JPH0775556A - Culture of alga containing docosahexaenoic acid - Google Patents

Abstract

PURPOSE:To provide a culture process capable of getting algal cells containing large amounts of oil and docosahexaenoic acid in the cell at an arbitrary culture stage and, accordingly, enabling the production of a docosahexaenoic acid- containing alga in a high efficiency at a low production cost. CONSTITUTION:An algal strain growing by heterotrophy and producing docosahexaenoic acid, e.g. Crypthecodinium cohnii is cultured by setting the pH of the culture liquid to a level higher than the pH suitable for the growth of alga by 0.1-7 during the culture, especially after the middle stage of the exponential growth phase.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the cultivation of algae capable of producing docosahexaenoic acid (hereinafter abbreviated as DHA), which grows under heterotrophic conditions, and efficiently accumulates DHA in the algal body at a high concentration. The present invention relates to a method for culturing the algae.

[0002]

2. Description of the Related Art Recently, DHA has been attracting attention as a kind of highly unsaturated fatty acid having various physiological activities. As algae capable of producing this, for example, algae of the genus Amphidinium (Amphidinium) and the like have been reported (J. Protozoal, Volume 17 (2), 213-219).
Page, 1970), and the production of DHA-producing algae belonging to the genus Isochrysis has been attempted (Japanese Patent Laid-Open No. 4-410).
346760). However, all of them are algae that grow autotrophically, and not only the culture cost is high, but the oil content in the alga body is low, and the satisfactory D
It has not been possible to produce HA-containing fats and oils. On the other hand, algae belonging to the genus Crypthecodinium are known as DHA-producing algae that grow by heterotrophy (Phytochemistry, Vol. 27 (6),
Pp. 1679-1683, 1988).

Conventionally, in the production of fats and oils by microorganisms and algae that grow heterotrophically, methods for accumulating fats and oils in fungi or algae include adjusting the C / N ratio of the medium composition and updating the medium. A culture control method has been used mainly by adjusting the nitrogen source of the medium components (for example, "oil and fat", Vol. 24 (5), 126-135, 1971). However, in these methods, the start time of the stage of accumulating oils and fats varies depending on the growth state of microorganisms and algae, contamination of various bacteria at the time of renewal of the medium, the yield of microbial cells or algae and the oil content are unstable. However, it was not a desirable method from the viewpoint of industrial production.

[0004]

In the conventional culture of DHA-producing algae, there are the above problems, and it has been difficult to produce DHA-containing algae which are efficient and have a low production cost. Therefore, an object of the present invention is to provide a culture method capable of efficiently accumulating oil and DHA at a high concentration in the cells of the alga in culturing a DHA-producing alga growing in heterotrophic, Consequently, it is to provide an efficient industrial method for producing DHA-containing algae with low production cost.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that during culturing of unicellular algae capable of heterotrophic culture and capable of producing DHA, It was found that it is possible to remarkably increase the oil content and DHA content in the alga at any culture time by increasing the pH of the medium, and completed the present invention. That is, the present invention relates to DH grown in heterotrophic
In culturing an alga capable of producing A, during the culturing, the pH of the culture solution is set to a value 0.1 to 7 higher than the pH suitable for the growth of the alga, and the culturing is performed to the alga. It is related.

The alga used in the present invention is an alga that can be heterotrophically cultivated and capable of producing and accumulating DHA in the alga body, and its species or strain is not particularly limited. Examples of algae corresponding to this include "Microbiology Dictionary 1
According to "1992 edition" (Gihodo Publishing), Crypthecodium corny (Crypthecod) belonging to the genus Chlorophyta, Dinoflagellate, Peridinium, and genus Crypthecodinium.
inium cohnii), and Thraustochytrium aureum. These algae are from the American Type Culture Collection (US,
Easily available from algae research institutes such as the abbreviation ATCC),
For example, ATCC30021, 30334, 30336, 50052 in the former, and ATCC28 in the latter.
211, 34304 and the like.

In the practice of the present invention, the algae that grow in the above-mentioned heterotrophic and have the ability to produce DHA are first cultivated in a medium by a conventional method, and then the pH of the medium is raised at an arbitrary time. That is, using a natural or artificial seawater medium, or a medium containing a carbon source such as glucose and sucrose, a nitrogen source such as an ammonium salt, a yeast extract, an amino acid or a salt thereof, various vitamins and minerals, and adjusting the pH to 5 The culture is preferably carried out at 7 to 6 and preferably at 20 to 35 ° C., preferably 25 to 32 ° C., suitably from the middle stage of exponential growth to the stationary phase in the growth curve of the alga. If the pH and temperature during this initial culture deviate from these ranges, the growth of algae tends to be inhibited. Then, at any culture time, preferably at or after the mid-exponential phase, for example, any phase from the logarithmic growth phase to the stationary phase, the pH of the culture solution
Increase the value. The pH increase value at this time is 0.1 to 7 preferably 1 to 4 than the pH suitable for the growth of the algae.
Set to a higher value and continue culturing at that pH value. If the increase in pH is less than 0.1, the oil content and DHA in the alga
The content cannot be increased, and raising the pH above 7 is not preferable because it inhibits the growth of algae.

The pH of the culture solution can be increased by adding an alkaline aqueous solution such as sodium hydroxide or potassium hydroxide, or by adding an appropriate buffer solution.
The present invention is not limited to these. The concentration of the alkaline aqueous solution is preferably 0.1 to 10N. The time for raising the pH of the medium may be any culture as described above, for example, when the growth amount of the alga body reaches a desired amount, or when it is necessary in the work of the culture process, etc. It is any stage after the middle stage of exponential growth of algae. In the culture stage before this, for example, pH
If it is a slight rise of about 0.1 to 1, it will be okay,
A sharply large rise may inhibit the growth of algae itself.

[0009] In this way, although it depends on the kind of algae in the culture solution of which the pH value is raised, it is usually cultivated for about 3 to 50 hours to obtain an algal body having a high oil content and DHA content, which is the object of the present invention. it can. If the culture time after raising the pH is less than 3 hours, the DHA content in the alga is small, and conversely, if the culture is continued for a long time exceeding 50 hours, the alga body yield is reduced. In the present invention, the lipid concentration of the algal cells obtained by culturing by raising the pH of the medium varies depending on the culturing time and the timing of starting the pH increase. For example, the oil content is about 24 hours after the start of the pH increase. Is 15 to 45% by weight, and the DHA content of the constituent fatty acids of the oil is 30 to 50% by weight. On the other hand, when the culture is carried out without increasing the pH, the oil content is 10 to 15% by weight, and DH in the constituent fatty acids is the same.
The A content remains around 20% by weight.

[0010]

【Example】

Example 1 Crypthecodinium coney
hnii) ATCC30336 was inoculated into 3 liters of the medium having the composition shown in Table 1 and subjected to aeration culture with a jar fermenter at 30 ° C while adjusting the pH to 6.8. During the culture, the weight of the wet matter of the grown algal cells was measured over time, and from the growth curve, 100 hours after the start of the culture, which corresponds to the mid-logarithmic growth phase, the p of the medium was diluted with 1N sodium hydroxide aqueous solution.
H was increased from 6.8 to 10.0 and the culture was continued for another 24 hours. After that, the cultured algal cells are collected by centrifugation,
The algal cells were freeze-dried to obtain 60 g of dried algal cells. It should be noted that 6.0 g of this dried algal cell was mixed with chloroform / methanol = 1: 1.
(Weight) After crushing the cells with a hyscotron in a mixed solvent, extraction was performed to obtain 1.8 g of oil. About 5 ml of sulfuric acid / methanol mixture was added to 200 mg of extracted oil, and the mixture was refluxed for 1.5 hours in a hot water bath to make methyl ester, and then 5 ml of hexane and 5 ml of water were added for extraction, and the fatty acid composition was analyzed by gas chromatography. When analyzed, the DHA content in the total fatty acids was 40% by weight. The oil content in the algal cells was 30% by weight.

[0011]

[Table 1]

[0012] * vitamin mix aqueous solution of biotin: 0.003g / liter - solution Thiamine: 1.000 〃 ** metal mix aqueous solution of Na ₂ EDTA: 1.00 g / liter - solution FeCl ₃ · 6H ₂ O: 0.05 〃 H ₃ BO _3: 1.00 〃 MnCl ₂ · 4H ₂ O: 0.15 〃 ZnCl _2: 0.01 〃 CoCl ₂ · 6H ₂ O: 0.005 〃

Example 2 Crypthecodinium co
hnii) ATCC30021 was cultivated under the same conditions as in Example 1, and the pH of the medium was adjusted 125 hours after the start of culture corresponding to the end of logarithmic growth thereof with a 1N aqueous sodium hydroxide solution.
Was increased from 6.8 to 11.0, and the culture was continued for further 36 hours. Then, the cultured algal cells were collected by centrifugation and freeze-dried to obtain 55 g of dried algal cells. Using a part of the dried algal cells, the oil content was extracted in the same manner as in Example 1, and the DHA content in the total fatty acid was analyzed. As a result, the oil content was 3
The content of DHA in the total fatty acids was 3% by weight and 39% by weight.

Comparative Example 1 Crypthecodinium coney
hnii) ATCC30336 was cultivated for 124 hours under the same conditions as in Example 1 except that the pH of the medium was maintained at 6.8. Then, the cultured algal cells were collected by centrifugation and freeze-dried to obtain only 20 g of dried algal cells. Further, using a part of the dried algal cells, the same analysis as in Example 1 revealed that the oil content and the DHA content were both as low as 10% by weight.

Example 3 Thraustochytrium
aureum) ATCC34304 was inoculated into 3 liters of a medium having the composition shown in Table 1, and the pH was adjusted to 6.8 at 25 ° C.
Aeration culture was carried out with a jar fermenter while adjusting to.
During the culture, the weight of the wet matter of the grown algal cells was measured with time, and from the growth curve thereof, the end of the logarithmic growth corresponding to the start of the culture 13
At 8 hours, the pH of the medium was raised from 6.8 to 10.0 using a 1N aqueous sodium hydroxide solution, and the culture was continued for another 24 hours. Thereafter, the cultured algal cells were collected by centrifugation, and the algal cells were freeze-dried to obtain 65 g of dried algal cells. In addition,
Using a part of this dried alga body, the oil content in the alga body determined by the same method as in Example 1 was 35% by weight, and the DHA content in the total fatty acids was 50% by weight.

Example 4 The same alga strain (ATCC34304) as in Example 3 was cultivated under the same conditions as in Example 3, and 1N sodium hydroxide aqueous solution was added at 115 hours after the start of culture corresponding to the middle logarithmic growth phase. Used to raise the pH of the medium from 6.8 to 10.0,
The culture was continued for another 24 hours. Then, the cultured algal cells were collected by centrifugation, and the algal cells were freeze-dried to obtain 56 g of dried algal cells.
Obtained. Using a part of this dried alga, the oil content in the alga determined by the same method as in Example 1 was 30% by weight, and the DHA content in the total fatty acids was 51% by weight.

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, DH grown in heterotrophic
In culturing an alga capable of producing A, since it is possible to obtain an algal cell containing a large amount of oil and DHA in the algal body at an arbitrary culturing time, it is possible to efficiently produce a DHA-containing alga with low production cost. Is possible.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 7/64 C12R 1:89)

Claims (4)

[Claims] 1. When culturing algae capable of producing docosahexaenoic acid that grows in heterotrophic culture, the pH of the culture solution is set to a value 0.1 to 7 higher than the pH suitable for growth of the algae during culturing. A method for culturing algae containing docosahexaenoic acid, which comprises culturing. 2. The culture method according to claim 1, wherein the pH suitable for the growth of algae is 5 to 7. 3. The culture method according to claim 1 or 2, wherein the time of increasing the pH value is after the middle exponential growth of the culture. 4. The algae is Crypthec
The culturing method according to any one of claims 1 to 3, which is a single cell alga belonging to the genus odinium).