [Objective] To provide a method for obtaining docosahexaenoic acid, which is industrially stable and inexpensive and has no fishy odor, by extracting docosahexaenoic acid from an algal body produced by continuously culturing marine microalgae. [Structure] A method for culturing marine microalgae, which comprises continuously culturing marine microalgae under aeration and stirring conditions to produce algal bodies of the marine microalgae, and a method for producing docosahexaenoic acid using the same .

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cholesterol lowering action, an anticoagulant action, an antitumor action, and further improvement of memory and learning ability in relation to the brain metabolic system, prevention of senile dementia, and treatment of Alzheimer's disease. TECHNICAL FIELD The present invention relates to a method for producing docosahexaenoic acid, which is one of the polyunsaturated fatty acids whose physiological action is drawing attention as a drug, a growth essential fatty acid for fry, and the like. In particular, the present invention relates to a method for producing docosahexaenoic acid, which comprises stably and inexpensively supplying docosahexaenoic acid from an algal body of a marine microalga, which is produced by continuously culturing marine microalgae efficiently and in large quantities. The present invention relates to a method for producing docosahexaenoic acid (hereinafter sometimes abbreviated as DHA) by extracting it from an algal body obtained by continuous culture of a marine microalga.

[0002]

It has been known that docosahexaenoic acid having many physiologically active functions is contained in fish oil, but its practical use has been delayed because it was difficult to separate and extract it from fish oil. In recent years, the discovery of raw materials containing high concentrations of docosahexaenoic acid in fish oil such as orbital fat of tuna and the development of advanced refining technology for fatty acids have led to research on the elucidation and practical application of the physiologically active function of docosahexaenoic acid. Became active. Examples of the purification method and use of docosahexaenoic acid include a prophylactic and therapeutic agent for thrombosis (JP-A-57-183716), a method for separating and purifying highly unsaturated fatty acid, or an ester thereof (JP-A-61-
37752), an anticancer composition containing a highly unsaturated fatty acid (JP-A-63-258816), and a method for separating and purifying a highly unsaturated fatty acid alkyl ester (JP-A-63-8).
356), an anticancer agent containing docosahexaenoyl monoglyceride as an active ingredient (JP-A-1-203322),
Brain function improving agent containing DHA and its ester as an active ingredient (JP-A-1-153629), brain function improving composition,
Learning ability enhancer, memory enhancer, dementia preventive agent, dementia therapeutic agent or functional food having a brain function improving effect
-49723) and many other reports have been made.

At present, most of docosahexaenoic acid or oils containing it are extracted and refined from fish oils such as tuna, sardines and skipjacks, but it is difficult to be a stable supply source due to migratory property of fish and the composition of fish oils. For the above reasons
It is difficult to separate DHA from other unsaturated fatty acids,
It has drawbacks such as a peculiar smell of fish oil. On the other hand, as a production method using raw materials other than fish oil, a method for producing highly unsaturated fatty acids by conversion of Mortierella spp.
No. 5389), a method for producing highly unsaturated fatty acid-enriched fats and oils by a microorganism capable of producing arachidonic acid (JP-A-1-3048).
92), a method for producing highly unsaturated fatty acid-containing lipids from marine microorganisms (JP-A-2-142486), Echinosporangi
Process for producing highly unsaturated fatty acids by um genus (Japanese Patent Laid-Open No. 2-23
878), Echinosporangium Transversalie ATTCC 1
Patent applications are known such as a production method (European No.-355972) for obtaining highly unsaturated fatty acids from cultures of 6960, 18036. In addition, bacteria (Delong, EF & Yayanos, AA: Ap
pl.Environ.Microbiol., 51 (4), 730 (1986)), fungus Thraus
tochytrium (Haskins, RH et al; Can.J.Microbiol.10,18
7 (1964)), Entomophthora (Tyrrell; Can.J.Microbiol., 1
3,755 (1967)), Japonochytrium sp. ATCC 28207 (JP-A-1-199588), and methods for using dinoflagellates and haptophytes for algae are known (Joseph. JD; Lipids, 1).
0,395 (1975) or Nichols, PD et al; Phytochemistr
y, 23,1043 (1984)), however, these are neither natural growth methods nor static cultures that have been devised to actively increase algal cells. In addition, these methods produced a small amount of DHA and were inferior to fish oil-derived products in terms of industrial availability.

In general, among the culture methods of microorganisms, in batch culture (batch culture) and fed-batch culture (fed-batch culture), since the growth of microorganisms is suppressed by the accumulation of secondary metabolites and waste products, high production is achieved. It is widely known that continuous culture is more advantageous in terms of production efficiency, while it is difficult to maintain the sexuality, while continuous culture in which the growth activity of the microbial flora can be maintained and controlled to a high level during the culture. This has already been reported in many reports such as the production process of many microbial proteins (SCP). However, in the culture of marine microalgae, almost all the cultures have been performed in static cultures that do not apply shear force due to stirring, because the algal bodies are not resistant to shear force due to stirring, and even shaking cultures have been investigated. There wasn't. Therefore, it has been considered that such continuous culture cannot be performed.

Regarding the shaking culture, a little
Environmental conditions affecting DHA formation in thecodinium cohnii (DHB
EACH et al; Biochimica et Biophysica Acta, 316,56-65
(1975), and books: David J.kyle et al; Industrial.
Application of Single CellOils, (American Oil Chemi
st's Society, 1992) chapter 16, 287-300 is known, but its productivity is low for industrial production. In addition, an invention relating to the production of docosahexaenoic acid by Crypthecodinium cohnii MK-8840 (ATCC 40750) and its content was recently filed (WO91 / 11918), and a shaking culture method for marine microalgae was disclosed. However, if an attempt is made to increase the DHA-containing lipids in the alga body by this method, the invention limits the nutrient components necessary for the growth of the alga body and sacrifices the growth of the alga body to increase the DHA-containing component in the alga body. Since it is a measuring method, depletion of nutrients is an essential requirement, so continuous culture is not possible and disadvantages in industrial production cannot be avoided. Therefore,
In order to put these known methods into practical use, it is necessary to overcome many technical problems, and it has been considered that industrial practical production is difficult because of economical profitability.

[0006] According to the conventional methods, algal cell productivity is increased in continuous culture, but the content of the desired product is reduced, and the production of secondary metabolites is batchwise in microorganisms with a low specific growth rate. It has been reported that it is disadvantageous in terms of cost compared to culture [Basics of fermentation technology: PF Stanbury, A.
Whitaker; Translated by Bunbayashi Ishizaki, p239 (1988) (Academic Publishing Center).

The marine microalga Crypthecodinium cohnii is generally known to grow both asexually and sexually. In asexual reproduction, after the trophozoites become spherical cells, the cytoplasm undergoes 1 to 3 cell divisions, releasing 2 to 8 migratory cells. As the cells divide, the alga solids increase.

On the other hand, in sexual reproduction, migrating cells act as gametes, and by joining two gametes, act as zygotes and have four flagella. Eventually, the flagella disappeared, transferred to spherical zygotes and meiotically performed, and individual cells became trophozoites and proliferated.

The present inventors focused on the life history of asexual reproduction of this marine microalga Crypthecodinium cohnii, and acquired a strain that stably grows for a long time only by asexual reproduction. The present invention has been completed by finding out the culture conditions for stable growth of this strain.

[0010]

Docosahexaenoic acid is expected to have many physiological activities such as a carcinostatic effect, but its supply is unstable and its quality is not constant because it depends on fish oil as a raw material. Furthermore, in order to remove the odor peculiar to fish oil, many steps and costs are required for refining. In addition, even in this process, the odor peculiar to fish oil cannot be sufficiently removed, which makes it difficult to use. For example, many technical problems to be solved are required, and thus the product is expensive.

In order to solve these problems, the object of the present invention is to carry out continuous culture using marine microalgae other than fish, which can be industrially stably produced and supplied with a certain quality, thereby continuously producing algae. It is to provide a method capable of dramatically increasing the content of docosahexaenoic acid in the body and the productivity of algal cells than ever before, and stably producing docosahexaenoic acid obtained from the algal cells at low cost.

[0012]

As a result of diligent efforts to solve these problems, the present inventor has succeeded in continuously treating marine microalgae in which algae were conventionally grown only by a batch culture method. Succeeded in producing a high concentration of algal cells by culturing, and further found that the content of docosahexaenoic acid in the algal cells could be dramatically increased by this method. The present invention has been completed on a method for producing docosahexaenoic acid, which comprises extracting and purifying docosahexaenoic acid from an algal body after drying.

That is, the present invention provides a method for culturing marine microalgae, which comprises continuously culturing marine microalgae under aeration and stirring conditions to produce an algal body of the marine microalgae, and docosahexaenoic acid produced from the algal body. Provide a way to do. Here, there is provided a method wherein the marine microalgae is Crypthecodinium cohnii. Also provided is a method for continuously culturing and propagating marine microalgae under aeration and stirring conditions. In addition, the Crypthecodinium cohnii
Provides a method which is resistant to surfactants, resistant to shaking culture, and resistant to continuous culture. Furthermore, in the medium,
Further provided is a method of adding polyols, pyruvic acid, triolein and / or amino acids. In addition, the Crypthecod
inium cohnii) is provided. Also provided is Crypthecodinium cohnii, which has resistance to continuous culture under aeration and stirring conditions and has the ability to produce docosahexaenoic acid. Furthermore, Crypthecodium corny (Crypthecodium corny), which is resistant to detergents, shake culture, and continuous culture, and has the ability to produce docosahexaenoic acid
inium cohnii) is provided.

[0014]

The present invention provides continuous culture by repeating single cell separation of a continuous culture-resistant algal cell strain that can be propagated by shaking culture or continuous culture, which has been conventionally difficult except for static culture, and continuous culture by acclimatization. Can withstand Crypthecodinium cohnii
The present invention has been accomplished by the successful discovery and breeding of algae strains. The effect of the present invention is an invention that could not be reached at all by the conventional method or technique in which the proliferation ability was decreased by the passage of liquid shaking culture or an idea on the extension of the conventional method or technique. It could have been possible to reach with great success.

The microorganism used in the present invention may be any alga that belongs to marine microalgae and produces docosahexaenoic acid, such as Crypthecodinium cornii. These are known algae, for example AT
It is available from preservation organizations such as CC (American Type Culture Collection). As a specific example, Crypthecodinium Cornie ATCC 30021, 3054
3, 30556, 30571, 30572, 3057
5, 50051, 50053, 50055, 5005
6, 5,0058, 50060 and the like. In particular, the Crypthecodinium cornii ATCC30021 strain is preferable.

For the alga strain of the above-mentioned algae, an alga body obtained by growing an alga strain having resistance to continuous culture by repeating single cell separation and acclimation to culture is used. As a method for growing such algal cells, the cultured algal cells are 1.0 to 3.5.
Diluted with 10% physiological saline to a cell number of 10 ² to 10 ³ cells / ml and placed on an agar medium containing 2 to 4% by weight of glucose and 0.2 to 0.5% by weight of yeast extract. Stain and incubate for 3-4 days to allow colonies to form. Next, 100 ml of a medium in which a surfactant is added to the liquid medium (II) having the composition shown in Table 4 so that the concentration in the medium is 0.01 to 1.0 g / L is placed in a 300 ml Erlenmeyer flask. Then, the resulting colonies are strained, and the medium (I
Inoculate the medium obtained by adding the surfactant to I) to 24 to 32
Culturing with shaking at 150 ° C. and 150 to 250 rpm for 2 to 8 days. The algal bodies are collected from the obtained culture solution by centrifugation, and docosahexaenoic acid is extracted from the algal bodies with an organic solvent to measure the docosahexaenoic acid-producing ability of each alga strain.

Further, the alga bodies obtained by these shaking cultures are suspended again in 2% physiological saline, and thereafter, the same operations as the above static culture and the above shaking culture are repeated 10 to 15 times for growth. A fast-moving alga strain having high surfactant resistance, shaking culture resistance, and high docosahexaenoic acid-producing ability is selected at each stage. Here, the continuous culture operation of the selected algal cells with a small jar fermenter of about 5 L is performed at 28 to 32 ° C., pH 7.0, stirring 200 to 200 by using a medium in which a surfactant is added to the above medium (II). 350
5 and under the conditions of rpm and aeration rate of 0.2 to 1.5 vvm
Repeat 10 times to breed an alga strain that has fast growth, surfactant resistance, shaking culture resistance, high docosahexaenoic acid production ability, and resistance to continuous culture.

As a medium used for single cell separation, about 2% by weight of agar is added to a medium containing glucose (2-4% by weight) and yeast extract (0.2-0.5% by weight) in 2% physiological saline. The medium used for acclimation to the culture using the added agar medium includes the medium used for continuous culture described below. In addition, for shaking culture and continuous culture for acclimation of culture, a surfactant or an antifoaming agent at a concentration in the medium,
It is added so as to be 0.01 to 1.0 g / L.

Next, continuous culture is carried out using the alga strain having resistance to continuous culture obtained as described above. The culture conditions in continuous culture will be described below.

As a medium for culturing algae used in the present invention, a medium having any composition can be used as long as the medium can grow well. Suitable carbon sources, nitrogen sources, inorganic salts and the like can be contained as medium components. As the carbon source, any carbon source that can be used by the alga of the present invention can be used. Organic substances that can be used as such a carbon source include organic compounds such as glycerin, glucose,
There are carbohydrates such as fructose and galactose, and organic acids such as citric acid and acetic acid, and culturing is possible using any substrate. Furthermore, polyols, pyruvic acid,
DH when triolein and / or amino acids are added
The productivity of A can be improved. Also, arginine,
Amino acids similar to the urea cycle such as glutamic acid are DHA
It is effective as a nitrogen source that is almost essential for production. The preferred addition amount of polyols, pyruvic acid, triolein and / or amino acids is 0.25 to 10 mM. The concentration of the carbon source in the medium is 0.1 to 0.1 during continuous culture.
It is 6.0% by weight, particularly 0.2 to 3.0% by weight,
It is preferable in that the amount of glucose remaining in the culture solution is reduced when the culture solution treatment operation is performed after the alga bodies are collected and separated, the cost is reduced, and the yield of docosahexaenoic acid is maintained.

As the nitrogen source, commonly used inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone, yeast extract, casein hydrolyzate, glutamic acid and corn steep liquor can be used. The concentration of the nitrogen source in the medium is preferably 1/10 to 1/5 of the carbon source in the medium during continuous culture.

As the inorganic salts, natural seawater is preferable, but various kinds of known artificial seawater artificially prepared can be widely used, and various sodium salts, phosphates, magnesium salts, potassium salts, borate salts, Carbonate can be used.
In addition, trace amounts of heavy metal salts, such as iron salts, manganese salts,
Cobalt salts, zinc salts, chlorine compounds and bromine compounds can be used. These heavy metal salts are also included in the inorganic salts in the method of the present invention.

As the culturing method, a continuous culturing method utilizing growth by asexual reproduction of Crypthecodinium cohnii is used. This dramatically increases the content of docosahexaenoic acid in the algal cells. The condition for asexual reproduction is a freshwater dinoflagellate, and it has been reported to be in the form of sexual reproduction under nutrient depletion conditions: Phycologia, 14 (1), 1-8 (1975) Nutrient depletion Keep the culture conditions to prevent In order to increase the productivity of docosahexaenoic acid, since docosahexaenoic acid is contained / accumulated in the algal body in Crypthecodinium cornie used in the present invention, the productivity of docosahexaenoic acid can be increased by increasing the productivity of the algal body. Can be increased. Furthermore, in the marine microalga Crypthecodinium cohnii used in the present invention, the content of the metabolite docosahexaenoic acid does not decrease even after continuous culture, as shown in Examples.

Cultivation for increasing the algal cell concentration to a constant concentration at the start of continuous culture, for example, 15 g / L, and the culture temperature during continuous culture can be carried out at 15 to 32 ° C., but 28 to 3
2 ° C is optimal. The pH can be determined in the vicinity of neutrality, and the number of days of culture can be determined by the remaining amount of carbon source and the amount of secreted substances into the medium. The sugar concentration in the medium is controlled such that the glucose concentration at which the specific growth rate of the alga strain is good is 6.0% by weight or less, particularly 0.2 to 3.0% by weight, and continuous culture is performed. Yeast extract is used as a nitrogen source at a concentration of 1/5 to 1/10 times the glucose concentration, and minerals are prepared by using a medium having the composition shown in Table 3.

Further, continuous culture and continuous culture with aeration and jar fermenter aeration are also carried out by adding a surfactant such as a fatty acid ester type or silicon type and an antifoaming agent. As a surfactant or antifoaming agent in the medium, glycerin monofatty acid esters, sorbitan fatty acid esters, silicon antifoaming agents, for example, glycerin monooleate, glycerin monostearate, glycerin monopalmitate, glycerin monolinoleate, Examples thereof include sorbitan monooleate and silicon KM-75. The content of the surfactant or antifoaming agent in the medium is preferably 0.01 to 1.0 g / L in order to maintain the DHA-producing ability of the algal cells and prevent foaming.

The method for carrying out the continuous culture of the present invention means that a jar fermenter or a large tank is used, and when the alga body concentration reaches a certain concentration, a new medium is continuously supplied, and an amount corresponding to this supply amount. It is a method of collecting the culture solution. The algal cells grown from the collected culture solution are collected by centrifugation, filtration, or the like. The dilution rate of the new medium is 0.25 in liquid hourly space velocity.
Continuous culture is possible at hr ^-1 or less, but 0.1 to 0.2 h
The highest productivity is obtained with r ⁻¹ . Above 0.25 hr ^-1 , the specific growth rate due to asexual reproduction of Crypthecodinium cornii is higher than that, and the culture solution is washed out and stable continuous culture cannot be performed. The dilution may be constant during continuous culture, but for example, a method of gradually increasing the dilution rate every 10 hours is preferable. The temperature of continuous culture is preferably 26 to 30 ° C., and aeration is 0.2 to 1.0.
VVM is preferable, and when the stirring blade is used, the stirring is preferably 50 to 250 cm / sec at the tip peripheral speed of the blade. Continuous culture is performed while adding a medium so that the above-mentioned culture conditions are kept constant. As the medium to be continuously injected, the medium (III) containing glucose and yeast extract shown in Table 5 can be used.

Collection of algae from the taken-out culture solution is carried out by a general method such as centrifugation or filtration, for example, 10 ° C., 8,
It is performed by centrifugation at 000 × g for 10 minutes. Docosahexaenoic acid can be extracted from the recovered alga bodies as raw alga or as dried alga bodies by freeze-drying, heating and ventilation drying.

Extraction and purification of docosahexaenoic acid from algae are preferably carried out after adding a fatty acid antioxidant such as α-tocopherol or in an inert gas such as nitrogen. In addition, methanol /
It can be performed by a method of extracting lipids by an extraction method using an organic solvent such as chloroform or a purification method of Folch, Lees, Stanley et al. The crude product thus obtained can also be purified by various methods such as column chromatography or recrystallization.

[0029]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 Crypthecodinium cohnii ATCC 30021 was diluted with 2% physiological saline to a cell number of 10 ² to 10 ³ cells / ml, and glucose was added at 2% by weight. The yeast extract was spread on an agar medium containing 0.2% by weight and incubated at 28 ° C. for 4 days to form colonies. Next, a liquid medium (II) having the composition shown in Table 4 was added with a surfactant (I) so that the concentration in the medium was 0.5 g / L.
V) 100 ml was taken in a 300 ml Erlenmeyer flask. Then, the resulting colonies are strained, and the medium (I
V) and incubate at 28 ° C and 180 rpm for 5
Culture was carried out with shaking for one day. The alga bodies are recovered from the obtained culture solution by centrifugation, and docosahexaenoic acid is extracted from the alga bodies using an organic solvent, and the docosahexaenoic acid productivity of each alga strain is measured, and the docosahexaenoic acid productivity is excellent. Selected algae strains.

Further, these selected algal cells were added to 2% again.
The suspension was suspended in physiological saline, and then the same operations as the above static culture and the above-mentioned shaking culture were repeated 12 times to obtain a fast-growing algae strain, which was resistant to surfactants, resistant to shaking culture, and capable of producing docosahexaenoic acid. High algae strains were selected at each stage. Here, the continuous culture operation of the selected algal cells with a 5 L small jar fermenter was performed according to the following medium (IV).
Repeated 7 times under the conditions of 28 ° C., pH 7.0, stirring 300 rpm, aeration 0.3 vvm, fast growth, surfactant resistance, shaking culture resistance, high docosahexaenoic acid productivity, and continuous. Algae strains resistant to culture were bred. For the following culture, use a continuous culture resistant strain,
It was performed under the condition that asexual reproduction was repeated.

The medium has the composition shown in Table 3 and contains 2% glucose.
(V / v), yeast extract 0.2% (v / v) were added with glucose and yeast extract, and 1 at 120 ° C.
After autoclaving for 5 minutes, the cooled product was used as the medium (II) (Table 4).

Crypthecodinium cohnii ATCC was added to 100 ml of the medium (II).
30021 of 1 platinum loop was inoculated and the initial pH was 5.5 to 7.5.
The cultivation was started at. The culture temperature was 28 ° C. and static culture was carried out for 7 days (hereinafter, the alga body obtained thereby is referred to as a static culture alga body). 10 ml of the obtained culture solution was added to 100 ml of the medium (II), and a 300 ml flask 5 was prepared.
The strain was inoculated into a book and cultured for 5 days with shaking at a rotary shaker at a temperature of 28 ° C., a pH of 6.8, and a rotation speed of 180 rpm (hereinafter, the alga body thus obtained is referred to as a shake culture alga body). Continuous culture was carried out in a 10 L jar fermenter using the culture solutions obtained from the five flasks as a mother bacterium (hereinafter, the alga thus obtained is referred to as a continuous culture alga).

For jar fermenter culture, 500 ml of the culture solution obtained by rotary shaker shaking culture was
A 10 L jar fermenter charged with L medium (II) was inoculated and cultured. The culture conditions for the jar fermenter culture are temperature 28 ° C., pH 7.0, and stirring 30.
The air flow rate was 0 rpm and the air flow rate was 0.3 V.VM. Further, 0.75 g of glycerin monooleate, which is a fatty acid ester-based surfactant, was used as the surfactant (antifoaming agent).

After the start of the culture, the concentration of algal cells was measured, and when the algal cell concentration reached 5 g / L at 105 ° C. for 5 hours, the medium (III) shown in Table 5 was diluted for 0.1 hr. ^-1 was continuously fed, and the other culture conditions were the same as those of the jar fermenter culture, and the continuous culture was performed. From the start of continuous culture, the dilution rate was gradually increased to 0.12 hr ^-1 ,
0.14 hr ^-1 , 0.16 hr ^-1 and 0.02 respectively, and after continuous feeding, continuous culture was performed while adjusting the dilution rate so as to keep the glucose concentration at 0.5 to 3.0% by weight. I went for 3 days. After the start of this continuous culture, no algal cells with multiple flagella were observed in the culture solution. The taken culture solution was collected by freeze-drying the algal cells by centrifugation at 8,000 xg for 15 minutes at 10 ° C. The cell yield was determined by drying at 105 ° C. for 5 hours, and the dry alga weight was 15.
The productivity of the algal cells was 0 g / L and 1.8 g / hr / L.

Extraction and purification of docosahexaenoic acid from the obtained freeze-dried algal cells are carried out by a conventional extraction method Bligh-using a warning blender containing 5 times the amount of algal cells in methanol / chloroform (1/2). The crude product was obtained according to the Dyer method (Shinsei Chemistry Laboratory 4 Lipid II p9- (1991) Tokyo Kagaku Dojin). The crude product was methyl-esterified (ibid., P. 54) and quantified by gas chromatograph analysis using aucoscentic docosahexaenoic acid as a standard substance. In addition, as usual, liquid chromatography (ODS column, mobile phase methanol: water =
95: 5, flow rate 1.0 ml / min, detection RI) D by method
HA was fractionated to obtain docosahexaenoic acid with a purity of 99.2% at a production rate of 0.12 g / L / hr.

The analysis values of the fatty acid composition in the algal cells by gas chromatography were as follows. Fatty acid static culture Alga body Shake culture Alga body Continuous culture Alga body C 12: 0 33.4% 5.7% 3.0% C 14: 0 40.2% 27.5% 16.2% C 16: 0 10.9% 22.7% 23.7% C 18: 0 0.9% 1.6% 1.8% C 18: 1 3.3% 8.0% 9.1% C 22: 6 11.3% 34.5% 45.2% (docosahexaenoic acid) ──────────────────────── ─ 100.0% 100.0% 100.0%

Example 2 Each of the following marine microalgae, Crypthecodinium cohnii (C. cohnii) ATCC, was cultured under the same conditions as in Example 1. Regarding the culture results, Table 1 shows the yield of algal cells and the yield of docosahexaenoic acid (DHA) per hour. (ATCC30021
Only the continuous culture for 3 days and the other one for 1 day are shown).

[0039]

(Comparative Example 1) (Fed-batch culture) Using Crypthecodinium cornii ATCC30021 as an alga cell strain to be used, static culture and shaking culture were carried out under the same conditions as in Example 1, and 500 ml of the obtained culture solution was used. Was used as a mother bacterium to inoculate a 10 L jar fermenter charged with 7 L of the medium (II) and cultured. jar·
In fermenter culture, 28 ℃, pH 6.8, stirring 300 until alga body concentration reaches 5g / L (dry alga body).
Aeration / stirring batch culture was performed at rpm and an aeration rate of 0.3 VVM, and 60 hours, 80 hours, and 100 hours after the start of the culture with the progress of the culture, glucose as a carbon source, yeast extract as a nitrogen source, and Fed-batch culture was performed in which minerals were added aseptically and a cumulative total of 10% by weight of carbon source, the same ratio (10% by weight) of nitrogen source, and minerals of the concentrations shown in Table 3 were added. The temperature was kept constant at 28 ° C., but the stirring was gradually increased from 300 rpm to increase the temperature to 315 rpm after 60 hours, 330 rpm after 80 hours, and 350 rpm after 100 hours. The amount is 0.3V.VM gradually, 0.3 hours after 60 hours
It was increased to 5v.vm, 0.4v.vm after 80 hours, and 0.5v.vm after 100 hours. As an antifoaming agent, 2.25 g (total amount) of sorbitan fatty acid ester was added at any time. 1
After 20 hours, 26.3 g of dried algal cells were obtained per liter. After culturing, algal cells with multiple flagella were observed in the culture medium. The obtained dried alga was extracted and treated in the same manner as in Example 1 to obtain 1.15 g of docosahexaenoic acid per liter. The productivity of dried algal cells per 1 L for 1 hour is
It was 0.21 g / hr / L, and the productivity of docosahexaenoic acid was 9.58 mg / hr / L.

(Comparative Example 2) The algal cell strain obtained in Comparative Example 1 was cultivated by the same culturing method as in Example 1, and the dilution ratio of 0 was reached when the algal cell dry concentration reached 15 g / L. The continuous culture was carried out at 0.35 / hr. However, 12 hours after the continuous culture was carried out, the continuous culture could not be carried out at a stable concentration because the growth rate of the algal cells could not catch up with the additional speed of the medium.

(Comparative Example 3) Table 2 shows the results of fed-batch culture under the same conditions as in Comparative Example 1, using each of the Crypthecodinium cohnii ATCC strains shown in Table 2.

[0043]

Table 3 Composition of medium (I) Aquamarine composition (Yasu Chemical Co., Ltd.) MgSO ₄ .6H ₂ O 11.11 g KBr 0.10 g CaCl ₂ .2H ₂ O 1.54 g H ₃ BO ₃ 0.03 g SrCl ₃ .6H ₂ O 0.04 g NaF 0.003 g KCl 0.69 g NaCl 24.53 g NaHCO ₃ 0.20 g Na ₂ SO ₄ 4.09 g distilled water 1.0 L pH 7.0 (adjusted with 1N-HCl) Table 4 Composition of medium (II) Glucose 2% (v / v) was added to the composition of medium (I) in Table 3.
Medium with glucose and yeast extract added to give yeast extract 0.2% (v / v) Table 5 Composition of medium (III) Glucose 24% (v / v) was added to the composition of medium (I) in Table 3.
v), a medium to which glucose and yeast extract were added so as to be 2.4% of yeast extract Table 6 Composition of medium (IV) 0.5 g / L of surfactant was added to the composition of medium (II) of Table 4. Cultured seawater Seawater from Kawasaki-cho, Chuo-ku, Chiba City was collected and used.

(Example 3) Under the same culture conditions as in Example 2,
Crypthecodinium coney
hnii) ATCC30021 was added to the medium and
Arginine 0.267 mM / L was added and continuous culture was performed to obtain DHA productivity of 0.16 g / L.

(Example 4) Under the same culture conditions as in Example 3,
4.56 g / L of glycerol was further added to the medium for continuous culture to obtain DHA productivity of 0.12 g / L.

(Example 5) Under the same culture conditions as in Example 3,
Further, 5.0 g / L of pyruvic acid was added to the medium and continuous culture was performed to obtain DHA productivity of 0.14 g / L.

[0048]

Industrial Applicability As described above, according to the method of the present invention, a natural product such as tuna or sardine oil is conventionally extracted from fish oil whose production amount and quality are unstable depending on the season and region.
Docosahexaenoic acid can be stably and inexpensively produced by extracting docosahexaenoic acid from algal cells produced from a liquid continuous culture of marine microalgae, which is capable of industrially producing docosahexaenoic acid at a high concentration stably. Can be supplied.

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Claims (12)

[Claims] 1. A method for culturing marine microalgae, which comprises continuously culturing marine microalgae under aeration and stirring conditions to produce algal bodies of the marine microalgae. 2. The method for culturing marine microalgae according to claim 1, wherein the marine microalgae is Crypthecodinium cohnii. 3. The Crypthecodinium Cornie (Cr
The method for culturing marine microalgae according to claim 2, wherein ypthecodinium cohnii) is surfactant-resistant, shake culture-resistant, and continuous culture-resistant. 4. The culture method according to claim 1, wherein polyols, pyruvic acid, trioleins and / or amino acids are further added to the medium. 5. The Crypthecodinium Cornie (Cr
The culturing method according to any one of claims 2 to 4, wherein the asexual reproduction stage of ypthecodinium cohnii) is utilized. 6. A method for producing docosahexaenoic acid, which comprises continuously culturing marine microalgae under aeration and stirring conditions to obtain docosahexaenoic acid from the algal bodies of the marine microalgae. 7. The method for producing docosahexaenoic acid according to claim 6, wherein the marine microalgae is Crypthecodinium cohnii. 8. The Crypthecodinium Cornie (Cr
The method for producing docosahexaenoic acid according to claim 7, wherein the ypthecodinium cohnii) is surfactant-resistant, shake culture-resistant, and continuous culture-resistant. 9. The production method according to claim 6, wherein polyols, pyruvic acid, trioleins and / or amino acids are further added to the medium. 10. The method according to any one of claims 7 to 9, wherein the asexual period of Crypthecodinium cohnii is used. 11. Crypthecod, which has a resistance to continuous culture under aeration and stirring conditions and an ability to produce docosahexaenoic acid.
inium cohnii). 12. Crypthecodinium cohnii, which is characterized by having resistance to surfactants, resistance to shaking culture, resistance to continuous culture, and the ability to produce docosahexaenoic acid.