JPH09284A - Novel microorganism capable of producing docosahexaenoic acid and production of docosahexaenoic acid with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for producing oil and fat containing docosahexaenoic acid and docosahexaenoic acid in a higher yield than that of the conventional processes by using a microorganism having high ability to produce docosahexaenoic acid in no need of complicated process in extremely high efficiency. SOLUTION: Schizochytrium SR21 strain is a microorganism having the ability to produce oil and fat containing docosahexaenoic acid and the content of the docosahexaenoic acid is <=1.0wt.%. This microorganism is cultured in a medium to collect oil and fat containing docosahexaenoic acid. Docosahexaenoic acid is produced by separating the acid from the oil and fat.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel microorganism capable of producing docosahexaenoic acid (DHA), and a method for producing docosahexaenoic acid-containing fats and oils and a microorganism using docosahexaenoic acid.

[0002]

2. Description of the Related Art Docosahexaenoic acid is a highly unsaturated fatty acid present specifically in the brain and retina of animals, and plays an important physiological role in these organs, as well as an anti-inflammatory effect and a blood cholesterol lowering effect. It also has the physiological activity of For this reason, docosahexaenoic acid is a useful substance that has attracted attention in the fields of medicine and food, and in recent years, its use has also been expanded in the field of functional foods such as health foods and baby milk.

[0003] Docosahexaenoic acid is contained in fish oil belonging to the blue fish, and in particular, oil derived from sardines and tuna contains about 20%. A major problem when using fish-derived docosahexaenoic acid-containing fats and oils in the food field is that a great deal of operation is required to remove fish odor. In addition, since fish-derived docosahexaenoic acid-containing fats and oils contain various highly unsaturated fatty acids such as arachidonic acid and icosapentaenoic acid (EPA), they are easily oxidized and it is difficult to obtain stable quality fats and oils. Furthermore, when docosahexaenoic acid is used in the field of pharmaceuticals, etc., it is necessary to separate and purify docosahexaenoic acid from docosahexaenoic acid-containing fats and oils, but it contains various highly unsaturated fatty acids similar in structure to docosahexaenoic acid. Therefore, separation and purification are difficult. In particular, in the preparation of infant milk, docosahexaenoic acid-containing oils and fats having a low content of icosapentaenoic acid are desirable, but when the source is fish oil, it is extremely difficult to efficiently remove only icosapentaenoic acid.

[0004] As a source of docosahexaenoic acid other than fish oil, a production method using microorganisms has been considered. Microorganisms that produce docosahexaenoic acid-containing fats and oils include Vibrio marinus, a bacterium isolated from the deep sea.
s) (ATCC 15381) and Vibrio spp. The algae Cyclotella cryptica
tica), and the production of docosahexaenoic acid-containing fats and oils by a culture method using these microorganisms has been studied (PKBajapai, P.Bajapai and OPWard, J. A.
m. Oil Chem. Soc., 68: 509 (1991) and JP-A-1-199588.
(See the official gazette). However, according to the conventionally known method using microorganisms, the production amount of docosahexaenoic acid-containing oil / fat per liter of medium is as small as about 100 to 700 mg. Therefore, the production amount of docosahexaenoic acid remains at an extremely low level of several tens to 500 mg per liter of medium.

[0005] The reason why the productivity of docosahexaenoic acid-containing oils and fats by the conventionally known microorganisms is extremely low is that the growth of these bacteria is extremely low. In other words, these bacteria have a low growth rate and a low concentration of the cells. The low growth of the bacteria is related to the low nutrients of these microorganisms, that is, they are susceptible to growth inhibition by substrates, so that the concentration of a carbon source such as glucose must be reduced to 1 liter of medium. 20 per
g, and can only be as low as about g, and the bacteria cannot be efficiently grown. As a result of the low bacterial growth, production of docosahexaenoic acid-containing fats and oils is also limited. In addition, the accumulation of docosahexaenoic acid-containing fats and oils in the cells of conventionally known microorganisms is low, that is, the content of docosahexaenoic acid-containing fats and oils is limited to at most 20% by weight per dry bacterial cell. Is one of the factors.

[0006] The content of icosapentaenoic acid in fats and oils obtained by using conventionally known microorganisms is relatively low as compared with fish oil, but is still as high as several weight% or more. Difficult to purify.

As described above, an industrial production method of docosahexaenoic acid using a microorganism having an ability to produce docosahexaenoic acid has not yet been established. Further, there is still no known microorganism capable of producing docosahexaenoic acid with high productivity and usable for industrial production.

[0008]

An object of the present invention is to provide a microorganism having a high docosahexaenoic acid-producing ability. Another object of the present invention is to provide a method for producing docosahexaenoic acid-containing fats and oils having a lower icosapentaenoic acid content ratio in a high yield without using a complicated process at a high yield by using a microorganism having a docosahexaenoic acid-producing ability. Is to provide. Another object of the present invention is to provide a method for efficiently producing docosahexaenoic acid.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, they have excellent proliferating properties and oil / fat accumulating properties, and have a low icosapentaenoic acid content,
A new microorganism having a high docosahexaenoic acid-containing fat / oil producing ability has been found, and it has been found that docosahexaenoic acid-containing fats and oils and docosahexaenoic acid can be efficiently produced using such a microorganism,
The present invention has been completed.

The present invention is the SR21 strain of the genus Schizochytrium, which is capable of producing docosahexaenoic acid.
In addition, the present invention relates to a microorganism belonging to the genus Schizochytrium and having a docosahexaenoic acid-containing oil / fat producing ability, wherein the microorganism having an icosapentaenoic acid content of 1.0% by weight or less in the fat / oil is used as a medium. A method for producing the docosahexaenoic acid-containing oil or fat, wherein the docosahexaenoic acid-containing oil or fat is collected from the culture. Furthermore, the present invention is a microorganism belonging to the genus Schizochytrium and having a docosahexaenoic acid-containing fat and oil producing ability, wherein the content ratio of docosahexaenoic acid to all n-3 fatty acids contained in the fat is 98% by weight or more. A method for producing a docosahexaenoic acid-containing oil or fat, which comprises culturing a microorganism capable of producing oil or fat in a medium, and collecting the docosahexaenoic acid-containing oil or fat from the culture. Furthermore, the present invention is a method for producing docosahexaenoic acid, characterized by separating docosahexaenoic acid from any of the above-mentioned fats and oils containing docosahexaenoic acid.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the method for producing docosahexaenoic acid-containing fats and oils of the present invention belongs to the genus Schizochytrium and has the ability to produce docosahexaenoic acid-containing fats and oils. Has an oil / fat producing ability with a content ratio of 1.0% by weight or less, and / or
Any strain may be used as long as it has a fat and oil producing ability in which the content ratio of docosahexaenoic acid to fatty acids is 98% by weight or more. Specifically, for example, the novel microorganism of the present invention, Schizochytrium genus SR21, can be used.

The Schizochytrium sp. SR21 strain has been isolated from seawater off the coast of Yap Island in the Federated States of Micronesia. The mycological properties of the Schizochytrium sp. SR21 strain are as follows.

The mycological properties of the SR21 strain were examined by the following two methods. First, a nutrient medium in which 2 g of glucose, 0.2 g of yeast extract and 0.5 g of sodium glutamate are added to 1 liter of artificial seawater (tropic marine) is placed in a small petri dish, and a pre-flask culture solution of the SR21 strain in the same medium is added.
Drops were inoculated and cell morphology was followed by an inverted microscope. In this case, the release of amoebic atypical cells was observed. Next, the same tracking was performed in filter-sterilized natural seawater. In this case, release of amoeba-like amorphous cells was not observed, and release of zoospores was observed from some cells of the vegetative cell mass after repeating two divisions. Alternatively, it was also observed that one vegetative cell directly differentiated into zoospores without undergoing two divisions.

Glutaraldehyde was added at 10% by volume to a sample from which zoospores were frequently released, and the zoospores were observed with an optical microscope. FIG. 1 is an optical micrograph showing the morphology of zoospores of Schizochytrium sp. SR21. Furthermore, electron microscope observation of the flagella was performed by a negative staining method using uranium acetate. FIG. 2 is a transmission electron micrograph showing the structure of flagella of zoospores of Schizochytrium sp. SR21. Figure 1 shows two flagella with different lengths.
Shows a three-part structure consisting of the base, shaft and apical hair of the flagella mastagonema. In addition, in the cell morphology observation by the above two inverted microscopes, it was found that the vegetative cells repeated two divisions to form a cell cluster and a cytoplasmic network. FIG. 3 is an optical micrograph showing a network of vegetative cell clusters and cytoplasm of Schizochytrium sp. SR21.

The colony formed by the SR21 strain on the agar plate medium has the same smooth ocher color as the yeast colony. When the SR21 strain is grown in a liquid medium, the turbidity of the culture solution increases due to the growth, but zoospores having two flagella with different lengths are observed in the initial stage (FIG. 1). Two
Among the flagella of the book, the hairy structure (mastigonema) in the long flagella takes on a three-part structure consisting of a base, an axis, and apical hair (Fig. 2). Thus, the SR21 strain has the chromista kingdom (Kingdom Chromista),
It belongs to the unequal hair gate (Phylum Heterokonta). Furthermore, the SR21 strain from the network-forming properties of the protoplasm and the scales derived from the Golgi apparatus belong to the class Labyrinthulea (Order Labyrinthulida). And it is clear that the SR21 strain belongs to the family Thraustochytriidae because the vegetative cells are spherical or oval and there is no gliding movement in the cytoplasmic network. Further, the vegetative cells of the SR21 strain repeat two divisions to form 8-32 vegetative cell clusters. Thereafter, the amoebic amorphous cells are released from some cells, gradually dissociate from the cell mass and become spherical cells after 1-2 hours. The spherical cells then differentiate into 8 to 16 zoospores as zoospores. At that time, the membrane of the zoosporangium is not observed. In addition, there are cells that differentiate directly into zoospores from one vegetative cell without dividing into two cells, or differentiate into zoospores without passing through amorphous cells after dividing into two cells into a vegetative cell mass. Has a complex life cycle.

The Thraustochytrium family is a porter (D. Po
rter, “Handbook of Protoctista”, Jones and Bartl
According to ett Publishers (1990)), it consists of 30 species of 7 genera.
Later, the genus Corallochytrium (Raghu
kumar, S., Botanica Marina, 30: 83 (1987)) and Moss, ST, “The Biology of Free-living
Heterotrophic lagellates ”, Oxford University Pr
According to ess (1991)), there are 33 species of 8 genera. The characteristics of the genus Thraustrium 8 are as follows. The vegetative cells of the genus Labyrinthuloides are spherical, but glide irregularly on the protoplasmic network. Also,
The genus Aplanochytrium is propagated by immobilized spores, ie spores without flagella. Arsonia
The genus (Althornia) has no protoplasmic network and is planktonic. The genus Japonochytrium produces extracellular apophysis. Ulkenia
The genus ia) differentiates into zoospores after the release of amoeboid amorphous cells from the zoospores. Also, Thraustochytrium
In the genus (Thraustochytrium), one zoospore becomes one vegetative cell, which forms one zoosporangium. In addition, the genus Schizochytrium (Schizochytrium) divides after one zoospore has formed, forming a plurality of vegetative cell masses,
Each becomes a zoospore. Corallochitorium
The genus chytrium forms spore-like spores and does not form flagellar zoospores.

Of the above eight genera, the genus Urkenia was described in 1977 by Gelertner (Gaertner, A., Veroff. Inst. M.
According to eeresforsch. Bremerh., 16: 139 (1977), a trait that differentiates into zoospores after the release of naked plasma masses (amoeba-shaped amorphous cells) from zoospores is used as a genus classification criterion. , Two genera that have been classified into the genus Thraustochytrium, Thraustochytrium visurgence (Thraustochytr
ium visurgense) (Ulken, A., Veroff. Inst. Meeresfor
sch. Bremerh., 9: 289 (1965)) and Thraustochytrium amoeboidum (Ba
hnweg, O. and Sparrow, FK, Jr. Am. J. Bot., 61: 754.
(1974)) to the genus Ulkenia, and to them, a new species, Ulkenia Minuta (Ulken
ia minuta) (Raghukumar, S., Veroff. Inst. Meeresfo
rsch. Bremer., 16: 159 (1977)) and three new species were combined to propose a new genus, Ulkenia, as six species of the genus Urkenia.

However, since then, no paper has been described describing a new species of the genus Urkenia. Curling
ing, JS, "Predominantly Holocarpic and Eucarpic
Simple Biflagellate Phycomycetes ", J. Cramer (198
1)) questioned whether the genus Urkenia would be formed as an independent genus and listed it as tentative. However, the above-mentioned description is described in the documents of Porter and Moss.

Since the SR21 strain forms amoebic amorphous cells, it is considered that it belongs to the genus Ulkenia if its traits are emphasized. However, lag coomers are Thraustochytrium sp.
(Thraustochytriumstriatum) have reported that a nutrient medium forms amoebic amorphous cells that prey on bacteria (Raghukumar, S., Marine Biology, 113:
165 (1992)). In addition, Ragcoomer is a new species of the genus Schizochytrium,
hytrium mangrovei) formed amoebic amorphous cells in a nutrient medium, but did not form amoebic amorphous cells when cultured in a nutrient-diluted medium containing only pine pollen in seawater (Raghukumar , S., Trans. Br. M
ycol. Soc., 90: 627 (1988)). Therefore, it was necessary to investigate this trait using a reference medium, because the trait that forms amoebic amorphous cells is affected by the composition of the medium and the culture conditions. As the reference medium, the above-mentioned seawater / pine pollen medium, which has been conventionally and often used, and often used for observing morphological characteristics in the original descriptions of genera and species. Are listed. All six species classified in the genus Ulkenia are known to form amoebic amorphous cells in this seawater / pine pollen medium. On the other hand, Thraustochytrium striatum and Schizochytrium mangrovii form amoebic amorphous cells in nutrient medium as described above, but they do not form amoeba-like amorphous cells in seawater / pine pollen medium. Not. Based on the above, the SR21 strain forms amoebic atypical cells in the nutrient medium, but this was not observed in the medium containing only seawater, so it may not be appropriate to classify it in the genus Ulkenia.

On the other hand, after one zoospore has settled, the vegetative cell repeats two divisions to form a plurality of vegetative cell masses,
The traits of zoosporangium are stable regardless of the medium composition, and are traits that are always observed in the life cycle of SR21 strain. The properties observed in SR21 strains, including this trait, are described by Goldstein, S. and Belsky,
M., Am. J. Bot., 51:72 (1964)), and Boots et al.
h, T. and Miller, CE, Can. J. Bot., 47: 2051 (1969)).
Does not conflict with the description of the genus Schizochytrium as reported by Therefore, it is judged that it is appropriate to classify the SR21 strain as Schizochytrium.

At present, the following four species are described in the literature within the genus Dizochytrium (Goldstein, S and Bels).
ky, M., Am. J. Bot., 51:72 (1964), Booth, T. and Mill
er, CE, Can.J. Bot., 47: 2051 (1969), Raghukumar, S.,
Trans. Br. Mycol. Soc., 90: 273 (1988), Raghukumar,
S., Trans. Br. Mycol. Soc., 90: 627 (1988)).

[0022] Schizochy aggregatum
In trium aggregatum, the vegetative cells form a clump of many cells adhered to each other by successive divisions. Of the cell mass, three, four or more cells differentiate into zoospores. One zoospore forms 16 to 64 zoospores. In addition, no zoospore release was noted from the two cells (Goldstein, S. and Belsk
y, M., Am. J. Bot., 51:72 (1964), Booth, T. and Mille
r, CE, Can. J. Bot., 47: 2051 (1969)).

Schizochytr minutum
ium minutum), like Schizochytrium aggregatum, results in the division of vegetative cells, forming 4 to 8 or hundreds of cell clusters, releasing two zoospores from each zoospore. The zoospores are bean-shaped and the length of the two flagella is about 8.5 μm and about 3.0 μm (Gaertner, A., Veroff. Inst. Meerestorsch.
Bremer., 19:61 (1981)).

In addition, Schizochytrium octosporum (S
chizochytrium octosporum) differs from Schizochytrium minutum in that eight zoospores are released from one zoospore (Raghukumar, S., Trans. Br.
Mycol. Soc., 90: 273 (1988)).

Furthermore, in 1987, the Thraustochytrium family organisms separated from decayed leaves of mangroves in Goa (India) by rag coomers were classified into the genus Schizochytrium because vegetative cells form a cell mass through continuous division. However, while the three zoospores described so far are all formed in a bag called zoospores, in this organism, continuous mitotic cell division by two causes
The cells became 4, 6, 8 or 12 cells, and each cell took a process of directly becoming a zoospore, and did not take the form of a zoosporangium. Ragcoomer has noticed this feature, and has developed a new species, Schizochytrium mangrovei.
(Schizochytrium mangrovei, Raghukumar, S.,
Trans. Br. Mycol. Soc., 90: 627 (1988)).

Ragcoomer proposed a lookup table for the genus Schizochytrium previously known in the same literature (Table 1). Original report and SR describing the search table shown in Table 1 and 4 types
Let's compare the mycological properties of 21 strains. First, Schizochytrium sp. SR21 is different from Schizochytrium mangrovai, in which divided vegetative cells do not take the form of zoospores, but become individual zoospores. The diameter of the zoospore is 14μm
In the following, when two zoospores are formed from each zoospore, they belong to Schizochytrium minutum, and when eight zoospores are also formed, they belong to Schizochytrium octosporum. It differs from either of these because it differentiates into 16 zoospores. In addition, if the zoospores have a diameter of 15 to 25 μm and 16 to 64 zoospores are formed from the zoospores (however, the original report has many expressions), Schizochytrium
Although it is considered to be aggregatum, the SR21 strain is different from this because no amoebic atypical cells have been observed in this species. Furthermore, in the SR21 strain, cells that differentiate into zoospores without passing through vegetative cells or amorphous cells that do not divide are also seen. From the above, it was confirmed that the SR21 strain did not correspond to the existing four species of the genus Schizochytrium and was a new species of the genus Schizochytrium.

[0027]

[Table 1]

The Schizochytrium sp. SR21 strain was deposited on March 6, 1995 with the National Institute of Bioscience and Human Technology, and obtained the accession number FERM BP-5034. Deposited with the Institute on March 17, 1995 and received accession number IFO 32693.

Further, the microorganisms used in the method for producing docosahexaenoic acid-containing fats and oils of the present invention are not limited to FERM BP-5034 or IFO 32693, but are substantially the same mycologically as the above-mentioned Thraustochytrium SR21 strain. Any strain can be used as long as it has a property. After culturing such a microorganism and accumulating docosahexaenoic acid-containing oils and fats in the cultured cells at a high concentration, the docosahexaenoic acid-containing oils and the like can be collected to produce docosahexaenoic acid-containing oils and the like.

The growth of the Schizochytrium sp.
21 strains are inoculated into an appropriate medium prepared with natural seawater or artificial seawater and cultured according to a conventional method. As the medium, any known medium can be used.
For example, examples of the carbon source include carbohydrates such as glucose, fructose, saccharose, and starch, oils and fats such as oleic acid and soybean oil, glycerol, and sodium acetate. These carbon sources are used, for example, at a concentration of 20 to 120 g per liter of medium. Examples of the nitrogen source include organic nitrogen such as yeast extract, corn steep liquor, polypeptone, sodium glutamate and urea, and inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate and ammonium nitrate. As the inorganic salt, potassium phosphate or the like can be appropriately used in combination. After the above medium is prepared, the pH is adjusted to 4.0 to 6.5 by adding an appropriate acid or base.
After being adjusted within the range, the mixture is sterilized by an autoclave. The cultivation of the bacterium is performed at a culture temperature of 10 to 35 ° C, preferably 17 to 35 ° C.
The culture is performed at 30 ° C. for 3 to 7 days by aeration, stirring, shaking, or stationary culture.

[0031] In this way, cells having a high concentration of docosahexaenoic acid-containing fats and oils in the culture are produced at a high concentration of about 15 to 40 g in terms of dry cell weight per liter of medium. As a method for separating the culture solution and the cells from the culture, a conventional method such as centrifugation or filtration can be used, but centrifugation is preferred.

Further, the cells isolated from the above culture are
For example, after crushing with ultrasonic waves or Dynomill,
For example, docosahexaenoic acid-containing fats and oils can be obtained by solvent extraction with chloroform, hexane, or the like. The content of docosahexaenoic acid-containing fats and oils per 100 g of dried cells is about 25 to 60 g, and the production amount of docosahexaenoic acid-containing fats and oils per liter of medium is as follows:
It reaches about 8 to 15 g. The content of docosahexaenoic acid in the fatty acid composition of fats and oils is as high as 25 to 45% by weight. Therefore, the production amount of docosahexaenoic acid per liter of the medium is as high as about 2.2 to 7.2 g.

In order to separate docosahexaenoic acid from docosahexaenoic acid-containing fats and oils, a conventional method such as a urea addition method is used in the state of mixed fatty acids or fatty acid esters.
Concentration and collection are performed by a cooling separation method, a high performance liquid chromatography method, a supercritical chromatography method, or the like.

When the Schizochytrium sp. SR21 strain is used as a microorganism, the fatty acid composition of the obtained docosahexaenoic acid-containing oil and fat is characterized by the fact that docosahexaenoic acid is contained at a high concentration of 25% by weight or more, and the total n-3 fatty acids contained in the oil and fat are The ratio of docosahexaenoic acid to is 98% by weight or more. Further, this fat is characterized in that it contains almost no arachidonic acid and eicosapentaenoic acid. Specifically, the content ratio of icosapentaenoic acid in the fat or oil is 1.0% by weight or less. Therefore, such fats and oils have an advantage in terms of concentration and separation of docosahexaenoic acid. In addition, it is advantageous that such fats and oils contain almost no fatty acid whose physiological activity is similar or antagonistic to docosahexaenoic acid in aiming at utilization in functional foods and pharmaceuticals.

[0035]

The present invention will be described more specifically with reference to the following examples. [Example 1] In Experiment Nos. 101 to 110 flasks, artificial seawater (tropic marine) having a concentration of 50% was added.
A medium was prepared by adding 1 liter and then adding corn steep liquor (CSL) as the carbon source and the nitrogen source shown in Table 2 in the amounts shown in Table 2, respectively. This was inoculated with SR21 strain of the genus Schizochytrium, and shake-cultured at 26 ° C. for 5 days. 2 ml of the obtained culture is collected, the cells are collected by centrifugation, washed, and placed in an oven at 110 ° C. for 5 minutes.
The dried cells were obtained by drying for an hour. Table 2 shows the results obtained by measuring the weight of the dried cells and determining the amount of cells per liter of the culture medium. Next, the fats and oils were directly extracted and fatty acid methyl esters were prepared from the dried cells in a conventional manner. That is, the dried cells were placed in a screw-mouth test tube for 10 minutes.
% Hydrochloric acid and dichloromethane were added, and the mixture was heated and reacted in a 60 ° C. water bath for 3 hours, and then a fatty acid methyl ester component was extracted with hexane. The fatty acid composition of the fatty acid methyl ester component was determined by gas chromatography analysis, and the content ratio of docosahexaenoic acid was determined. Also,
By the internal standard method by adding a known amount of an internal standard substance at the time of the reaction, the total fatty acid amount contained in the cells was determined from the total fatty acid methyl ester amount detected by gas chromatography analysis, and docosahexaene was used. The amount of docosahexaenoic acid produced was determined from the amount of acid methyl ester detected. Thus, the total fatty acid content per liter of the culture medium, the fatty acid content ratio per dry bacterial cell, the docosahexaenoic acid (DHA) content ratio in the total fatty acids, and the docosahexaenoic acid (DHA) content per liter of the medium were determined. Table 2 shows the results. Docosahexaenoic acid was compared with a standard substance by gas chromatography-mass spectrometry,
confirmed.

[0036]

[Table 2]

The amount of carbon source per liter of medium is 60 to 12
Good cell growth was achieved even with a medium as high as 0 g, from 21.9 to 35.
9 g of cells were obtained. In addition, the amount of fatty acid per liter of medium is 8.2 to 14.6 g, and 30.
It was shown that docosahexaenoic acid-containing fats and oils were accumulated at a high content of 3 to 58.0% by weight. Further, the content of docosahexaenoic acid (DHA) in the total fatty acids is 25.5 to 25.5.
It was as high as 9.7% by weight, indicating that the production amount of docosahexaenoic acid (DHA) per liter of medium was as high as 2.43 to 4.20 g.

Example 2 Experiment Nos. 201 to 204: In a flask were placed 1 liter of artificial seawater of 50% concentration, 60 g of glucose as a carbon source, 4.0 g of monopotassium phosphate, 1.0 g of yeast extract, and corn steep as an organic nitrogen source. 1.0 g of liquor (CSL), and as an inorganic nitrogen source, Table 3
Was added in the amounts shown in Table 3 to prepare a medium. This was inoculated with the Schizochytrium sp. SR21 strain and shake-cultured at 25 ° C. for 4 days. In the same manner as in Example 1, the amount of cells per liter of culture medium after culturing, the amount of total fatty acids per liter of culture medium, the percentage of fatty acid content per dry cell, and the docosahexaenoic acid (DH)
A) The content ratio and the amount of docosahexaenoic acid (DHA) per liter of the medium were determined. Table 3 shows the results.

[0039]

[Table 3]

From these results, it can be seen that the SR21 strain can grow well even when inorganic nitrogen is used as a nitrogen source.
21 strains were shown to have high docosahexaenoic acid productivity.

Example 3 Experiment Nos. 301-302 60 g glucose, 20 g polypeptone, 10 g yeast extract and 1 liter 50% artificial seawater (A), or 90 g glucose, 10 g polypeptone, 10 g corn steep liquor and Medium consisting of 1 liter of 50% artificial seawater
Using (B), cultivation was performed in a jar fermenter (culture tank capacity: 5 liters, medium volume: 3 liters). Culture was performed at a culture temperature of 25 ° C., an aeration rate of 0.5 vvm, and a stirring speed of 200 rpm. After the culture, the cells were collected by centrifugation, freeze-dried, and the amount of cells per liter of medium was determined by a gravimetric method. The results are shown in Table 4. A mixed solution of chloroform / methanol (2: 1 v / v) was added to the dried cells and homogenized in the presence of glass beads to disrupt the cells and extract oils and fats. After the extract was washed by the Folch method, the solvent was distilled off to obtain a purified oil and fat, and the amount of generated oil and fat was determined by a gravimetric method. Fatty acid methyl esters were prepared from a part of the obtained fats and oils by a conventional method, and the fatty acid composition and the content of docosahexaenoic acid (DHA) in all fatty acids were determined by gas chromatography. The amount of docosahexaenoic acid (DHA) produced was determined as a value obtained by multiplying the amount of produced fat and oil by the content ratio of docosahexaenoic acid (DHA). Table 4 shows the results.

[0042]

[Table 4]

From the above results, it was shown that the Schizochytrium SR21 strain shows good growth even in the aeration and agitation culture which is a practical culture method, and efficiently accumulates oils and fats having a high content of docosahexaenoic acid (DHA). It was The production amount of docosahexaenoic acid (DHA) reaches a maximum of 7.2 g per liter of medium, and the productivity is extremely excellent.

Example 4 Content ratios of n-3 fatty acids such as docosahexaenoic acid (DHA) and icosapentaenoic acid (EPA) contained in the fats and oils obtained in Examples 1 to 3 and total n-3 fatty acids. Table 5 shows the ratio of docosahexaenoic acid (DHA). Schizochytrium SR21
The docosahexaenoic acid (DHA) -containing fat or oil obtained from the strain has a content ratio of icosapentaenoic acid (EPA), which is abundant in fish oil, of 1.0% by weight or less, and a content ratio of docosahexaenoic acid (DHA) to all n-3 fatty acids of 98% by weight. %
That is all. These facts indicate that fish oil has an advantage of facilitating the operation of concentration, separation and purification of docosahexaenoic acid (DHA), compared to the case where fish oil contains about 10% by weight of icosapentaenoic acid (EPA). It is.

[0045]

[Table 5]

Reference Example The docosahexaenoic acid-producing ability of a conventionally known microorganism and that of the Schizochytrium genus SR21 strain of the present invention were compared. Table 6 shows the case where culture was performed using Thraustochytrium aureum (ATCC 34304) as a microorganism to produce docosahexaenoic acid (DHA) [P.
Bajapai, PK Bajapai and OP Ward, Appl.Miocro
biol. Biotechnol. 35: 706 (1991), A. Kendric and C.
Ratledge, Lipids, 27:15 (1992) and PKBajapai, P.
Bajapai and OPWord, J. Am. OilChem. Soc., 68: 509.
(1991)], Japonokitorium s as a microorganism
p. (ATCC28207) to produce docosahexaenoic acid (DHA) (see JP-A-1-199588), and as a microorganism Schizochytrium aggregatam (ATCC 28209) [A. Kendric and C. . Ratledge, Li
pids, 27:15 (1992)] and cultivation using the Schizochytrium sp. strain SR21 of the present invention to produce docosahexaenoic acid (DHA).
2 shows the amount of cells per liter of medium, the content of fats and oils or fatty acids per dry cell, the content of docosahexaenoic acid (DHA) in all fatty acids, and the amount of docosahexaenoic acid (DHA) per liter of medium.

[0047]

[Table 6]

As shown in Table 6, when cultivation was carried out using the SR21 strain of the genus Schizochytrium of the present invention, the amount of cells per culture medium was much larger than that of conventionally known microorganisms, and the SR21 strain had a high growth potential. It turns out that it is excellent. In addition, the Schizochytrium SR21 strain of the present invention has a very high oil / fat content as compared with conventionally known microorganisms. Further, according to the present invention, since the content ratio of docosahexaenoic acid in all fatty acids is as high as 30% by weight, the amount of docosahexaenoic acid per liter of the culture medium is 10 to 100 as compared with the case where a conventionally known microorganism is used.
It is clear that the SR21 strain has an extremely high docosahexaenoic acid-producing ability, which is about twice as high. Furthermore, according to conventionally known microorganisms, fats having an icosapentaenoic acid content of several weight% can be obtained, whereas according to Schizochytrium sp. Strain SR21, an icosapentaenoic acid content of 1.0% is obtained.
It is understood that fats and oils as low as not more than% by weight can be obtained.

[0049]

Industrial Applicability The microorganism of the present invention is excellent in growth and fat accumulation, and has high docosahexaenoic acid (DHA) production ability. Therefore, by using the microorganism of the present invention, it is possible to efficiently produce oils and fats having a high docosahexaenoic acid content, which are useful in the fields of foods and pharmaceuticals, with an unprecedented high yield. Moreover, since the docosahexaenoic acid-containing oil and fat obtained by the production method of the present invention has a low content of icosapentaenoic acid, separation and purification of docosahexaenoic acid from the oil and fat are easy. Therefore, docosahexaenoic acid can be efficiently produced from the docosahexaenoic acid-containing fat or oil. That is, according to the present invention, an excellent method for producing docosahexaenoic acid-containing fats and oils containing a docosahexaenoic acid-containing oil having a very low content of icosapentaenoic acid, which is a docosahexaenoic acid-containing fat or oil by a culture method, which replaces the docosahexaenoic acid-containing fats and oils currently separated and purified from fish oils. Could be provided.

[Brief description of drawings]

FIG. 1 is an optical micrograph showing the morphology of zoospores of Schizochytrium genus SR21.

FIG. 2 is a transmission electron micrograph showing the structure of flagella of zoospores of Schizochytrium sp. SR21.

FIG. 3 is an optical micrograph showing a network of vegetative cell clusters and cytoplasm of Schizochytrium genus SR21.

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[Procedure amendment]

[Submission date] June 24, 1996

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

[Figure 3]

[Fig. 2]

Claims (5)

[Claims] 1. An SR21 strain of the genus Schizochytrium having the ability to produce docosahexaenoic acid. 2. A microorganism belonging to the genus Schizochytrium and having an ability to produce docosahexaenoic acid-containing fats and oils, wherein the content of icosapentaenoic acid in the fats and oils is 2.
A method for producing the docosahexaenoic acid-containing oil or fat, which comprises culturing a microorganism having an oil or fat producing ability of 1.0% by weight or less in a medium, and collecting the docosahexaenoic acid-containing oil or fat from the culture. 3. A microorganism belonging to the genus Schizochytrium and having an ability to produce docosahexaenoic acid-containing fats and oils, wherein the content of docosahexaenoic acid with respect to all n-3 fatty acids contained in said fats and oils is 98% by weight or more. A method for producing the docosahexaenoic acid-containing oil or fat, which comprises culturing a microorganism having a productivity in a medium and collecting the docosahexaenoic acid-containing oil or fat from the culture. 4. The method according to claim 1, wherein the microorganism is Schizochytriu.
m) The method for producing a docosahexaenoic acid-containing oil or fat according to claim 2 or 3, which is of the genus SR21. 5. A method for producing docosahexaenoic acid, comprising separating docosahexaenoic acid from the docosahexaenoic acid-containing fat or oil according to any one of claims 2 to 4.