JPH0787960A - Method for culturing marine fine algae - Google Patents

Abstract

PURPOSE:To efficiently culture marine fine algae stably at a high concentration without containing highly unsaturated fatty acids by culturing, in a medium in the presence of acidic amino acid (salt), etc., marine fine algae capable of producing docosahexaenoic acid. CONSTITUTION:In producing docosahexaenoic acid from algae proliferated by culturing algae belonging to marine fine algae and capable of producing docosahexaenoic acid such as Cryptecodinium conii ATCC 3021, the culture is conducted in a medium in the presence of at least one kind selected from acidic amino acids (salts 2), basic amino acids (salts), amides thereof and imides thereof (e.g. sodium aspartate, sodium glutamate, lysine).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culturing method for favorably growing marine microalgae capable of producing docosahexaenoic acid and enhancing 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 polyunsaturated fatty acid that has been reported to have various physiological effects, has been investigated for its origin other than fish oil and for selective production by microorganisms. Above all, it has been conventionally studied to produce docosahexaenoic acid by growing Crypthecodinium cornii, which belongs to the marine microalgae.

The medium of marine microalgae, such as Crypthecodinium cornii, has different physiological properties depending on the place where it is collected, and seawater which forms a precipitate by sterilization is basically added to this nutrient substance which is easily deficient. Synthetic media that do not form precipitates and that also guarantee reproducibility of experiments by autoclaving are preferred over natural media.

[0004] Some examples of the treatment of Crypthecodinium cohnii are given. In the study by Earl James Henderson et al., A synthetic medium, AXM medium, was used. The biosynthesis of ¹⁴ C-labeled docosahexaenoic acid has been reported (Phytochemistry, 27 (6), 1679-1.
683 (1988)) only shows the proportion of docosahexaenoic acid in the triglyceride fraction and the phospholipid fraction, and does not mention the actual yield of docosahexaenoic acid.

Further, in the study by R.C.Tatull et al., Aiming at optimization of the growth rate of algal cells, the amount of the components was optimized based on MLH medium which is a synthetic medium, and the generation time was changed. Effects have been reported (Phycol
Ogia, 14 (1), 1-8 (1975)), but do not mention the effects and effects on the composition and content of fatty acids such as docosahexaenoic acid.

[0006] Further, in the study by Martech, for the purpose of increasing the yield of docosahexaenoic acid, it was reported that the culture was carried out in a medium based on natural seawater or artificial seawater and containing glucose and yeast extract (WO91 / 1191).
8). However, no mention is made of the effects of individual components of the synthetic medium on algal growth and docosahexaenoic acid accumulation.

On the other hand, the inventors of the present invention have filed Japanese Patent Application No. 04-344.
In No. 279, stable growth of algal cells of marine microalgae and high productivity of docosahexaenoic acid were achieved by using a medium containing essential components containing specific sugars, organic nitrogen sources, inorganic salts and heavy metal elements. Indicated.

[0008]

The object of the present invention is to stably grow an alga that belongs to a marine microalgae and has the ability to produce docosahexaenoic acid, and extract from the algal body of the alga. It is intended to provide a medium having a simple and effective composition for increasing the content of docosahexaenoic acid in lipids.

[0009]

Means for Solving the Problems In the present invention, as a result of extensive studies to solve these problems, algae which belong to marine microalgae and have the ability to produce docosahexaenoic acid were identified as α-amino acids. Acidic amino acids,
By culturing in a medium in the presence of its salt or its amide, basic amino acid or its salt, or imino acid, it was found that the stable growth of algal cells and the content of docosahexaenoic acid in the lipid significantly increase, The present invention has been completed.

That is, the present invention, when producing docosahexaenoic acid from an algal body grown by culturing an alga that belongs to a marine microalgae and has the ability to produce docosahexaenoic acid, Provided is a method for culturing marine microalgae, which comprises culturing in a medium in the presence of acidic amino acids or salts thereof, basic amino acids or salts thereof, amides thereof, or imino acids. Further, it is preferable that the α-amino acids are sodium aspartate, sodium glutamate, asparagine or glutamine. The α-amino acids are preferably lysine, arginine or arginine hydrochloride. Then, as the α-amino acids, sodium glutamate at a concentration of 2.0 to 30.0 mM and asparagine at 1.0
It is preferred that arginine hydrochloride is present at a concentration of ˜20.0 mM or arginine hydrochloride at a concentration of 0.25 to 10.0 mM. Also, the imino acid is preferably proline.
Further, it is preferable that the marine microalgae is Crypthecodinium cornii ATCC30021.

As the marine microalgae used in the culturing method of the present invention, strains belonging to Crypthecodinium cornii and the like, acidic amino acids, salts or amides thereof, basic amino acids or salts thereof as α-amino acids, Alternatively, culturing in a medium in the presence of imino acid not only shows very good growth of algal cells, but also it is possible to highly increase the proportion of docosahexaenoic acid in the lipid as a highly unsaturated fatty acid, and at the same time improve productivity. It should be noted that it can be improved.

The present invention will be described in detail below. The alga used in the present invention may be any as long as it belongs to a marine microalgae and produces docosahexaenoic acid, for example, Crypthecodynium cohnii (Crypth
ecodinium cohnii) and the like.

These are ATCC (American Type Cultur)
It is also possible to use publicly known ones available from various preservation organizations such as e Collection). Specifically, Crypthecodinium cohnii
ATCC30021, 30543, 30556, 30571, 30572, 30775, 5005
1, 50053, 50055, 50056, 50058, 50060 and the like. Among them, Crypthecodinium cohnii ATCC 30021 is preferable because it has a high DHA content. In addition, the use of a mutant strain obtained by subjecting the microorganism to known mutation treatment such as ultraviolet irradiation or treatment with various mutagens is also included in the present invention.

It is essential that the medium used in the culture method of the present invention contains α-amino acid as an essential component.
And, as an α-amino acid, an acidic amino acid or a salt thereof, a basic amino acid or a salt thereof, an amide thereof,
Alternatively, an imino acid may be used.

Examples of acidic amino acids or salts thereof include glutamic acid, sodium glutamate, aspartic acid,
Sodium aspartate and the like can be mentioned. Basic amino acids or salts thereof include lysine, lysine hydrochloride,
Examples include arginine, arginine hydrochloride, histidine and the like. Examples of these amides include glutamine and asparagine. Examples of the imino acid include proline and the like.

One of these α-amino acids or imino acids may be present alone or two or more of them may be present together in the medium used in the culture method of the present invention. When the content of these α-amino acids or imino acids in the medium is sodium glutamate, the content is 2.0 to
It is preferably 30.0 mM. If it is less than 2.0 mM, it is difficult to obtain a high yield of algal cells, and therefore it is difficult to obtain docosahexaenoic acid, and if it exceeds 30.0 mM, the growth is significantly slowed or the amount of lipids produced by algal cells is low. This is not preferable because the yield of docosahexaenoic acid is greatly reduced. Further, since it is an expensive amino acid, it is preferably 30.0 mM or less in consideration of the economical viewpoint.

In the case of asparagine, 1.0-20.
It is preferably 0 mM. If it is less than 1.0 mM, a high yield of algal cells cannot be obtained. Therefore, it is difficult to obtain docosahexaenoic acid, and if it exceeds 20.0 mM, the growth is remarkably slowed or the amount of lipids produced by algal cells is increased. This is not preferable because the yield of docosahexaenoic acid is greatly reduced. Further, since it is an expensive amino acid, it is preferably 30.0 mM or less in consideration of the economical viewpoint.

In the case of arginine hydrochloride, 0.25-
It is preferably 10.0 mM. If it is less than 0.25 mM, it is difficult to obtain a high yield of algal cells, and therefore it is difficult to obtain docosahexaenoic acid, and if it exceeds 10.0 mM, the growth is remarkably slowed or the amount of lipids produced by algal cells is reduced. This is not preferable because the yield of docosahexaenoic acid is greatly reduced. Further, since it is an expensive amino acid, it is preferably 30.0 mM or less in consideration of the economical viewpoint.

The medium used in the culture method of the present invention may further contain an organic nitrogen source, sugars, inorganic salts, heavy metal element-containing substances and the like.

As the organic nitrogen source used in the present invention,
Various extracts such as yeast extract, beef extract and peptone,
Agricultural product residues such as molasses and corn steep liquor are listed, and it is possible to combine these.

The sugars used in the present invention include glucose and galactose, and it is possible to combine them.

As the inorganic salt used in the present invention, it is possible to use a commercially available artificial seawater concentrate, but it is also possible to use a combination of, for example, sodium chloride, magnesium sulfate and the like.

Examples of the heavy metal element-containing substance used in the present invention include substances containing each element of iron, manganese, cobalt and zinc.
It may be a salt, a hydrate or the like.

In addition to the above, addition of boric acid or ethylenediaminetetraacetic acid is preferable for stabilizing heavy metal salts, although it does not directly affect the growth of algal cells and the yield of docosahexaenoic acid. Further, as a defoaming agent, fatty acid monoglyceride, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester or the like may be added.

The pH of the medium used in the present invention is usually 5 to 5.
9, preferably 6-8. For stabilizing the pH, it is also preferable to add a buffering agent such as trishydroxymethylaminomethane or morpholinoethanesulfonic acid.

As the culturing method used in the present invention, a static culturing method can be used. However, considering the algal cell productivity of microalgae and the content of docosahexaenoic acid in lipids, the shaking culturing method or deep aeration is used. Culture by the stirring culture method is preferred. Shaking culture may be reciprocal shaking or rotary shaking. Shaking culture and deep aeration stirring culture method,
For example, the method described in Japanese Patent Application No. 04-077189 can be used. It is possible to produce algal cells at a culture temperature of usually 15 to 34 ° C.

When the marine microalgae are cultivated by the culturing method of the present invention as described above, not only the stable growth is shown, but also the proportion of docosahexaenoic acid as a highly unsaturated fatty acid in the lipid is extremely high. Algae are obtained. After the completion of the culture, the recovery of the algal cells from the culture solution as a method for obtaining such crude lipid is carried out by a general method, for example, 10 ° C., 8
It can be carried out by a centrifugation method at 000 rpm for 10 minutes, a filtration method using a filter paper and a glass filter, or the like. The algal cells collected in this way can be used as they are,
Alternatively, a dried alga can be obtained by a freeze-drying method, a hot-air drying method, or the like. From the obtained algal cells or their 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 algal body, an ordinary lipid extraction method can be used. In particular, the Folch method or Bligh-Dyer method is used.
It is possible to use a general extraction method using an organic solvent such as chloroform / methanol system represented by the method.

Purification of docosahexaenoic acid from the crude lipid can be carried out by a conventional method. For example, after the crude lipid is saponified with NaOH or the like, it can be used as it is, or can be converted into an alcohol ester by an acid or alkali catalyst to perform column chromatography or fractionation,
It can be easily obtained as a pure product by a method such as distillation or supercritical extraction. This is because the algal cells do not contain docosahexaenoic acid and highly unsaturated fatty acids that have very similar physical properties at the same time.Compared with conventional purification from fish oil, docosahexaenoic acid is very simple and efficient. It is possible to obtain the acid.

As described above, according to the present invention, when culturing marine microalgae, acidic amino acids, salts thereof or amides thereof, basic amino acids or salts thereof, or imino acids are used as α-amino acids. By culturing in an existing medium, it is possible to increase the content of docosahexaenoic acid with stable growth of algal cells and other polyunsaturated fatty acids, but it is usually carried out in accordance with the gist of the present invention. Modifications are included in the present invention.

[0031]

The present invention will be described in more detail with reference to the following examples, 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 represented by the weight of the dried algal cells after culturing, and the content of docosahexaenoic acid was calculated from the dried algal cells by chloroform / methanol (2: 1). )
The crude lipids extracted in step 1 were converted into fatty acid methyl esters with a boron trifluoride methanol complex, and docosahexaenoic acid produced using heptadecanoic acid as an internal standard was quantified by gas chromatography.

Examples 1 to 7 and Comparative Examples 1 to 6 are shown in Table 1 below.
The medium (I) described in 1. was used.

(Examples 1 and 2) Each of the acidic amino acids shown in Table 2 below was added to the medium (I) shown in Table 1 above at 2.67 m.
100 ml of each of the medium contained so as to give M
It was put in a 300 ml Erlenmeyer flask and sterilized. After cooling, glucose 10g / l, yeast extract (Dif
Co.) 2 g / l was dissolved in artificial seawater aquamarine (manufactured by Yasu Pharmaceutical Co., Ltd.) and cultivated at 28 ° C. for 7 days in a culture medium adjusted to pH 7.4.
Inoculate 5 ml of the culture solution of Cornie ATCC30021,
As main culture, spin shaking culture at 180C for 5 days (180 r
pm) was performed. The dry algal weight, crude lipid yield and docosahexaenoic acid yield obtained from the cultured alga were obtained as shown in Table 2.

(Examples 3 and 4) The medium (I) shown in Table 1 above was mixed with the acidic amino acid amide shown in Table 2 below.
The culture was performed in the same manner as in Examples 1 and 2 except that the medium contained at a concentration of 67 mM was used.
The results shown in are obtained.

(Examples 5 and 6) In the medium (I) shown in Table 1 above, the basic amino acids shown in Table 2 below were added to 2.67.
Culturing was carried out in the same manner as in Examples 1 and 2 except that the medium contained at a concentration of mM was used, and the results shown in Table 2 were obtained.

Example 7 Medium (I) shown in Table 1 above
Example 1 except that the medium containing the imino acid shown in Table 2 below at a concentration of 2.67 mM was used.
Culturing was carried out in the same manner as in Examples 1 and 2, and the results shown in Table 2 were obtained.

Comparative Examples 1 to 6 Aliphatic amino acids, hydroxyamino acids, sulfur-containing amino acids, aromatic amino acids, and heterocyclic amino acids shown in Table 2 below were added to the medium (I) shown in Table 1 above at 2.67 mM. The culture was carried out in the same manner as in Examples 1 and 2 except that a medium containing the above-mentioned concentration was used, and the results shown in Table 2 were obtained.

[0038]

[Table 1]

Note) Corn steep liquor: manufactured by Wako Pure Chemical Industries, Ltd. −: Indicates that it is not contained.

(Examples 8 to 11) 100 ml of each medium containing sodium glutamate at the concentration shown in Table 3 below.
Was placed in a 300 ml Erlenmeyer flask for sterilization. After cooling, glucose 10g / l, yeast extract (Di
2 g / l (manufactured by fco) was dissolved in artificial seawater aquamarine (manufactured by Yasu Pharmaceutical Co., Ltd.), and the medium was adjusted to pH 7.4.
5 ml of a culture solution of Crypthecodinium cornii ATCC30021 previously statically cultured at 28 ° C. for 7 days was inoculated, and the main culture was carried out at 28 ° C. for 5 days by rotary shaking culture (18
0 rpm) was performed. The dry algal weight and docosahexaenoic acid content obtained from the cultured algal bodies were as shown in Table 3.

Comparative Example 7 As shown in Table 3 below, the culture was carried out in the same manner as in Examples 8 to 11 except that a medium having a composition containing no glutamic acid was used as the organic nitrogen source. I got the result. The result of Comparative Example 7 is
It is also described in Tables 4 and 5 for comparison with Examples 12 to 15 and Examples 16 to 19.

(Examples 12 to 15) Culture was performed in the same manner as in Examples 8 to 11 except that a medium containing asparagine at the concentration shown in Table 4 below was used instead of sodium glutamate, and the results shown in Table 4 were obtained. Got

(Examples 16 to 19) Cultures were carried out in the same manner as in Examples 8 to 11 except that a medium containing arginine hydrochloride at the concentration shown in Table 5 below was used instead of sodium glutamate. The results shown were obtained.

[0044]

[Table 2]

[0045]

[Table 3]

[0046]

[Table 4]

[0047]

INDUSTRIAL APPLICABILITY In the method for culturing marine microalgae of the present invention, algae having an ability to produce docosahexaenoic acid are used as α-amino acids, and acidic amino acids, salts thereof or amides thereof, basic amino acids or salts thereof, Alternatively, by culturing in a medium in which imino acid is present, the growth of algal cells can be stably performed and the content of docosahexaenoic acid can be significantly increased without containing other polyunsaturated fatty acids such as eicosapentaenoic acid. Furthermore, the supply of raw materials is unstable and the quality is not constant, and docosahexaenoic acid, which was obtained by extraction from fish oil with a unique odor and advanced separation and purification technology, can be stably produced at a high concentration, and, This is an industrially effective effect in that highly pure products can be supplied by a very simple separation and purification technique.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:89)

Claims (6)

[Claims] 1. When producing docosahexaenoic acid from an algal body grown by culturing an alga that belongs to a marine microalgae and has an ability to produce docosahexaenoic acid, α
-A method for culturing marine microalgae, which comprises culturing in a medium in which acidic amino acids or salts thereof, basic amino acids or salts thereof, amides thereof, or imino acids are present as amino acids. 2. The method for culturing marine microalgae according to claim 1, wherein the α-amino acid is sodium aspartate, sodium glutamate, asparagine or glutamine. 3. The method for culturing marine microalgae according to claim 1, wherein the α-amino acid is lysine, arginine or arginine hydrochloride. 4. The imino acid is proline.
The method for culturing marine microalgae according to 1. 5. As the α-amino acids, sodium glutamate at a concentration of 2.0 to 30.0 mM, asparagine at a concentration of 1.0 to 20.0 mM, or arginine hydrochloride at 0.25 to 10. The method for culturing marine microalgae according to any one of claims 1 to 3, wherein the method is present at a concentration of 0 mM. 6. The marine microalgae is Crypthecodinium cornii ATCC 30021.
The method for culturing marine microalgae according to any one of 1.