JPH1072590A - (N-6) Docosapentaenoic acid-containing fats and oils, method for producing the fats and oils, and use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oil or fat having a high content of (n-6) docosapentaenoic acid (DPA), a method for producing the oil or fat and (n-6) DPA, and various kinds of oils and fats to which the oil or fat is added. Food, feed and feed. SOLUTION: By culturing a SR21 strain of the genus Schizochytrium having the ability to produce (n-6) system DPA and a microorganism belonging to the same species as the strain SR21 or having substantially the same mycological properties, Fats and oils with a high content of (n-6) -based DPA can be obtained. Since the fats and oils thus obtained also contain a high content of the (n-3) docosahexaenoic acid (DHA), the fats and oils are added to various foods, feeds or feeds, and the (n-6) These polyunsaturated fatty acids can be stably and efficiently supplied to a subject in need of DPA and / or (n-3) DHA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an oil or fat containing (n-6) docosapentaenoic acid (DPA), the oil or fat,
The present invention relates to a method for producing (n-6) -based DPA, various foods, feeds and feeds to which the fats and oils are added.

[0002]

2. Description of the Related Art Docosahexaenoic acid is used in animals.
Highly unsaturated fatty acids such as (DHA) and docosapentaenoic acid (DPA) are considered to have various physiological activities. It is known that these highly unsaturated fatty acids can be classified into (n-3) and (n-6) systems depending on the difference in the position of the unsaturated bond. (N-3) and (n-6) in animals
The highly unsaturated fatty acids in the system belong to different metabolic pathways, and animals require both as essential fatty acids.

The (n-3) polyunsaturated fatty acids include, for example, eicosapentaenoic acid [20: 5 (n-3)] and docosahexaenoic acid [22: 6 (n-3)]. Is known to have physiological activities such as anti-inflammatory activity and anti-thrombotic activity, and is attracting attention as a material for functional foods and pharmaceuticals. On the other hand, (n-6) polyunsaturated fatty acids include, for example, γ-linolenic acid [18: 3 (n-6)], dihomo-γ-linolenic acid [20: 3 (n-6)], arachidone The acid [20: 4 (n-
6)] and these are attracting attention as intermediate metabolites of one or two groups of eicosanoids such as prostaglandins and leukotrienes called local hormones.

In the animal body, the (n-3) system contains docosahexaenoic acid and (n-6)
In the system, arachidonic acid is the final metabolite. For example, the fatty acid composition of the phospholipids of human erythrocytes is (n-3) eicosapentaenoic acid 0.70%, docosapentaenoic acid 2.09%, docosahexaenoic acid 4.37%, while (n-3) 6) The system is 12.67% linoleic acid, 0.62% dihomo-γ-linolenic acid, 16.93% arachidonic acid, 0.86% docosapentaenoic acid [Hardy et al., Biochem. J., v.
ol. 274, p133 (1991)], and the amount of (n-6) docosapentaenoic acid is extremely small.

[0005] The (n-3) docosahexaenoic acid (DHA), which is the final metabolite of the (n-3) system, is specifically present in the brain and retina of animals, and is considered to play some function in these organs. Have been. This (n-3) DHA is contained in fish oil belonging to the blue fish, and particularly about 20% in sardine and tuna derived oil. In recent years, raw materials containing high concentrations of DHA, such as tuna orbital fat, have been discovered, and advanced purification technology for fatty acids has led to the development of DHA.
Research on the elucidation of the physiologically active function of and the practical application thereof has been actively promoted. As the physiologically active functions of DHA, cholesterol lowering action, anticoagulant action, anticancer action, improvement of memory and learning ability in relation to brain metabolic system, prevention of senile dementia,
A remedy for Alzheimer's disease, essential fatty acids for growth of fry, etc. have been clarified, and are used as materials for health foods and baby milk.

On the other hand, when the composition of (n-6) docosapentaenoic acid (DPA) increases in the animal body, it is considered to be a price for deficiency of (n-3) essential fatty acids. For example,
The fatty acid composition of the optic choroid plexus of the third generation rat of the third generation rats, which continued to feed on a diet containing extremely much (n-6) -series safflower oil, showed that the (n-3) -series DHA decreased to 1/3. On the other hand, the (n-6) system DPA quadrupled [Homayoun et al., J. Ne.
urochem., vol.51, p.45 (1988)]. In addition, vitamin A
(N-6) system D in liver microsomes of deficient rats
A sharp increase in the PA composition from 0.9% to 10.5% of the normal value [Hamm et al., Biochem. J., vol. 245, p907 (1987)], and a low (n-3) palm oil (N-3) DHA decreased and (n-6) DPA increased in rats fed [Rebhung et al., Biosci. Biotech. Biochem., Vol.
8, p314 (1994)].

[0007] As described above, it is considered that the (n-3) -system DHA plays a certain function in the brain and retina of animals.
Production of (n-6) -series DPA in vivo as a price suggests that (n-6) -series DPA has some physiological role and is also expected to be an arachidonic acid antagonist it can.

Further, the currently known (n-6) DP
As a method of using A, a tranquilizer is used as a base for making it easier to carry to the brain (Japanese Patent Application Laid-Open No. 61-204136).
No.) and diseases in which the (n-6) series unsaturated fatty acids having 22 carbon atoms are lower than normal values, for example, viruses,
Infection, especially by the wart virus; leukemia, breast cancer and other types of cancer; premenstrual syndrome and benign breast disease; hypertension, hyperlipidemia and obesity, dry eye
Syndromes; scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis and other forms of autoimmune and inflammatory diseases;
(N-6) DPA (n-6) docosatetraenoic acid for the treatment of infertility; diabetes; and psychiatric disorders including schizophrenia and alcoholism (including the effects of both excessive and abstinence) To be used in combination with JP-A-60-3
No. 8324).

This (n-6) DPA is not present at all in the fats and oils generally supplied, and is only slightly contained in fish oil together with the (n-3) DPA. . A method for separating and concentrating (n-6) DPA from fish oil has been applied for a patent [Japanese Patent Laid-Open No. 1-180849], but the content of (n-6) DPA in fish oil is as small as about 1%, Arachidonic acid,
Rich in polyunsaturated fatty acids having similar structures such as eicosapentaenoic acid and docosahexaenoic acid, and (n-
3) Since the system DPA is contained at a content several times higher than that of the (n-6) system DPA, efficient separation / concentration is difficult, such as necessity of multi-stage chromatography.

As described above, fish oils include (n-3) -based DHA and (n-6) -based DPA which have remarkable physiological functions, but (n-3) -based DHA and particularly (n-)-based DHA 6) System DPA
Fats and oils containing a large amount of are not yet known. Furthermore, fish oil has drawbacks in that it is difficult to become a stable supply source due to the migratory properties of fish and the like, and that it has a peculiar odor peculiar to fish oil. Also, fish oil contains highly unsaturated fatty acids such as arachidonic acid (AA) and eicosapentaenoic acid (EPA), so that it is easily oxidized and it is difficult to obtain stable quality oils and fats. Furthermore, high purity (n-3) DHA or (n-3)
-6) When trying to obtain system DPA, its separation and purification are difficult. In particular, when added to infant milk, it is desirable that the content of EPA is low, but when the source is fish oil, it is extremely difficult to efficiently remove only EPA.

(N-3) DHA other than fish oil or (n-6)
Various microorganisms are candidates for the source of the system DPA. For example, as a microorganism having an (n-3) DHA-producing ability, a bacterium Vibrio marinus (V.
ibrio marinus) (ATCC 15381) and Vibrio spp. bacteria isolated from the intestine of deep-sea fish, and microalgae Cyclotella cryptica and Crypthecodinium cohnii (Tokuheihei 5-503425) , The flagellated fungus Thraustochytrium aureum (AT
CC 34304) [Kendrick, Lipids, vol.27, p15 (19
92)] and Japonochytrium sp.
sp.) (ATCC 28207) (JP-A-1-199588) and the like.

[0012] Among these microorganisms, it is known that docosapentaenoic acid is contained in some of microalgae and in flagellated fungi such as Thraustochytrium aureum (ATCC 34304) and Japonokitorium sp. (ATCC 28207). However, in the above literature, these are reported to be (n-3) systems. That is, it is not known that (n-6) docosapentaenoic acid is present in a sufficient amount in fats and oils produced by these microorganisms.

[0013]

DISCLOSURE OF THE INVENTION The present inventors have sought out a wide variety of marine microorganisms for microorganisms which highly produce oils and fats having a high content of the above (n-6) docosapentaenoic acid (DPA). Was.

[0014]

As a result, the present inventors have found that certain marine microorganisms (a new species belonging to the genus Schizochytrium) produce oils and fats having a high (n-6) DPA content. I found Further, this microorganism is represented by (n-6)
Fats and oils having not only a high content of DPA but also a high content of (n-3) DHA and a low content of EPA, that is, a fatty acid composition useful for addition to various foods, feeds or feeds. It has been found that oils and fats having high production are obtained.

That is, the present invention relates to a Schizochytrium genus SR2 having the ability to produce (n-6) docosapentaenoic acid.
(N-) wherein one strain and a microorganism belonging to the same species as the SR21 strain or having substantially the same mycological properties are cultured in a medium, and an oil or fat is collected from the culture. 6) A method for producing a fat containing docosapentaenoic acid is provided. Further, the present invention provides a method for producing (n-6) docosapentaenoic acid, further comprising a step of isolating (n-6) docosapentaenoic acid from the fat or oil. . Further, the present invention relates to (n-6) docosapentaenoic acid in an amount of 5% by weight or more based on all fatty acids in the fat or oil,
An (n-6) -based docosapentaenoic acid-containing fat or oil comprising 20% by weight or more of (n-3) -type docosahexaenoic acid and 2% by weight or less of eicosapentaenoic acid. The present invention also provides various foods, feeds and feeds to which the fats and oils are added, and methods for using the fats and oils as additives for various foods, feeds and feeds.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Note that the terms “oil and fat”, “lipid”, and “oil” described herein have the same meaning. Marine microorganism SR21 used in the present invention
The strain was isolated from seawater off the coast of Yap Island, Federated States of Micronesia.

Initially, this SR21 strain was considered to be a microorganism belonging to the genus Thraustochytrium. However, as a result of a detailed examination of the mycological properties, this strain was found to be a microorganism recognized as a new species of the genus Schizochytrium. The mycological properties of the Schizochytrium sp. SR21 strain are as follows.

The mycological properties of the SR21 strain were examined by culturing in a nutrient medium and seawater. First, a nutrient medium obtained by adding 2 g of glucose, 0.2 g of yeast extract and 0.5 g of sodium glutamate to 1 L of artificial seawater (tropic marine) was placed in a small petri dish, and SR21 was used in the same medium.
One drop of a flask preculture of the strain was inoculated, and the cell morphology was followed by an inverted microscope. In this case, release of amoeboid amorphous cells was observed. Next, the same tracking was performed in filter-sterilized natural seawater. In this case, no release of amoeba-like amorphous cells was observed, and release of zoospores was observed from some cells of the vegetative cell mass after repeating two divisions. In addition, it was also observed that one vegetative cell was directly differentiated into a zoospore without dividing into two cells.

Glutaraldehyde was added at 10% by volume to the sample in which the release of zoospores was observed, and the zoospores were observed with an optical microscope. FIG. 1 is an optical micrograph showing the morphology of zoospores of the SR21 strain, showing flagella with two different lengths. Further, electron microscope observation of the flagella was performed by a negative staining method using uranium acetate. FIG.
Is a transmission electron micrograph showing the structure of flagella of zoospores of the SR21 strain, showing a three-part structure consisting of the base, axis, and apical hair of the flagella mastagonema. In addition, the above 2
Observation of the cell morphology using an inverted microscope showed that the vegetative cells repeatedly divided into two cells and formed a network of cell mass and cytoplasm. FIG. 3 is an optical micrograph showing a network of the vegetative cell mass and the cytoplasm of the SR21 strain.

The colony formed by the SR21 strain on the agar plate medium exhibits a smooth ocher color similar to the yeast colony. When this SR21 strain was grown in a liquid medium, zoospores having two flagella with different lengths were observed in the initial stage (FIG. 1), and the hairy structure in the long flagella of the two flagella was observed. (Mastigonema) has a three-part structure: base, shaft, and top hair
(FIG. 2). Based on these facts, SR21 strain was obtained from Kingdom Chromista, Phylum Heterokont (Phylum Heterokont).
belongs to a). Furthermore, from the network-forming properties of the protoplasm and the scales derived from the Golgi apparatus, the SR21 strain was identified as Labyrinthura.
(Class Labyrinthulea) Labyrinthula (Order Labyri
nthulida). And it is clear that the SR21 strain belongs to the family Thraustochytriidae because the vegetative cells are spherical or elliptical and there is no gliding movement in the protoplasmic network.

Furthermore, the vegetative cells of the SR21 strain repeat two divisions to form 8-32 vegetative cell masses. Thereafter, the amoebic amorphous cells are released from some of the cells, gradually dislodge 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 this time, no zoospore membrane is observed. In addition, there are cells that differentiate directly from one vegetative cell into zoospores without dividing into two cells, or differentiate into zoospores without passing through amorphous cells after dividing into two vegetative cell clusters. , Have a complicated life cycle.

The Thraustochytrium family is a porter [DP
orter, “Handbook of Protoctista”, Jones and Bartl
According to ett Publishers (1990)], it consists of 30 species of 7 genera.
Later, the genus Corallochytrium [Raghuk
umar, S., Botanica Marina, 30:83 (1987)] and Moss, ST, “The Biology of Free-living H
eterotrophic Iagellates ", Oxford University Press
According to (1991)], there are 33 species of 8 genera.

The characteristics of these eight genera of the Thraustochytrium family are as follows. Labyrinthul
Vegetative cells of the genus oides) are spherical, but glide irregularly on the protoplasmic network. Aplanocytrium (Aplanoc
hytrium) multiplies by immobilized spores, ie spores without flagella. The genus Althornia does not form a protoplasmic network and is planktonic. The genus Japonochytrium produces extracellular apophysis. The Ulkenia genus differentiates into zoospores after amoebic amorphous cells are released from the zoospores. In the genus Thraustochytrium, one zoospore turns into one vegetative cell, which forms one zoosporangium. The genus Schizochytrium divides after one zoospore has formed and forms two or more vegetative cell clusters, each of which becomes a zoosporangium. The genus Corallochytrium forms slime-like spores and does not form flagellar zoospores.

Among the above eight genera, the genus Urkenia is described by Gertner [Gaertner, A., Veroff. Inst. Meeresfors].
ch.Bremerh., 16: 139 (1977)] uses the trait that differentiates into zoospores after the release of a naked protoplasmic mass (amoeba-shaped amorphous cells) from zoospores, Thraustochytrium visurgens, previously classified in the genus Thraustochytrium
e) [Ulken, A., Veroff.Inst.Meeresforsch.Bremerh.,
9: 289 (1965)] and Thraustochytrium amoeboidum (Bahnweg, O. and Sparrow, FK, Jr. Am. J. Bot., 61: 754 (1974)). Transfer to them, a new species of rug coomer Ulkenia minuta [Ulkenia minuta] [Raghukumar,
S., Veroff.Inst.Meeresforsch.Bremerh., 16: 158 (19
77)] and three new species, and a new genus Ulkenia was proposed as six species of Ulkenia.

However, no paper has been described since that time about a new species of the genus Urkenia. Curling [Kar
ring, 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 amoeba-shaped irregular cells, it is considered that the SR21 strain belongs to the genus Urkenia if its traits are emphasized. However, rag coomers have been reminded of the Thraustochytrium spp.
hraustochytrium striatum) have been reported to form amoebic amorphous cells that prey on bacteria in nutrient media [Raghukumar, S., Marine Biology, 113: 1.
65 (1992)]. In addition, RagCoomer is a new species of the genus Schizochytrium,
izochytrium mangrovei) formed amoebic amorphous cells in 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.Myc
ol. Soc., 80: 627 (1988)].

[0027] Therefore, rag coomers determined that it is necessary to investigate this trait using a standard medium, because the trait of forming amoeboid cells is affected by the medium composition and culture conditions. As the reference medium, the aforementioned seawater / pine pollen, which has been traditionally and often used, and has been often used for observing the morphological traits in the original descriptions of genera and species. The medium is listed. All six species classified in the genus Ulkenia,
It is known to form amoebic amorphous cells in this seawater / pine pollen medium. On the other hand, Thraustochytrium striatam and Schizochytrium mangrovai form amoebic amorphous cells in the nutrient medium as described above, but do not form amoeba-like amorphous cells in seawater / pine pollen medium. Not classified. Based on the above, the SR21 strain forms amoebic amorphous cells in a nutrient medium, but this was not observed in a medium containing only seawater, so it may not be appropriate to classify it into the genus Urkenia.

On the other hand, after one zoospore has settled, the vegetative cells repeat two divisions to form a plurality of vegetative cell masses,
The traits that each become zoospores are stable regardless of the medium composition, and are traits that are always observed in the life cycle of the SR21 strain. This trait and other properties observed in the SR21 strain are described by Goldstein et al. [Goldstein, S. and Belsky, M., Am. J. Bot., 51:72 (1964)] and Boots et al. And Miller, CE, Can. J. Bot., 47: 2051
(1969)] is consistent with the description of the genus Schizochytrium. Therefore, it is judged that it is appropriate to classify the SR21 strain into the genus Schizochytrium.

At present, Schizochytrium microorganisms include:
The following four are described in the literature. In Schizochytrium aggregatum, the vegetative cells form a mass in which many cells adhere to each other by successive divisions. Of the cell mass, three to four or more cells differentiate into zoospores. One zoospore forms 16 to 64 zoospores. In addition, 2
No zoospore release is reported from individual cells [Goldstein, S. and Belsky, M., Am. J. Bot., 51:72.
(1964), Booth, T. and Miller, CB, Can.J.Bot., 4
7: 2051 (1969)].

Schizochytr minutum
ium minutum), like Schizochytrium aggregatam, divide vegetative cells to form 4-8 or hundreds of cell clumps, releasing two zoospores from each zoospore. The zoospores are bean-shaped, and the length of the two flagella is 8.5 μm and 3.
[Gaertner, A., Veroff. Inst. Meerestor
sch. Bremer., 19:61 (1981)].

In addition, Schizochytrium octosporum (S
chizochytrium octosporum) is 8
Differs from Schizochytrium minutum in the release of zoospores [Raghukumar, S., Trans.Br.Mycol.So
c., 90: 273 (1988)].

Furthermore, the microorganism of the family Thraustochytrium isolated by rag coomers from decayed leaves of mangroves in Goa (India) in 1987 was classified into the genus Schizochytrium because vegetative cells form a cell mass through continuous division. . However, all three zoospores described so far are formed in a bag called zoosporangia, whereas in this microorganism, continuous mitosis of vegetative cells causes 4, 6 zoospores. , 8 or 12 cells, and each cell took the process of directly becoming a zoospore, and did not take the form of a zoospore. RagCoomer has noticed this feature and has developed a new species, Schizochytrium mangrove.
mangrovei) (Raghukumar, S., Trans.Br.Mycol.S
oc., 90: 627 (1988)].

In the same document, Ragcoomer proposed a search table for the known Schizochytrium.
(Table 1).

[Table 1] Table 1. Search table for four species of Schizochytrium (by rag coomer) 1. The settled zoospores form a cell mass through repeated two divisions, and each cell differentiates into zoospores .... 2 1. The settled zoospores form a cell mass by repeated two divisions, and each cell differentiates into zoospores. mangro
vei 2. The diameter of the zoospores is 15 to 25 μm, and the zoospores form 16 to 64 zoospores. aggreg
atum 2. 2. The zoospores have a diameter of 14 μm or less, and the zoospores form 2 or 8 zoospores . ···· S.octosporum 3 zoospores sac to form an eight zoospores. The zoospores form two zoospores ... S.minutum

A comparison is made between the search table shown in Table 1 and the original report describing four known species and the mycological properties of the SR21 strain. First, the Schizochytrium genus SR21 strain is different from Schizochytrium mangrovai, in which divided vegetative cells do not take the form of zoospores and become individual zoospores. In addition, when the diameter of the zoospores is 14 μm or less and two zoospores are formed from each zoospore, the Schizochytrium minutum is formed. -Although each belongs to octosporum, the SR21 strain differs from both of these because it differentiates into 8 to 16 zoospores. further,
The diameter of the zoospores is 15-25 μm, and
When 6 to 64 zoospores are formed (however, only the expression "many" is used in the original report), it is called Schizochytrium aggregatam. In this species, amoeboid amorphous cells are observed. SR2
One strain is also different. Furthermore, in the SR21 strain, cells that differentiate into zoospores without passing through vegetative cells or amorphous cells that do not divide are also found. From the above, SR21 strain did not correspond to the existing four species of the genus Schizochytrium, and was confirmed to be a new species of the genus Schizochytrium.

The Schizochytrium genus SR21 strain is
"The marine fungus SR21"
The strain has been deposited on March 6, 1995 under the name of "strain", and has the accession number FERM BP-5034. Furthermore, it has been deposited with the Fermentation Research Institute on March 17, 1995, and has accession number IFO 32693.

The microorganism used in the method for producing a DPA-containing fat or oil of the present invention is the aforementioned FERM BP-5034 or IF
Not only O32693 but also Schizochytrium sp.
Any strain which belongs to the same species as the SR21 strain or has substantially the same bacteriological properties in light of the bacteriological properties of the R21 strain, for example, any strain recognized as belonging to a subspecies Can also be used. It is preferable to use SR21 strain of the genus Schizochytrium or a strain recognized to belong to the same species as the SR21 strain.

As described above, the microorganism used in the present invention produces (n-6) docosapentaenoic acid at a high level, and also produces (n-3) docosahexaenoic acid at a high level. Produces low levels of eicosapentaenoic acid. If the microorganism used in the present invention has such a polyunsaturated fatty acid-producing ability, it belongs to the above-mentioned Schizochytrium sp. SR21 strain or the same species as the SR21 strain or has substantially the same mycological properties. It may be a mutant or recombinant strain of a microorganism (wild strain) having properties. That is, the use of mutants and recombinants designed to produce higher levels of (n-6) docosapentaenoic acid and / or (n-3) docosahexaenoic acid is all within the scope of the present invention. It is in. Such a mutant or recombinant strain has a (n-6) docosapentaenoic acid and / or (/) in oil or fat compared to the amount produced by the original wild-type strain when cultured using the same substrate. (n-3) -based docosahexaenoic acid and those designed with a view to increasing the total amount of fats and oils or both are included. Furthermore, a fat and oil containing the same amount (n-6) docosapentaenoic acid and / or (n-3) docosahexaenoic acid as the corresponding wild type is produced by efficiently using a cost-effective substrate. Microorganisms designed to include such microorganisms.

Microorganisms capable of producing oils and fats containing (n-6) docosapentaenoic acid and (n-3) docosahexaenoic acid used in the present invention are selected, for example, by the following screening method. be able to. That is, the collected seawater is filtered and collected using a sterile 0.4 μm filter, and the filter is stuck on an agar medium composed of 90% natural seawater, glucose, yeast extract, and peptone, and cultured at 20 to 30 ° C. . The colony formed on the filter of this agar plate medium was cultured on an agar medium having the same composition as above, and the obtained cells were collected with a spatula, and fatty acids were directly methyl-esterified from the cells according to a conventional method, and the The composition is analyzed by gas chromatography, and strains producing (n-6) docosapentaenoic acid and (n-3) docosahexaenoic acid are selected. Furthermore, the fats and oils in the cells are 10% by weight or more per dry cells,
Preferably accumulate in an amount not less than 20% by weight and / or not more than 2% by weight of eicosapentaenoic acid in the total fatty acids;
Preferably 1% by weight or less, more preferably 0.5% by weight
The following strains can be selected:

The Schizochytrium genus SR21 strain can accumulate 20% by weight or more of fats and oils per dry cell. In addition, 6 to 11% by weight of (n-6) -based docosapentaenoic acid and 25 to 45% by weight of (n-3) -based docosahexaenoic acid, based on the total fatty acids in the fat and oil, and the content of eicosapentaenoic acid Is 1% by weight or less. Further, 98% by weight or more of (n-3) docosahexaenoic acid is contained in all (n-3) fatty acids in the fat or oil.

Therefore, a strain belonging to the same species or having substantially the same mycological properties as the Schizochytrium sp. SR21 strain used in the present invention preferably accumulates 10% by weight or more of fats per dry cell. , More preferably at least 20% by weight, most preferably at least 30% by weight. Further, the content of (n-6) docosapentaenoic acid per total fatty acids in the fat or oil is 5% by weight or more, preferably 6% by weight or more, more preferably 6 to 11% by weight. The content of (n-3) docosahexaenoic acid per total fatty acids in the fat or oil is 20% by weight or more, preferably 25% by weight or more, and more preferably 25 to 45% by weight. The content of eicosapentaenoic acid in the fat or oil is 2% by weight or less, preferably 1% by weight or less, more preferably 0.5% by weight or less. Further, the fat or oil of the present invention contains (n-3) -based docosahexaenoic acid in the (n-3) -based fatty acid in an amount of 90% by weight or more, preferably 95% by weight or more.

The oils and fats of the present invention can be obtained by inoculating the above-mentioned microorganisms into an appropriate medium prepared using natural seawater or artificial seawater, and culturing in accordance with a conventional method. As a carbon source to be added to the medium, glucose, fructose, xylose, saccharose, maltose, other carbohydrates such as soluble starch, oleic acid, oils and fats such as soybean oil, molasses, glycerol, mannitol,
Examples include sodium acetate, but are not limited thereto. These carbon sources can be used, for example, at a concentration of 20 to 120 g per liter of medium.

As a nitrogen source, in addition to natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid and corn steep liquor, sodium glutamate;
Organic nitrogen sources such as urea or ammonium acetate, ammonium sulfate, ammonium chloride, ammonium nitrate,
An inorganic nitrogen source such as sodium sulfate can be exemplified.
It is not limited to these.

In addition, if necessary, phosphates such as potassium phosphate and potassium dihydrogen phosphate, inorganic salts such as ammonium sulfate, sodium sulfate, magnesium sulfate, iron sulfate, copper sulfate, magnesium chloride and calcium chloride, and vitamins Can also be used as a micronutrient source. These medium components are not particularly limited as long as they do not impair the growth of microorganisms.

After preparing the medium, the pH is adjusted to a range of 4.0 to 6.5 using an appropriate acid or base and sterilized by an autoclave. The cultivation of the bacterium is carried out at 10 to 35 ° C., preferably 17 to 30 ° C., usually for 3 to 7 days, by aeration-agitation culture, shaking culture or static culture.

In order to promote the production of (n-6) docosapentaenoic acid and / or (n-3) docosahexaenoic acid, (n-6) docosapentaenoic acid and / or (n-3) ) A precursor of docosahexaenoic acid can be added to the medium. As the precursor, a hydrocarbon such as tetradecane, hexadecane, octadecane, a fatty acid such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid, or oleic acid, or a salt thereof (for example, a sodium salt or a potassium salt), a fatty acid ester, or a fatty acid Fats and oils contained as components (eg, olive oil, soybean oil, cottonseed oil, coconut oil) and the like can be mentioned, but not limited thereto.

The conditions for producing the oil and fat of the present invention in a commercially viable yield include the following conditions.
By studying the culture conditions of Schizochytrium sp. SR21 strain,
The SR21 strain and a strain belonging to the same species or having substantially the same bacteriological properties can be used in natural seawater or artificial seawater or a medium containing half the concentration of natural seawater or artificial seawater at pH3. It was found that the cells grew well at a pH of 0.5 to 6.0, preferably pH 4.0 to 4.5.

The carbon source and nitrogen source to be added to the medium may be those commonly used as described above. The nitrogen source may be either organic nitrogen or inorganic nitrogen. If the nitrogen concentration is kept constant, the organic nitrogen can be added without affecting the cell growth, lipid content, DHA and DPA accumulation. And the ratio of inorganic nitrogen can be changed. When these are added at a concentration used for ordinary culture of microorganisms, good growth can be obtained. Good growth can be achieved using phosphate at concentrations used for normal microbial culture.

By increasing the concentration of the nitrogen source together with the concentration of the carbon source in the medium at the same rate, high-concentration culture is possible. Dry cell mass according to the increase rate of carbon source and nitrogen source,
The amount of lipids also increases, and the production of DHA and DPA also increases.

At the time of the high-concentration culture, at the start of the culture, only the concentration of the carbon source, for example, glucose is increased, and the nitrogen source, for example, corn steep liquor / ammonium sulfate, is added in a usual amount to increase the glucose consumption. Depending on the method, a method of adding an insufficient amount later can be used. In the case of high-concentration culture, the carbon source and the nitrogen source may be set at a low concentration at the start of the culture, and the carbon source and the nitrogen source may be increased later according to the consumption of glucose.

Cultivation under the above conditions can be carried out using an ordinary stirred fermenter. In addition, a bubble column type culture device can also be used. As the conditions for the aeration stirring culture, ordinary culture conditions for microorganisms can be used. In aeration and agitation culture, when the number of revolutions is increased and the amount of dissolved oxygen is increased, a remarkable increase in growth rate and yield of bacterial cells are observed as compared with flask culture. In the early stage of the culture, it is particularly important to keep the dissolved oxygen level high to increase the growth rate.

In this manner, the cells containing the (n-6) docosapentaenoic acid and the (n-3) docosahexaenoic acid-containing fats and oils in the culture are added in an amount of 10 g or more, preferably 20 g or more per liter of the culture medium. And more preferably at a high concentration of 40 g or more. In addition, this fat or oil is satisfactorily accumulated in the cells after the middle stage of the culture, and can be 30% by weight or more, preferably 50% by weight or more, more preferably 60% by weight or more per dry cell.

As a method for collecting cells from the culture, a conventionally used method such as centrifugation or filtration can be used. The collected cells are crushed by, for example, dynomill or ultrasonic waves, and then subjected to solvent extraction with chloroform, hexane, methanol, ethanol, or the like, thereby obtaining the (n-6) docosapentaenoic acid and (n-3). ) Based docosahexaenoic acid-containing fats and oils can be obtained. (N-6) docosapentaenoic acid and (n-3) per 1 g of dry cells
The amount of the fat containing docosahexaenoic acid is preferably about 0.3 g or more, more preferably 0.6 g or more.

The fats and oils of the present invention may be fats and oils obtained from the Schizochytrium SR21 strain or a strain belonging to the same species or having substantially the same mycological properties. The fat or oil of the present invention contains (n-6) docosapentaenoic acid in an amount of 5% by weight or more, preferably 6% by weight or more, and 20% by weight or more of the (n-3) docosahexaenoic acid per fatty acid in the fat or oil. , Preferably 25% by weight or more, and eicosapentaenoic acid in an amount of 2% by weight or less, preferably 1% by weight or less, more preferably 0.5% by weight or less. Further, the lipid properties of the fats and oils of the present invention are usually as follows. The proportion of neutral lipids is extremely high, accounting for over 90% by weight of the total lipids. The fatty acid composition in the neutral lipids was 45-55% by weight of palmitic acid, (n-
3) 33 to 43% by weight of docosahexaenoic acid, 7 to 10% by weight of (n-6) docosapentaenoic acid, 0 to 1% by weight of (n-3) eicosapentaenoic acid, and 0 to 0.6 of arachidonic acid.
% By weight, and about 10 to 20% by weight of other fatty acids.

About 85% by weight or more, preferably 90% by weight or more of the obtained neutral lipid is triglyceride, and contains little diglyceride and monoglyceride. Further, free sterols and sterol esters are contained in an amount of 2 to 3%. The molecular species of triglyceride in fats and oils having the above fatty acid composition is mainly 14: 0-1.
6: 0-16: 0, 16: 0-16: 0-0-16: 0, 14: 0
-16: 0-22: 6, 16: 0-16: 0-22: 5,1,
6: 0-16: 0: 0-22: 6, 16: 0-22: 5-22:
6, 16: 0-22: 6-22: 6 (the binding position of the fatty acid residue is not limited). The above description “14: 0” indicates that 14 represents the number of carbon atoms of the fatty acid, and 0 represents the number of double bonds of the fatty acid. For example, “16: 0” represents a double bond having 16 carbon atoms. Represents a fatty acid without.

Further, the triglycerides may be those in which (n-3) type DHA is bonded only to position 2 of glycerin, or (n-3) type DHA may be those in positions 1 and 2 of glycerin,
Alternatively, those bonded to the 1 and 3 positions are included.

As polar lipids, phosphatidylcholine accounts for the majority at 60 to 80% by weight, and also includes phosphatidylethanolamine at 5 to 20% by weight, phosphatidylinositol at 2 to 8% by weight, and the like. The molecular species of the phosphatidylcholine is 16: 0-22:
6, 16: 0-22: 5, 22: 5-22: 6, 22:
6-22: 6.

In order to separate the (n-6) -docosapentaenoic acid from the (n-6) -docosapentaenoic acid-containing oil or fat, a mixed fatty acid or a fatty acid ester can be separated by a conventional method.
For example, concentration and collection are performed by a urea addition method, a cooling separation method, a column chromatography method, or the like. Separation of (n-6) -docosapentaenoic acid-containing triglyceride from oils and fats collected from cultured cells and the like is performed by a conventional method, for example, a cooling separation method, a column chromatography method, or the like. When the Schizochytrium sp. SR21 strain of the present invention is used, arachidonic acid, EP
Since almost no A is contained, it is easy to concentrate and collect (n-6) docosapentaenoic acid, which is convenient for high-concentration production.

The fats and oils of the present invention can be used in various feed, feed or food products such as (n-6) -based DPA and (n-
3) It can be used as a source of system DHA. In utilizing the fats and oils of the present invention in products, fats and oils collected from cultured cells or fats and oils obtained by purifying the same can be used. Solution or a sterilized culture solution thereof, or a culture solution at the end of the culture or a sterilized culture solution thereof, or a cultured bacterial cell collected therefrom or a dried product thereof, or after collecting the oil or fat from the culture solution or the bacterial cell. Residues containing the fats and oils can also be used.

The present invention relates to an animal feed containing the fat or oil of the present invention. Examples of the animal feed of the present invention include pet food such as dog food and cat food, feed for poultry such as chicken, feed for livestock such as pigs and cows, and feed for fish farming. (n-6) DPA and (n-
3) Microorganisms or cultured cells of microorganisms producing and accumulating fats and oils containing DHA are preferable because the fats and oils are protected in the cells to prevent oxidation and are stable to heat sterilization. In addition, (n-
6) Extraction residue after extracting fats and oils containing system DPA and (n-3) system DHA can also be used for the animal feed of the present invention. This extraction residue is composed of (n-6) DPA or (n-3)
It is preferable because it contains proteins, ash, carbohydrates, etc. in addition to the system DHA. The present invention further includes an animal feed additive containing the oil or fat of the present invention.

Further, the present invention relates to a feed for a microfeed, comprising a cultured bacterial cell or a culture solution which has produced and accumulated the oil and fat of the present invention. Conventionally, in the cultivation of fish and shellfish and crustaceans, seeds (larvae) are produced using microprey organisms (zooplankton such as rotifer, brine shrimp), and these are first used for larval fish culture. It is necessary to cultivate small creatures. In the case of culturing these micro organisms, the feed given to the micro prey organisms is determined in consideration of the nutritional requirements of the larvae to be fed later as feed. The microbial organism is fed with a cultured microbial cell or a culture solution containing the oil or fat of the present invention, whereby an (n-6) -system DPA is obtained.
And (n-3) type DHA, whereby a microprey organism that satisfies the nutritional requirements of the juveniles can be obtained. The present invention further includes a feed for fish and shellfish containing the above-mentioned microprey organism.

Further, the present invention relates to the use of
6) utilizing the DPA and (n-3) DHA for the production of enriched poultry eggs, and (n-6) DPA and
(n-3) Use of DHA-enriched egg yolk oil for production. Poultry eggs enriched with (n-6) -based DPA and (n-3) -based DHA of the present invention are produced by feeding the above-mentioned animal feed to egg-collecting poultry, especially chickens, for rearing. By extracting oils and fats from such poultry eggs, particularly egg yolks, according to a conventional method, the (n-6) system D of the present invention can be obtained.
Egg yolk oil enriched with PA and (n-3) DHA is obtained. The present invention also includes the addition of the yolk oil to infant formula, premature infant formula, infant food, and maternal food.

The present invention further relates to infant formula, premature infant formula, infant food, and maternal food containing the oil or fat of the present invention. In particular, attempts have been made to make the components of human milk powder as close as possible to human milk as long as possible, and each of the main components of human milk, such as protein, fat, and carbohydrate, is similar to human milk. Has become an important issue. Particularly with regard to fat, there is a problem that polyunsaturated fatty acids originally contained in breast milk are deficient in conventional infant formula. Various reports have been made on the composition of unsaturated fatty acids in breast milk, for example, see “INFORM” [Vol. 6, No. 8, pp. 940-946 (August 1995)
)], The composition of polyunsaturated fatty acids in breast milk of Americans, Europeans and Africans is described in “JJPEN” [Vol.13,
No. 9, pp. 765-772 (1991)] describes the composition of polyunsaturated fatty acids in Japanese breast milk.

Recently, it has been reported that arachidonic acid and DHA are also contained in human milk and are useful for the development of infants [“Advances in Polyunsaturated Fatty Acid Re.”
search ", Elsevier Science Publishers, pp.261-264,
(1993)]. In addition, its importance in fetal height and brain development has been reported [Proc. Natl. Acad. Sci. USA, 90, 107].
3-1077 (1993), Lancet, 344, 1319-1322 (1994)].

Thus, there is a movement to add arachidonic acid and DHA, which are the major differences in the fatty acid composition between human milk and prepared milk powder, to prepared milk powder. Thus, for the purpose of adding DHA, prepared milk powder to which fish oil is added has been put on the market. However, EPA contained in fish oil is hardly contained in mother's milk. Recent studies have shown that EPA is inconvenient for growing premature babies [“Advances in
Polyunsaturated Fatty Acid Research ", Elsevier S
cience publishers, pp. 261-264, (1993)] and U.S. Pat. No. 5,374,657 discloses fats and oils for infant formula addition which combine DHA-containing cell edible oil with low EPA and arachidonic acid-containing cell edible oil. Have been. However, it is completely known that conventionally known milk and fats and oils for adding milk contain (n-6) -based DPA and (n-3) -based DHA-containing fats and oils originally contained in mother's milk. Had not been.

The fats and oils of the present invention, for example, Schizochytrium sp.
Fats and oils derived from the R21 strain or a strain belonging to the same species or having substantially the same mycological properties are (n-
6) Three to three parts by weight of (n-3) system DHA to 1 part by weight of system DPA
Since it contains 6 parts by weight, contains little EPA, and 85% or more is triglyceride, it is suitable for producing infant formula similar to breast milk.

Further, the present invention relates to foods such as nutritional supplements, foods for the aged, health foods and the like containing the fats and oils of the present invention. The food of the present invention is used for supplementing (n-6) DPA and (n-3) DHA, and is used for maintaining health. The form may be a solid or liquid food or a luxury item. As foods containing oils and fats, for example, natural foods such as meat, fish and nuts, foods to which oils and fats are added at the time of cooking Chinese foods, ramen, soups, etc., as a heat medium such as tempura, fried, fried, fried rice, donuts, sugar and the like Food with butter, margarine,
Spray or apply fats and oils at the time of finishing such as mayonnaise, dressing, chocolate, instant noodles, caramel, biscuits, cookies, cakes, ice creams, etc. And the like. However, the food of the present invention is not limited to foods containing fats and oils, and for example, agricultural products such as bread, noodles, rice, confectionery (candy, chewing gum, gummy, tablet confectionery, Japanese confectionery), tofu and processed products thereof Food,
Fermented foods such as sake, medicinal liquor, mirin, vinegar, soy sauce, and miso; livestock foods such as yogurt, ham, bacon, and sausage; fishery foods such as kamaboko, fried heaven, and starch;
Fruit juice drinks, soft drinks, sports drinks, alcoholic drinks,
Drinks such as tea can also be mentioned.

The food of the present invention can be processed and produced by a general production method by blending a predetermined amount of the fat or oil of the present invention with food ingredients. The compounding amount varies depending on the dosage form and the form of the food, and is not particularly limited, but is generally preferably 0.001 to 50% by weight based on the total amount of the food.

Further, the present invention relates to a functional food (including a food for specified health use) containing the fat or oil of the present invention. The functional food of the present invention comprises (n-6) DPA and (n-3) DHA
It is a food for the purpose of exerting the physiologically active function possessed by the present invention, for restoring and maintaining a degraded state to a healthy state, or for preventing a degraded state. The form may be in the form of a pharmaceutical preparation, or, for example, protein (proteins such as milk protein, soy protein, egg albumin, etc., with high nutritional value and amino acid balance are most widely used as protein sources. In addition to these degradation products, egg white oligopeptides, soy hydrolysates, etc., a mixture of amino acids alone is also used), sugars, fats, trace elements, vitamins, emulsifiers, fragrances, etc. Natural liquid foods, semi-digestive nutritional foods and component nutritional foods, drinks, processing forms such as enteral nutritional can be mentioned,
It may be in the form of the aforementioned food or drink.

The functional foods and dietary supplements of the present invention can be prepared by using the fats and oils of the present invention to prepare powders, granules, tablets, capsules, troches, liquids for internal use, suspensions, emulsions, syrups,
It can be manufactured as a food or drink having a form such as a drink, a natural liquid diet, a semi-digestive nutritional diet, a component nutritional diet, and an enteral nutritional supplement. At this time, any nutrient component or functional component may be blended with the fat or oil of the present invention. Also,
Under the supervision of a nutritionist under the direction of a physician, the fats and oils of the present invention were added to any food at the time of cooking a hospital meal, and the meal adjusted on the spot was converted to a (n-6) -series DPA and (n-3) It can also be given to patients with reduced systemic DHA.

The present invention further relates to the use of the fat or oil of the present invention for the production of a pharmaceutical product alone. That is, using the fat or oil of the present invention as a starting material, (n-6) -based DPA or (n-3)
The production of the system DHA or derivatives thereof. The (n-6) -based DPA or (n-3) -based DHA or a mixture thereof may be in free form or as a pharmaceutically acceptable salt, such as a sodium, potassium, lithium or other salt. Salt forms of other metals such as alkali metal salts, zinc salts, calcium salts, magnesium salts, monoglycerides, diglycerides, triglycerides,
It may be in various forms such as esters of lower alcohols, phospholipids, glycolipids, amides and the like. Here, the lower alcohol refers to a monohydric alcohol having 6 or less carbon atoms, and examples thereof include methanol, ethanol, propanol, isopropanol, butanol, pentanol, and hexanol.

The present invention further relates to cosmetics containing the fats and oils of the present invention. The cosmetics of the present invention can be prepared by blending the fats and oils of the present invention with various types of bases known as ordinary cosmetics according to a conventional method. Examples of the form of the cosmetic include, but are not particularly limited to, cosmetics such as emulsions, creams, lotions, packs, dispersions, and cleaning agents. As the base of the cosmetic, a base corresponding to the form of the cosmetic, for example, purified water, lower alcohols, polyhydric alcohols, fats and oils, surfactants, various cosmetic ingredients, ultraviolet absorbers, thickeners , Dyes, preservatives, fragrances and the like can be used.

The present invention further relates to a detergent containing the fat or oil of the present invention. As the cleaning agent of the present invention, regardless of medicinal or non-medical use, soaps, shampoos, facial creams generally used to keep the body clean,
Rinsing and the like are included, as well as bath additives and the like. In addition, detergents such as utensils used in everyday homes such as tableware may be used.

[0073]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Production of fats and oils by Schizochytrium sp. SR21 strain (1) Glucose 60 g, polypeptone 20 g, yeast extract 10
medium (A) consisting of 1 g of artificial seawater having a concentration of g and 50%,
Alternatively, cultivation was carried out in a jar fermenter (culture tank capacity 5 L, medium volume 3 L) using a medium (B) comprising 90 g of glucose, 10 g of polypeptone, 10 g of corn steep liquor and 1 L of artificial seawater having a concentration of 50%. Culture is
Culture temperature 25 ° C, aeration rate 0.5vvM, stirring speed 20
Performed at 0 rpm. After the culture, the cells were collected by centrifugation and freeze-dried, and the amount of cells per 1 L of the medium was determined by a gravimetric method. Next, 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 fats and oils. After the extract was washed by the Folch method, the solvent was distilled off to obtain a purified oil and its weight was determined. In order to evaluate the fatty acid composition of the obtained refined fat and oil, a part of the fat and oil was dissolved in an equal mixture of a methanol solution containing 10% HCl and dichloromethane, and heat-treated at 60 ° C. for 2 hours to obtain fatty acid methyl. Esters were prepared and the fatty acid composition of the fats and oils was analyzed by gas chromatography. The separation conditions for gas chromatography are as follows. Separation conditions 1) Column: Capillary column, TC-70 manufactured by GL Science Co., Ltd. (GL Science Co., Ltd.) 0.25 mm inner diameter × 30 m length 2) Flow rate: 0.8 ml / min, 100 kPa (column head pressure) 3) Carrier gas: Nitrogen gas (purity 99.999%) 4) Column temperature: Temperature rising mode, 170-220 ° C (4 ° C)
/ Min) 5) Detection: FID

The results are shown in Tables 2 and 3.

[Table 2] Table 2 Experiment No. Medium Culture days Number of bacterial cells Total fat content Fat content (days) (g) (g) (wt%) * 1) * 1) * 2) 301 (A) 7 35.0 8.7 24.8 302 (B) 14 39.6 21.2 53.5 * 1) Amount per 1 L of culture medium * 2) Percentage per dry cell

[Table 3] Table 3. Fatty acid composition (% by weight) Experiment 14: 0 15: 0 16: 0 17: 0 18: 0 20: 4 20: 5 22: 5 22: 6 No. (AA) (EPA) (DPA) (DHA) 301 1.6 10.1 25.0 1.8 1.0 − 0.4 11.1 43.7 302 2.8 6.7 44.6 1.6 1.3 − 0.4 8.5 34.0

From the above results, it was found that Schizochytrium sp.
It was shown that one strain showed good growth even in aeration and agitation culture, which is a practical culture method, and efficiently accumulated fats and oils. As the highly unsaturated fatty acids, docosahexaenoic acid (DHA) is contained at an extremely high concentration, and docosapentaenoic acid (DPA) is also contained, but arachidonic acid (AA) and eicosapentaenoic acid (EPA) are contained. It was shown to contain almost no. This result is significantly different from fish oil containing about 10% by weight of these fatty acids.

Example 2 The oils and fats obtained in Example 1 such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)
Table 4 shows the content ratio of each (n-3) fatty acid and the ratio of DHA to all (n-3) fatty acids.

[Table 4] Table 4. (n-3) fatty acid composition (% by weight) Experiment EPA DPA DHA Ratio of DHA to No. of all (n-3) fatty acids 301 0.4 0.2 43.7 98.6 302 0.4 0.1 34.0 98.6

As can be seen from the table, Schizochytrium S
The fats and oils obtained from the R21 strain are EPA rich in fish oil
Is not more than 1.0% by weight, and all (n-3)
The DHA content relative to the system fatty acid is 98% by weight or more. These facts show that the strain has an advantage of facilitating the operation of concentration, separation and purification of DHA as compared to fish oil containing around 10% by weight of EPA.

Reference Example Comparison with Known Microorganisms DHA of Known Microorganisms with SR21 of Schizochytrium
And EPA productivity. Table 5 shows that Thraustochytrium aureum (ATCC34) was used as a microorganism.
304) to produce DHA [P. Bajapai, PKBajapai and OPWard, Appl.
obiol. Biotechnol. 35: 706 (1991), A. Kendric and
C. Ratledge, Lipids 27:15 (1992), and PKBajapa
i, P. Bajapai and OPWard, J. Am. Oil Chem. Soc., 6
8: 509 (1991)], and culture was carried out using Japonokitorium sp. (ATCC28207) as a microorganism.
A (see Japanese Patent Application Laid-Open No. Hei 1-199588), and cultivation using Schizochytrium aggregatam (ATCC28209) as a microorganism
A production [A. Kendric and C. Ratledge, Li
pids 27:15 (1992)], and the above-mentioned experiments No. 301 and 302, which were cultured using Schizochytrium sp. The content of fats and oils or fatty acids per body, the content of DHA in all fatty acids, the content of EPA, and the amount of DHA per liter of medium are shown.

[0079]

[Table 5] Table 5. Comparison of DHA production ability with known microorganisms Microorganisms References Microbial cell amount Fats and oils or DHA EPA DHA or fatty acid content Content No. Content ratio Proportion (g) (% by weight) (% by weight) (% by weight) (% by weight) (mg) * 1) * 2) * 3 ) * 3) * 1) Cyanophonochytrium sp. (ATCC28207) (a) 1.7 8.2 30.0 6.4 42 Thraustochytrium aureum (ATCC34304) (b) 5.0 20.2 51.0 No description 511 Thraustochytrium aureum (ATCC34304) (c) ) 3.8 16.5 48.5 3.6 270 Thraustochytrium-Aureumu (ATCC34304) (d) 4.0 10.0 24.1 9.3 96 Shizo Kitoriumu-Agu Legatum (ATCC28209) (d) 1.4 1.7 6.0 6.1 1 Shizo Kitoriumu genus SR21 strain No.301 35.0 24.8 43.7 0.4 3800 Shizo Kitoriumu the genus SR21 strain No.302 39.6 53.5 34.0 0.4 7200 * 1) Amount per 1 L of culture medium * 2) Percentage per dry cell (SR21: Fatty acid content; Other strains: Fat content) * 3) Total fatty acids (A) JP-A-1-199588 (b) PKBajap ai, P. Bajapai and OPWard, J. Am. Oil Chem. Soc., 68: 509 (1991) (c) P. Bajapai, PKBajapai and OPWard, Appl. Microbiol. Biotechnol. 35: 706 (1991) (d) A. Kendric and C. Ratledge, Lipids 27:15 (1992)

As shown in Table 5, Schizochytrium SR
When culturing is performed using 21 strains, the amount of cells per medium is much larger than that of known microorganisms, indicating that the SR21 strain has excellent growth properties. Also, Schizochytrium SR
The 21 strains also have a very high oil and fat content compared to known microorganisms. Further, since the DHA content ratio in the total fatty acids is as high as 30% by weight, the amount of DHA per 1 L of the culture medium is as high as about 10 to 100 times as compared with the case where a conventionally known microorganism is used. Has a very high DHA-producing ability. Furthermore, according to known microorganisms, fats and oils having an EPA content of several weight% are obtained, whereas according to Schizochytrium sp.
It can be seen that a fat and oil having a PA content of as low as 0.5% by weight or less can be obtained.

Example 3 Production of fats and oils by Schizochytrium sp. SR21 strain (2) Glucose 60 g / L, Corn steep liquor 0.5 g /
L, potassium phosphate 3g / L, ammonium sulfate 2g / L
Using a medium consisting of 50% artificial seawater and a jar fermenter (5 L capacity, medium volume 3 L), culture was performed for about 60 hours. The culture conditions were controlled at a culture temperature of 28 ° C., an aeration rate of 1.0 v. Vm, a stirring speed of 300 rpm, and a pH of 4 with 10% sodium hydroxide. After completion of the culture, the cells were collected by centrifugation, lyophilized, and weighed to obtain about 20 g of dried cells per liter of medium. Extraction of oils and fats was performed by mixing chloroform / methanol (2: 1, v / v) with the dried cells and homogenizing in the presence of glass beads according to a conventional method. The amount of crude oil extracted from 60 g of the dried cells was 36 g. This crude oil and fat was converted into a methyl ester according to a conventional method, and the fatty acid composition was examined by gas chromatography.
The composition shown in FIG.

[0082]

[Table 6] Table 6. Fatty acid composition in crude oils and fats Fatty acids 14: 0 15: 0 16: 0 17: 0 18: 0 20: 4 20: 5 22: 5 22: 6 n-6 n-3 AA EPA DPA DHA content (%) 2.3 5.8 44.6 2.0 1.4 0.6 0.8 8.4 34.0

Example 4 Isolation and Identification of Docosapentaenoic Acid To increase the concentration of highly unsaturated fatty acids in the crudely extracted fatty acids, urea was added to remove the saturated fatty acids according to a conventional method. That is, 20 g of urea and 200 ml of methanol were added to about 9 g of the crude extracted fatty acid prepared by the method shown in Example 3, and
After heating at 3 ° C. for 3 hours, the mixture was gradually cooled to 10 ° C. After filtering the precipitated urea crystals, the non-crystal solution was concentrated to obtain about 4 g of a non-adduct of urea. The fatty acid composition of this non-urea adduct is docosapentaenoic acid 17.7%, docosahexaenoic acid 77.
The content of other fatty acids was 9% or less. The urea non-adduct obtained by the above treatment was subjected to liquid chromatography (ODS column, mobile phase acetonitrile: water = 97.5: 2.5, detection UV) to collect docosapentaenoic acid. As a result, about 3.2 g of docosapentaenoic acid having a purity of 99% or more was obtained.

Further, in order to determine the double bond position of docosapentaenoic acid, the fatty acid was converted into a piconyl ester derivative according to a conventional method, and analyzed by GC / MS. That is, 100 μl of thionyl chloride was added to 20 μg of the urea non-added fatty acid obtained above, left at room temperature for 1 minute, and then dried under a nitrogen stream. To this was added 10 μl of 10% 3-pyridylcarbonyl, and the mixture was left at room temperature for 1 minute.
Analysis was performed by MS. As a result, as shown in FIG.
Docosapentaenoic acid produced by SR21 strain (22: 5)
Has unsaturated bonds at △ 4,7,10,13,16
It was identified as (n-6) unsaturated fatty acid.

Example 5 Fatty Acid Analysis of Schizochytrium Species SR21 Strain The crude oil extracted and obtained according to Example 3 was used.
The neutral lipid and the polar lipid were separated from the crude oil / fat by liquid / liquid distribution using hexane and 90% methanol in a conventional manner. The amount of lipid obtained was 1.8 g of polar lipid,
The amount of neutral lipid was 32.9 g. After hydrolyzing this lipid, it was methyl-esterified, and the fatty acid composition was analyzed by gas chromatography.

The analysis results are as follows.

[Table 7] Table 7. Polar / neutral lipid composition in crude oils and fats Polar lipid (%) Neutral lipid (%) 14: 0 1.3 3.2 15: 0 0.6 1.0 16: 0 32.4 49.2 20: 4 (n-6) 0.20 .6 20: 5 (n-3) 0.6 0.8 22: 5 (n-6) 10.6 7.9 22: 6 (n-3) 52.1 33.7

Example 6 Analysis of Fatty Acid Residues of Phosphatidylcholine Phosphatidylcholine in the polar lipid obtained in Example 5 was separated and fractionated by liquid chromatography. The separation conditions were as follows. 1) Column: silica gel ram, type; TSK gel silica-60, manufacturer; Tosoh Corporation, shape; diameter 2
0 mm x length 250 mm 2) Flow rate: 10 ml / min 3) Mobile phase (volume ratio): acetonitrile / methanol / phosphoric acid = 95.0 / 5.0 / 0.5 4) Column temperature: 25 ° C 5) Inspection Source: Differential refraction, 25 ° C

The separated phosphatidylcholine was separated into molecular species by liquid chromatography. After separating them, they were subjected to hydrolysis and methyl esterification, and fatty acid residues were determined by gas chromatography. The separation conditions for liquid chromatography were as follows. 1) Column: ODS gel column, type;
(Inertsil) ODSII, manufacturer; GL Science Co., Ltd. Shape; diameter 4.6 mm × length 250 mm 2) Flow rate: 1 ml / min 3) Mobile phase (volume ratio): methanol / H ₂ O = 95.0 /
5.0 4) Column temperature: 25 ° C. 5) Detection: differential refraction, 25 ° C. Separation conditions for gas chromatography were as follows. 1) Column: type; capillary column, TC-7
0, Manufacturer: GL Sciences, Shape: 0.25m in diameter
mx length 30m 2) Flow velocity: 0.8ml / min, 100kPa (column head pressure) 3) Carrier gas: nitrogen gas 4) Column temperature: temperature rising mode, 170-220 ° C (4 ° C)
5) Detection: FID As a result, the molecular species of the phosphatidylcholine of the SR21 strain were mainly 16: 0-22: 6, 16: 0-22: 5,
22: 5-22: 6 and 22: 6-22: 6.

Example 7 Lipid Analysis of Schizochytrium sp. SR21 The neutral lipid and polar lipid obtained in Example 5 were analyzed by thin-layer chromatography according to a conventional method. Color development was performed using sulfuric acid, and the identification of spots obtained was determined by the Rf value with each standard lipid. More than 90% of the neutral lipids were triglycerides. The polar lipids are mostly phosphatidylcholine (60-80% by weight),
Then phosphatidylethanolamine (5-20% by weight), phosphatidylinositol (2-8% by weight)
Met. The triglyceride in the neutral lipid was separated from the molecular species by liquid chromatography (ODS column, mobile phase acetone: acetonitrile = 3: 2, differential refractometer) according to a conventional method (FIG. 5). Hydrolysis methyl esterification,
Fatty acid residues were determined by gas chromatography.

The results are as follows.

[Table 8] Table 8. Analysis of triglycerides in neutral lipids Peak molecular species Weight ratio (%) No. 1 14: 0-16: 0-0-16: 0 3.4 2 16: 0-16-16: 0-16: 0 8.0 3 14: 0-16-16: 0-22: 6 4.0 4 16: 0- 16: 0-22: 5 7.8 5 16: 0-16: 0-22: 6 27.4 6 16: 0-22: 5-22: 6 8.4 7 16: 0-22: 6-22 : 6 16.9 These seven types of triglycerides accounted for more than 70% by weight of the total triglycerides. The largest species of this triglyceride was 16: 0-16: 0-22: 6, accounting for about 27% by weight of the total triglyceride.

Example 8 Determination of Binding Position of Triglyceride to Fatty Acid Residue Triglyceride fractionated in Example 7 (molecular species 16: 0-1)
6: 0-22: 6) was dried, treated with a lipase specific to the 1,3-position, and the resulting 2-monoglyceride was trimethylsilylated, and then the fatty acid residues were determined by GC / MS. Lipase treatment was performed using 2 mM of 50 mM acetate buffer (pH 5.5).
1, 1000 units of lipase, 35 ° C., 30 minutes.
After completion of the reaction, the mixture was extracted with ether, and 2-monoglyceride was trimethylsilylated using a commercially available trimethylsilylating agent. As a result, as shown in FIG. 6, a fragment peak corresponding to the molecular weight of monoglyceride to which 22: 6 was bound was obtained, and this triglyceride has a 22: 6 fatty acid residue bound to the 2-position of the glycerol skeleton. 1
6: 0-22: 6-16: 0.

Example 9 Effect of Nitrogen Concentration Per liter of medium, 60 g of glucose, 3 g of potassium dihydrogen phosphate, and 0.5 g of corn steep liquor were added.
Ammonium sulfate was added at a concentration shown in Table 9 to an artificial seawater medium having a 1 / concentration, and 3 L of this medium was placed in a 5-L jar fermenter. To this medium, 60 ml of a preculture liquid prepared in the same manner as in Example 3 was added and cultured. The culture was performed at 28 ° C., 1 v.vm, 300 rpm, and pH 4.0. After consumption of glucose, 100 ml of the culture solution was collected, and the cells were collected by centrifugation. The cells were washed, and dried cells were obtained using a freeze-drying apparatus. The weight of the dried cells was measured to determine the amount of cells per liter of medium. Next, in the same manner as in Example 3, the amount of cells per 1 L of the medium was determined.
The total fatty acid content ratio per 1 L of the medium, and the DHA and DPA amounts per 1 L of the medium were determined. Table 9 shows the results.

[0093]

[Table 9] Table 9. result of analysis Experiment (NH ₄ ) ₂ SO ₄ cell mass Total fat Fatty acid DHA content DHA content DPA content DPA content No. Acid content Content ratio Proportion (g) (g) (wt%) (wt%) (g) (wt% ) (g) 401 0.5 6.5 4.8 74.5 31.4 1.5 7.9 0.38 402 1.0 8.6 6.0 69.8 32.0 1.9 8.0 0.48 403 2.0 15.3 9.1 59.6 35.0 3.2 8.8 0.81 404 3.0 20.5 10.7 52.2 35.8 3.8 8.9 0.90 Ammonium sulfate concentration 2.0-3.0g / L showed good growth. Further, when the ammonium sulfate concentration was set to 0.5 to 1.0 g / L, the fatty acid content increased to 70% or more.

Example 10 Influence of Organic Nitrogen Source and Inorganic Nitrogen Source To 1 L of medium, 60 g of glucose, 3 g of potassium dihydrogen phosphate and 0.5 g of corn steep liquor were added.
The concentration of the nitrogen source was kept constant at the concentration shown in Table 10, and the ratio of corn steep liquor (CSL) as the organic nitrogen source and ammonium sulfate as the inorganic nitrogen source were changed to two parts of the artificial seawater medium having a concentration of 1 / . 3 L of this medium was placed in a 5 L jar fermenter, and 60 ml of a preculture solution prepared in the same manner as in Example 3 was added, followed by culturing under the same conditions as in Example 3. After consumption of the glucose, the culture 100
ml was collected and cells were collected by centrifugation. The cells were washed, and dried cells were obtained using a freeze-drying apparatus. Table 10 shows the results obtained by measuring the weight of the dried cells and determining the amount of cells per 1 L of the culture medium. Next, in the same manner as in Example 3, the amount of cells per 1 L of medium, the total fatty acid content per 1 L of medium, and the amount of DHA and DPA per 1 L of medium were determined. Table 10 shows the results.

[0095]

[Table 10] result of analysis Experiment CSL Sulfuric acid bacteria Total fat Fatty acid DHA containing DHA DPA containing DPA No. Ammonium Amount of acid Amount Percentage Amount Ratio Amount (g) Aum (g) (g) (g) Total (%) (Weight%) (g) (Wt%) (g) 501 0.7 2.0 21.0 14.8 70.5 31.8 4.7 8.1 1.2 502 2.0 1.7 20.0 13.7 68.5 31.7 4.3 7.3 1.0 503 3.3 1.3 23.2 14.5 62.4 30.7 4.5 6.9 1.0 504 5.3 0.7 21.0 13.7 68.5 29.9 4.3 8.0 1.1 505 8.0 0 20.6 13.4 65.2 27.9 3.8 6.7 0.9 It shows good growth at any composition ratio, and the fungus grows irrespective of organic nitrogen or inorganic nitrogen. Also,
The productivity of DHA and DPA has not changed much.

Example 11 High-Concentration Culture Glucose, corn steep liquor, and ammonium sulfate were added at the concentrations shown in Table 11 to a half-concentrated artificial seawater medium containing 3 g of potassium dihydrogen phosphate per liter of the medium. . 3 L of this medium was placed in a 5 L jar fermenter and cultured under the culture conditions described in Example 3. After consumption of glucose, 100 ml of the culture solution was collected, and the cells were collected by centrifugation. The cells were washed, and dried cells were obtained using a freeze-drying apparatus. The weight of the dried cells was measured to determine the amount of cells per 1 L of the culture medium.
The amount of cells per liter of medium, the amount of total fatty acids per liter of medium, the percentage of fatty acids per dry cell, the percentage of DHA and DPA in all fatty acids, and the amounts of DHA and DPA per liter of medium were determined in the same manner as described above. I asked. Table 11 shows the results.

[0097]

[Table 11] Table 11. result of analysis Experiment Glu CSL Sulfuric acid culture Dried whole fat fatty acid DHA DHA DPA DPA No. Coarse amount Ammonium time Bacterial fatty acid content Content Content Amount Amount Amount Amount Ratio Ratio (g) Ratio (g) (g) (g) (hour) ) (g) (g) (% by weight) (% by weight) (% by weight) 601 60 0.7 2.0 60 21.9 14.8 67.6 34.5 5.11 6.8 1.01 602 80 0.93 2.7 84 32.0 21.9 68.6 34.7 7.62 7.1 1.55 603 100 1.17 3.3 92 37.7 31.15 82.6 33.3 10.37 6.3 2.03 604 120 1.4 4.0 108 48.1 37.3 77.5 35.6 13.26 7.4 2.76 605 150 1.75 5 120 59.2 41.6 70.3 37.3 15.52 8.2 3.43 From these results, it can be seen that the bacterium increases glucose by increasing the concentration of carbon and nitrogen sources. As the concentration increases, DH
A. It became clear that the production of DPA also increased.

Example 12 Influence of Stirring Speed Under the same medium composition and culturing conditions as in Example 3, culturing was performed with the stirring speed of the culturing tank set to 100 rpm and 300 rpm. Glucose consumption time was about 100 hours at 100 rpm, but about 300 times at 50 rpm.
60 hours. On the other hand, the amount of fatty acid per liter of medium,
The DHA and DPA amounts were slightly higher at 300 rpm (Table 12).

[Table 12] Table 12. result of analysis Number of rotations Bacterial cell content Total fat Fatty acid DHA content DHA content DPA content DPA content Acid content Content ratio Proportion (rpm) (g) (g) Total (%) (%) (g) (g) (g) 100 15.3 9.1 59.6 35.0 3.2 8.4 0.76 300 20.5 10.7 52.2 35.8 3.9 8.5 0.91

Example 13 A culture solution prepared according to Example 11 was collected, and the solution was removed with a filter press and dried to obtain 10 kg of cells containing 10% of water. Extract the cells with hexane according to a standard method,
After removing hexane, 5.9 kg of oil was obtained. After drying the cells after hexane extraction to remove hexane,
3.9 kg of oil extraction residual cells were obtained. The fat contained 35% DHA and 8% DPA. The oil-extracted cells contained 1.2% DHA and 0.3% DPA.

Example 14 A group of five layers of laying hens for 200 meals of Isa Brown breed was divided into two groups. One group was bred with a normal feed for 25 days as a control group. The remaining group was an experimental group, and the fats and oils obtained in Example 13 were added to a normal feed in an amount of 5 g daily and bred for 25 days. Egg weight for three eggs on day 25, yolk weight,
The DHA concentration, DPA concentration, egg yolk shine, and egg yolk taste were measured. Table 13 shows the results. By rearing with the addition of fats and oils, the DHA and DPA contents in the yolk clearly increased. In addition, it was possible to obtain a bright and melting egg yolk.

[Table 13] Table 13 Weight in the experimental group the control group eggs (3 min) g 213 211 yolk weight g 55 52 DHA (%) 3.5 1.5 DPA (%) 0.8 trace yolk irradiation shine is good rough felt yolk Taste melty watery

Example 15 Egg-laying hens of 200 meals of Isa Brown breed were divided into two groups. 1
As a control group, five birds were bred on a normal feed for 25 days. The remaining group was bred for three days as an experimental group, and the normal feed was reduced by 50 g from the control group, and 50 g of the oil-extracted residual cells obtained in Example 13 was added to supplement the amount by 25 g. Egg weight, egg yolk weight, DHA for three eggs on day 25
The concentration, DPA concentration, shine of egg yolk, and taste of egg yolk were measured.
Table 14 shows the results. By rearing the cells with the oil extract residual cells, the DHA and DPA contents in the egg yolk clearly increased. In addition, it was possible to obtain a bright and melting egg yolk.

[Table 14] Table 14 Experimental group Control group Egg weight (for 3 pieces) g 212 211 211 Yolk weight g 53 52 DHA (%) 2.1 1.5 DPA (%) 0.2 Trace amount Yolk good illuminated Egg yolk Taste melty watery

Example 16 Preparation of Milk Containing (n-6) Docosapentaenoic Acid and DHA (n-6) Docosa extracted from the egg yolk obtained in Example 14 to 100 g of powdered milk raw material according to a conventional method. By mixing 6 g of pentaenoic acid and DHA-containing egg yolk oil [0.8 of (n-6) -based docosapentaenoic acid, containing 3.5% of DHA]
An (n-6) -based docosapentaenoic acid and DHA-containing milk were prepared. The ratio of (n-6) docosapentaenoic acid to the total fatty acids in this milk was 0.19%, and the ratio of DHA was 0.1%.
The ratio was 84%, and the (n-6) docosapentaenoic acid and DHA which were insufficient in the conventional formula could be brought close to the mother's milk.

Example 17 Preparation of Milk Containing (n-6) Docosapentaenoic Acid and DHA The crude extract oil obtained in Example 5 was subjected to liquid / liquid partitioning with hexane and 90% methanol according to a conventional method. Neutral lipid separated into neutral lipid and polar lipid and containing (n-6) docosapentaenoic acid and DHA [7.9% (n-6) docosapentaenoic acid, containing 33.7% DHA] I got 0.6 g of this oil was mixed with 100 g of powdered milk raw material to prepare a milk containing (n-6) docosapentaenoic acid and DHA. (N-
6) The ratio of the system docosapentaenoic acid was 0.19%, and the ratio of DHA was 0.80%, which was insufficient in the conventional formula.
(n-6) docosapentaenoic acid and DHA could be brought close to breast milk.

Example 18 Preparation of Sepnel Containing (n-6) Docosapentaenoic Acid and DHA

[Table 15] Table 15. Capsule Gelatin 70.0% Glycerin 22.9% Methyl parahydroxybenzoate 0.15% Propyl paraoxybenzoate 0.51% Water qty 100% In a soft capsule shell consisting of the above ingredients, Example 1
(N-6) docosapentaenoic acid and DH obtained in 1
300 mg of the A-containing microbial oil was filled in a conventional manner to obtain a soft capsule.

Example 19 Preparation of Beverage Containing (n-6) -Based Docosapentaenoic Acid and DHA 50 g of commercially available plain yogurt was placed in a container.
(N-6) docosapentaenoic acid and DHA obtained in
Containing 50% microbial oil and 1 g of β-cyclodextrin
And stirred for about 3 minutes to emulsify, W / O /
An emulsion mixed with W, O / W / O type and the like was obtained.

Example 20 Production of Micro-Feed Biological Feed Containing DHA and DPA A culture solution produced according to Example 3 was collected, and the solution was removed with a filter press to obtain cells. The obtained cells were added to 105
The mixture was dried by heating at a temperature of 3 ° C. for 3 hours and powdered with a coffee mill. Using the resulting powder or baker's yeast as a control, rotifers and brine shrimp were cultured. The culture method is as follows: 200 L of seawater is 300 L
At 23 ° C under aeration conditions, 100 rotifers per ml, brine shrimp 20 per ml.
Individuals were allowed to feed, and the above-mentioned bacterial cell powder or baker's yeast was fed as feed at a rate of 1 g / 10 ⁶ rotifers per day and 1 g / 10 ⁵ brine shrimp per day. Ingest these in rotifers or brine shrimp and grow them,
As a result of examining the constituent fatty acid composition by sampling on the third day, the results are as shown in Tables 16 and 17.

As shown in the results, the accumulation of DHA and DPA was higher in rotifers and brine shrimp than in baker's yeast.

[Table 16] Rotifer Fatty acid composition (%) DHA DPA control (baker's yeast) 00 test (SR21 strain) 265

[Table 17] Table 17. Brine shrimp Fatty acid composition (%) DHA DPA control (baker's yeast) 00 test (SR21 strain) 234

[0108]

The marine microorganisms used in the present invention include:
It has excellent growth and fat accumulation properties, has high (n-3) -based DHA and (n-6) -based DPA production ability, and has extremely low EPA production. Therefore, using this microorganism, (n-
3) Fats and oils having a high content of the system DHA and the (n-6) system DPA and a low content of the EPA can be produced in high yield. In addition, a highly pure (n-3) DH
A or (n-6) DPA can also be separated. The fats and oils of the present invention have various physiological activities (n-3)
Since the (D-6) DPA is contained at a high concentration together with the DHA, the fats and oils are added to various foods, feeds and feeds, and the (D-3) DHA and the (n-6) DPA are required. These highly unsaturated fatty acids can be supplied stably and efficiently to the target. In particular, in animal feeds or animal feed additives, micro-feed biological feeds, DHA and D
It is very economical because the extraction residue of the cultured cells containing PA can also be used. In addition, by feeding the above-mentioned animal feed to poultry, it is possible to obtain poultry eggs or yolk oil enriched with DHA and DPA, which have never been obtained before.

[Brief description of the 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 morphology of flagella of zoospores of Schizochytrium genus SR21.

FIG. 3 is an optical micrograph showing the morphology of a vegetative cell mass and protoplasm network of Schizochytrium sp. SR21.

FIG. 4. (n-6) derived from Schizochytrium sp. SR21 strain
4 is a chart showing the results of measuring the GC / MS spectrum of a system docosapentaenoic acid.

FIG. 5 is a chart showing the results of separating triglycerides in neutral fats and oils derived from Schizochytrium sp. SR21 by liquid chromatography.

FIG. 6 shows that GC / M after triglyceride (molecular species 16: 0-16: 0-22-22: 6) derived from Schizochytrium sp. SR21 was subjected to lipase treatment and then trimethylsilylation.
4 is a chart showing the results measured in S.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location A23L 1/30 A23L 1/30 Z 2/52 A61K 31/23 ADD A61K 31/23 ADD 35/74 G 35/74 C11B 15/00 C11B 15/00 C12P 7/64 C12P 7/64 2115-4H C07C 57/12 // C07C 57/12 A23L 2/00 F (C12P 7/64 C12R 1:90) (72 Inventor: Toshihiro Yokochi 1-3-1, Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute (72) Inventor Toshiro Nakahara 1-3-1, Higashi, Tsukuba, Ibaraki Pref. 72) Inventor Takanori Higashihara 1-3-3 Higashi, Tsukuba City, Ibaraki Pref., National Institute of Advanced Industrial Science and Technology (72) Inventor: Gohiro Tanaka Koichi Ichinomiya-cho, Shiso-gun, Hyogo Sentence 400 (72) Inventor Toshiaki Yaguchi 23-6 Inarimae, Tsukuba, Ibaraki Pref.

Claims (26)

[Claims] 1. An amount of (n-6) -based docosapentaenoic acid of 5% by weight or more, (n-3) -based docosahexaenoic acid of 20% by weight or more, and eicosapentaenoic acid of 2% by weight, based on the total fatty acids in the fat or oil. It is characterized by containing the following amount
(n-6) -based docosapentaenoic acid-containing fats and oils. 2. An amount of at least 6% by weight of (n-6) docosapentaenoic acid, at least 25% by weight of (n-3) docosahexaenoic acid, and 1% by weight of eicosapentaenoic acid, based on the total fatty acids in the fat or oil. The fat or oil according to claim 1, which is contained in the following amount. 3. The fat or oil according to claim 1, wherein the proportion of the neutral lipid in the fat or oil is 90% by weight or more. 4. The fat or oil according to claim 3, wherein 85% by weight or more of the neutral lipid is triglyceride. 5. The method according to claim 5, wherein the fat or oil is a Schizochytrium genus SR21 strain (FER) capable of producing (n-6) docosapentaenoic acid.
MBP-5034) or an oil or fat obtained by culturing a microorganism belonging to the same species or having substantially the same mycological properties as the SR21 strain in a medium, and collecting from the culture. Item 5. The fat or oil according to any one of Items 1 to 4. 6. The microorganism to be cultured is Schizochytrium sp. SR.
The fat or oil according to claim 5, which is a microorganism recognized to belong to the same species as the 21 strain or the SR21 strain. 7. The microorganism to be cultured is Schizochytrium sp. SR.
The fat and oil according to claim 6, which is 21 strains. 8. The fat or oil according to any one of claims 5 to 7, wherein the fat or oil is a fat or oil obtained by purifying a fat or oil obtained by collecting from a culture of a microorganism. 9. The method according to claim 9, wherein the oil or fat is a culture solution during the production of the oil or fat by cell culture, or a sterilized culture solution thereof, or a culture solution at the end of the culture or a sterilized culture solution thereof, or a cultured bacterial cell collected from each of them. The oil or fat according to any one of claims 5 to 7, wherein the oil or fat is contained in a dried product thereof or a residue obtained after collecting the oil or fat from a culture solution or cells. 10. A dietary supplement containing the fat or oil according to any one of claims 1 to 9. 11. The dietary supplement according to claim 10, wherein the fat or oil is encapsulated in a gelatin capsule. 12. The dietary supplement according to claim 10, wherein the dietary supplement is a drink or a food or drink in the form of a granule or tablet. 13. An infant formula containing the fat or oil according to any one of claims 1 to 9. 14. A formula for premature infants, comprising the fat or oil according to claim 1. 15. An infant food containing the fat or oil according to any one of claims 1 to 9. A food for pregnant women containing the fat or oil according to any one of claims 1 to 9. 17. A food for the elderly containing the fat or oil according to any one of claims 1 to 9. 18. An enteral nutrient containing the fat or oil according to any one of claims 1 to 9. 19. An animal feed containing the fat or oil according to any one of claims 1 to 9. 20. An animal feed additive containing the fat or oil according to any one of claims 1 to 9. 21. A feed for living organisms, comprising the oil or fat according to any one of claims 1 to 9. A poultry egg obtained by breeding the fat or oil according to any one of claims 1 to 9. 23. A Schizochytrium sp. SR21 strain (FERM BP) capable of producing (n-6) docosapentaenoic acid.
(N-5034) or a microorganism belonging to the same species as the SR21 strain or having substantially the same mycological properties in a medium, and collecting the fat or oil from the culture, wherein (n- 6) A method for producing a fat containing docosapentaenoic acid. 24. The microorganism to be cultured is Schizochytrium sp.
The method according to claim 23, wherein the microorganism is a microorganism recognized to belong to the same species as the R21 strain or the SR21 strain. 25. The microorganism to be cultured is Schizochytrium sp.
The method according to claim 24, which is an R21 strain. 26. The method according to claim 23, wherein the (n-6) docosapentaenoic acid is isolated from the (n-6) docosapentaenoic acid-containing oil or fat. A method for producing (n-6) docosapentaenoic acid.