JP2000060587A - Production and use of new oil and fat composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a new oil and fat composition useful as a dietary supplement a health food, etc., by treating oils and fats containing (n-6)-based docosapentaenoic acid and docosahexaenoic acid, etc., with a lipase derived from a bacterium in the presence of a medium chain length fatty acid. SOLUTION: Oils and fats containing at least one of (n-6)-based docosapentaenoic acid and docosahexaenoic acid obtained from a culture product of a bacterium [e.g. Ulkenia SAM 2,179 strain (FERM BP-5,601), etc.], belonging to the genus Ulkenia, Schizochytrium, Thraustochytrium, etc., capable of producing at least one of (n-6)-based docosapentaenoic acid and docosahexaenoic acid are treated with a lipase derived from a bacterium (e.g. Pseudomonas KW1-56 strain, etc.), belonging to the genus Pseudomonas in the presence of a medium chain length fatty acid to efficiently give the objective oil and fat composition containing at least one of (n-6)-based docosapentaenoic acid and docosahexaenoic acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a structural lipid containing (n-6) -type docosapentaenoic acid and / or a structural lipid containing docosahexaenoic acid, and an oil / fat composition containing the structural lipid obtained by this method. And the use of this oil and fat composition.

[0002]

2. Description of the Related Art Recent researches in the field of physiochemistry show that medium-chain fatty acids (C8) are present at 1- and 3-positions rather than fats and oils in which long-chain fatty acids (C18 or higher) are bonded at 1- and 3-positions of triglycerides.
~ C12) -bound fats and oils are more easily decomposed by pancreatic lipase in vivo and are therefore more easily absorbed in the intestine [Oil Chemistry, Vol. 37, p781, 1].
995]. Among fatty acids, docosahexaenoic acid (D
Highly unsaturated fatty acids such as HA) and docosapentaenoic acid (DPA) are considered to have various physiological activities in the animal body. Therefore, the structured lipid in which the medium-chain fatty acid is bound to the 1- and 3-positions and the polyunsaturated fatty acid is bound to the 2-position is a fat or oil to which the highly unsaturated fatty acid which is a useful fatty acid is bound and which is easily absorbed in the intestine. It is getting attention.

Polyunsaturated fatty acids are classified into (n-3) type highly unsaturated fatty acids and (n-6) type highly unsaturated fatty acids depending on the difference in the position of the unsaturated bond contained therein.
In the animal body, (n-3) type polyunsaturated fatty acids and (n
-6) Belongs to a metabolic pathway other than the system's highly unsaturated fatty acids. Linoleic acid having 18 carbon atoms [18: 2 (n-6)]
And α-linolenic acid [18: 3 (n-3)] are widely present as the main components of vegetable oils and fats. On the other hand, for animals,
Inability to biosynthesize linoleic acid and α-linolenic acid. Therefore, animals require these (n-3) and (n-6) polyunsaturated fatty acids as essential fatty acids. Ingested highly unsaturated fatty acids such as linoleic acid are further desaturated and carbon chain extended to be converted into highly unsaturated fatty acids having higher carbon number and higher carbon number. However, in patients with adult diseases, infants, elderly people, etc., the function of the desaturation reaction in biosynthesis is often reduced, so that highly unsaturated fatty acids having a high degree of unsaturation and a large number of carbon atoms tend to be deficient.

Examples of the (n-3) type highly unsaturated fatty acids include eicosapentaenoic acid [20: 5 (n-3)] and docosahexaenoic acid [22: 6 (n-3)]. These (n-3) type polyunsaturated fatty acids are known to have physiological activities such as anti-inflammatory activity and antithrombotic activity, and are therefore attracting attention as materials for functional foods and pharmaceuticals.

On the other hand, examples of (n-6) type highly unsaturated fatty acids include γ-linolenic acid [18: 3 (n-6)] and dihomo-γ-linolenic acid [20: 3 (n-6)]. ], Arachidonic acid [20: 4 (n-6)] and the like. These (n-6) type polyunsaturated fatty acids are attracting attention as intermediate metabolites into one or two groups of eicosanoids such as prostaglandins and leukotrienes, which are called local hormones.

In the body of an animal, docosahexaenoic acid is generally used as the (n-3) type polyunsaturated fatty acid, and arachidonic acid is used as the (n-6) type polyunsaturated fatty acid. It is the final metabolite. In the animal body, the content of the (n-6) type docosapentaenoic acid is extremely low.

Docosahexaenoic acid (DHA), which is the final metabolite of the (n-3) system, is specifically present in the brain and retina of animals. DHA is thought to perform some function in these organs. In recent years, the discovery of raw materials containing high concentrations of DHA, such as orbital fat of tuna, and the development of advanced purification technology for fatty acids, have led to research on the elucidation and practical application of the physiologically active function of DHA. It is being actively promoted. The physiologically active functions of DHA include a blood cholesterol lowering action, an anticoagulant action, an antitumor action, and an action for improving memory and learning ability and preventing senile dementia in relation to the brain metabolic system. Became clear. from these things,
DHA is also expected as a therapeutic drug for Alzheimer's disease. It was also clarified that DHA is an essential fatty acid for growing juvenile fish. For practical use, DHA
Is used as a material for health foods, baby milk, and the like.

It accounts for the composition of fatty acids in the animal body (n-
When the content of 6) docosapentaenoic acid (DPA) is high, the (n-3) essential fatty acid is often deficient. This is deficient (n-3)
It is considered that the (n-6) type highly unsaturated fatty acid compensates for the function of the type highly unsaturated fatty acid. In particular, as an alternative to DHA, there have been several reports of (n-6) -type DPA being produced in vivo. This suggests that the (n-6) system DPA has some physiological role. (N-
The 6) system DPA can also be expected as an antagonist of arachidonic acid.

The (n-6) type DPA does not exist at all in the oils and fats generally supplied, but is slightly contained in the fish oil together with the (n-3) type DPA. However, since the content of the (n-6) type DPA in fish oil is as small as about 1%, it is not easy to efficiently separate and concentrate the (n-6) type DPA.

As described above, fish oil has DHA and (n-6) type DPA which have remarkable physiological functions, and many findings have been obtained regarding the purification method thereof. On the other hand, fish oil contains DHA and (n-6) DP
The content of A is low; mainly migratory fish are used as raw materials, but the amount of migratory fish caught varies with the seasons, making it difficult to provide a stable supply; having a unique offensive odor; and arachidonic acid (AA) and eicosapentaene Since it also contains highly unsaturated fatty acids such as acid (EPA), it is easily oxidized,
There are drawbacks such as difficulty in obtaining oil and fat of stable quality.

At least one of DPA and DHA,
Sources other than fish oil include fats and oils (microbial oil) accumulated in cultured cells of microorganisms having the ability to produce these highly unsaturated fatty acids. Examples of these microorganisms are the bacterium Vibrio marinus ATCC 15381 isolated from the deep sea, the genus Vibrio bacterium isolated from the intestines of deep-sea fish, and the flagellate Thraustochytrium aureum ATCC.
34304, Thraustochytrium s
p.) ATCC 28211, ATCC 20890 and ATCC 20891, Schizochytrium sp. ATCC 20888 and AT
CC 20889 [US Pat. No. 5,340,742], Thraustochytrium sp. SR21 strain [Journal of the Japanese Society of Agricultural Chemistry, Volume 69, Extra number, July 5, 1995], Japonochrytrium (Japonochytrium) sp.) AT
CC 28207 [JP-A-1-199588], Cyclotella cryptic which is a microalga
a), Crypthecodini
um cohnii) [JP-A-5-503425] and Emilia [JP-A-5-308978] are known.

Each of the above-mentioned microorganisms has drawbacks such as a low DHA and / or DPA production rate, a long culture time, and a special medium or culture conditions required for production. Especially when using algae such as Emilia, D
Even if the production amount of HA is high, there are drawbacks such that the process is complicated because light is required for culturing and it is not suitable for industrial production.

Further, regardless of which of the above-mentioned fats and oils is used, conventionally, a structured lipid containing at least one of DPA and DHA, in particular, any one of these highly unsaturated fatty acids mainly at only the 2-position of triglyceride. It has been difficult to efficiently produce bound structured lipids.

[0014]

The present invention is intended to solve the above problems, and efficiently produces a structured lipid containing at least one of (n-6) -type docosapentaenoic acid and docosahexaenoic acid. The present invention aims to provide a method suitable for use on an industrial scale. Another object of the present invention is to provide an oil and fat composition containing the structured lipid obtained by this method, and a food using the oil and fat composition.

[0015]

Means for Solving the Problems As a result of diligent efforts to solve the above problems, the inventors of the present invention have found that fats and oils containing at least one of (n-6) type docosapentaenoic acid and docosahexaenoic acid, By treating the produced fat with a lipase derived from bacteria, (n-6)
It was found that a structured lipid containing at least one of docosapentaenoic acid and docosahexaenoic acid can be produced in a good yield, and the present invention has been completed.

The present invention is a method for producing a structured lipid containing at least one of (n-6) type docosapentaenoic acid and docosahexaenoic acid, wherein at least one of (n-6) type docosapentaenoic acid and docosahexaenoic acid is used. There is provided a method comprising a step of treating a fat or oil containing a bacterium with a lipase of bacterial origin in the presence of a medium chain fatty acid.

In one embodiment, the lipase is a Pseudomonas-derived lipase. In a preferred embodiment, the lipase is a Pseudomonas sp. Strain KWI-56.

In another embodiment, the fat is (n-
6) Obtained from a culture of a microorganism capable of producing at least one of docosapentaenoic acid and docosahexaenoic acid. In a preferred embodiment, the microorganism is a microorganism belonging to the family Thraustochytrium. In an even more preferred embodiment, the microorganism is a microorganism belonging to the genus Ulkenia, the genus Schizochytrium, or the genus Thraustochytrium. In an even more preferred embodiment, the microorganism is a microorganism belonging to the genus Ulkenia. In an even more preferred embodiment, the microorganism is Ulkenia sp. SAM2179 strain.

The present invention also provides an oil and fat composition containing the structured lipid obtained by any of the above methods.

In one embodiment, the structured lipid is 1
It is a triglyceride in which medium-chain fatty acids are bound only at positions 3, 3 or only at positions 1 and 3. In a preferred embodiment, the medium chain fatty acid is caprylic acid.

The present invention further includes dietary supplements, health foods, or functional foods, infant foods, infant formulas or premature infant formulas, and geriatrics containing any of the above oil and fat compositions. Provide food.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

1. Definition of Terms As used herein, unless otherwise specified, the term "docosapentaenoic acid" refers to the (n-6) -based docosapentaenoic acid [22: 5 (n-6)]. DPA is an abbreviation for docosapentaenoic acid.

In the present specification, the term "docosahexaenoic acid" means (n-3) type docosahexaenoic acid [22: 6 (n-3)] unless otherwise specified. DHA
Is an abbreviation for docosahexaenoic acid.

In the present specification, the term "lipid" means a simple lipid containing a compound (for example, glyceride) in which a fatty acid and an alcohol are ester-bonded, or an analog thereof (for example, cholesterol ester), and further phosphoric acid, The term includes complex lipids containing sulfuric acid, sugars, amino groups, etc., and derivatized lipids that are hydrolysates of lipids and are insoluble in water.

In the present specification, "oil and fat" and "oil"
And the terms "triglyceride" are used interchangeably.

As used herein, "structural lipid" refers to a non-natural triglyceride modified to include a mixture of short and / or medium chain fatty acids and long chain fatty acids. Here, the short-chain fatty acid refers to a fatty acid having about 7 or less carbons, the medium-chain fatty acid refers to a fatty acid having about 8 to 12 carbons, and the long-chain fatty acid is about 13 or more. Refers to a fatty acid having

In the present specification, the "oil and fat composition" means
Refers to a composition containing fats and oils. The oil or fat composition usually contains about 10% by weight or more of oil or fat, preferably about 20% by weight or more, more preferably about 50% by weight or more, even more preferably about 70% by weight, based on the weight of the entire composition. %
It contains the above fats and oils.

2. Materials 2.1 Lipase Lipase catalyzes various reactions such as ester hydrolysis, ester synthesis, and ester transfer (acidolysis, alcoholysis, transesterification, aminolysis). In the present invention, the fact that bacterial lipase can catalyze an acidolysis reaction with high activity is utilized to efficiently generate a structured lipid. The acidolysis reaction is represented by the following reaction formula 1.

[Chemical 1]

In the present invention, any bacterial lipase can be used. Bacterial-derived lipases usually have no regioselectivity in the acidolysis reaction with triglycerides, and have a significantly low activity for catalyzing the acidolysis reaction of long-chain fatty acids having about 20 or more carbons. It is considered that this is because bacteria themselves do not naturally synthesize long-chain fatty acids having about 20 or more carbons, and thus it is difficult to catalyze the reaction of such long-chain fatty acids. Therefore, DPA and /
Alternatively, when a triglyceride containing DHA is used as a substrate for the acidolysis reaction, these DPA and / or D
It can selectively catalyze reactions for shorter chain fatty acids while substantially maintaining HA. Examples of bacteria for collecting lipase include Pseudomona spp.
s), genus Alcaligenes, Bacillus (Ba)
cillus), Staphylococcus
Genus, bacteria belonging to the genus such as Acinetobacter (Acinetobacter) genus, and the like, but preferably bacteria belonging to the genus Pseudomonas, more preferably Pseudomonas sp. KWI-56 strain .

As the lipase, one obtained by culturing bacteria and collected or commercially available one may be used. When collecting lipase from bacteria, methods known in the art can be used. Purified lipase is preferred, and its purity can be preferably about 40% or more, more preferably about 60% or more, but may be unpurified lipase.

The lipase may be used without being immobilized.
However, since it is advantageous in that the enzyme reaction can be continuously performed and that the enzyme can be recovered and reused after the reaction,
Immobilized enzymes are preferred. Any method can be used for immobilization as long as the activity of lipase is not significantly reduced. Examples of immobilization methods include carrier binding methods known in the art,
Cross-linking methods, encapsulation methods, and combinations thereof.

When the lipase is immobilized by the carrier binding method, examples of the carrier include calcium carbonate (CaCO ₃ ) powder, ion exchange resin, polyurethane foam, ceramics, and polysaccharides (eg, cellulose, dextran, agarose). Derivative, polyacrylamide, activated carbon,
Examples include bentonite, diatomaceous earth, chitin and chitosan. The carrier is preferably calcium carbonate powder, ion exchange resin, diatomaceous earth, ceramics, bentonite, chitosan, and more preferably calcium carbonate powder.

When the lipase is immobilized by the crosslinking method,
For cross-linking, a water-soluble bifunctional reagent having two or more functional groups is usually used. Examples of such reagents include glutaraldehyde, isocyanate derivatives,
Examples thereof include bisdiazobenzidine, N, N'-polymethylenebisiodoacetamide, N, N'-ethylene bismaleimide. As bifunctional reagents, glutaraldehyde and isocyanate derivatives are preferred.

When the lipase is immobilized by the entrapping method, for example, a lattice-type entrapping method using polyacrylamide gel, polyvinyl alcohol gel, silicon resin, starch matrix, konjak powder, urethane resin polymer, calcium alginate, agar, and nylon, It can be immobilized by a microcapsule encapsulation method using polyurea, a ladder polymer of phenylsiloxane, polystyrene, ethyl cellulose, collodion, cellulose nitrate, cellulose butyl acetate and the like. As a comprehensive method, a method using polyacrylamide gel, urethane resin polymer, calcium alginate, and agar is preferable. The method for immobilizing an enzyme is described in detail, for example, in “Immobilized Enzyme”, edited by Ichiro Chibata, Kodansha, Ltd., pages 9-85, 1975.

2.2 (n-6) system DPA and / or D
Fats and Oils Containing HA In the present invention, as fats and oils containing at least one of (n-6) docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), any fat or oil containing a desired amount of these highly unsaturated fatty acids is used. Can be used. In fats and oils, that is, triglycerides, at least 2-position (n-
6) The system DPA or DHA is preferably bound. Therefore, oils and fats containing DPA or DHA only in the 2-position, 1-position and 2-position, or 2-position and 3-position are preferred.

Examples of such fats and oils include (n-6)
Fats and oils obtained from a culture of a microorganism capable of producing at least one of the systems DPA and DHA are mentioned. However, the present invention is not limited to oils and fats derived from microorganisms, and fish oils collected from sardines, mackerels, saury, salmon, etc., animal oils collected from seals, etc. can also be used.

As an example of the microorganism to be cultivated to collect oils and fats, a microorganism belonging to the family Thraustochytiidae, which has the ability to produce at least one of (n-6) type DPA and DHA, is preferable. Include a microorganism classified into the genus Ulkenia, the genus Schizochytrium, or the genus Thraustochytrium, more preferably a microorganism classified into the genus Ulkenia. Preferable examples of the microorganism of the genus Ulkenia include the SAM strain 2179 of the genus Ulkenia and SA isolated from seawater by the present inventors.
This is the M2180 strain (WO 98/03671). Among these strains, it is the SAM2179 strain that can produce the (n-6) type DPA and DHA at a particularly high content. The Ulkenia SAM2179 strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, dated July 23, 1996, and the deposit number F
Obtained ERMBP-5601.

Ulkenia SAM2179 strain and SA
The mycological properties of the M2180 strain are as follows: These microorganisms were treated with KMV liquid medium [Fuller. M. and A. Jawor.
ski (ed): Zoosporic Fungi in Teaching & Research
VII + 303 pp., 1987, Southeastern Publishing Corporat
Ion, Athens], spherical or oval cells are observed when cultured in the dark at 20 ° C, and zoospores with two flagella are also observed. However, no net-like naked protoplasts are observed. Therefore, these microorganisms are
Kazuko Konno: Based on the illustration of Japanese aquatic fungi [p. 169, published by the author in 1986], it is judged to be a fungus belonging to Thraustochytriales. Furthermore, these microorganisms are
In MV liquid medium, forming roots, lacking cysts,
It is considered that the fungus belongs to the genus Ulkenia because it forms amoebic cells.

The microorganism used in the present invention is not only a microorganism of the genus Urkenia (wild strain) isolated from nature, but also
It may be a mutant strain or a recombinant strain. That is, (n
-6) The use of mutant strains and recombinant strains designed to produce even higher levels of fats and oils containing at least one of the systems DPA and DHA is within the scope of the invention. Such mutant strains or recombinant strains contain at least one of (n-6) -type DPA and DHA in oil and fat when compared with the amount produced by the original wild strain when cultured using the same substrate. Those designed to have a high content or a high total amount of fats or oils, or both are included. Further included are microorganisms designed to efficiently use cost effective substrates to produce the same amount of at least one of the (n-6) series DPA and DHA as the corresponding wild strain.

In order to culture the microorganism used in the present invention, a preculture liquid obtained by previously culturing the strain is inoculated into a suitable liquid medium or solid medium and then cultured. Medium components such as carbon source, nitrogen source, micronutrient source added to the medium,
The culture conditions such as pH and temperature are selected from known conditions. These conditions are not particularly limited as long as the content of at least one of the (n-6) type DPA and DHA is not adversely affected.

In order to promote the production of at least one of the (n-6) series DPA and DHA, its precursor can be added to the medium. Examples of the precursor include hydrocarbons such as tetradecane, hexadecane and octadecane,
Alternatively, a fatty acid such as oleic acid, linoleic acid, α-linolenic acid, or a salt (for example, sodium salt or potassium salt) or ester thereof can be mentioned. further,
Oils and fats containing these fatty acids as constituents (for example, olive oil, soybean oil, cottonseed oil, coconut oil) and the like can also be added. These can be used alone or in combination.

The carbon source, nitrogen source, precursor and the like can be added to the medium before or during the start of the culture. Moreover, these can be added once or multiple times, or continuously. (N-6) system DPA and D
In order to obtain fats and oils containing at least one of HAs in a practically preferable yield, it is preferable to carry out aeration stirring culture using a liquid medium. For culturing, an ordinary stirred fermenter or bubble column type culturing device can be used.

By culturing in this manner, (n-6) type DPA
Oils and fats containing at least one of DHA and DHA are produced and accumulated in the cells. When a liquid medium is used, the culture solution in the process of producing an oil or fat by cell culture or its sterilized culture solution, the culture solution at the end of the culture or its sterilized culture solution, or the cells was collected from each culture solution. From cultured cells or dried products thereof, (n-6) system D
Oils and fats containing at least one of PA and DHA are collected. Collection of oils and fats from cultured cells is performed, for example, as follows.

After completion of the culture, the culture cells are obtained from the culture medium by a conventional solid-liquid separation means such as centrifugation or filtration. Wash the cells thoroughly with water. Preferably, the separated bacterial cells are dried.
Drying can be performed by freeze-drying, air-drying and the like. The dried cells are crushed by, for example, Dynomill or ultrasonic waves, and then preferably subjected to extraction treatment with an organic solvent under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane,
Petroleum ether or the like can be used. Good results can also be obtained by alternate extraction of methanol and petroleum ether, and extraction using a single-layer solvent of chloroform-methanol-water. By distilling off the organic solvent from the extract under reduced pressure, (n-6) type DPA and DH
An oil and fat containing at least one of A in a high concentration can be obtained.

Instead of the above method, wet cells may be used for the extraction. In this case, a solvent compatible with water such as methanol or ethanol, or a mixed solvent compatible with water composed of these alcohols and water and / or another solvent is used. Other procedures are the same as above.

Among the lipids thus obtained, (n
-6) At least one of the systems DPA and DHA is present in the form of neutral lipids (eg triglycerides) and polar lipids (eg phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol). The oil or fat containing at least one of (n-6) type DPA and DHA can be purified from this lipid by a conventional method, for example, a cooling separation method, a column chromatography and the like.

2.3 Medium Chain Fatty Acids Medium chain fatty acids are saturated or unsaturated fatty acids having about 8-12 carbons. Saturated fatty acids are preferred. Examples of saturated medium chain fatty acids include caprylic acid [8: 0], capric acid [10: 0], and lauric acid [12: 0]. The medium-chain fatty acid is available as a natural oil / fat raw material, for example, a fraction derived from coconut oil or palm kernel oil. In the present invention, caprylic acid is particularly preferable as the medium chain fatty acid because it can be obtained abundantly and cheaply from natural fats and oils.

3. Production of Structured Lipid (n-6) -based Oil and fat containing at least one of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), lipase, and medium-chain fatty acid are mixed, and stirred or shaken to advance the acidolysis reaction. As a result, the target structured oil and fat is obtained.

The amount of the medium chain fatty acid is usually about 10 to 1000 parts by weight, preferably about 150 to 500 parts by weight, more preferably about 200 to 300 parts by weight, based on 100 parts by weight of the fat and oil. Parts by weight. The amount of lipase is usually about 100 to 100,000 per 1 g of oil and fat.
U, preferably about 1000-50000 U, more preferably about 5000-10000 U.

With regard to lipase, the amount of enzyme that produces 1 μmol / min of fatty acid by hydrolysis is defined as 1 U. The enzyme amount of lipase is measured, for example, by the following method. First, add olive oil 2.0 to a 100 ml sample bottle.
ml, 1.0 M of 0.05 M CaCl ₂ aqueous solution, and 10 ml of 0.1 M acetate buffer (pH 5.6) are added. This mixed solution is placed in a reactor at 30 ° C ± 0.1 ° C and preheated for 10 minutes while stirring at 500 rpm. The enzyme sample is added to the sample bottle, the stirring is continued, and after reacting for 1 hour, 40 ml of ethanol is added to stop the reaction. After stopping the reaction, titration is performed by adding 0.05N sodium hydroxide solution to the sample bottle until the pH reaches 10. At this time, a blank test is simultaneously performed by the same procedure except that the sample is not added. After the measurement, the hydrolysis activity of the enzyme is calculated by the following formula: A = (ab) × f × 50 × 1/60 × 1 / S where A: hydrolysis activity (U / mg or U / ml) a: 0.05N sodium hydroxide solution (ml) required for sample measurement b: 0.05N sodium hydroxide solution (ml) required for blank test measurement f: Factor of 0.05N sodium hydroxide solution S: sample Amount (mg or ml) 50: 0.05 Amount of fatty acid (μmol) corresponding to 1 ml of N sodium hydroxide solution 60: Reaction time (min).

The mixture of oil and fat, lipase, and medium chain fatty acid may further contain other substances. For example, the mixture may be dissolved in a solvent such as hexane, ether or ester. Since the organic solvent adversely affects the human body, it is necessary to remove it after the reaction, which is costly and troublesome. Therefore, it is preferable to carry out the reaction in a solvent-free system.

The temperature for treating the mixture of fats and oils, lipase, and medium chain fatty acid is not particularly limited, but is usually about 10
℃ ~ 50 ℃, preferably about 20 ℃ ~ 40 ℃, more preferably about
25-35 ° C. The time for reacting the above mixture is not particularly limited, but is usually about 1 to 500 hours, preferably about 10 to 300 hours, more preferably about 20 to 250 hours. During the reaction, it is preferable to replace the air in the vessel containing the reaction liquid with nitrogen in order to prevent the oxidation of unsaturated fatty acids and the mixing of water. If water enters the reaction system,
This is not preferable because the hydrolysis reaction proceeds in parallel with the acidolysis reaction. By advancing the acidolysis reaction between the fat and oil and the medium chain fatty acid under such conditions, (n-
6) Structural lipids containing at least one of the systems DPA and DHA are produced. Depending on the degree of progress of the reaction and the amounts of the (n-6) type DPA and DHA contained in the fat and oil used in the reaction, the medium chain fatty acid is bound to either the 1-position or the 3-position,
A triglyceride having (n-6) type DPA or DHA bound to the 2-position; and a medium chain fatty acid bound to the 1- and 3-positions and a triglyceride bound to the (n-6) -type DPA or DHA at the 2-position obtain.

After completion of the reaction, it is preferable to remove the free fatty acid by washing the reaction solution with alkali or the like.
Purification of the produced structured lipid can be carried out, if necessary, in the same manner as in the purification of fats and oils described in 2.2 above. Therefore, the structured lipid is usually obtained as an oil / fat composition containing other components, but it is also possible to isolate the target structured lipid as a pure product.

In the produced structured lipid, the total amount of fatty acids contained in the structured lipid is 100 mol%, and is generally about 20 to 80 mol%, preferably about 40 to 70 mol%, and more preferably about 60 mol%.
It contains ~ 70 mol% of medium chain fatty acids. The medium chain fatty acid is preferably incorporated at the 1- and 3-positions of triglyceride. Medium chain fatty acids (MA) in the 1 and 3 position
, And a triglyceride having (n-6) type DPA or DHA bound to the 2-position (MA-DPA / DHA-MA)
Of the medium-chain fatty acids at positions 1 and 2 and (n-
6) The ratio of the system DPA or DHA-bonded triglyceride (MA-MA-DPA / DHA) (the ratio of the former when the sum of the two is 100%) is typically 60% or more, preferably 65%. These may be the case, but are not limited to these.

4. Utilization of structural lipids (n-6) type DPA and DH can be obtained by adding a structural oil containing at least one of (n-6) type DPA and DHA to various foods or feeds or feeds.
There is provided an oil / fat-containing food or the like that can make up for the shortage of A. According to this, at least one of the (n-6) type DPA and DHA can be ingested in a state in which it is easily decomposed by the pancreatic lipase in vivo and therefore easily absorbed in the intestine.

Examples of the above foods include dietary supplements,
Examples include health foods, functional foods, infant foods, infant formulas, premature infant formulas, and aged foods. As used herein, food is a generic term for solids, fluids and liquids and mixtures thereof that are edible.

The dietary supplement refers to a food fortified with a specific nutritional component. Healthy food refers to food that is considered to be healthy or healthy, and includes dietary supplements, natural foods, diet foods, and the like. The functional food is a food for supplementing nutritional components that fulfill the body's regulation function, and is synonymous with food for specified health uses. What is infant food?
A food for children up to about 6 years old. Geriatric food refers to food that has been treated so that it is easier to digest and absorb as compared to untreated food. Infant formula refers to formula for infants up to about 1 year of age. Premature infant formula refers to modified milk that is fed to a premature infant until about 6 months after birth.

Examples of the forms of these foods include meat,
Natural foods such as fish and nuts (treated with fats and oils); Foods to which fats and oils are added during cooking such as Chinese food, ramen and soups; Tempura, fried foods, fried rice, doughnuts, sugar, etc. Foods; butter, margarine, mayonnaise, dressing, chocolate, instant ramen, caramel, biscuits, cookies, cakes, ice cream, and other oil-based foods; Occasionally, a food or the like sprayed or coated with oils and fats can be mentioned. However, the food of the present invention is not limited to foods that originally contain fats and oils, for example, bread, noodles, rice, confectionery (candies, chewing gum, gummies, tablets, Japanese confectionery), tofu and the like. Agricultural foods such as processed foods; fermented foods such as sake, medicinal liquor, mirin, vinegar, soy sauce and miso; livestock foods such as yogurt, ham, bacon and sausages;
It may be a fishery product such as kamaboko, fried tempura, and rice paste; a juice drink, a soft drink, a sports drink, an alcoholic drink, a drink such as tea, and the like.

The food of the present invention is also most widely used in the form of pharmaceutical preparations, or as a protein source (proteins such as milk protein, soy protein, egg albumin, etc., which have a good amino acid balance and high nutritional value). , These hydrolyzates, egg white oligopeptides, soybean hydrolysates, mixtures of various amino acids, etc.), sugars, fats, trace elements, vitamins, emulsifiers, flavors and the like were blended with the oil of the present invention. It may be a processed form such as a natural liquid food, a semi-digestive nutrition food and a component nutrition food, a drink, an enteral nutrition. When the food of the present invention is in the form of a pharmaceutical preparation, the food of the present invention is a powder, a granule, a tablet, a capsule, a troche, an internal solution, a suspension, an emulsion,
It may be in the form of a syrup, a drink, a natural liquid diet, a semi-digestive nutritional diet, a component nutritional diet, an enteral nutritional supplement, and the like.

These foods can be manufactured by known methods. The oil / fat composition of the present invention is added at any stage as long as it does not substantially modify or decompose the structured lipid. The amount of the oil / fat composition added is not particularly limited, and is appropriately set so that the desired structured lipid has a desired content.

[0062]

EXAMPLES Next, the present invention will be described more specifically by way of examples.

<Example 1> Production of fats and oils by Ulkenia spp. Microorganisms (1) Ulkenia sp. SAM2179 strain and SAM2180 strain were each placed in a jar fermenter type culture tank having a volume of 5 L and a medium having the following composition (1). Culture was performed using 3 L and the following culture conditions (2).

(1) Medium composition (g / L) 1) Glucose: 60 2) Potassium phosphate: 3 3) Ammonium sulfate: 2 4) Corn steep liquor: 0.7 5) 50% artificial seawater: 1L 6) pH: 4.0 (2) Culture conditions 1) Culture temperature (° C): 28 2) Aeration rate (VVM): 0.5 3) Agitation speed (rpm): 300 4) pH adjustment: 10% (w / v) pH with sodium hydroxide
Hold at 4.

After culturing for 3 days, the cells were collected by centrifugation and freeze-dried to determine the amount (weight) of cells per 1 L of the medium. Next, a mixture of chloroform / methanol (2: 1, v / v) was added to the dried cells at 100 v based on the weight of the cells.
/ W ratio, and homogenization was performed in the presence of glass beads to crush the cells and extract fats and oils.
After the extract was washed by the Folch method, the solvent was distilled off to obtain a refined oil and fat, and its weight was measured.

In order to evaluate the fatty acid composition of the obtained refined oil and fat, a part of the oil and fat was dissolved in an equal volume mixture of a methanol solution containing 10% HCl and dichloromethane, and heat treated at 60 ° C. for 2 hours. A fatty acid methyl ester was prepared. This was introduced into a gas chromatograph to analyze the fatty acid composition. The separation conditions of gas chromatography (GC) were as follows.

(3) Separation condition 1) Column: Capillary column, TC-70 GL Science Co., Ltd, inner diameter 0.25 mm
× Length 30 m 2) Flow rate: 0.8 ml / min, 100 kPa (column head pressure) 3) Carrier gas: Nitrogen gas 4) Column temperature: temperature rising mode, 170-220 ° C (4 ° C / min) 5) Detection: = FID The results are shown in Tables 1 and 2 below.

[Table 1]

[0068]

[Table 2]

As described above, the SAM2179 strain and the SAM2180 strain produced oils and fats having a high DPA content and a high DHA content in the medium containing artificial seawater.

<Example 2> Production of fats and oils by microorganisms of the genus Ulkenia (2) For the SAM2179 strain of the genus Ulkenia, in a jar fermenter type culture tank having a volume of 5 L, 3 L of the medium having the composition (1) below and the following ( It culture | cultivated using the culture conditions of 2).
The test was performed twice (tests 1 and 2).

(1) Medium composition (g / L) 1) Glucose: 60 2) Potassium phosphate: 3 3) Ammonium sulfate: 2 4) Magnesium chloride: 1.3 5) Sodium sulfate: 16) Calcium chloride: 0.3 7) Corn steep liquor: 0.7 8) pH: 4.0 (2) Culture conditions 1) Culture temperature (° C): 28 2) Aeration rate (VVM): 0.5 3) Agitation speed (rpm): 300 4) pH adjustment: 10% ( w / v) pH 4 with sodium hydroxide
Held in.

After culturing for 3 days, purified fats and oils were obtained under the same conditions as in Example 1 and their weights were measured. The fatty acid composition of the obtained refined oil and fat was analyzed by GC in the same manner as in Example 1. The results are shown in Tables 3 and 4 below.

[Table 3]

[0073]

[Table 4]

As described above, the SAM2179 strain had a DPA content and a DHA content even in a medium containing no artificial seawater.
Produced high fat content.

Example 3 (n-6) Production of Structural Lipid Containing DPA and DHA Powdered lipase (PSL) derived from Pseudomonas sp. KWI-56 strain
(Manufactured by Kurita Industry Co., Ltd .: hydrolysis activity = 56.5 U / mg) 50
By dissolving 0 mg in 5 ml of distilled water, 5600 U / ml
A PSL solution of was prepared. To 5 ml of this PSL solution, 2.5 g of CaCO ₃ as an immobilization carrier was added, and the mixture was stirred for 10 minutes while cooling with ice. The immobilized enzyme was precipitated by adding 40 ml of cold acetone to this solution, followed by centrifugation at 8000 rpm and 4 ° C. for 20 minutes, and the supernatant was discarded to obtain a residue. The residue was dried to obtain the immobilized enzyme to be used. The specific activity of this immobilized enzyme was 9.3 U / mg. As a control, Rhizomucor miehei-derived lipase (Lipozyme) was used. Lipozyme (registered trademark) is an immobilized enzyme having an ion exchange resin as a carrier and is sold by Novo Nordisk.

Obtained in Test 2 of Example 2, (n-6)
1 g of refined oil and fat containing docosapentaenoic acid and docosahexaenoic acid, 540 mg of immobilized PSL (or Lipozyme 1
00 mg), and caprylic acid 1, 2, or 3 g were mixed. By stirring or shaking this mixed solution at 30 ° C., the acidolysis reaction proceeded. During the reaction, the reaction solution was sampled over time. According to a conventional method, the reaction mixture was washed with alkali to remove fatty acids, and triglyceride was recovered by silica gel chromatography. The fatty acid composition of the recovered triglyceride was analyzed.

Analysis of fatty acids by GC was performed in the same manner as in Example 1. The results are shown in Table 5 below and FIGS.
And shown in FIG.

In Table 5, the results for the immobilized PSL show the composition of fatty acids contained in the structural lipid obtained after the reaction for 144 hours with the weight ratio of fat and oil to caprylic acid being 1: 3. The results for Lipozyme show the composition of the fatty acids contained in the structural lipids obtained after 144 hours of reaction with a fat / fat to caprylic acid weight ratio of 1: 2.

When using Lipozyme, the uptake of caprylic acid was about 23%, whereas when using immobilized PSL, the uptake of caprylic acid was about 65%. That is,
It was shown that immobilized PSL can increase the uptake of caprylic acid into triglycerides by up to about 3-fold compared to Lipozyme.

[Table 5]

FIG. 1 is a graph showing the influence of the weight ratio of fats and oils and caprylic acid on the amount of caprylic acid taken up. Figure 1a shows the results with immobilized PSL,
Figure 1b shows the results with Lipozyme. The weight ratio of the fats and oils used in the reaction to caprylic acid was 1:
The case of 1 (◯), the case of 1: 2 (Δ), and the case of 1: 3 (□) are shown.

FIG. 2 shows the progress of the acidolysis reaction.
It is a graph which shows the change of the fatty acid composition contained in fats and oils.
In FIG. 2a, the weight ratio of fat and oil to caprylic acid is 1: 3,
The result when using immobilized PSL is shown. FIG. 2b shows the results when Lipozyme was used with a weight ratio of fat and oil to caprylic acid of 1: 2.

From FIGS. 1 and 2, it can be seen that when Lipozyme is used, the amount of caprylic acid incorporated into fats and oils is limited to about 23%, and even if an excess amount of caprylic acid is used, the amount incorporated is not increased. On the other hand, when immobilized PSL is used, the amount of caprylic acid incorporated into fats and oils is significantly increased. When the weight ratio of fat and oil to caprylic acid was 1: 3, about 77% of caprylic acid was taken into the fat and oil in 216 hours.

FIG. 3 is a chromatogram by GC. FIG. 3a shows the results of a reaction with immobilized PSL for 144 hours with a weight ratio of fat and oil to caprylic acid of 1: 3.
In FIG. 3b, the weight ratio of fat and oil to caprylic acid is 1: 2,
The result of having reacted for 144 hours using Lipozyme is shown. Figure 3
c shows the results for untreated fats and oils. In FIG. 3, C27 indicates 8-8-8 and C35 indicates 8-8-16.
, C41 represents 8-8-22 or 8-14-16, and C49 represents 8-16-22 or 14-16-16.
, C51 indicates 16-16-16, and C55 indicates 8
22-22 or 14-16-22, C57 is 16-16-22, and C63 is 16-22-
22 is shown. (Here, 8 is caprylic acid, 14 is myristic acid, 16 is palmitic acid, 22 is DHA or DP.
A is shown. However, the order of the numbers is independent of the position of attachment in the triglyceride. ) The integrated intensity ratios of these peaks are shown in Table 6 below.

[Table 6]

FIG. 3 and Table 6 show that when Lipozyme was used, the content of the C41 compound containing the desired structural lipid was about 19%, and a large amount of other C49 and C55 compounds remained. . On the other hand, fixed PSL
When C. was used, the content of the C41 compound was about 38%, and C49 and C55 were reduced to about 7 to 8%, respectively. Also, the content of C27 (8-8-8) increased to about 29%. From this, it is shown that compared with the case of using Lipozyme, when the immobilized PSL is used, it is possible to produce a structured lipid in which the target C41 compound is about twice as many as the starting oil and fat.

Example 4 Molecular Species Analysis of Structural Lipids by HPLC The structural lipids obtained in Example 3 were analyzed for the molecular species of triglycerides by HPLC using a silver ion-bonded column.

(1) Analytical conditions 1) Column: CHROMSPHER 5 LIPIDS (250 × 4.6 mm) manufactured by CHROMAPACK 2) Flow rate: 0.65 ml / min 3) Eluent (volume ratio): A) Hexane: Propanol: Acetonitrile = 350 :Ten
0: 2.75 B) Hexane: Propanol: Acetonitrile = 350: 10
0:10 0 to 3 minutes A100% 3 to 13 minutes A100 → 0% / B0% → 100% 13 to 33 minutes B100% 4) Detection: UV206nm The results are shown in FIG. FIG. 4a shows the results of a 144-hour reaction using immobilized PSL with a weight ratio of fat and oil to caprylic acid of 1: 3. FIG. 4b shows the results of a 144-hour reaction using Lipozyme with a weight ratio of fat and oil to caprylic acid of 1: 2. In FIG. 4, the peaks with symbols respectively represent the following triglycerides: 1 = only saturated fatty acids 9 = 16-DPA-DPA triglycerides 10 = 8-DPA-DPA 2 = 8-16-DPA 11 = 16 -DHA-DPA 3 = 8-DPA-8 12 = 8-DHA-DPA 4 = 8-8-DPA 13 = 16-DHA-DHA 5 = 8-DHA-16 14 = 8-DHA-DHA 6 = 8- 16-DHA or 16-8-DHA 15 = DPA-DPA-DHA 7 = 8-DHA-8 16 = DPA-DHA-DHA 8 = 8-8-DHA 17 = DHA-DHA-DHA (where 8 is Caprylic acid, 16 indicates palmitic acid, and peaks 9 to 16 have no distinction of positional isomers.)

From FIG. 4, it is a molecular species having DPA or DHA at the 2-position and incorporating caprylic acid at the 1- and 3-positions by using immobilized PSL as compared with the case of using Lipozyme. It is shown that structural fats (peaks 3 and 7) are obtained in large amounts.

<Example 5> Preparation of milk containing (n-6) type DPA and DHA 1.2 g of the oil / fat composition obtained in Example 3 was mixed with 100 parts of powdered milk.
(n-6) system DPA and D
HA-containing milk was prepared. The ratio of (n-6) type DPA to the total fatty acids of this milk was 0.27% and the ratio of DHA was 0.84%. Therefore, a formula containing the (n-6) type DPA and DHA as structural lipids, which was insufficient in the conventional formula, was obtained.

Example 6 Preparation of (n-6) -based DPA and DHA-containing capsules

[Table 7]

300 mg of the fat / oil containing the structural lipid containing the (n-6) type DPA and DHA obtained in Example 3 was filled in a soft capsule consisting of the components shown in Table 7 by a conventional method to give a soft capsule. Got The soft capsule itself is useful as a dietary supplement or as a raw material for a dietary supplement.

Example 7 Preparation of Beverage Containing (n-6) -Based DPA and DHA 50 g of commercially available plain yogurt, (n-6) -based DPA and DHA obtained in Example 3 50 g of fats and oils containing structural lipids containing at least one, and β-
1 g of cyclodextrin was added. This was stirred for about 3 minutes and emulsified to obtain an emulsion in which W / O / W, O / W / O type and the like were mixed. 500 this emulsion with water
A functional beverage was obtained by doubling the dilution.

[0092]

According to the manufacturing method of the present invention, the (n-6) type D
A step of treating an oil or fat containing a large amount of at least one of PA and DHA with a lipase derived from a bacterium in the presence of a medium chain fatty acid is included. By using a lipase derived from bacteria, which has a high catalytic activity for the acidolysis reaction, (n-
6) A structured lipid mainly containing at least one of DPA and DHA in the 2-position can be efficiently produced.

The oil / fat composition of the present invention is a highly unsaturated fatty acid capable of exerting an important physiological activity in the living body (n-6).
It contains at least one of the systems DPA and DHA as a structural lipid that is particularly highly absorbable in vivo. Therefore, the oil composition of the present invention, various products that require at least one of these highly unsaturated fatty acids (baby formula, premature formula milk, infant food, elderly food, nutritional supplement, function Sex foods, enteral nutrients, animal feed or animal feed additives, micro-feeding biological feed, etc.) (n-
6) At least one of the systems DPA and DHA can be stably and efficiently supplied as a structural lipid.

[Brief description of drawings]

FIG. 1 is a graph showing the weight ratio of fat and oil to caprylic acid,
It is a graph which shows the influence on the amount of caprylic acid uptake. FIG. 1a shows the results with immobilized PSL and FIG.
Shows the results when Lipozyme was used.

FIG. 2 is a graph showing changes in the composition of fatty acids contained in fats and oils as the acidolysis reaction progresses. Figure 2
In a, the weight ratio of fats and oils and caprylic acid is set to 1: 3, and the result when immobilized PSL is used is shown. FIG. 2b shows the results when Lipozyme was used with a weight ratio of fat and oil to caprylic acid of 1: 2.

FIG. 3 is a chromatogram by GC. FIG. 3a shows the results of a reaction with immobilized PSL for 144 hours with a weight ratio of fat and oil to caprylic acid of 1: 3. Figure 3b
Is Lipozy with a weight ratio of fats and oils to caprylic acid of 1: 2.
The result of reaction for 144 hours using me is shown. Figure 3c shows
The result about untreated fat is shown.

FIG. 4 is a chromatogram by HPLC. FIG. 4a shows the results of a 144-hour reaction using immobilized PSL with a weight ratio of fat and oil to caprylic acid of 1: 3.
In FIG. 4b, the weight ratio of fat and oil to caprylic acid is 1: 2,
The result of having reacted for 144 hours using Lipozyme is shown.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Miho Narita             180 Naka-Ishikicho Market, Tsushima City, Aichi Prefecture F-term (reference) 4B001 AC17 AC25 BC14 EC05                 4B018 LB07 MS03                 4B026 DC05 DG20 DH01 DL09                 4B064 AD85 AD88 CA02 CA21 DA10                 4H059 BA26 BA33 BB02 BB05 BB07                       BC04 BC06 BC48 CA37

Claims (15)

[Claims] 1. A method comprising the step of treating an (n-6) -based fat or oil containing at least one of docosapentaenoic acid and docosahexaenoic acid with a lipase derived from a bacterium in the presence of a medium chain fatty acid. -6) A method for producing a structured lipid containing at least one of docosapentaenoic acid and docosahexaenoic acid. 2. The method according to claim 1, wherein the lipase is derived from Pseudomonas. 3. The lipase is Pseudomonas sp. KWI-
The production method according to claim 2, which is a lipase derived from 56 strains. 4. The production method according to claim 1, wherein the fat or oil is obtained from a culture of a microorganism capable of producing at least one of (n-6) -type docosapentaenoic acid and docosahexaenoic acid. 5. The production method according to claim 4, wherein the microorganism is a microorganism belonging to the family Thraustochytrium. 6. The method according to claim 5, wherein the microorganism is a microorganism belonging to the genus Ulkenia, the genus Schizochytrium, or the genus Thraustochytrium. 7. The production method according to claim 6, wherein the microorganism is a microorganism belonging to the genus Ulkenia. 8. The microorganism is Ulkenia sp. SAM21.
The production method according to claim 7, which is 79 strains. 9. An oil and fat composition containing the structured lipid obtained by the method according to any one of claims 1 to 8. 10. The oil and fat composition according to claim 9, wherein the structured lipid is a triglyceride in which medium-chain fatty acids are bound only at the 1-position, 3-position, or 1- and 3-positions. 11. The medium chain fatty acid is caprylic acid,
The oil and fat composition according to claim 10. 12. A dietary supplement, a health food, or a functional food containing the oil composition according to any one of claims 9 to 11. 13. An infant food containing the oil composition according to any one of claims 9 to 11. 14. A modified milk for infants or a modified milk for premature infants, which comprises the oil or fat composition according to any one of claims 9 to 11. 15. An aged food containing the oil or fat composition according to any one of claims 9 to 11.