JP7262536B2 - Lipid composition, use thereof and method for producing the same - Google Patents

Description

IPOD FERM P-22324

The present invention provides a lipid composition containing n-3 docosapentaenoic acid (n-3DPA), uses thereof,
and its manufacturing method.

Polyunsaturated fatty acids (PU
FA) are important components in human and animal nutrition.

PUFAs are classified as n-3 (or ω-3) PUFAs, depending on the position of the first double bond in the carbon chain.
, n-6 (or ω-6) PUFA, etc. Examples of n-3 PUFA include eicosapentaenoic acid (EPA, C20:5n-3), n-3 docosapentaenoic acid (n-3DPA, C
22:5n-3), docosahexaenoic acid (DHA, C22:6n-3), and the like. Arachidonic acid (ARA, C20:4n-6), n-6 docosapentaenoic acid (n-6DPA, C22:5n-6) and the like are known as n-6PUFA.

Since n-3 PUFA and n-6 PUFA are not converted to each other in metabolism in the human body, each must be ingested. n-3 PUFA and n-6 PUFA are about 1: 4
It is recommended to take in a balance of (for example, Patent Document 1). However, in Japan today, n-3 PUFA and n-6 PUFA are ingested at a ratio of about 1:15 to 1:25, which is not a desirable balance.

On the other hand, EPA, which is one of the n-3 PUFAs, is known to have antiarteriosclerotic activity. Since EPA administered in the human body is converted to n-3DPA on the inner wall of blood vessels, n-3DPA is E
It is considered to be the active substance of PA's antiarteriosclerotic activity (Non-Patent Documents 1 to 3).

As a conventional method for producing n-3DPA, Patent Document 2 discloses that EPA is produced by adding EPA to a medium for culturing a transformant into which a mammal-derived fatty acid chain elongase gene has been introduced.
A method for producing n-3DPA from is disclosed. Non-Patent Document 4 discloses a method of synthesizing n-3DPA from EPA by organic chemical synthesis. Patent Document 3 discloses extracting lipids from the soft bodies or eggs of specific molluscs such as gastropods (snail nets) and collecting lipid components containing n-3DPA as a constituent fatty acid.

US Pat. No. 6,200,001 discloses at least 50% (a/a) EPA, substantially in free acid form;
A pharmaceutical composition comprising at least 15% (a/a) DHA, substantially in free acid form, and at least 1% (a/a) DPA (n-3), substantially in free acid form things are disclosed.

US Pat. No. 6,200,000 discloses a method of reducing triglyceride levels from baseline levels in a human patient comprising from 160 mg/day to about 600 mg/day of omega-3DPA, optionally further comprising DHA, if DHA. discloses a method of administering a composition that does not exceed 2:1 DHA:n-3DPA.

Patent Document 6 discloses providing a transformation method for obtaining a stramenopile with improved ability to acidify unsaturated fatty acids by genetically engineering a gene to disrupt it or suppress its expression. Thraustochytriu as a stramenopile
m), etc. are exemplified. In Example 8-6 of Patent Document 6, Thra
Disruption of the Δ4 desaturase gene in the OrfA-disrupted strain of ustochytrium aureum ATCC 34304 resulted in little biosynthesis of C22:5n-6 (n-6DPA) and C22:6n-3 (DHA) and C22:4n-6 (DTA).
) and C22:5n-3 (n-3DPA) accumulated, and Figure 64 shows the composition of the lipids produced as C22:4n-6 (DTA) at 11.01%, C22:5n-6 (DTA) at 11.01%;
: 5n-3 (n-3DPA) is 15.76%, C22: 5n-6 (n-6DPA) is 0.
21% and C22:6n-3 (DHA) was 0.51%.

Patent Document 7 describes an oil and fat composition that has excellent absorbability into the body of n-3 polyunsaturated fatty acids, is easy to process without precipitating oil crystals even at room temperature, and has a good flavor. Among the constituent fatty acid residues, it contains 20 to 60% by weight of n-3 polyunsaturated fatty acids, 10 to 30% by weight of caprylic acid and/or capric acid, and 15 to 40% by weight of palmitic acid, ranking second. A triglyceride in which the amount of palmitic acid residues bound/total amount of palmitic acid residues (weight ratio) is 0.4 to 0.95, and all fatty acids bound in the entire oil and fat composition are caprylic acid and/or capric acid. content of 10% by weight or less is disclosed.

JP 2014-159576 A JP 2003-116566 A JP 2013-40235 A US2013/0209556 US 8,906,964 WO2012/043826 Japanese Unexamined Patent Application Publication No. 2016-202001

Arterios cl. Thromb. Vas. 1994, 14(3), 471 Am. J. Epidemiol. 1995, 142(5), 469 Prostag. Leucotr. Ess. 1996, 54(5), 319 Lipids, 2006, 144(2), 172 J. Lipid Res. 2012, 53(8), 1543 J. Clin. Invest. 2015, 125(12), 4544 Can. J. Biochem. Phys. 1959, 37(8), 911 Mycoscience, 2007, 48, 199

The present invention provides a lipid composition comprising n-3 PUFA such as n-3DPA, a pharmaceutical composition, a food composition, or a feed composition comprising said lipid composition, a method for producing said lipid composition, n-3DPA
Provided are a method for producing the contained lipid, and a method for producing the pharmaceutical composition, the food composition, the feed composition, or the feed composition.

The present invention includes the following inventions.
(1) A lipid composition comprising
As a peak area ratio in the chromatogram obtained when analyzed by GC-FID (gas chromatography using a flame ionization detector as a detector),
For the total amount of fatty acid components,
In terms of methyl ester,
10% or more of n-3DPA (n-3 docosapentaenoic acid);
containing at least 30% or more DHA (docosahexaenoic acid),
The total content of n-6 polyunsaturated fatty acids is 15% or less,
A lipid composition characterized in that the content of palmitic acid is 10% or less.
(2) The lipid according to (1), which contains 30 parts or more of n-3DPA to 100 parts of DHA as a peak area ratio in a chromatogram obtained when analyzed by GC-FID, in terms of methyl ester. Composition.
(3) A pharmaceutical composition, food composition, or feed composition comprising the lipid composition of (1) or (2).
(4) A method for producing a lipid composition containing n-3DPA as a fatty acid component,
a culturing step of culturing a microorganism belonging to the genus Aurantiochytrium;
A lipid composition obtaining step of obtaining a lipid composition from the culture obtained in the culturing step,
The culturing step is
Cultivating the microorganism in a medium containing a carbon source at a concentration of 0.5% by mass or more at the start, and reducing the carbon source in the medium to 0.1% by mass or less by consumption of the carbon source by the microorganism. 1 step;
A second step of culturing for 48 hours or more in a medium with a carbon source concentration of 0.1% by mass or less,
the aforementioned method.
(5) The first step includes culturing the microorganism in a medium containing a carbon source at a concentration of 0.5% by mass or more, causing the microorganism to consume the carbon source, and then adding 0.5% by mass of the carbon source. The method according to (4), further comprising adding the carbon source to the medium so as to obtain a concentration of 10% or higher, and further culturing the microorganism to consume the carbon source.
(6) The method according to (4) or (5), wherein the medium used in the culturing step contains one or more components selected from the group consisting of ascorbic acid and isoascorbic acid.
(7) The lipid composition to be produced is the lipid composition according to (1) or (2), (4
) to (6).
(8) A method for producing an n-3DPA-containing lipid containing n-3DPA as a fatty acid component,
(4) A lipid composition manufacturing step for manufacturing a lipid composition by the method according to any one of (7);
A method comprising a separation step of separating n-3DPA-containing lipids from said lipid composition produced.
(9) A method for producing an n-3DPA-containing lipid containing n-3DPA as a fatty acid component,
A method comprising a separation step of separating the n-3DPA-containing lipid from the lipid composition according to (1) or (2).
(10) A method for producing a pharmaceutical, food, or feed composition containing a lipid composition containing n-3DPA as a fatty acid component,
(4) A lipid composition production step for producing a lipid composition containing n-3DPA as a fatty acid component by the method according to any one of (4) to (7);
and a blending step of blending the manufactured lipid composition.
(11) A method for producing a pharmaceutical composition, food composition, or feed composition containing n-3DPA-containing lipids containing n-3DPA as a fatty acid component,
(4) A lipid composition production step for producing a lipid composition containing n-3DPA as a fatty acid component by the method according to any one of (4) to (7);
a separation step of separating n-3DPA-containing lipids from the produced lipid composition;
and a blending step of blending said separated n-3DPA-containing lipid.
(12) D42-16625 (accession number: FERM P-22324).

According to the present invention, a lipid composition comprising n-3PUFA such as n-3DPA, a pharmaceutical composition, a food composition, or a feed composition comprising said lipid composition, a method for producing said lipid composition, n- 3
A method for producing a DPA-containing lipid and a method for producing the pharmaceutical composition, the food composition, the feed composition or the feed composition are provided.

<1. Lipids and Lipid Compositions>
In the present invention, lipid is a general term for lipophilic compounds containing at least a fatty acid component, including fatty acids, fatty acid triglycerides, fatty acid diglycerides, fatty acid monoglycerides, phospholipids and the like.

In the present invention, a lipid composition refers to a composition containing one or more lipids, may contain two or more lipids, and may further contain other components.

<2. Features of lipid composition>
One aspect of the present invention is
Based on the total amount of fatty acid components,
In terms of methyl ester,
10% or more n-3DPA;
containing at least 30% or more DHA,
The total content of n-6PUFA is 15% or less,
It relates to a lipid composition characterized in that the content of palmitic acid is 10% or less. Hereinafter, the lipid composition according to this aspect is referred to as "the lipid composition of the present invention".

In this specification, unless otherwise specified, the ratio (%) of fatty acids in the lipid composition of the present invention refers to a sample obtained by methyl-esterifying the fatty acid of the lipid composition using a GC-FID (flame ionization type detector as a detector). It means the ratio of a predetermined fatty acid to the total amount of fatty acid components in terms of methyl ester as a peak area ratio in a chromatogram obtained when analyzed by gas chromatography using a detector. Analysis of the fatty acid composition in the lipid composition can be performed by the following method: the culture solution is collected by centrifugation, disrupted, and then Blig
It is extracted by the h & Dyer method (Non-Patent Document 7). A sample obtained by converting the extracted lipid composition into methyl esters is analyzed for fatty acid composition by GC-FID. As specific conditions for GC-FID, the conditions described in Examples can be exemplified.

In the lipid composition of the present invention, "the total content of n-6 PUFA is 15% or less" means that n-6P
It means that UFA is not contained or, even if it is contained, the content is 15% or less.

Similarly, in the lipid composition of the present invention, "the content of palmitic acid is 10% or less" means that palmitic acid is not contained, or even if it is contained, the content is 10% or less. It means that there is

Similarly, in this specification, when only specifying that the content of a certain component is equal to or less than a predetermined upper limit, the component is not included, or even if it is included, the content is It means that it is less than or equal to the upper limit.

The lipid composition of the present invention contains, as n-3PUFA, 10% or more n-3DPA and 30%
Since it contains at least DHA, it contains at least 40% or more of n-3 PUFA. The total n-6 PUFA content is less than 15%. That is, the lipid composition of the present invention contains n-3 PUFA in an amount that is 2.6 times greater than n-6 PUFA. By ingesting the lipid composition of the present invention, it is possible to correct the balance between n-3PUFA and n-6PUFA, which tend to be biased toward n-6PUFA. In addition, by ingesting the lipid composition of the present invention, n-3DPA and DHA, which are known to have beneficial physiological actions, can be efficiently ingested. As such, the lipid compositions of the present invention are useful as ingredients in pharmaceutical compositions, food compositions, or feed compositions.

In the lipid composition of the present invention, the content of n-3DPA is more preferably 15% or more,
More preferably 20% or more, more preferably 24% or more. When the content of n-3DPA in the lipid composition of the present invention is within this range, n-3DPA and n-3PUFA can be efficiently ingested by ingesting the lipid composition of the present invention. Although the upper limit of the content of n-3DPA in terms of methyl ester is not particularly limited, it is usually 50% or less,
48% or less, or 46% or less.

In the lipid composition of the present invention, the content of DHA is more preferably 35% or more, more preferably 40% or more. When the DHA content in the lipid composition of the present invention is within this range, DHA and n-3PUFA can be ingested more efficiently. Although the upper limit of the DHA content in terms of methyl ester is not particularly limited, it is usually 65% or less, 60% or less, or 55% or less.

The lipid composition of the present invention is preferably 30 parts or more, more preferably 40 parts or more, per 100 parts of DHA in terms of methyl ester as a peak area ratio in a chromatogram obtained when analyzed by GC-FID. , more preferably 50 or more parts of n-3DPA. A lipid composition having a content of n-3DPA relative to DHA in this range contains n-3DP
Advantageous physiological activities of A such as antiarteriosclerotic activity can be effectively exhibited.

In the lipid composition of the present invention, the total content of n-6 PUFA is 15% or less, and n
-6 PUFA may not be included. n-6PUFA is n-6PUF having 16 to 24 carbon atoms and 2 to 6 unsaturated bonds, such as n-6 docosapentaenoic acid (n-6DPA)
A, specifically linoleic acid (LA, 18:2), γ-linolenic acid (GLA, 18
:3), calendic acid (18:3 (12-position geometric isomer of GLA)), n-6 eicosadienoic acid (EDA, 20:2), dihomo-γ-linolenic acid (DGLA, 20:3), arachidonic acid (ARA, 20:4), n-6 docosadienoic acid (DDA, 22:2), adrenic acid (2
2:4), n-6DPA (22:5), n-6 tetracosatetraenoic acid (24:4), and tetracosapentaenoic acid (24:5). Since the content of n-6PUFA is within the above range, in the lipid composition of the present invention, n-3PUFA is 2.6% of n-6PUFA.
It is preferable because it will be contained more than twice as much. The content of individual fatty acids of n-6PUFA is not particularly limited, but for example, the content of n-6DPA in the lipid composition of the present invention can be 15% or less, or 12% or less.

The lipid composition of the present invention typically contains arachidonic acid (AR
A), does not contain linoleic acid (LA), γ-linolenic acid (GLA).

The content of palmitic acid in the lipid composition of the present invention is 10% or less, and may be free of palmitic acid. The content of palmitic acid is more preferably 8% or less,
More preferably, it is 6% or less. The lipid composition of the present invention can prevent vascular calcification caused by endoplasmic reticulum stress induced by saturated fatty acids because the content of palmitic acid is within this range (Non-Patent Documents 5 and 6 ). More preferably, the lipid composition of the present invention has a total content of saturated fatty acids including palmitic acid of 16% or less, and may contain no saturated fatty acids.

The lipid composition of the present invention may contain n-3 PUFA other than n-3DPA and DHA. Another n-3 PUFA is EPA (eicosapentaenoic acid).
The content of EPA in the lipid composition of the present invention is, for example, 10% or less, 8% or less, 6
% or less, or 5% or less.

The lipid composition of the present invention more preferably comprises
Based on the total amount of fatty acid components,
In terms of methyl ester,
10% or more and 50% or less of n-3DPA;
At least 30% or more and 65% or less DHA,
The total content of n-6PUFA is 15% or less,
The content of palmitic acid is 10% or less,
The content of EPA is 10% or less.

A more preferred range for each fatty acid can be limited to the range described above for each fatty acid.
For example, the lipid compositions of the invention are
Based on the total amount of fatty acid components,
In terms of methyl ester,
20% or more and 50% or less of n-3DPA;
At least 35% or more and 60% or less DHA,
The total content of n-6PUFA is 15% or less,
The content of palmitic acid is 8% or less,
The content of EPA may be 6% or less.

<3. Pharmaceutical composition, food composition, or feed composition>
Another aspect of the present invention is
It relates to a pharmaceutical composition, food composition or feed composition containing the lipid composition of the invention as described above.

A pharmaceutical composition of the present invention can be prepared by combining the lipid composition of the present invention described above with one or more other pharmaceutically acceptable ingredients. The pharmaceutical composition of the present invention may be in any form such as solid, liquid or semi-solid. The pharmaceutical composition of the present invention is preferably in the form of oral intake. The amount of the lipid composition of the present invention in the pharmaceutical composition of the present invention is not particularly limited. can do.

The food composition of the present invention can be prepared by blending the lipid composition of the present invention described above with one or more other food-acceptable ingredients. The food composition of the present invention may be in any form such as solid, liquid or semi-solid. The food composition of the present invention can be used as a dietary supplement. The amount of the lipid composition of the present invention in the food composition of the present invention is not particularly limited.
It can be blended so as to contain 0% by mass.

Feed compositions of the present invention can be prepared by combining the lipid compositions of the present invention described above with one or more other ingredients that are acceptable as feed for non-human animals. The feed composition of the present invention may be in any form such as solid, liquid, semi-solid and the like. The amount of the lipid composition of the present invention in the feed composition of the present invention is not particularly limited. For example, the lipid composition of the present invention contains 1 to 50% by mass of n-3DPA in terms of methyl ester can be compounded.

<4. Method for producing lipid composition>
Another aspect of the present invention is
A method for producing a lipid composition containing n-3DPA as a fatty acid component,
a culturing step of culturing a microorganism belonging to the genus Aurantiochytrium;
A lipid composition obtaining step of obtaining a lipid composition from the culture obtained in the culturing step,
The culturing step is
Cultivating the microorganism in a medium containing a carbon source at a concentration of 0.5% by mass or more at the start, and reducing the carbon source in the medium to 0.1% by mass or less by consumption of the carbon source by the microorganism. 1 step;
a second step of culturing for 48 hours or more in a medium with a carbon source concentration of 0.1% by mass or less,
It relates to said method.

The present inventors have found that microorganisms belonging to the genus Aurantiochytrium have a small proportion of n-3DPA-containing lipids in the lipids produced under normal culture conditions, but under conditions of carbon source starvation stress. Based on the surprising finding that the ratio of n-3DPA-containing lipids remarkably increases when cultured, the method for producing the n-3DPA-containing lipid composition of the present invention has been completed.

Aurantiochytrium (A
urantiochytrium) genus, when the culture step is performed,
In the first step, the content of n-3DPA in the fatty acid component in the lipid composition in the microorganism when the concentration of the carbon source in the medium reaches 0.1% by mass (in terms of methyl ester Refers to the peak area ratio in the chromatogram in GC-FID analysis.The same applies below)
Compared to the lipid composition in the microbial body at the end of the second step, it may be a microorganism that exhibits a higher content of n-3DPA in the fatty acid component, and more preferably, the second step. In step 1, compared with the content of n-3DPA in the fatty acid components in the lipid composition in the microorganism at the time when the concentration of the carbon source in the medium was 0.1% by mass,
n-accounting for fatty acid components in the lipid composition in the microorganism at the end of the second step
A microorganism whose 3DPA content is 5 times or more, more preferably 10 times or more, and more preferably 10% or more in terms of methyl ester as a lipid composition in the microorganism at the end of the second step. n-3DPA as a fatty acid component.

A preferred specific example of the microorganism belonging to the genus Aurantiochytrium used in the present invention and having the ability to produce a lipid composition containing n-3DPA is Aurantiochytrium s
p. (Aurantiochytrium sp.) Microorganism strain D42-16 classified
625 (accession number: FERM P-22324).

In the present invention, the microbial strain is a concept that includes mutant strains thereof, which have the ability to produce a lipid composition containing n-3DPA. A mutant strain of the microbial strain is
It is a mutant strain obtained by subjecting the microbial strain to mutagenesis. Mutagenic treatment can be performed using any suitable mutagen. The term "mutagen" is here to be understood to include not only agents with a mutagenic effect, but also treatments with a mutagenic effect, such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogues such as bromouracil and acridines, but if appropriate: Other mutagens can also be used.

Microorganisms belonging to the genus Aurantiochytrium can be judged by the following indicators: Microscopic observation shows that the cell walls are thin, spherical and orange in shape, the ectoplasmic net is underdeveloped, and astaxanthin is used as a pigment. , which contains phenoxanthin and accumulates triglycerides containing docosahexaenoic acid (DHA) as the main fatty acid component as oil droplets (Non-Patent Document 8). More specifically, when cultured in a nutrient-rich medium, lipids containing about 40% DHA, about 30% palmitic acid, and about 5% n-6DPA as fatty acid components in terms of methyl esters are formed in cells. It is a microorganism that accumulates as

In the first step, the microorganism is cultured in a medium containing a carbon source at a concentration of 0.5% by mass or more at the start, and the consumption of the carbon source by the microorganism reduces the carbon source in the medium to 0.1 mass. %
Decrease to

At this time, a liquid medium or a solid medium (agar medium, etc.) containing at least a carbon source can be used as the medium.

The concentration of the carbon source at the start of the first step is not particularly limited as long as it is 0.5% by mass or more in the medium, and for example, 0.5 to 20% by mass, or 1.0 to 10% by mass is suitable. . As carbon sources, sugars such as glucose, fructose, galactose and xylose, and alcohols such as glycerol can be used. You may use these carbon sources in combination of 2 or more types.

In the first step, the carbon source is consumed by the microorganism to reduce the carbon source in the medium. At this time, the carbon source concentration in the medium is reduced to 0.1% by mass or less.

In the second step, in a state where the concentration of the carbon source is 0.1% by mass or less in the first step, further 4
Culture is continued for 8 hours or longer, preferably 72 hours or longer, more preferably 96 hours or longer, and more preferably 120 hours or longer.

The duration of the culture step including the first and second steps depends on the initial carbon source concentration, but the total
One day or less, eg 4-11 days is suitable. In the culture process, the culture temperature is 16-3
7°C is suitable. The initial pH of the medium is 3.0 to 12.0, preferably 3.0 to 10.0,
More preferably, 6.0 to 9.5 are appropriate. Cultivation may be performed stationary, or may be cultured with shaking or agitation.

Preferably, the medium further contains a nitrogen source. Nitrogen sources include natural nitrogen sources such as peptone, polypeptone, yeast extract, malt extract, meat extract, casamino acids, corn steep liquor, and soybean meal; organic nitrogen sources such as sodium glutamate and urea; One or more selected from inorganic nitrogen sources such as ammonium chloride and ammonium nitrate can be used, but are not limited to these.

Preferably, the medium further contains natural sea salt or artificial sea salt. When natural sea salt or artificial sea salt is contained in the medium, its concentration is not particularly limited, but for example, sodium chloride concentration is 0
. A medium containing natural sea salt or artificial sea salt in an amount of 5% to 4.1% can be exemplified.

The medium may further optionally contain inorganic salts such as iron sulfate, ammonium sulfate, sodium sulfate, magnesium sulfate, copper sulfate, magnesium chloride, calcium chloride, sodium chloride and potassium chloride, potassium phosphate, potassium dihydrogen phosphate and the like. Phosphate and one or more other nutritional ingredients selected from vitamins may be included.

In the method of the present invention, more preferably, the first step includes culturing the microorganism in a medium containing a carbon source at a concentration of 0.5% by mass or more to allow the microorganism to consume the carbon source, and then
The method includes additionally adding a carbon source to the medium so as to have a concentration of 0.5% by mass or more, and further culturing the microorganism to consume the carbon source. By making additional additions, n-3D
The yield of the lipid composition of the invention containing PA can be increased.

Here, the "medium containing the carbon source at a concentration of 0.5% by mass or more" may be the medium at the start of the first step, or when the additional addition of the carbon source is performed multiple times, the previous A medium to which a carbon source is additionally added may be used. Additional addition can be performed N times (where N is an integer of 1 or more), and when N is 2 or more, after the (n-1)th (n is 2 to N) additional addition of the carbon source , after the carbon source is consumed by the microorganism, the n-th additional addition is performed, and the carbon source is further consumed by the microorganism.

After the final N-th additional addition, the consumption of the carbon source by the microorganism reduced the carbon source in the medium to 0.1% by mass or less, and the concentration of the carbon source was 0.1% by mass or less. Further, in the medium, as the second step, the cells are cultured for preferably 48 hours or longer, more preferably 72 hours or longer, more preferably 96 hours or longer, and more preferably 120 hours or longer. Even when additional addition is performed, the total time of the culture process including the first step and the second step is 11 days or less, for example, 4 to 11 days
A suitable culture temperature is 16 to 37°C. The initial pH of the medium is 3.0-1
2.0, preferably 3.0 to 10.0, more preferably 6.0 to 9.5 are appropriate. Cultivation may be performed stationary, or may be cultured with shaking or agitation.

In the 1st to N-th additional addition of the carbon source, the concentration of the carbon source in the medium immediately before is not particularly limited, but is preferably 0.1% by mass or less.

The amount of the carbon source added in the 1st to Nth additional additions is not particularly limited, but is preferably
The carbon source is added to the medium at a concentration of 0.5% by mass or more, and more preferably, the concentration of the carbon source in the medium is 0.5 to 20% by mass, or 1.0 to 10% by mass. Add so that The type of carbon source to be additionally added can be selected from the same range as the carbon source in the medium at the start of culture.

In the culture step including the first step and the second step, it is preferable to further add ascorbic acid or isoascorbic acid to the medium. The present inventors have found that n-
A surprising effect was found that the content of 3DPA was remarkably improved. A suitable concentration of ascorbic acid or isoascorbic acid is 0.5 to 1.5% by mass in the medium. The forms of ascorbic acid and isoascorbic acid are not particularly limited, and they may be free forms or salts with alkali metals such as sodium.

In the lipid composition obtaining step, a lipid composition is obtained from the culture obtained in the culturing step.
Here, the culture is a mixture of microorganisms and medium obtained by culturing. The culture may be sterilized or may not be sterilized.

The method of obtaining the lipid composition from the culture is not particularly limited. Typically, the culture is separated by solid-liquid separation means such as centrifugation and filtration to collect microbial bodies, and the lipid composition is obtained from the collected microbial bodies. The collected microbial body is further selected from washing, drying and crushing 1
The above processing may be performed. Drying of the microorganism can be performed by freeze-drying, air-drying, or the like. The crushing of microorganisms can be carried out by treatment with a mill (for example, treatment with a bead mill using glass beads), ultrasonic treatment, or the like.

The method for obtaining the lipid composition from the collected microbial organism is not particularly limited, but preferably
A lipid composition is extracted from the microbial organism with an organic solvent. As an extraction method using an organic solvent, the Bligh & Dyer method can be exemplified (Non-Patent Document 7). Examples of organic solvents that can be used include chloroform, chloroform/methanol mixed solvent, and normal hexane used in the Bligh & Dyer method. By evaporating the solvent from the extract under reduced pressure, a highly concentrated lipid composition is obtained.

In addition, it is not essential to separate the lipid composition from the culture obtained in the culture step,
The culture itself containing the lipid composition or its processed material (dry product, concentrate, crushed product, etc.) may be used for the application of the lipid composition.

The lipid composition produced by the n-3DPA-containing lipid composition production method of the present invention contains at least n-3DPA as a fatty acid component, and preferably further contains DHA. More preferably, the lipid composition produced by the n-3DPA-containing lipid composition production method of the present invention is the lipid composition of the present invention described above.

<5. Method for producing n-3DPA-containing lipid>
Another aspect of the present invention is
A method for producing an n-3DPA-containing lipid containing n-3DPA as a fatty acid component,
A method comprising a separation step of separating n-3DPA-containing lipids from a lipid composition containing n-3DPA as a fatty acid component.

The n-3DPA-containing lipid is a lipid compound containing an n-3DPA component in the molecule, and includes n-3DPA as a free fatty acid, a fatty acid triglyceride containing one or more n-3DPA components as an acyl chain, and an n-3DPA component as an acyl chain. , fatty acid monoglycerides containing n-3DPA components as acyl chains, phospholipids containing one or more n-3DPA components as acyl chains, lower alcohol esters of n-3DPA, and the like.

A method for separating n-3DPA-containing lipids from a lipid composition containing n-3DPA as a fatty acid component is not particularly limited. For example, a lipid composition containing n-3DPA as a fatty acid component is esterified with an alcohol such as fatty acid methyl ester, fatty acid ethyl ester, etc., and n-3DPA is extracted from the resulting fatty acid ester. Methods for separating the esters by chromatography or distillation can be used.

The n-3DPA-containing lipid obtained by the separation step is blended with other ingredients to form a pharmaceutical composition,
It can be in the form of a food composition or a feed composition.

The pharmaceutical composition to be produced may be in any form such as solid, liquid, semi-solid, etc.
Although the amount of the 3DPA-containing lipid to be blended is not particularly limited, for example, n-3DPA can be blended so as to contain 1 to 50% by mass in terms of methyl ester.

The food composition to be produced may be in any form such as solid, liquid, semi-solid, etc.
Although the amount of the 3DPA-containing lipid to be blended is not particularly limited, for example, n-3DPA can be blended so as to contain 1 to 50% by mass in terms of methyl ester.

The forage composition to be produced may be in any form, such as solid, liquid, semi-solid, etc. n
The amount of the -3DPA-containing lipid to be blended is not particularly limited, but, for example, n-3DPA can be blended so as to contain 1 to 50% by mass in terms of methyl ester.

<1. Aurantiochytrium microorganism>
In experiments described later, as a strain of a microorganism belonging to the genus Aurantiochytrium, "D42-1
6625" was used. This strain is a mutant strain derived from a microorganism strain classified as a microorganism belonging to the genus Aurantiochytrium based on the nucleotide sequence encoding 18S rRNA. being classified.

In addition, this strain was cultured with shaking in a liquid medium having the composition shown in Table 1 at a temperature of 28° C. and a shaking speed of 300 rpm for 48 hours. Lipids of the lyophilized cultured microorganisms were converted to methyl esters by the method described later and analyzed by gas chromatography. became like
This strain contains about 40% docosahexaenoic acid (DHA) and about 30% palmitic acid as fatty acids.
%, and about 5% of n-6DPA, confirming that it is a microorganism of the genus Aurantiochytrium.

This strain is Aurantiochytrium sp. (Aurantiochytrium sp.
. ), and deposited as an identification mark "D42-16625" at the National Institute of Technology and Evaluation Patent Organism Depositary Center (zip code 292-0818, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) and has been assigned an accession number: FERM P-22324 (
Acceptance date: January 6, 2017).

<2. Culture and lipid extraction>
(1) Culture A liquid medium shown in Table 1 above is prepared, dispensed into test tubes, and autoclave sterilized (121
°C for 20 minutes) was applied.

This strain was inoculated into the liquid medium in each test tube, and cultured with shaking at a temperature of 28° C. and a shaking speed of 300 rpm.

The liquid medium contains 1.9% by mass of carbon source (glucose) at the start of culture.
The maximum culture time was 168 hours.

In some experiments, a carbon source (glucose or glycerol) was added to the liquid medium at 48 hours or 72 hours after the initiation of culture, and the culture was continued.

In some experiments, the liquid medium further added with 1% by mass of sodium ascorbate or sodium isoascorbate was used as the liquid medium before starting the culture.
The test conditions for Tests 1 to 7 are as follows.

(2) Lipid Extraction After a predetermined period of time, the culture solution was centrifuged, and the supernatant was removed to isolate the microorganism.
The isolated microorganisms were pulverized by pulverization treatment with a bead mill using glass beads.
Lipids were extracted from the microbial pulverized product by the Bligh & Dyer method (Non-Patent Document 7).

(3) Lipid Analysis The lipid extract obtained by the Bligh & Dyer method was concentrated to dryness, and methanol and 1N aqueous sodium hydroxide solution were added to convert fatty acid components into methyl esters. After neutralization with 1N hydrochloric acid, the fatty acid methyl ester was extracted with chloroform and analyzed by gas chromatography (GC-FID) using a hydrogen flame ionization detector as a detector under the following conditions. In addition, mass spectrometry (MS) was further performed as necessary for the analysis of fatty acids in the components separated by gas chromatography.

In the GC-FID chromatogram obtained under the above conditions, the ratio (%) of the peak area of each fatty acid methyl ester to the total peak area of all detected components (all presumed to be fatty acid methyl esters) was calculated. .

Identification of each fatty acid methyl ester was performed by comparison with a commercially available known sample and comparison with mass spectrometry (MS) patterns and publicly known information.

In Tests 1-3, the lipid composition was analyzed 168 hours after initiation of culture by the above procedure.
In Tests 4 and 5, the lipid composition was analyzed 72 hours after the start of culture and after 96 hours of culture after addition of glucose 72 hours after the start of culture, by the above procedure.

In Test 6, the lipid composition was analyzed 48 hours, 72 hours, and 168 hours after the initiation of culture by the above procedure.

In test 7, 48 hours after the start of the culture and an additional 24 hours after the addition of glucose after 48 hours of the start of the culture.
The lipid composition after 12 hours, 48 hours and 96 hours of culture was analyzed by the procedure described above.

Furthermore, in Tests 4, 5, 6 and 7, tricosane was used as an internal standard substance to determine the concentration of n-3DPA (in terms of methyl ester) in the culture after completion of the culture.

(4) Analysis Results Table 5 shows the analysis results of Tests 1-5.
The analytical results of Tests 6 and 7 are shown in Table 6.

In Tables 5 and 6, the ratio (%) of each fatty acid methyl ester is the ratio to the total peak area of all detected components (all presumed to be fatty acid methyl esters). Tables 5 and 6
However, in some cases, the apparent sum of the numerical values of the ratio of fatty acid methyl esters does not appear to be 100%, but this is the result of rounding off each numerical value.

The extracted lipid contains n-6DPA and adrenic acid among the 11 types of n-6PUFA described above in the ratios shown in Tables 5 and 6, and arachidonic acid, linoleic acid, and γ-linolenic acid. It was confirmed that it does not contain Also, in Tables 5 and 6, "Other, n-3, n-
7” is a component identified to be n-3PUFA or n-7PUFA from comparison with standard substances and publicly known information, and “other, saturated” is saturated from comparison with standard substances and publicly known information. A component identified to be a fatty acid.

(5) Analysis of carbon source concentration In tests 1 to 7, the glucose concentration in the medium was measured by high performance liquid chromatography (HPLC).
, detection wavelength UV 505 nm). The glucose concentration in the medium at the start of culture was 1.9% by mass as described above.

In Tests 1 to 7, the glucose concentration in the medium after 24 hours of culture was in the range of 1.2 to 1.9% by mass, and although there was considerable variation, glucose was not depleted after 24 hours of culture. It can be seen that there is a sufficient amount of On the other hand, the glucose concentration in the medium 48 hours after the initiation of culture was 0.1% by mass or less in all of Tests 1 to 7. From this, it can be seen that glucose is depleted 48 hours after the start of culturing of this strain.

Tests 1 to 3 are examples of culturing for an additional 120 hours (168 hours in total) from 48 hours after the start of culture, when the initial glucose is considered to be depleted, and as shown in Table 5, the n-3DPA ratio is remarkably high. A lipid composition resulted.

Tests 4 and 5 added an additional carbon source 72 hours after the start of culturing of this strain, and further added 96
culturing for hours (168 hours in total) yielded lipid compositions with significantly higher n-3DPA ratios, as shown in Table 5. Also, the concentration of n-3DPA in the culture was remarkably high.

The n-3DPA ratios in Tests 1 and 2, which were cultured for 168 hours, were 24.4% and 45.2%. was 5%. From this fact, continuous culture of this strain under starvation conditions resulted in n-3 in lipids.
A significant increase in the DPA ratio was shown.

Both Tests 6 and 7 are examples in which the glucose concentration in the medium was 0.1% by mass or less 48 hours after the start of culture.

As shown in Table 6, in Test 6, an increase in the n-3DPA ratio was observed after 24 hours of culture (72 hours in total) from 48 hours after the start of culture, when glucose was considered to be depleted, and 48 hours after the start of culture. When cultured for an additional 120 hours (total of 168 hours), n-3DPA
It was confirmed that the ratio increased significantly.

In Test 7, 48 hours after the start of the culture, when the glucose concentration was 0.1% by mass or less and exhausted, 2.0% by mass of glucose was added to the medium, and the glucose concentration in the medium was increased after 24 hours of culture. was 0.1% by mass or less again. That is, it is considered that the additionally added glucose was exhausted in 24 hours. As shown in Table 6, in Test 7, 24 hours after the additional glucose was added (48+24 in total) when the additional glucose was considered exhausted.
An increase in the n-3DPA ratio was observed when cultured for an additional 24 hours (48 + 48 hours in total), and increased from 24 hours (48 + 24 hours in total) to an additional 72 hours (48 + 24 hours in total) after additional glucose addition.
It was confirmed that the n-3DPA ratio significantly increased when cultured for a total of 48+96 hours.

As described above, from Tests 6 and 7, by continuously culturing this strain under starvation conditions, n-
It was shown that the 3DPA ratio was significantly increased.

The lipid composition provided by the present invention can be used in the fields of pharmaceuticals, foods, feeds and the like.

Claims (11)

A method for producing a lipid composition containing n-3DPA as a fatty acid component,
a culturing step of culturing a microorganism belonging to the genus Aurantiochytrium;
A lipid composition obtaining step of obtaining a lipid composition from the culture obtained in the culturing step,
The culturing step is
Cultivating the microorganism in a medium containing a carbon source at a concentration of 0.5% by mass or more at the start, and reducing the carbon source in the medium to 0.1% by mass or less by consumption of the carbon source by the microorganism. 1 step;
a second step of culturing for 48 hours or more in a medium with a carbon source concentration of 0.1% by mass or less ,
The medium contains mannitol, a nitrogen source, and a fructose-producing microorganism capable of converting mannitol into fructose and secreting it extracellularly, or the fructose-producing microorganism is cultured in a medium containing mannitol and a nitrogen source. is not a medium prepared by a method comprising
the aforementioned method. In the first step, the microorganism is cultured in a medium containing a carbon source at a concentration of 0.5% by mass or more to consume the carbon source by the microorganism, and then the carbon source is added at a concentration of 0.5% by mass or more. 2. The method of claim 1, further comprising adding to said medium to achieve a concentration and further culturing to consume the carbon source by said microorganism. 3. The method according to claim 1 or 2, wherein said medium used in said culturing step contains one or more components selected from the group consisting of ascorbic acid and isoascorbic acid. The lipid composition produced is
As a peak area ratio in the chromatogram obtained when analyzed by GC-FID (gas chromatography using a flame ionization detector as a detector),
In terms of methyl ester, for the total amount of fatty acid components,
10% or more of n-3DPA (n-3 docosapentaenoic acid);
containing at least 30% or more DHA (docosahexaenoic acid),
The total content of n-6 polyunsaturated fatty acids is 15% or less,
The method according to any one of claims 1 to 3, wherein the lipid composition has a palmitic acid content of 10% or less. Claim 4, wherein the lipid composition contains 30 parts or more of n-3DPA to 100 parts of DHA in terms of methyl ester as a peak area ratio in a chromatogram obtained when analyzed by GC-FID. described method. A method for producing an n-3DPA-containing lipid containing n-3DPA as a fatty acid component,
A lipid composition manufacturing step for manufacturing a lipid composition by the method according to any one of claims 1 to 5,
A method comprising a separation step of separating n-3DPA-containing lipids from said lipid composition produced. A method for producing an n-3DPA-containing lipid containing n-3DPA as a fatty acid component,
As a peak area ratio in the chromatogram obtained when analyzed by GC-FID (gas chromatography using a flame ionization detector as a detector),
In terms of methyl ester, for the total amount of fatty acid components,
10% or more of n-3DPA (n-3 docosapentaenoic acid);
containing at least 30% or more DHA (docosahexaenoic acid),
The total content of n-6 polyunsaturated fatty acids is 15% or less,
A method comprising a separation step of separating n-3DPA-containing lipids from a lipid composition having a palmitic acid content of 10% or less. Claim 7, wherein the lipid composition contains 30 parts or more of n-3DPA to 100 parts of DHA in terms of methyl ester as a peak area ratio in a chromatogram obtained when analyzed by GC-FID. described method. A method for producing a pharmaceutical, food, or feed composition containing a lipid composition containing n-3DPA as a fatty acid component, comprising:
A lipid composition production step for producing a lipid composition containing n-3DPA as a fatty acid component by the method according to any one of claims 1 to 5;
and a blending step of blending the manufactured lipid composition. A method for producing a pharmaceutical, food, or feed composition containing n-3DPA-containing lipids containing n-3DPA as a fatty acid component, comprising:
A lipid composition production step for producing a lipid composition containing n-3DPA as a fatty acid component by the method according to any one of claims 1 to 5;
a separation step of separating n-3DPA-containing lipids from the produced lipid composition;
and a blending step of blending said separated n-3DPA-containing lipid. D42-16625 (accession number: FERM P-22324).