JP3506740B2 - Method for culturing algae containing docosahexaenoic acid - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the cultivation of algae capable of producing docosahexaenoic acid (hereinafter abbreviated as DHA), which grows under heterotrophic conditions, and abundantly accumulates DHA-containing polar lipids in the algal body. 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 and the like have been reported (J.
Protozoal, Vol. 17 (2), pp. 213-219, 1
970), and also DH of the genus Isochrysis
Attempts have been made to produce A-producing algae (Japanese Patent Laid-Open No. 4-346760).
Issue). However, all of them are algae that grow autotrophically, and not only the culture cost is high, but also the oil content in the algal body is low, and satisfactory DHA-containing fats and oils cannot be produced. On the other hand, algae belonging to the genus Crypthecodinium are known as DHA-producing algae that grow by heterotrophy (Phytochemistry, Vol. 27 (6), No. 1679-1).
683, 1988).

Conventionally, in the production of fats and oils by microorganisms and algae that grow heterotrophically, there has been interest in a technique for accumulating fats and oils in fungi or algae, and research has been promoted. That is, the accumulation of oil in the living cells is generally carried out mainly in the form of triglycerides, so-called neutral lipids, and when accumulated in large amounts, these triglycerides are accumulated in the cells in the form of oil droplets. And as a method for accumulating a large amount of oil in cells, C / N of the medium composition is
Methods such as increasing the ratio or artificially giving a deficiency state of the nitrogen source have been used. ("Fat and oil", Vol. 24 (5), pp. 126-135, 1971).

That is, in heterotrophically growing algae,
It has been known that most of the oil content is mainly accumulated in the algal body as neutral lipids. For example, the above-mentioned Phytochemistry,
According to Volume 27 (6), pp. 1679-1683, 1988, Crypthecodinium Cornie (Crypthec
About 72% of the oil content in the cultured algal cells of odinium cohnii)
Is a neutral lipid consisting mainly of triacylglyceride, about 28
% Exists as a polar lipid mainly composed of phospholipids such as phosphatidylcholine, and the main fatty acids constituting fatty acids of such phospholipids are highly unsaturated fatty acids such as DHA.

Therefore, of the fatty acids that make up the oil,
Fatty acids such as palmitic acid and oleic acid other than highly unsaturated fatty acids such as DHA mainly exist as neutral lipids such as triacylglycerides, while highly unsaturated fatty acids such as DHA are polar lipids such as phospholipids and glycolipids. It is considered that they tend to exist in the alga body in the form of. However, no attempt has been made so far for algae capable of producing DHA to accumulate a large amount of DHA-containing polar lipid in the alga body.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to cultivate DHA-producing algae that grow in heterotrophic nutrients.
An object of the present invention is to provide a culturing method capable of abundantly accumulating HA-containing polar lipids in the cells of the algae, and further to provide an efficient and industrially low-cost industrial DHA-containing algae production method. .

[0007]

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 by increasing the salt concentration of the culture solution, it is possible to significantly increase the polar lipid concentration such as phospholipids and glycolipids in the algal cells and the DHA content at any culturing time. Completed the invention. That is, the present invention sets the salt concentration of the culture solution to a value higher by 0.1 to 10% by weight than the salt concentration suitable for the growth of algae during the culture when culturing algae capable of producing DHA that grow in heterotrophic. The present invention relates to a method for cultivating algae, which comprises culturing the algae.

The algae used in the present invention are heterotrophically culturable algae capable of producing and accumulating DHA in the alga body, and the species or strain thereof is not particularly limited. Examples of algae corresponding to this include “Microbiology Dictionary 19
1992 edition ”(Gihodo Publishing Co., Ltd.), Crypthecodinium corny (Crypthecodin) belonging to the genus Chlorophyta, dinoflagellate, Peridinium, and genus Crypthecodinium.
ium cohnii), Thraustochytrium aureum (Th
raustochytrium aureum) and the like. These algae can be easily obtained from algae research institutes such as American Type Culture Collection (US, abbreviated as ATCC). For example, ATCC30021, 30334, 30
336, 50052, and in the latter ATCC 2821
1 and 34304.

In carrying out 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 salt concentration of the medium is increased at an arbitrary time. That is, a natural or artificial seawater medium having a salt concentration of 1.2 to 2.5% by weight, or a carbon source such as glucose and sucrose, an ammonium salt, a yeast extract, a nitrogen source such as an amino acid or a salt thereof, and various vitamins. Using a medium containing algae and minerals, the culture is carried out at a pH of 5 to 7 at 20 to 35 ° C., preferably until the middle exponential growth phase in the growth curve of the algae. P during this initial culture
If H, temperature and salt concentration deviate from this range, the growth of algae tends to be inhibited.

Then, the salt concentration of the culture solution is increased at an arbitrary culture time, preferably after the exponential growth phase, for example, at any time from the logarithmic growth phase to the stationary phase. The increase value of the salt concentration at this time is 0.1 to 10% by weight, preferably 0.1 to 3% by weight higher than the salt concentration suitable for the growth of the algae, that is, 1.2 to 2.5% by weight. And continue the culture at that salt concentration. The range of increase in salt concentration is 0.
If it is less than 1% by weight, the DHA-containing polar lipid cannot be accumulated at a high concentration, and if the salt concentration exceeds 10% by weight, the growth of algae is inhibited, which is not preferable. In the present invention, when increasing the salt concentration in the medium, not only the salt concentration but also other seawater constituents other than the salt may be added at the same time as the salt to increase the seawater concentration. It is convenient to use a concentrate of natural seawater as the salt and seawater constituents used for this purpose.

The time for increasing the salt concentration of the medium may be any culture time as described above, for example, when the growth amount of the algal cells reaches a desired amount or when it is necessary in the work of the culture process. However, it is preferably any stage after the middle exponential growth of algae. In the culture stages prior to this,
A rapid increase in salt concentration may inhibit the growth of algae, which is not preferable. Note that it is preferable to apply this method to cultured algal cells in a stationary phase, since algal cells with a high concentration of polar lipids such as phospholipids and glycolipids can be obtained in high yield.

[0012] In the culture solution thus increased in salt concentration,
Although it depends on the type of algae, the alga body containing a large amount of the DHA-containing polar lipid, which is the object of the present invention, can be obtained by generally culturing for about 3 to 50 hours. If the culture time after raising the salt concentration is less than 3 hours, the content of polar lipids in the alga body is small, and conversely, if the culture is continued for a long time exceeding 50 hours, the alga body yield decreases.

In the present invention, the lipid content of the algal cells obtained by culturing by increasing the salt concentration of the medium varies depending on the culturing time and the time when the increase of the salt concentration is started. In 24 hours, 50 to 65% by weight of polar lipids and 35 to 50% by weight of neutral lipids are contained in the oil. The breakdown of polar lipids is that phospholipids are 2
0-40%, glycolipids 30-50%, other ingredients about 3
It is 0%. Of the constituent fatty acids in total lipids, DHA
The content is 45 to 50% by weight, and the DHA content of the constituent fatty acids in the polar lipid is 60 to 75% by weight. On the other hand, when the culture is carried out without increasing the salt concentration, the polar lipid content is 20 to 30% by weight and the neutral lipid content is 70 to
80% by weight, and D among the constituent fatty acids in total lipids
The HA content is about 20% by weight, and the DHA content of the constituent fatty acids in the polar lipid is 40 to 50% by weight.

[0014]

【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, at 100 hours after the start of culture, which corresponds to the mid-logarithmic growth phase, 20 g / 1 L of salt was added to determine the salt concentration in the medium. It was increased by 2% by weight, and the culture was continued for another 24 hours.
Then, the cultured algal cells were collected by centrifugation and freeze-dried to obtain 62 g of dried algal cells.

6.2 g of the dried algal cells were crushed by a hiscotron in a mixed solvent of chloroform / methanol = 1: 1 (weight) and then extracted to obtain 0.5 g of oil. The extracted oil was developed by thin layer chromatography (TLC) using a silica gel plate with a mixed solvent of chloroform / methanol / acetic acid, and separated into a polar lipid fraction and a neutral lipid fraction. Then, each spot was scraped off from this silica gel plate, and chloroform / methanol = 1:
When re-extracted with 1 (weight) mixed solvent, the polar lipid content was 52% by weight and the neutral lipid content was 48% by weight.
Among polar lipids, phospholipids are 30% and glycolipids are 40%.
% And other were 30%.

Further, to 200 mg of extracted oil, about 5 ml of a sulfuric acid / methanol mixture was added and refluxed for 1.5 hours in a water bath, methyl esterification was performed, and then 5 ml of hexane and 5 ml of water were added for extraction to obtain a fatty acid composition. When analyzed by gas chromatography (GLC), the DHA content in the total fatty acids was 46% by weight. Further, the constituent fatty acid composition of the polar lipid was similarly treated and subjected to GLC analysis. As a result, the content of DHA in the constituent fatty acid in the polar lipid was 70% by weight.

[0017]

[Table 1]

[0018] * 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 〃

Comparative Example 1 The same algae strain (ATCC30336) as in Example 1 was inoculated in 3 liters of the medium having the composition shown in Table 1, and aerated with a jar fermenter at 30 ° C. while adjusting the pH to 6.8. did. The salt concentration was not adjusted during the culture, and the
At the time, the cultured algal cells were collected by centrifugation and freeze-dried to obtain 60 g of dried algal cells. This dried alga body 6.0 g was extracted with a chloroform / methanol = 1: 1 (weight) mixed solvent to obtain 0.75 g of an oil component. When the extracted oil was analyzed in the same manner as in Example 1, the polar lipid content was 25% by weight and the neutral lipid content was 75% by weight. The DHA content in the total fatty acids of the extracted oil was 18% by weight, and the DHA content in the constituent fatty acids of the polar lipid was 45% by weight.

Example 2 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 over time, and from the growth curve thereof, which corresponds to the middle logarithmic growth phase, 1
At 15 hours, 20 g / 1 L of salt was added to increase the salt concentration in the medium by 2% by weight, and 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 55 g of dried algal cells.

5.5 g of this dried algal cell was extracted with a mixed solvent of chloroform / methanol = 1: 1 (weight) to give 0.6
g oil was obtained. When the extracted oil was analyzed in the same manner as in Example 1, the polar lipid content was 58% by weight and the neutral lipid content was 42% by weight. Of the polar lipids, phospholipid was 35%, glycolipid was 35%, and other components were 30%. In addition, the DHA content in the total fatty acids of the extracted oil is 50
% By weight, and the DHA content in the constituent fatty acids of the polar lipid was 65% by weight.

Comparative Example 2 The same algae strain as in Example 2 (ATCC34304) was inoculated in 3 liters of a medium having the composition shown in Table 1, and aerated at 25 ° C. with a jar fermenter while adjusting the pH to 6.8. did. The salt concentration was not adjusted during the culture, and 139 after the start of the culture.
At the time, the cultured algal cells were collected by centrifugation and freeze-dried to obtain 52 g of dried algal cells. 5.2 g of this dried algal cell was extracted with a mixed solvent of chloroform / methanol = 1: 1 (weight) to obtain 0.5 g of an oil component. When the extracted oil was analyzed in the same manner as in Example 1, the polar lipid content was 30% by weight and the neutral lipid content was 70% by weight. The DHA content in the total fatty acids of the extracted oil was 22% by weight, and the DHA content in the constituent fatty acids of the polar lipid was 50% by weight.

EXAMPLE 3 Crypthecodinium co
hnii) ATCC30336 was cultivated under the same conditions as in Example 1 and 100 g of the culture was started to add 30 g / 1 L of salt to increase the salt concentration in the medium by 3% by weight, and further 2
Culture was continued for 4 hours. Thereafter, the same treatment as in Example 1 was carried out to obtain 55 g of dried algae, from 5.5 g of which was 0.5.
4 g of oil was obtained. The polar lipid content in this oil content was 60% by weight, and the neutral lipid content was 40% by weight. The DHA content in the total fatty acids of the extracted oil was 46% by weight, and the DHA content in the constituent fatty acids of the polar lipid was 74% by weight.

Example 4 In Example 3, 130 after the start of culture corresponding to the stationary phase.
All were treated under the same conditions except that 3 g / L of sodium chloride was added at the time to obtain 80 g of dried alga bodies, and 8.0 g to 0.8 g of oil was obtained. Content of polar lipids in this oil: 5
The content was 1% by weight and the content of neutral lipid: 49% by weight. Also,
The DHA content in the total fatty acids of the extracted oil is 45% by weight, and the DHA content in the constituent fatty acids of the polar lipid is 70% by weight.
Met.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, DH grown in heterotrophic
In the culture of algae capable of producing A, the polar lipid concentration is high, the amount of DHA is high, and the DH
Since it is possible to obtain algal cells that accumulate a large amount of polar lipids with high A content, DHA is efficient and has a low manufacturing cost.
It becomes possible to produce the contained algae.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ identifier FI (C12P 7/64 C12R 1:89) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/12 BIOSIS ( DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for culturing algae capable of producing docosahexaenoic acid, which grows in heterotrophic culture, comprising culturing in a culture solution having a salt concentration of 1.2 to 2.5% by weight.
And the salt concentration of the culture solution in the culture is raised from 1.2 to 2.5 wt% 0.1 to 10 wt%, culturing is continued for brine concentration after rising, docosahexaenoic acid-containing, characterized in that Method of culturing algae. 2. A salt concentration of 1.2 to 2.5% by weight and a value of 0.
Time to increase to 10% by weight, the method of culturing docosahexaenoic acid-containing algae according to claim 1 which is exponential growth phase after or stationary phase of algae in culture. 3. The algae, chestnut-flops Seco di chloride (Crypthec
A single cell alga belonging to the genus odinium).
The method for culturing algae containing docosahexaenoic acid according to 1. 4. Docosahexaenoic acid grown in heterotrophic
Docosahexaenoic acid obtained by culturing viable algae
A method for producing algae containing: Cultivated in a culture solution with a salt concentration of 1.2 to 2.5% by weight
Then During cultivation, the salt concentration of the culture solution should be 1.2-2.5% by weight.
0.1 to 10% by weight increase, It is characterized in that the culture is continued at the increased salt concentration.
A method for producing algae containing docosahexaenoic acid. 5. A salt concentration of 1.2 to 2.5% by weight and a value of 0.
The time to increase 1 to 10% by weight is the index of algae in culture
The docosa according to claim 4, which is after the growth phase or in the stationary phase.
Method for producing hexaenoic acid-containing algae. 6. The algae is krypsecodinium ( Crypthec
odinium ) A unicellular alga belonging to the genus, or
5. The method for producing an alga containing docosahexaenoic acid according to 5.