JP2010042037A - Method for producing triglyceride formed out of three residues of highly unsaturated fatty acid of one kind and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide fats and oils containing a new triglyceride, a method for producing the same, and a microorganism used for producing the same. <P>SOLUTION: In this method for producing the fats and oils so composed that a ratio of the triglyceride formed by combining three hydroxy groups of a glycerol with highly unsaturated fatty acids of the same kind is 20 wt.% or more based on an total amount of triglycerides, the microorganism capable of producing the fats and oils is cultured and the fats and oils are collected, when desired. Thus, the fats and oils obtained by the method and the method for producing the fats and oils are provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a triglyceride composed of three types of highly unsaturated fatty acid residues, and the use thereof, and in particular, a method for producing the above triglyceride efficiently and stably, and this production method. It relates to the triglyceride obtained and its typical use.

  Polyunsaturated fatty acid (PUFA) here is a fatty acid having 18 or more carbon atoms and two or more double bonds. Since PUFA has various unique physiological activities, it is added to various foods and animal feeds to increase its functionality. The main ones are linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), Examples include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In use, it may be used as a free fatty acid type or a phospholipid type, but it is mainly used as a triglyceride type, and the acyl residue often contains PUFA as a constituent component.

  In general, chemical synthesis of PUFA itself is difficult and its cost is very high, so its practical source is limited to extraction from biological resources. In general, it is often extracted from oil plants, seafood, microorganisms, microalgae and the like. These organisms often contain many types of PUFAs, and few biological resources consisting of a single PUFA are known.

  In using PUFA in the triglyceride form, it has been known in recent years that its functionality is further enhanced by using it as a so-called “structured lipid” in which the binding position and the number of bonds of PUFA are specified. In order to produce structured lipids, regiospecific chemical methods may be used, but in many cases, they are mainly used as foods, and in acyl group conversion reactions using enzymes such as lipases. It is often manufactured (International Publication W0 03/004667). In this case, although it depends on the design of the reaction system, in general, the fatty acid bonded to a specific position in the raw triglyceride is not cleaved, and only the fatty acid bonded to the other position is subjected to hydrolysis reaction or acylation. It is often removed by a group exchange reaction. Reaction Formula 1 shows an example in which triglyceride containing caprylic acid and free fatty acid are generated at the 1 and 3 positions by a lipase reaction between a PUFA-containing triglyceride mixture and caprylic acid (A to I: fatty acid, 8: caprylic acid). ).

  In this case, the target PUFA content in the structured lipid as a product depends on the target PUFA content contained in the fatty acid residue that is not cleaved (in this case, position 2) in the starting triglyceride. In many cases, the by-product free fatty acid has a high utility value, but the type and ratio of the fatty acid contained in the free fatty acid is the fatty acid residue to be cleaved in the raw triglyceride (in this case, the 1st and 3rd positions). Depends on the type and proportion of fatty acids contained therein.

  When a structured lipid containing a specific PUFA at a specific position is produced by a conversion reaction of an acyl group using a lipase or the like using a triglyceride containing the PUFA as a starting material, and a by-product fatty acid is also used, When the starting material is a mixture containing many types of PUFAs, the following points are problematic. (1) Fatty acids other than the target PUFA are mixed into the acyl group that is desired to remain in the state of being bonded to the glycerol skeleton. (2) Fatty acids produced as by-products from the glycerol skeleton become a mixture of many types of fatty acids.

  In order to solve the above (1) and (2) at the same time, a triglyceride in which all three fatty acid residues constituting the triglyceride are composed of one target PUFA may be used as a starting material. By doing so, both the acyl group that is desired to remain in the state bonded to the glycerol skeleton and the fatty acid that is generated as a by-product from the glycerol skeleton are all made of only one type of target PUFA, and the added value is increased.

  In addition, when humans or animals ingest lipids containing PUFAs in anticipation of the physiological effects of specific PUFAs, they are often taken as triglycerides from the viewpoint of physical properties such as stability and hypoallergenicity to living bodies. However, since it is a lipid, there may be a concern that the calories are high. In this case, if you ingest triglyceride consisting of all of the target PUFAs with all three fatty acid residues that make up triglyceride, you don't need to ingest any other fatty acid, so ingest to get the same amount of target PUFA. The effect that the calorie which must be done becomes the lowest is also expected.

  Thus, triglyceride, in which the three fatty acid residues constituting triglyceride are all composed of one target PUFA, is a highly value-added substance. However, there is no known triglyceride that has been obtained from the natural world or by fermentation, and contains a high proportion of triglycerides consisting of one PUFA. Mortierella alpine has been cultured. Only about 19% of the triglyceride obtained in this way was known to be composed of triarachidonoylglycerol (see Example 2).

  When the triglyceride mixture containing the target PUFA contains even a small amount of triglyceride molecular species consisting of one type of target PUFA, it may be separated and purified using high performance liquid chromatography or the like. Or, after converting the lipid containing the target PUFA to the form of free fatty acid or fatty acid alcohol ester, it is obtained by separating and purifying one type of target PUFA using high performance liquid chromatography etc., and further by chemical synthesis or enzymatic conversion In principle, it is possible to obtain a triglyceride in which all three fatty acid residues constituting the triglyceride are composed of one target PUFA by combining with glycerol.

However, the above conventional technique has a problem that it is difficult to efficiently and stably produce a triglyceride composed of three PUFA residues of one kind.
Further, as described above, generally, chemical synthesis of PUFA itself is difficult and its cost is very high, so that its practical supply source is limited to extraction from biological resources. Major sources include linoleic acid and α-linolenic acid from oil plants, arachidonic acid and dihomo-γ-linolenic acid from microorganisms, and EPA and DHA from seafood and microalgae. The PUFAs contained in these sources often exist in the triglyceride form.

  However, in many cases, the triglyceride contains many types of PUFAs, and the binding positions thereof are often distributed at all of the 1st, 2nd, and 3rd positions of the glycerol skeleton. Thus, since there are so many kinds of molecular species in terms of the triglyceride molecular species that distinguishes the types of the three fatty acid residues of triglyceride, all the three fatty acid residues constituting the triglyceride are all present. The molecular species that is triglyceride consisting of one type of PUFA of interest is hardly contained or is present only in a small amount, but all three fatty acid residues constituting triglyceride are of the target PUFA1. It is difficult to separate and purify the molecular species, which is a triglyceride composed of types, from the above-mentioned sources. Moreover, in order to separate and purify a specific triglyceride molecular species from a triglyceride mixture, it is necessary to use an analytical instrument such as high performance liquid chromatography, and it is very difficult to mass-produce industrially.

  In addition, one type of PUFA of interest is obtained by separation and purification in the form of free fatty acid or fatty acid alcohol ester, and then combined with glycerol by chemical synthesis or enzymatic conversion, so that all three fatty acid residues constituting triglyceride can be obtained. It is also possible to obtain triglycerides consisting of one type of PUFA of interest. However, the target PUFA itself can still be obtained from the above sources, extraction of fats and oils, conversion to free fatty acid or fatty acid alcohol ester, separation and purification of the target PUFA, coupling with glycerol by chemical synthesis or enzymatic conversion, Because it has to go through many steps such as separation and purification of triglyceride consisting of one kind of PUFA, there was a big problem in cost and yield and it was not practical at all.

  The present invention has been made in view of the above problems, and its object is to provide a technique capable of efficiently and stably supplying a triglyceride composed of three PUFA residues of one kind, and a typical example thereof. Is to provide the best use technology.

  As a result of intensive studies in view of the above problems, the present inventor has performed mutation treatment on a lipid-producing bacterium that produces triglyceride containing the target PUFA, so that all three fatty acid residues constituting the triglyceride are one type of target PUFA. That a triglyceride containing a high concentration of triglyceride can be obtained, and that the triglyceride consisting of all three fatty acid residues constituting the triglyceride can be easily extracted from the cells of the mutant strain. The headline and the present invention have been completed.

  That is, according to the present invention, a method for producing a triglyceride composed of three PUFA residues of one type obtains a cell by culturing a lipid-producing bacterium capable of producing a triglyceride composed of three types of PUFA residues. , A method for producing triglyceride consisting of three types of PUFA residues from the cells, characterized by culturing a lipid-producing bacterium capable of producing triglycerides consisting of three types of PUFA residues Yes.

Therefore, the present invention provides a method for producing fats and oils in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides. Provided is a method characterized by culturing a microorganism that can be produced, and collecting the oil if desired.
In the above method, the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to the three hydroxyl groups of glycerol is preferably 30% by weight or more based on the total triglycerides.

More preferably, in the above method, the ratio of triglyceride formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 40% by weight or more based on the total triglycerides, and more preferably glycerol. The ratio of the triglyceride formed by bonding the same highly unsaturated fatty acid to the three hydroxyl groups is 50% by weight or more based on the total triglycerides.
In the above method, the same polyunsaturated fatty acid is, for example, arachidonic acid.

The microorganism used in the above method is preferably a microorganism belonging to the genus Mortierella, and more preferably a microorganism belonging to the genus Mortierella (Mortierella). Preferably, the microorganism is a microorganism of the species Mortierella alpina. Usually, the above-mentioned microorganism is a mutant strain of a microorganism capable of producing fats and oils containing highly unsaturated fatty acids.
The present invention also relates to fats and oils produced by the above method.

  The present invention further provides a maltierella that can produce fats and oils in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to the three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides ( Mortierella) microorganisms, preferably, can produce oils and fats in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 30% by weight or more based on the total triglycerides A microorganism of the genus Mortierella is provided. More preferably, the microorganism is, for example, Mortierella alpina. The above microorganism is preferably a mutant, for example, an artificial mutant.

  The present invention also provides a fat or oil in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides; Oils and fats in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acids to hydroxyl groups is 30% by weight or more based on the total triglycerides; more preferably, the highly unsaturated fatty acids identical to the three hydroxyl groups of glycerol The ratio of the triglyceride formed by binding the same highly unsaturated fatty acid to the three hydroxyl groups of glycerol, and more preferably, the ratio of the triglyceride formed by binding to the triglyceride is 40% by weight or more based on the total triglycerides Provides oils and fats that are 50% by weight or more based on total triglycerides.

  The present invention further provides a microorganism cultured microbial cell comprising fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides. provide. Preferably, the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to the three hydroxyl groups of glycerol is 30% by weight or more based on the total triglycerides, and more preferably, the three hydroxyl groups of glycerol. The ratio of the triglyceride formed by bonding the same highly unsaturated fatty acid to the triglyceride is 40% by weight or more based on the total triglycerides, for example, the triglyceride formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol. The ratio is 50% by weight or more based on the total triglycerides.

EFFECT OF THE INVENTION When the triglyceride consisting of three PUFA residues of one kind according to the present invention is produced efficiently and stably and the triglyceride can be easily obtained, the following effects are produced.
When a structured lipid containing a specific PUFA at a specific position is produced by a conversion reaction of an acyl group using lipase or the like and a by-product fatty acid is also used, one kind of PUFA residue 3 according to the present invention is used. By using triglyceride consisting of individual as starting materials, all of the acyl groups that are desired to remain in the state of being bound to the glycerol skeleton and the fatty acids that are separated from the glycerol skeleton as a by-product are all of a single type of PUFA of high purity. As a result, the added value of the main product and by-product is greatly increased.

  In addition, when humans and animals ingest lipids containing PUFA in anticipation of the physiological action of a specific PUFA, if they ingest triglyceride consisting of one target PUFA, all three fatty acid residues constituting the triglyceride, Since it is not necessary to consume other fatty acids, it is expected that the calorie that must be consumed in order to obtain the same amount of the PUFA of interest is the lowest.

An embodiment of the present invention will be described as follows, but the present invention is not limited to this.
According to the present invention, a method for producing a triglyceride consisting of three PUFA residues of one type is obtained by culturing a lipid-producing bacterium capable of producing a triglyceride consisting of three types of PUFA residues. A production method for extracting triglycerides consisting of three PUFA residues of one type from bacterial cells, characterized by culturing a lipid-producing bacterium capable of producing triglycerides consisting of three types of PUFA residues.

  The lipid-producing bacteria used in the present invention are not particularly limited, but include Mortierella genus, Conidiobolus genus, Pythium genus, Phytophthora genus, Penicillium ( Penicillium genus, Cladosporium genus, Mucor genus, Fusarium genus, Aspergillus genus, Rhodotorula genus, Entomophthora genus, Echinosporangium genus, Echinosporangium genus And at least one selected from the genus Saprolegnia is preferably used.

Among these, when the genus Mortierella is used as the lipid-producing bacterium, the bacterium of the genus Mortierella is preferably the genus Mortierella, and the bacterium of the genus Mortierella is Mortierella alpina. More preferably. In the Mortierella (Mortierella) belonging to the genus Mortierella (Mortierella) microorganisms belonging to the subgenus, for example Mortierella elongata (Mortierella elongata), Mortierella exigua (Mortierella exigua), Mortierella Figurofira (Mortierella hygrophila), Mortierella alpina ( Mortierella alpina ).

More specifically, Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina 221266266, CC41, 266266CC Examples include CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, CBS754.68 and the like.
In addition to the Mortierella subgenus, strains capable of producing AA-PL include Echinosporangium transversalis ATCC 16960, Conidiobolus heterosporus CBS138.57, Saproregnia raponica. (Saprolegnia lapponica) CBS284.38 etc. can be mentioned.

  All of these strains are obtained from Osaka City Foundation for Fermentation (IFO), American Type Culture Collection (ATCC), and Centrralbureau voor Schimmelcultures (CBS) without any restrictions. can do. The strain Mortierella alpina SAM2268 (FERM P-17762) isolated from soil by the research group of the present invention can also be used, but is not limited to these strains. Although strains belonging to these type cultures or strains isolated from the natural world can be used as they are, natural mutations having different properties from the original strains obtained by performing growth and / or isolation once or more are used. be able to.

  Furthermore, by subjecting these lipid producing bacteria to mutation treatment and selection, it is possible to select lipid producing bacteria having an increased ability to produce triglyceride consisting of three kinds of PUFA residues. Mutation treatment is not particularly limited as long as it can be applied to the above-mentioned lipid-producing bacteria, but radiation (X-ray, gamma ray, neutron ray) irradiation, ultraviolet irradiation, high heat treatment, etc., and microorganisms Suspension in a suitable buffer, etc., add mutagen, incubate for a certain period of time, dilute appropriately and inoculate on agar medium to obtain mutant mutant colonies .

  Mutagens include nitrogen mustard, methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), alkylating agents, base analogs such as 5-bromouracil, mitomycin C, etc. Examples thereof include antibiotics, base synthesis inhibitors such as 6-mercaptopurine, pigments such as proflavine, certain carcinogens such as 4-nitroquinoline-N-oxide, and compounds such as manganese chloride and formaldehyde. The microorganism used may be a growing cell (mycelium) or a spore.

  The method for selecting a lipid-producing bacterium having an increased ability to produce triglyceride consisting of three PUFA residues from one mutated lipid-producing bacterium is not particularly limited, but the mutated lipid-producing bacterium is not limited. It is preferable to analyze the triglyceride produced by the above by liquid high performance chromatography or the like.

  As shown in Example 1, when about 3,000 strains subjected to mutation treatment were selected as described above, fats and oils containing a high proportion of triglycerides consisting only of the same PUFA residues relative to other triglycerides were selected. Three mutants were obtained that produced. This means that an average of 1 target mutant was obtained per approximately 1,000 strains subjected to mutation treatment. Therefore, the frequency with which the mutant strain of the present invention is obtained is much higher than in the case of the random selection method by general mutation treatment-selection, and the above-mentioned characteristics equivalent to the three mutant strains actually obtained in the present invention A mutant strain having can be easily obtained by repeating the method described in Example 1 of the present invention.

  A specific method for culturing a lipid-producing bacterium having an increased ability to produce triglyceride consisting of three PUFA residues of one kind is not particularly limited as long as the bacterium can grow and produce triglyceride. What is necessary is just to culture | cultivate by a well-known culture method according to the kind of lipid production microbe. In general, a spore, mycelium of a strain of a lipid-producing bacterium to be cultured, or a preculture solution obtained in advance is inoculated into a liquid medium or a solid medium and cultured. When it is desired to obtain a large amount of cells, liquid culture is often preferable. The culture equipment is not particularly limited, and in the case of a small amount of culture, a liquid medium is charged into various test tubes and flasks and shaken, or inoculated on an agar plate and statically cultured. In the case of a large amount of culture, various fermenters and jar fermenters may be used.

The type of medium used for culture is not particularly limited, and may be prepared by appropriately selecting known components according to the type of lipid-producing bacteria. Alternatively, a medium having a known composition or a commercially available medium may be used as it is.
When the medium is a liquid medium, the carbon source is not particularly limited, and general saccharides can be suitably used. Specific examples include glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol and the like. These carbon sources may be used alone or in combination of two or more.

  The nitrogen source is not particularly limited, and a known one can be suitably used. Specifically, for example, natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, soy protein, defatted soybean, cottonseed residue; organic nitrogen source such as urea; sodium nitrate, And inorganic nitrogen sources such as ammonium nitrate and ammonium sulfate. In the present invention, although depending on the type of strain to be cultured, among the above, in particular, a natural nitrogen source obtained from soybean, specifically soybean, defatted soybean, soybean flake, edible soybean protein, okara, soy milk Kinako flour and the like can be preferably used. Among these, it is possible to use a product obtained by heat-modifying defatted soybean, more preferably a product obtained by heat treating defatted soybean at about 70 to 90 ° C. and further removing ethanol-soluble components. These nitrogen sources may be used alone or in appropriate combination of two or more.

  Components other than the above carbon source and nitrogen source are not particularly limited, and known micronutrient sources and the like can be appropriately selected and added as necessary. Micronutrient sources include, for example, inorganic acid ions such as phosphate ions; alkali metal or alkaline earth metal ions such as calcium ions, sodium ions, magnesium ions, and calcium ions; VIIB such as iron, nickel, cobalt, and manganese Group VIII metal ions; ions of Group IB to IIB metals such as copper and zinc; various vitamins;

    The content rate (addition rate) of each component described above in the liquid medium is not particularly limited, and may be within a known range as long as the concentration does not inhibit the growth of lipid-producing bacteria. In practice, generally, the total amount of carbon source added is preferably in the range of 0.1 to 40% by weight, more preferably in the range of 1 to 25% by weight. The total amount of nitrogen source added is preferably in the range of 0.01 to 10% by weight, and more preferably in the range of 0.1 to 10% by weight. Furthermore, when feeding the culture medium, the initial carbon source should be added in the range of 1 to 5% by weight, and the initial nitrogen source should be added in the range of 0.1 to 6% by weight. preferable. The components of the medium fed during the culture may be both the carbon source and the nitrogen source, but more preferably only the carbon source is fed.

  In the production method according to the present invention, an unsaturated fatty acid precursor may be added to the medium for the purpose of increasing the yield of triglyceride consisting of three PUFA residues of one kind. Specific examples of the unsaturated fatty acid precursor include hydrocarbons such as hexadecane and octadecane; fatty acids such as oleic acid and linoleic acid or salts thereof; fatty acid esters such as ethyl ester, glycerin fatty acid ester, and sorbitan fatty acid ester Oils and fats such as olive oil, soybean oil, rapeseed oil, cottonseed oil and coconut oil; and the like, but are not particularly limited. These precursors may be used alone or in appropriate combination of two or more.

The amount of the unsaturated fatty acid precursor added is not particularly limited, but in general, it may be in the range of 0.001 to 10% with respect to the total weight of the medium, and is 0.5 to 10%. It is preferable to be within the range. Alternatively, lipid-producing bacteria may be cultured using these precursors as the sole carbon source.
The culture conditions are not particularly limited, and may be set as appropriate according to the type of strain to be cultured. For example, the culture temperature may generally be in the range of 5 to 40 ° C, and preferably in the range of 20 to 30 ° C. Moreover, after culture | cultivating within the range of 20-30 degreeC previously, and growing a microbial cell, you may continue culture | cultivating within the range of 5-20 degreeC. If such temperature control is performed, that is, culturing at a relatively high temperature first, and then culturing in a temperature range lower than the initial culturing temperature, the polyunsaturated fatty acid (PUFA) in the unsaturated fatty acid produced ) Ratio can be increased.

The pH of the medium is not particularly limited, but generally, it may be in the range of pH 4 to 10, more preferably in the range of pH 5 to 9. Although the culture period is not particularly limited, it may be usually within a range of 2 to 30 days, preferably within a range of 5 to 20 days, and more preferably within a range of 5 to 15 days. The external treatment applied to the medium during the culture is not particularly limited, and a known culture method such as aeration and agitation culture, shaking culture, or stationary culture may be appropriately selected.
When culturing in solid culture, use bran, rice bran, rice bran or the like to which 50 to 100% by weight of water is added based on the weight of the solid, using 5 to 40 ° C., preferably at the above temperature, for 3 to 14 days. Incubate. In this case, a nitrogen source, inorganic salts, and a trace nutrient source can be added to the medium as necessary.

  In the present invention, a hydrocarbon such as tetradecane and hexadecane, a fatty acid such as tetradecanoic acid and hexadecanoic acid, or a salt thereof (for example, a sodium salt or a potassium salt) is used to increase the amount of triglyceride consisting of three PUFA residues of one kind. Etc.) and esters, or fats and oils (for example, coconut oil and palm kernel oil) containing the fatty acid as a constituent component can be added as a substrate.

  The culture conditions are not particularly limited, and may be set as appropriate according to the type of strain to be cultured. The external treatment applied to the medium during the culture is not particularly limited, and a known culture method such as aeration and agitation culture, shaking culture, or stationary culture may be appropriately selected. It can also be cultured in solid culture.

  The PUFA constituting the triglyceride consisting of three PUFA residues of one kind according to the present invention may be any fatty acid having 18 or more carbon atoms and having two or more double bonds. Specifically, eicosadienoic acid; eicosatrienoic acid such as dihomo-γ-linolenic acid and mead acid; eicosatetraenoic acid such as arachidonic acid (AA); eicosapentaenoic acid; docosadienoic acid; docosatrienoic acid; Tetraenoic acid; docosapentaenoic acid; docosahexaenoic acid (DHA); tetracosadienoic acid; tetracosatrienoic acid; tetracosatetraenoic acid; tetracosapentaenoic acid; tetracosahexaenoic acid; Among the PUFAs, arachidonic acid (AA) is more preferably used.

  In the PUFA, at least one of the carbon-carbon double bond structures (—C═C—) contained in the structure may be a conjugated double bond. This conjugated double bond may be conjugated with a carbonyl group (C═O), or may be conjugated with adjacent carbon-carbon double bonds.

  The ratio of the triglyceride composed of three target PUFA residues to the total triglyceride produced by the lipid-producing bacterium is not particularly limited, but is preferably 30% by weight or more, If it is more than weight%, it is more preferable, and if it is 45 weight% or more, it is still more preferable. When the triglyceride consisting of three PUFA residues of one type is triarachidonoylglycerol, the proportion of triarachidonoylglycerol in the total triglycerides is not particularly limited, but is preferably 30% by weight or more. 33 wt% or more is more preferable, and 45 wt% or more is more preferable.

  In the present invention, the oil and fat extraction step is performed on the cells collected by the lipid-producing bacterium culture step. In the oil and fat extraction step, the cells can be used as they are after being collected, that is, live bacteria, or can be used after sterilization. Moreover, you may process as a culture solution, without collecting bacteria. The collected cells may be used after being processed into an arbitrary shape. The method for collecting the cells is not particularly limited, and when the cultured cells are in a small amount, they may be centrifuged using a general centrifuge. In the case of a large amount, it is preferable to separate by continuous centrifugation, but this may be combined with filtration through a membrane or the like.

In addition, the collected cells may be wet cells or may be used as dry cells obtained by drying the wet cells. In the present invention, it is particularly preferable to use dry cells. Thereby, fats and oils can be extracted efficiently. The method for drying the wet cells is not particularly limited, and examples thereof include known drying treatments such as air blowing, heat treatment, reduced pressure treatment, and freeze drying.
In the oil extraction step, the method for extracting the oil from the cells is not particularly limited, and a known extraction method can be used. Specifically, at least one of compression extraction by pressurization, extraction by rendering using hot water or steam, extraction by various extraction media, and supercritical carbon dioxide can be used.

The pressure extraction by pressurization is not particularly limited as long as it is a method of applying pressure to the raw material to squeeze out the oil content in the cells, and specifically, for example, a batch hydraulic press, a continuous type And a method using an apparatus such as an expeller.
Extraction by the rendering may be either dry or wet, and is not particularly limited, and specific examples include a dry method using an open flame and a steam rendering (wet method) using an autoclave.

The dry method will be specifically described. For example, fats and oils are eluted from the cells by direct fire, jacket steam heating, or the like. The steam rendering will be described in detail. When heated steam is blown into the cells and heated and stirred, the oil is obtained in the form of an emulsion together with moisture, protein and the like. This is separated into waste water by a centrifuge and filtered if necessary to obtain crude oil. The conditions for steam rendering are not particularly limited, and examples include conditions for elution with 3 to 4 kg / cm ² of heated steam for 4 to 6 hours.

  The extraction medium used for the extraction with the extraction medium is not particularly limited. Generally, an extraction liquid of at least one of an aliphatic organic solvent and water, or a supercritical carbon dioxide gas is used. be able to. Among the above extracts, as the aliphatic organic solvent, specifically, for example, saturated hydrocarbons such as hexane and petroleum ether (an organic solvent containing pentane and hexane as a main component); ketones such as acetone; methanol, Examples thereof include alcohols such as ethanol, propanol and butanol; esters such as ethyl acetate; halogenated hydrocarbons such as chloroform and dichloromethane; cyanide hydrocarbons such as acetonitrile; ethers such as diethyl ether;

Of the above extract, water may be used as an aqueous solution in which a known solute is dissolved. Only one type of these extracts may be used, or two or more types may be appropriately selected and used. Among the above extract solutions, saturated hydrocarbons such as hexane and petroleum ether are preferably used, and hexane is more preferable in order to efficiently extract fats and oils such as triglyceride.
The extraction process using the extraction medium may be performed in a batch manner or a continuous manner. In addition, the extraction conditions with the extraction medium are not particularly limited, and an appropriate temperature and an appropriate extraction medium are selected according to the type of oil and fat such as triglyceride to be extracted and the amount (volume and weight) of the bacterial cells. The amount may be extracted at an appropriate time. At the time of extraction, it is preferable that the bacterial cells are dispersed in an extraction medium and then gently stirred. This enables efficient extraction.

The fats and oils extracted by the fat and oil extraction step can be used as they are, or further refined to increase the triglyceride content. The purification method is not particularly limited as long as it is a method used for general oil refining, and examples thereof include degumming, deoxidation, decolorization, deodorization, filtration, wintering, molecular distillation, and steam distillation. . These processes may be used alone or in combination.
In this way, a triglyceride with a high proportion of triglycerides consisting of three target PUFA residues can be obtained, but the ratio of a triglyceride consisting of three target PUFA residues can be further increased. Is possible. The method is not particularly limited, and examples thereof include column chromatography, urea inclusion, and molecular distillation.

The method of using the present invention is not particularly limited, but a typical example is the use of a triglyceride consisting of three PUFA residues of one kind according to the present invention as a substrate (raw material) for an enzyme reaction. It is done.
The other substrate is not particularly limited, and triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid alcohol ester and the like can be used. Preferably, animal and vegetable oils and fats, microbial oils and fats, fine algal fats and oils, medium chain fatty acid triglycerides, fatty acids having 2 to 24 carbon atoms, and alcohol esters of fatty acids having 2 to 24 carbon atoms are used. More preferably, tricapryloylglycerol, caprylic acid, and ethyl caprylate are used, but not limited thereto.

  The enzyme reaction is not particularly limited and may be any reaction, but representative examples include a lipase reaction, a phospholipase reaction, and an esterase reaction. The products obtained by the reaction are functional fats and oils, free fatty acids and the like, and functional fats and oils are not particularly limited. Typically, however, structural lipids in which highly unsaturated fatty acids are bound only at the 2-position, , Structural lipids in which highly unsaturated fatty acids are bonded only at the 3-position, and structured lipids composed of highly unsaturated fatty acids and medium chain fatty acids. Preferably, caprylic acid at positions 1, 3 and a structural lipid having PUFA bound thereto at position 2, caprylic acid at positions 1, 3, and arachidonic acid, eicosatrienoic acid or eicosapentaenoic acid at position 2, are more preferable. Or the structure lipid which the docosahexaenoic acid couple | bonded is mentioned. Examples of the free fatty acid obtained by the reaction include highly unsaturated highly unsaturated fatty acid derived from triglyceride consisting of three PUFA residues of one kind according to the present invention.

  Moreover, the use which is used as a nutritional composition for replenishing triglyceride which consists of one type of PUFA residue 3 concerning this invention is also mentioned. The organism to be used for the nutritional composition is not particularly limited and may be any organism, but is typically a human, and other than that, domestic animals, laboratory animals, etc. are listed. be able to. The nutritional composition can be taken in any form, but the method of oral intake is most preferred. Therefore, the present invention also includes foods containing triglycerides composed of three PUFA residues of one kind described above.

  Various additives can be added to the triglyceride consisting of three PUFA residues of one kind according to the present invention in order to improve functionality. Specifically, for example, sterols, sterol esters, glycolipids, sphingolipids, waxes, pigments, carotenoids, tocopherols, vitamin E, tocotrienol, sesamin, sesaminol, sesamol, astaxanthin, astaxanthin esters, sterols, carotenes, etc. However, it is not particularly limited. Since the lipid composition concerning this invention can be utilized for foodstuffs etc. as a nutritional composition, it is possible to add all the additives which can be added to foodstuffs.

  Since the food concerning this invention should just contain the triglyceride which consists of one type of PUFA residue of 3 types concerning this invention, the kind is not specifically limited. Specifically, bread, Japanese and Western confectionery (including frozen confectionery, etc.), side dish foods, dairy products, cereal foods, tofu and fried foods, noodles, lunch boxes, seasonings, processed agricultural products such as flour and meat, long-term preserved foods General foods such as soups such as canned foods (frozen foods, frozen foods, retort foods, etc.), soft drinks, milk drinks, soy milk, and potage soups are not particularly limited. The method for adding triglyceride consisting of three PUFA residues of one kind to these general foods is not particularly limited, and any known appropriate method can be adopted depending on the kind of general food.

  In addition, examples of the food according to the present invention include functional foods used for specific uses other than general foods such as health foods and nutritional foods. Specific examples include dietary supplements such as various supplements and foods for specified health use. The triglyceride consisting of three PUFA residues of one type according to the present invention can be used as it is by simply processing it into an appropriate shape in the case of a supplement or the like. The processing shape at this time is not particularly limited. Specifically, the lipid composition (or food) according to the present invention may be in a liquid or powder form, a capsule form, or a tablet or tablet form. Moreover, all the techniques which can be used with respect to general fats and oils, such as melt | dissolution in general fats and oils-type foodstuffs and powdering, can be applied.

  Further, the present invention can be used not only in the food field as described above but also in the pharmaceutical field. That is, the triglyceride consisting of one type of three PUFA residues according to the present invention may be used as a pharmaceutical product. The specific example in the case of utilizing as a pharmaceutical is not specifically limited, either, A well-known technique should just be utilized according to the objective.

EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to this.
Example 1. Mortierella alpina (Mortierella alpina) SAM2268 acquisition Mortierella alpina avian arachidonoylglycerol highly productive strain by mutagenesis treatment (Mortierella alpina) SAM2268 (FERM P -17762) and Czapek agar medium (0.2% NaNO ₃ , 0.1% K ₂ HPO ₄ , 0.05% MgSO ₄ · 7H ₂ O, 0.05% KCl, 0.01% FeSO ₄ · 7H ₂ O, 3% sucrose, 2% agar, pH 6.0) in a large slant bottle Inoculated and cultured at 28 ° C. for 2 weeks.

After incubation, 50 mL of sterilized water was added, shaken, filtered through quadruple gauze, centrifuged at 8,000 × g for 10 minutes, and then suspended in 50 mM Tris buffer solution (pH 7.5) to prepare a spore suspension. Add 0.5 mL of 0.5% NTG (N-methyl-N′-nitro-N-nitrosoguanidine) solution to 1.5 mL of 1 × 10 ⁶ / mL spore suspension, and perform mutation treatment at 28 ° C. for 15 minutes. went. After adding ₃ mL of 10% Na ₂ S ₂ O ₃ and centrifuging at 5,500 × g for 10 minutes, it was washed with sterilized water to obtain an NTG-treated spore suspension.

  NTG-treated spore suspension was applied to GY agar medium (1% glucose, 0.5% yeast extract, 0.005% Triton X-100, 1.5% agar, pH 6.0), and colonies grown at 28 ° C were separated from other GY agar. Transferred to medium. A part of the grown cells was dried, and triglyceride was extracted with hexane according to a conventional method, and the triglyceride obtained by distilling off hexane was analyzed by high performance liquid chromatography. As a result of examining about 3,000 colonies, three mutant strains (# 1, # 2 and # 3) with an increased proportion of triarachidonoylglycerol in the total triglycerides were obtained.

  The percentages (weight%) of triarachidonoylglycerol in the total triglycerides of # 1, # 2, and # 3 were 17.0%, 15.1%, and 12.2%, respectively, but 8% for all other colonies. It was the following. The ratio (weight%) of triarachidonoylglycerol in the total triglycerides of # 1, Strain # 2, and Strain # 3 was determined according to a conventional method using a COSMOSIL 5C18-MA column (4.6 × 250 mm) as an analytical column. Acetonitrile / acetone (1: 1) was used as a mobile phase (1.0 mL / min) and detected with a differential refractometer.

Example 2. Comparison of triarachidonoylglycerol production ability
One platinum loop of three Mortierella alpina strains shown below in 10 mL of liquid medium (2% glucose, 1% yeast extract, 1% palmitoleic acid or none) in a 50 mL Erlenmeyer flask Were respectively inoculated and cultured with shaking at 28 ° C. and 120 rpm for 7 days.
(1) Mortierella alpine # 1 (mutant strain obtained in Example 1)
(2) Mortierella alpina SAM 2268
(3) Mortierella alpina IFO 8568

  After cultivation, the cells were collected by filtration and dried. A part of the grown cells was dried, and triglyceride was extracted with hexane according to a conventional method, and hexane was distilled off to obtain triglyceride. The obtained triglyceride was subjected to liquid high-performance chromatography, and the triglyceride molecular species were analyzed to determine the ratio of triarachidonoylglycerol (tri-AA). Further, a part of the grown cells was dried, and the fatty acid in the cells was methyl esterified with hydrochloric acid methanol according to a conventional method, followed by extraction with hexane, and hexane was distilled off to obtain a fatty acid methyl ester. The fatty acid methyl ester obtained was subjected to gas chromatography, and the ratio of arachidonic acid in the total fatty acid was determined. The results are shown in Table 1.

  The proportion of tri-AA in the parent strain SAM 2268 was 10.2%, while the proportion of tri-AA in mutant strain # 1 reached 33.3%. In addition, the proportion of tri-AA of IFO that had the same proportion of arachidonic acid in the total fatty acid was only 18.5%.

Example 3. Time course of triarachidonoylglycerol production
Inoculate a 10 ml liquid medium in a 50 mL Erlenmeyer flask (2% glucose, 1% yeast extract, 1% palmitoleic acid or none) with one platinum loop of Mortierella alpina # 1. The culture was shaken at 28 ° C. and 120 rpm for 6, 8, and 10 days. After cultivation, the cells were collected by filtration, and the ratio of triarachidonoylglycerol (tri-AA) and the ratio of arachidonic acid in the total fatty acids were determined in the same manner as in Example 2. The results are shown in Table 2.

  The proportion of tri-AA was 30.1% on the 6th day, but reached 45.9% on the 8th day and 62.5% on the 10th day.

Mass production Mortierella alpina Example 4. triarachidonoyl glycerol (Mortierella alpina) # 1 loopful was inoculated into seed medium A100mL, reciprocal shaking 100 rpm, 28 ° C., were precultured for three days with the conditions. Next, sterilize 5 L of the main medium D in a 10 L volume aeration and agitation culture tank, inoculate the entire amount of the above pre-cultured solution, and culture for 10 days at 26 ° C., aeration volume of 1 vvm, and agitation speed of 300 rpm did. Depending on the glucose consumption, 1% of glucose was appropriately flowed down. The obtained cultured cells were sterilized, the medium was removed and then dried to obtain 75 g of dried cells.

  To 75 g of the dried cells, 225 mL of hexane was added and stirred gently at room temperature for 3 hours, followed by filtration to obtain a hexane layer. Thereafter, 150 mL of hexane was added again to the dried cells, and the mixture was gently stirred at room temperature for 3 hours, and then a hexane layer was obtained by filtration. After the hexane layers were combined, hexane was distilled off to obtain 35 g of crude extracted oil. Furthermore, activated clay was applied to obtain 33 g of triglyceride. About a part of the obtained triglyceride, the ratio of triarachidonoylglycerol (tri-AA) and the ratio of arachidonic acid in the total fatty acids were determined in the same manner as in Example 2, and they were 55.0% and 59.7%, respectively. . About a part of obtained triglyceride, 10-g tri-AA was obtained by fractionating a tri-AA fraction with the high performance liquid chromatography shown in Example 1. FIG.

Example 5 Lipase reaction ion exchange resin carrier using triarachidonoyl glycerol (Dowex MARATHON WBA: Dow Chemical, trademark) 10 g was suspended in 8 ml of Rhizopus delemar lipase 12.5% aqueous solution (Tallipase powder: Tanabe Seiyaku Co., Ltd.). And dried under reduced pressure to obtain an immobilized lipase.
Next, 8 g of caprylic acid triglyceride (MCT), 600 mg of the above immobilized lipase and 240 μl of water were reacted at 30 ° C. for 48 hours with stirring (130 rpm). After completion of the reaction, the reaction solution was removed to obtain an activated immobilized enzyme.

1 g of triarachidonoyl glycerol (tri-AA) obtained in Example 4, 2 g of caprylic acid, 150 mg of the above immobilized enzyme ( Rhizopus delemar lipase, carrier: Dowex MARATHON WBA) was stirred (130 rpm) at 30 ° C. for 48 hours. Reacted. In the reaction fats and oils from which the immobilized enzyme has been removed, there is arachidonic acid cut out from the 1,3-position of tri-AA and excess caprylic acid, which is a reaction substrate, and these fatty acids are removed by alkaline extraction. Thus, a once-treated oil was obtained. 1 g of the obtained once-treated oil and fat, 2 g of caprylic acid, and 150 mg of the recovered immobilized enzyme were reacted at 30 ° C. for 48 hours with stirring (130 rpm). By removing caprylic acid and the like by the same treatment as described above, 0.8 g of oil treated twice was obtained. This twice-treated oil was 96 mol% 1,3-capryloyl-2-arachidonoyl-glycerol.

Example 6 Preparation of Capsules Containing Triarachidonoyl Glycerol Gelatin (Nitta Gelatin) and glycerin for food addition (Kao) were mixed at a weight ratio of 100: 35 and water was added. A gelatin film having a viscosity of 2000 cp was prepared by dissolving in a temperature range of 50 to 60 ° C. Next, triglyceride containing 55.0% of triarachidonoyl glycerol (tri-AA) obtained in Example 4 and vitamin E oil (manufactured by Eisai Co., Ltd.) were mixed at a weight ratio of 100: 0.05 to prepare a content. did. Using these, capsules were molded and dried by a conventional method to produce soft capsules containing 180 mg of content per grain. All of these soft capsules were suitable for oral intake.

Example 7 Preparation of Beverage Containing Triarachidonoyl Glycerol Triglyceride containing 55.0% of triarachidonoyl glycerol (tri-AA) obtained in Example 4 and soy lecithin (smoked oil) were mixed at a weight ratio of 9: 1. And uniformly dispersed in water to obtain a liposome dispersion. Each of the above-mentioned beverages as a food according to the present invention is prepared by adding 1/100 volume each of this liposome dispersion to orange juice, carbonated water, coffee beverage, milk, soy milk, or potage soup beverage ( Manufactured). These beverages were all suitable for oral intake.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments obtained by appropriate combinations are also included in the technical scope of the present invention.

Industrial Applicability As described above, in the present invention, triglyceride composed of three PUFA residues of one kind can be efficiently and stably produced by fermentation technology. Therefore, the present invention can be used not only widely in industries related to functional foods, but also in industries related to general foods and pharmaceuticals.

Claims (18)

  Microorganism capable of producing fats and oils in the method for producing fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides And then collecting the fats and oils if desired.   The method according to claim 1, wherein the ratio of triglyceride formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 30% by weight or more based on the total triglycerides.   The method according to claim 2, wherein the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 40% by weight or more based on the total triglycerides.   The method according to claim 3, wherein the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 50% by weight or more based on the total triglycerides.   The method according to any one of claims 1 to 4, wherein the same highly unsaturated fatty acid is arachidonic acid.   The method according to claim 1, wherein the microorganism is a microorganism of the genus Mortierella.   The method according to claim 6, wherein the microorganism is a microorganism of the genus Mortierella (Mortierella).   8. The method of claim 7, wherein the microorganism is a microorganism of the species Mortierella alpina.   The method according to any one of claims 1 to 8, wherein the microorganism is a mutant strain of a microorganism capable of producing fats and oils containing highly unsaturated fatty acids.   Oils and fats produced by the method according to claim 1.   A Mortierella microorganism capable of producing fats and oils in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides.   A Mortierella genus microorganism capable of producing fats and oils in which the ratio of triglycerides formed by bonding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 30% by weight or more based on the total triglycerides.   13. The microorganism according to claim 11 or 12, which is Mortierella alpina.   The microorganism according to any one of claims 11 to 13, which is a mutant strain.   A microorganism cultured microbial cell comprising fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 20% by weight or more based on the total triglycerides.   A microorganism cultured microbial cell comprising fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 30% by weight or more based on the total triglycerides.   A microorganism cultured microbial cell comprising fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 40% by weight or more based on the total triglycerides.   A microorganism cultured microbial cell comprising fats and oils in which the ratio of triglycerides formed by binding the same highly unsaturated fatty acid to three hydroxyl groups of glycerol is 50% by weight or more based on the total triglycerides.