JPH0965871A - Culture of maritime fine algae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably proliferate alga cell in a short time at a high density using a simple method by culturing maritime fine algae having docosahexaenoic acid-producing ability using a specific method. SOLUTION: In culturing algae (preferably Crypthecodinium cohnii ATCC 30021) belonging to maritime fine algae and having ability producing docosahexaenoic acid, a nutrient containing a carbon source and a nitrogen source is continuously or intermittently added to a culture medium during culture to control concentration of the carbon source and the nitrogen source in the culture medium. Preferably, the algae are cultured in an initial culture medium having 15-25g/l glucose concentration as the carbon source, 0.1-5g/l nitrogen source concentration, 0.1-100g/l inorganic salts concentration and 0.001-50mg/l heavy metal-containing component concentration and the above nutrient is added thereto at the point of time when algae concentration becomes >=3×10<6> cell/ml. After adding the nutrient, the carbon source content in the culture medium is preferably <=150g/l and the nitrogen source content is preferably <=15g/l.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a culturing method for favorably growing marine microalgae capable of producing docosahexaenoic acid to enhance the productivity of docosahexaenoic acid. Docosahexaenoic acid is a highly unsaturated fatty acid, which has been recently reported to have various physiological effects such as a cholesterol lowering effect, an anticoagulant effect, and a learning function improving effect.

[0002]

2. Description of the Related Art Docosahexaenoic acid, which is a highly unsaturated fatty acid that has been reported to have various physiological effects, has been studied for selective production by microorganisms and the like in search of its origin other than fish oil. Among them, it has been studied to produce docosahexaenoic acid by growing Crypthecodinium cornii, which belongs to the marine microalgae.

Since Crypthecodinium cornii is a heterotrophic organism, it is necessary to increase the concentration of nutrients such as carbon sources and nitrogen sources that can be biological components in order to increase the yield of algal bodies and docosahexaenoic acid yield. . For this purpose, a method of increasing the concentration of the nutrient source from the start of the culture and a method of newly adding the nutrient source during the culture are considered. However, in the former method, it was often observed that the culture itself became unstable, for example, the initial growth rate was decreased or the growth itself was not performed due to the high concentrations of carbon source and organic nitrogen. On the other hand, as the latter method, in the study by Martech, a method of adding a carbon source or a nitrogen source during the culture is attempted for the purpose of increasing the yield of docosahexaenoic acid (Japanese Patent Publication No. 05-503425). However, it cannot be said that algal growth is sufficient.

[0004] Some examples of the culture of Crypthecodinium cohnii are shown below.
・ Tatull et al. (See Phycologia, 14 (1), 1-8 (1975))
Reported the effects of temperature, pH, and irradiation light intensity on the generation time during culture, but did not mention the effect of newly adding a nutrient source during culture.

On the other hand, the inventors of the present invention disclosed in Japanese Patent Laid-Open No. 05-2769.
No. 63 discloses a shaking culture method and a deep aeration stirring culture method for marine microalgae capable of producing docosahexaenoic acid, and in Japanese Patent Laid-Open No. 06-253817, a specific carbon source is allowed to exist at an optimum concentration. It was shown that extremely good algal growth and docosahexaenoic acid productivity were obtained. Japanese Patent Application No. 07-29367 discloses 50 g for the purpose of improving the yield of marine microalgae.
It was shown that stable growth and high yield of algal cells can be obtained by controlling the pH value even in the presence of a relatively high concentration of carbon source exceeding 1 / l. However, it cannot be said that the initial growth rate of any of them is at a high level, and it was often observed that sufficient culture time was required to reach a certain algal cell yield.

[0006]

DISCLOSURE OF THE INVENTION The object of the present invention is to enhance the algae belonging to marine microalgae and having the ability to produce docosahexaenoic acid for the purpose of improving the yield of algal cells in a short period of time. It has been desired to develop a simple and effective culture method for stably growing algal cells at a high density (high concentration).

[0007]

Means for Solving the Problems In the present invention, as a result of intensive investigations in order to solve these problems, as a result of growing marine microalgae having the ability to produce docosahexaenoic acid for the purpose of improving the yield of algal cells, We found that stable growth of algal cells can be obtained in high density in a short period of time by culturing under the condition that new nutrients with high concentration are added continuously or intermittently to the culture medium during culturing. The present invention has been completed.

That is, the present invention relates to a method for cultivating an alga that belongs to a marine microalgae and has an ability to produce docosahexaenoic acid, wherein a nutrient solution containing a carbon source and a nitrogen source is continuously added to a culture solution during the culturing. The present invention provides a method for culturing marine microalgae in which the concentration of a carbon source and a nitrogen source in the culture medium is adjusted to be added intermittently or intermittently. further,
It is preferred to start adding nutrients when the marine microalgae in the culture reaches 3 × 10 ⁶ cells / ml. And
An alga that belongs to the marine microalgae and has an ability to produce docosahexaenoic acid is Crypthecodinium cohnii ATCC3002.
It is preferably 1.

The method of culturing of the present invention is to grow algae such as Crypthecodinium cornii as marine microalgae under the condition that a new nutrient source is added continuously or intermittently during the culture. In addition to exhibiting highly stable, reproducible and highly stable growth in a short period of time, productivity can be improved while the ratio of docosahexaenoic acid alone as a highly unsaturated fatty acid in the lipid is highly increased. It is worth noting in terms.

The microorganism used in the present invention may be any alga that belongs to marine microalgae and produces docosahexaenoic acid, such as Crypthecodinium cohnii. ATCC (American Type C
It is also possible to use publicly known ones available from various preservation organizations such as the ulture collection). As a specific example, Crypthecodinium Corney ATCC3002
1, 30543, 30556, 30571, 3067
2,30775, 50051, 50053, 5005
5, 50056, 50058, 50060, etc., but among them, Crypthecodinium Cornie ATC
C30021 is excellent in that it has a high proliferation rate in the early stage of culture of algae. In addition, the use of a mutant strain that has been subjected to a known mutation treatment such as ultraviolet irradiation or treatment with various mutagens is also included in the present invention as such a microorganism.

In the method of the present invention, for the high-density growth of marine microalgae in a short period of time, it is important to add a new nutrient source during the culture. In the method of the present invention, the medium (starting medium) used at the start of the main culture may be a medium containing the same nutrient as that to be newly added as described later. However, in the initial medium, carbon source, especially glucose and, nitrogen source, especially corn steep liquor, sodium glutamate to a low concentration below the specific concentration below, the concentration of the cultured alga body, when the specific concentration reached The culture medium is cultivated by increasing the concentration of the carbon source and the nitrogen source by adding the additive solution to the medium. Furthermore, it is preferable that the inorganic salts other than the carbon source and the nitrogen source, the heavy metal elements, and the like be kept constant before and after the addition of the additive solution, and the culture is continued. Before performing the main culture, a medium in which glucose, yeast extract or the like is added to artificial seawater, for example, a liquid medium that has been preliminarily subjected to liquid shaking culture in a medium similar to the initial medium of the following medium example 1 is used. Good.

The concentration of glucose as a carbon source in the initial medium is 150 g / l or less, further 50 g / L or less, 3
It is preferably 0 g / L or less, and particularly preferably 15 to 25 g / L from the viewpoint of alga body productivity. Further, the concentration of the nitrogen source in the initial medium is preferably 15 g / L or less, and particularly preferably 0.1 to 5 g / L from the viewpoint of productivity of algal cells. The concentration of the inorganic salts in the initial medium is preferably 0.1 to 100 g / L, and the concentration of the heavy metal-containing component is preferably 0.001 to 50 mg / L. An example of the starting medium having a preferable composition is illustrated in the starting medium of the following medium example 1.

Next, the concentration of algal cells is not less than a specific concentration, especially 3
At a time of × 10 ⁶ cells / ml or more, a new addition solution is added to the culture solution during the culture. In the method of the present invention, the addition solution is added to the culture solution to increase the concentration of nutrient sources, particularly carbon source and nitrogen source, in the culture solution, whereby the alga can be sufficiently grown.

The nutrient source contained in the newly added medium (addition liquid) used in the present invention is preferably a combination of carbon sources, nitrogen sources, inorganic salts, and components containing heavy metal elements, and May be used alone. These nutrient sources are essential as biological constituents.

Examples of the carbon source include carbohydrates such as galactose, glucose and hydrolysates of lactose, oils and fats such as fish oil and soybean oil, organic acids such as lactic acid and acetic acid,
Examples thereof include alcohols such as ethanol, and it is also possible to combine these. The content of the carbon source after the addition liquid is added is preferably 150 g / L or less in the culture liquid in terms of high productivity and not requiring pH control.

Examples of the nitrogen source include organic nitrogen such as yeast extract, beef extract, peptone, molasses and corn steep liquor, and inorganic nitrogen such as potassium nitrate, ammonium chloride and sodium glutamate, which may be combined. It is possible. The content of the nitrogen source after the addition of the additive solution is preferably 15 g / L or less in the culture solution in terms of high productivity.

As the inorganic salt, it is possible to use a commercially available concentrate of artificial seawater, but it is also possible to use a combination of sodium chloride, magnesium sulfate, calcium chloride, potassium nitrate, potassium hydrogenphosphate and the like. is there. Examples of the component containing a heavy metal element include simple substances such as iron, manganese, cobalt, and zinc, ions, chlorides, sulfates, nitrates, and various salts such as iron chloride, manganese chloride, cobalt chloride, and zinc sulfate. Is mentioned. In addition to the above, it is also possible to use boric acid or ethylenediaminetetraacetic acid, for example, to stabilize the component containing the heavy metal element.

The pH of the medium is preferably 5 to 8, for example. A buffer such as trishydroxymethylaminomethane or morpholinoethanesulfonic acid may be used for stabilizing the pH. Examples of the additive solution having a preferable composition include the additive solution of the following medium example 1. Furthermore, an additive solution containing at least one of components containing a carbon source, a nitrogen source, an inorganic salt and a heavy metal, particularly a component containing a carbon source, a nitrogen source, an inorganic salt and a heavy metal is intermittently added during the culture. Or continuously added to adjust the concentration of carbon source and nitrogen source in the culture solution, and keep the concentration of carbon source, inorganic salts other than nitrogen source, components containing heavy metals, etc. at the same level as the initial medium. It is preferable to improve the productivity of algal cells. The concentration of each component of the adjusted culture solution in the culture is, for example, 0.1 to 150 g / L of carbon source, 0.1 to 15 g / L of nitrogen source, 0.1 to 100 g / L of inorganic salts,
The content of the heavy metal-containing component is preferably 0.001 to 50 mg / L.

As a method of adding the nutrient source, either a method of continuously adding a fixed amount from the start of the culture or a method of intermittently adding a fixed amount after the start of the culture can be used. Multiple sources of nutrients may be added simultaneously.
It is preferable to start adding the nutrient source when the concentration of the marine microalgae reaches 3 × 10 ⁶ cells / ml or more. If the addition of the nutrient source is started at a time lower than this concentration, a high growth rate cannot be obtained, and the effect of adding a new nutrient source is diminished, which is not preferable.

The culture time of the entire main culture is 24 to 240 hours, although it varies depending on the conditions such as the concentration at the start of culture of the algal cells used for the culture, the composition of the medium, the amount of the medium and the like. The ratio of the culturing time from the start of culturing to the time before adding the additive solution, at the time of adding, and from the time of adding to the end of culturing is as follows: before adding: during adding: after adding =
A ratio of 1: 1: 8 to 1: 8: 1 is preferred.

The method of culturing is preferably deep aeration stirring culture, rotary shaking culture, aeration stirring culture, shaking culture and the like.

It is possible to carry out algal production at a culture temperature of usually 15 to 34 ° C. After completion of the culture, the alga body is recovered from the culture solution by a general method, for example, at 10 ° C., 80
It can be performed by a centrifugation method at 00 rpm for 10 minutes, a filtration method using a filter paper and a glass filter, or the like. In this way, the collected algal cells can be used as they are, or after freeze-drying, hot-air drying, etc., to obtain dried algal cells,
It is possible to extract crude lipids highly containing docosahexaenoic acid. As a method for extracting a crude lipid highly containing docosahexaenoic acid from an alga body, Folch
It is possible to use a general extraction method using an organic solvent such as a chloroform / methanol system represented by the method or the Bligh-Dyer method.

Purification of docosahexaenoic acid from crude lipid can be carried out by a conventional method. For example, the crude lipid is saponified with NaOH or the like and then is used as it is, or is converted into an alcohol ester by an acid or alkali catalyst, followed by column chromatography or fractionation, distillation,
It can be easily obtained as a pure product by a method such as supercritical extraction. This is because algae do not contain docosahexaenoic acid and polyunsaturated fatty acids that have very similar physical properties at the same time, which makes docosahexaenoic acid much easier and more efficient than conventional refining from fish oil. It is possible to obtain.

As described above, according to the present invention, when a marine microalga having the ability to produce docosahexaenoic acid is cultivated for the purpose of improving the yield of algal cells, a condition in which a nutrient source is newly added during culturing It was found that stable culture of algal cells can be obtained in a high density in a short period of time by culturing at 1. However, the modifications that are usually performed according to the gist of the present invention are included in the present invention.

[0025]

[0026]

EXAMPLES The present invention will be described in more detail with reference to examples below, but it goes without saying that these examples do not limit the scope of the present invention.

In the following examples, the algal cell productivity of marine microalgae is shown by the weight of the dried algal cells after culturing, and the content of docosahexaenoic acid in the dried algal cells is chloroform /
The crude lipid extracted with methanol (2: 1) was converted to a fatty acid methyl ester with a boron trifluoride methanol complex, and docosahexaenoic acid produced using heptadecanoic acid as an internal standard was quantified by gas chromatography for measurement.

(Example 1) Using the media shown in Table 1 below,
3 L of the medium shown in Table 1 below was placed in a 5 L jar fermenter for sterilization. After cooling, glucose 1 was added to this medium.
A culture solution of Crypthecodinium cornii ATCC30021, which was prepared by dissolving 0 g / l and 2 g / l of yeast extract in artificial seawater aquamarine (manufactured by Yasu Pharmaceutical Co., Ltd.) and preliminarily shaking for 5 days in a medium with liquid shaking. 300 ml was inoculated and the main culture was carried out at 28 ° C for 7 days with a stirring speed of 25.
Deep aeration agitation culture was carried out at 0 rpm, an aeration amount of 0.67 vvm, and without controlling the pH value during the culture. From 30 hours after the start of culture (algal cell concentration 5.0 × 10 ⁶ cells / L), 500 mL of the additive solution shown in Table 1 was added in equal amounts over 5 hours every 6 hours during 38 hours. The yield of dried algal cells obtained from the cultured algal cells was as shown in Table 1.

Example 2 Using the medium shown in Table 1 below,
Deep aeration agitation culture was performed in the same manner as in Example 1 except that 500 mL of the additive solution shown in Table 1 was continuously added from 24 hours after the start of culture. The yield of dried algal cells obtained from the cultured algal cells was as shown in Table 1.

(Comparative Examples 1 to 4) Deep aeration agitation culture was carried out in the same manner as in Example 1 except that the medium shown in Table 2 below was used and no new medium was added after the start of the culture. It was The yield of dried algal cells obtained from the cultured algal cells was as shown in Table 2.

Example 3 100 mL of the medium shown in Table 3 was placed in a 300 mL Erlenmeyer flask for sterilization. After cooling, glucose 10 g / l, yeast extract 2 was added to this medium.
g / l artificial seawater aquamarine (manufactured by Yasu Pharmaceutical Co., Ltd.)
Crypthecodinium cornii ATCC3, which was cultivated with liquid shaking in a medium adjusted to pH 7.4 for 5 days in advance with liquid shaking
10 ml of the culture solution of 0021 was inoculated to obtain 2 main cultures.
The culture was carried out at 8 ° C. for 7 days with stirring at a rotation speed of 200 rpm without controlling the pH value during the culture. After 30 hours from the start of the culture, 15 ml of the additive solution shown in Table 3 was added every 6 hours in 5 batches. The yield of dried algal cells obtained from the cultured algal cells was as shown in Table 3.

Comparative Example 5 Rotational shaking culture was carried out in the same manner as in Example 3 except that the medium shown in Table 3 below was used and no new medium was added after the start of the culture. The yield of dried algal cells obtained from the cultured algal cells was as shown in Table 3.

[0033]

[0034]

[0035]

[0036]

According to the culture method of the present invention, a marine microalga having the ability to produce docosahexaenoic acid is cultured under the condition that a nutrient source is newly added during the culture for the purpose of improving the yield of algal cells. It was found that a stable growth of high-density algal cells can be obtained in a short period of time. Conventionally, the supply of raw materials was unstable and the quality was not constant, and extraction from fish oil with a unique odor and advanced Docosahexaenoic acid obtained by various separation and purification techniques can be stably produced at a high concentration, and a very simple separation and purification technique can supply highly pure products, which is an industrially effective effect. .

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Claims (3)

[Claims] 1. A method for culturing an alga that belongs to a marine microalgae and has an ability to produce docosahexaenoic acid, wherein a nutrient solution containing a carbon source and a nitrogen source is continuously or intermittently added to a culture solution during the culture. The method for culturing marine microalgae is characterized in that the concentration of the carbon source and the nitrogen source in the culture solution is adjusted by adding the same. 2. The method for culturing marine microalgae according to claim 1, wherein when the marine microalgae in the culture solution reaches 3 × 10 ⁶ cells / ml, the nutrient source is added. 3. An alga that belongs to the marine microalgae and has an ability to produce docosahexaenoic acid is Crypthecodinium cohnii A.
The method for culturing marine microalgae according to claim 1 or 2, which is TCC30021.