JP4850153B2 - Novel triglyceride and composition containing the same - Google Patents

Description

  The present invention relates to a new triglyceride and a composition containing the same, and in particular, a triglyceride having a saturated fatty acid having 16 to 18 carbon atoms at the 2-position, and ω6, ω9 and / or ω3-unsaturated fatty acid at the 1- and 3-positions. Relates to a triglyceride having

  Most of the lipids we ingest are neutral fats, a mixture of triglycerides in which various fatty acids are ester-linked at positions 1, 2 and 3 of triglycerides. And it has been pointed out that the absorption and physiological activity differ depending on the binding position of fatty acids, and lipids (structural lipids) in which specific fatty acids are bound to fixed positions of triglycerides have recently been particularly noted. ing.

  For example, Japanese Patent Publication No. 4-12920 discloses a triglyceride having good digestibility and absorption in which a fatty acid having 8 to 14 carbon atoms is bonded to the 2nd position of the triglyceride and a fatty acid having 18 or more carbon atoms bonded to the 1st and 3rd positions. ing. In addition, since 2-monoglyceride is known to be the form most easily absorbed by the human body, JP-B-5-87497 contains a ω3 or ω6 highly unsaturated fatty acid having a physiological function at the 2-position. Triglycerides are disclosed which are bound and combined with saturated fatty acids which are easily hydrolyzed by enzymes of the digestive tract at positions 1 and 3. However, they do not disclose or suggest the relationship with unsaturated fatty acids in human breast milk and / or triglycerides containing the unsaturated fatty acids as constituent fatty acids.

  On the other hand, when looking at the physiological functions of fatty acids, arachidonic acid and docosahexaenoic acid have attracted attention in recent years. These fatty acids are contained in breast milk and have been reported to be useful for infant growth ("Advances in Polyunsaturated Fatty Acid Research", Elsevier Science Publishers, 1993, pp.261-264), as well as fetal height and brain There are reports that it is important for development (Proc. Natl. Acad. Sci. USA, 90, 1073-1077 (1993), Lancet, 344, 1319-1322 (1994)).

  Recommended public intakes were published by several public institutions (premature infants: arachidonic acid 60, docosahexaenoic acid 40; normal infants: arachidonic acid 20, docosahexaenoic acid 20 mg / kg body weight / day (WHO-FAO (1994) ) In some European countries, premature infant formulas containing arachidonic acid fermented in combination with docosahexaenoic acid as triglycerides are commercially available, but the triglycerides arachidonic acid and / or docosahexaenoic acid added to the formula No consideration is given to the bonding position.

  Triglyceride structure in human breast milk has a high ratio of palmitic acid (16: 0) binding to the 2nd position of triglyceride and a high ratio of highly unsaturated fatty acids or medium chain fatty acids to the 1st and 3rd positions. (Christie, WW (1986) The Positional Distribution of Fatty Acids in Triglycerids. Analysis of Oils and Fats in (Hamilton, RJ, and Russell, JB, eds.) Pp. 313-339, Elsevier Applied Science, London) However, this is only an analogy from the results of fatty acid analysis of triglycerides, and isolation and structural analysis of triglycerides in human breast milk have not yet been performed.

  In addition, as described above, arachidonic acid-containing triglycerides produced by fermentation have been added to the formula to bring the fatty acid composition close to that of breast milk, and the structure of this arachidonic acid-containing triglyceride includes palmitic acid. Saturated fatty acids are bonded to the 1 and 3 positions, and unsaturated fatty acids are bonded to the 2 position (JJ Myher, A. Kuksis, K. Geher, PW Park, and DA Diersen-Schade, Lipids 31, 207-215 (1996)), which is different from the presumed structure of triglycerides in human breast milk.

  Therefore, lipids presumed to be human breast milk triglyceride structures, that is, saturated fatty acids having 16 to 18 carbon atoms are bonded to the 2nd position of triglycerides, and highly unsaturated fatty acids or medium chain fatty acids are bonded to the 1st and 3rd positions. There is a strong demand for the development of structured lipids whose structures are clearly confirmed.

  Therefore, in the present invention, the triglyceride is a saturated fatty acid having 16 to 18 carbon atoms in the 2nd position, an unsaturated fatty acid is bonded to the 1st and 3rd positions, and at least one of the unsaturated fatty acids is an ω6, ω9 or ω3 unsaturated fatty acid. A novel triglyceride or a triglyceride in position 2 is a saturated fatty acid having 16 to 18 carbon atoms, one of positions 1 and 3 is a saturated fatty acid having 4 to 18 carbon atoms, and the other is ω6, ω9 or It is intended to provide a novel triglyceride which is an ω3 unsaturated fatty acid and a composition comprising the novel triglyceride.

  As a result of intensive studies to solve the above problems, the present inventors have used glycerides in which a saturated fatty acid having 16 to 18 carbon atoms is bonded to the 2nd position, and the 1st and 3rd positions. A lipase that specifically acts on an ester bond and an unsaturated fatty acid or fatty acid ester of the ω6, ω9, or ω3 system are allowed to act, and only the fatty acid at the 1 and 3 position is converted to an ω6, ω9, and / or ω3 system by transesterification. By finding unsaturated fatty acids, it is possible to produce triglycerides presumed to be the target human breast milk type, and by comparing the obtained triglycerides with triglycerides obtained from human breast milk, Triglyceride with a structure in which a saturated fatty acid having 16 to 18 carbon atoms is bonded to the 2nd position of triglyceride and a highly unsaturated fatty acid is bonded to the 1st and 3rd positions. It was completed for the first time clear to to the present invention which are present definitely.

The present invention relates to a novel triglyceride and a food composition, animal feed, therapeutic nutritional product, and pharmaceutical composition using the triglyceride.
According to the invention, the following general formula (I):

(Wherein R ¹ and R ² represent an acyl group of an unsaturated fatty acid having 18 to 22 carbon atoms, this acyl group may be oxidized, and n represents an integer of 14 to 16)
Wherein at least one of R ¹ or R ² is a ω6, ω9 or ω3 unsaturated fatty acid; or the following general formula (II):

(Wherein R ³ represents an acyl group of an unsaturated fatty acid having 18 to 22 carbon atoms, the acyl group may be oxidized, and n is an integer of 14 to 16, m is an integer of 2 to 16) Is represented).
The fatty acids constituting the 1 and 3 positions of the triglyceride obtained in the present invention are ω3, ω6 and / or ω9 unsaturated fatty acids. Specifically, ω3 unsaturated fatty acids include 9,12,15-octadecatrienoic acid (α-linolenic acid) [18: 3, ω3], 6,9,12,15-octadecatetraenoic acid (Stearidonic acid) [18: 4, ω3], 11,14,17-eicosatrienoic acid (dihomo-α-linolenic acid) [20: 3, ω3], 8,11,14,17-eicosatetraene Acid [20: 4, ω3], 5,8,11,14,17-eicosapentaenoic acid [20: 5, ω3], 7,10,13,16,19-docosapentaenoic acid [22: 5, ω3 4,7,10,13,16,19-docosahexaenoic acid [22: 6, ω3].

  The ω6 unsaturated fatty acids include 9,12-octadecadienoic acid (linoleic acid) [18: 2, ω6], 6,9,12-octadecatrienoic acid (γ-linolenic acid) [18: 3 , ω6], 8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid) [20: 3, ω6], 5,8,11,14-eicosatetraenoic acid (arachidonic acid) [20: 4, ω6], 7,10,13,16-docosatetraenoic acid [22: 4, ω6], 4,7,10,13,16-docosapentaenoic acid [22: 5, ω6] it can. Furthermore, the ω9 unsaturated fatty acids include 6,9-octadecadienoic acid [18: 2, ω9], 8,11-eicosadienoic acid [20: 2, ω9], and 5,8,11-eicosatrienoic acid. (Mead acid) [20: 3, ω9] can be mentioned. Furthermore, the acyl residue may be a hydroxylated, epoxidized or hydroxyepoxidized acyl residue.

The fatty acid constituting the 2-position of the novel triglyceride of the present invention comprises a fatty acid having 16 to 18 carbon atoms, and examples thereof include palmitic acid (16: 0) and stearic acid (18: 0).
The novel triglyceride of the present invention reacts with a triglyceride that clearly has a saturated fatty acid having 16 to 18 carbon atoms bonded to the 2-position by acting on a lipase that acts only on the ester bond at the 1,3-position of the triglyceride. It can be produced by transesterification with an ω3, ω6 or ω9 unsaturated fatty acid or fatty acid ester added to the system.

  Examples of triglycerides in which a saturated fatty acid having 16 to 18 carbon atoms is bound to the 2nd position include, for example, tripalmitin (all positions 1, 2 and 3 are palmitic acid (16: 0)), tristearin ( 1, 2 and 3 are all stearic acid (18: 0)), but all the fatty acids ester-linked to the triglycerides need not be the same, and the triglycerides have 16 to 18 carbon atoms at the 2nd position. As long as the saturated fatty acid is bonded, any fatty acid having 4 to 18 carbon atoms may be bonded to the 1 and 3 positions, and any combination may be used. In addition, fats and oils having a saturated fatty acid as a constituent fatty acid have a high melting point and a high reaction temperature when the constituent saturated fatty acid has 16 or more carbon atoms.

  For example, when tripalmitin is used, the reaction must be performed at 50 to 70 ° C., depending on the composition of the reaction solution. Therefore, when these high-melting-point triglycerides are used as a substrate raw material, the reaction should be carried out at a high temperature. However, at high temperatures, enzyme deactivation and the modification of unsaturated fatty acids added for transesterification are problematic. Before exchanging the 1st and / or 3rd fatty acid with the desired unsaturated fatty acid, the fatty acid bonded to the 1st and / or 3rd position of the raw triglyceride is, for example, 8 to 12 carbon atoms such as caprylic acid. It is preferable to transesterify to about one medium chain fatty acid or a fatty acid having a low melting point such as oleic acid or linoleic acid to lower the melting point of triglyceride and to react at a temperature of 45 ° C. or lower.

  In addition, the triglyceride having a saturated fatty acid bonded to the 2-position means that, for the purpose of the present invention, if a saturated fatty acid having 16 to 18 carbon atoms is bonded to the 2-position, the 1- or 3-position is ω3, ω6 or ω9 unsaturated fatty acids may be bound. When these substrates are used, ω3, ω6 or ω9 unsaturated fatty acids are introduced by transesterification at the position where the unsaturated fatty acids are not bound. be able to.

For example, the 2-position triglycerides unsaturated fatty acids in any of the 1 and 3 positions with saturated fatty acids are bonded, Crypthecodinium Kode hexafluorophosphate (Crypthecodenium) genus Thraustochytrium (Thraustochytrium) genus Schizochytrium (Schizochytrium) genus Ulkenia ( Oils obtained by culturing microorganisms of the genus Ulkenia , Japonochytorium and Haliphthoros can be used.

  From these, for example, 1,2-dipalmitoyl-3-docosahexanoyl triglyceride can be isolated, and a 1,3-position specific lipase is allowed to act on this triglyceride as a substrate, and ω3, ω6, or ω9 unsaturated. When transesterified with a fatty acid or a fatty acid ester thereof, docosahexaenoic acid is not transesterified, and only the 1-position palmitic acid is transesterified with an unsaturated fatty acid. When arachidonic acid is used as the unsaturated fatty acid, a triglyceride in which docosahexaenoic acid is bonded to one of the 1 and 3 positions, arachidonic acid is bonded to the other, and palmitic acid is bonded to the 2 position can be produced.

The present invention, the 1, 3-position specific lipase of triglycerides can be used as a catalyst, not particularly limited, for example, Rhizopus (Rhizopus) genus Rhizomucor (Rhizomucor) genus Mucor (Mucor) genus , Penicillium (Penicillium) genus Aspergillus (Aspergillus) genus Humicola (Humicola) genus Fusarium (Fusarium) lipases microorganisms such as genus produce, such as porcine pancreatic lipase and the like. About this lipase, a commercially available thing can be used.

For example, lipase of Rhizopus delemar (Rhizopus delemar) (Tanabe Seiyaku Co., Ltd.; Talipase), lipase of Rhizomucor Miihai (Rhizomucor miehei) (manufactured by Novo Nordisk (Ltd.); ribozyme IM), Aspergillus niger (Aspergillus lipase from Niger (manufactured by Amano Pharmaceutical Co., Ltd .; lipase A), lipase from Humicola lanuginosa (manufactured by Novo Nordisk Co., Ltd .; lipolase), lipase from Mucor javanicus (Amano Pharmaceutical) Lipase M) manufactured by (Co., Ltd.), Fusarium heterosporum lipase, and the like. Use forms of these lipases may be used as they are, or lipases immobilized on celite, ion exchange resin, ceramic carrier or the like may be used.

  The amount of water added to this reaction system is extremely important. If no water is contained, transesterification does not proceed, and if the amount of water is large, hydrolysis occurs, resulting in a decrease in triglyceride recovery rate or formation. In partial glycerides, spontaneous acyl group transfer occurs, and the saturated fatty acid at the 2nd position is transferred to the 1st or 3rd position. Therefore, when using an immobilized enzyme that does not have bound water, before performing the main reaction, first activate the enzyme using a substrate to which water has been added, and use a substrate to which water has not been added in the main reaction. It is effective. To activate in a batch reaction, the enzyme is pretreated with a substrate containing water in an amount of 0 to 1,000% (wt%) of the added enzyme. The substrate should be continuously flowed.

For example, the amount of water when activated by a batch reaction using Rhizopus delemar lipase (Tanabe Seiyaku Co., Ltd .; Talipase) immobilized on celite or a ceramic carrier is 10% of the amount of enzyme added. ~ 200% (% by weight). However, the amount of water necessary for the activation of the transesterification reaction greatly depends on the type of enzyme used. For example, if the lipase of Rhizomucor miehei (manufactured by Novo Nordisk Co., Ltd .; ribozyme IM) Little water is required, but rather excess water must be removed. For the removal of excess water, triglyceride that does not interfere with the main reaction is selected as a substrate, and this may be hydrolyzed with an immobilized enzyme.

The amount of lipase used in the batch reaction may be appropriately determined depending on the reaction conditions, and is not particularly limited. For example, lipase of Rhizopus delemar ( Rhizopus delemar ) immobilized on celite or ceramic carrier, or Rhizomucol Miihai ( When Rhizomucor miehei ) lipase is used, 1-30% (wt%) of the reaction mixture is adequate.

  The transesterification reaction in the batch reaction is performed by the following method. That is, an ω3, ω6, or ω9 unsaturated fatty acid or a fatty acid ester thereof is added to triglyceride in which a saturated fatty acid having 16 to 18 carbon atoms is bonded to the 2-position. Examples of fatty acid esters that can be used include methyl esters, ethyl esters, propyl esters, and butyl esters. The appropriate ratio of the triglyceride / fatty acid or triglyceride / fatty acid ester ratio used as a raw material is 1: 0.5 to 20. An amount appropriate for this substrate (usually 5,000 to 50,000 U / g; lipase 1U is an enzyme amount that liberates 1 μmol of fatty acid per minute using olive oil as a substrate) 1 , 3-position specific lipase is added, and transesterification is carried out at 20 to 70 ° C. for 2 to 100 hours with stirring or shaking.

The immobilized enzyme can be used repeatedly. That is, the reaction can be continued by leaving only the immobilized enzyme in the reactor after the reaction and exchanging the reaction solution with a newly prepared substrate. In the transesterification by the column method, the substrate may be continuously flowed at 0.05 to 20 ml / hr per gram of enzyme.
Moreover, the target triglyceride content can be raised by repeating transesterification. That is, in the presence of an ω3, ω6, or ω9 unsaturated fatty acid or a fatty acid ester thereof, the 1,3-position specific lipase of triglyceride is allowed to act so that the fatty acids at the 1st and 3rd positions are ω3, ω6 and / or ω9 unsaturated. A reaction liquid transesterified with a saturated fatty acid is obtained.

  Next, triglyceride is purified from the reaction solution by a method described later, and the transesterification is performed again with the ω3, ω6, or ω9 unsaturated fatty acid or its fatty acid ester using the purified triglyceride as a raw material. The target triglyceride content can be dramatically increased by this repeated esterification reaction, and the number of repetitions is preferably 2 to 5 times.

  In a conventional transesterification reaction using an immobilized lipase, acyl group transfer of a fatty acid bonded to the 2-position of a partial glyceride generated by a hydrolysis reaction occurring as a side reaction was induced. However, in the present invention, the hydrolysis reaction can be suppressed almost completely, and the amount of partial glyceride produced is about 1%, thus solving the conventional problems. In addition, if the moisture content contained in the substrate is several thousand ppm or less, the hydrolysis that occurs as a side reaction can be ignored, and there is no need to precisely control the amount of moisture contained in the substrate. is doing.

  Furthermore, in the reaction in the organic solvent using the conventional immobilized enzyme or the reaction at 50 ° C. or higher, the enzyme activity decreased after several uses, whereas the reaction system of the present invention containing no organic solvent. When the reaction is carried out at 45 ° C. or lower, the enzyme is not deactivated, and the enzyme can be used continuously for several tens of batch reactions or more than 100 days in a column reaction.

  In the present invention, since the substrate is simple, the triglyceride obtained by the reaction is composed of several molecular species. Therefore, the target triglyceride can be easily isolated by a conventional method such as liquid chromatography, molecular distillation, falling film distillation, precision distillation, or a combination thereof. The post-reaction triglyceride produced in the present invention is a triglyceride in which an unsaturated fatty acid is bonded to the 1-position and / or the 3-position. The triglyceride, the unreacted raw material, the unreacted unsaturated fatty acid or fatty acid ester, and the ester exchanged are obtained. It exists as a mixture with the fatty acid or fatty acid ester bound to the 1 and / or 3 position of the resulting triglyceride.

  Therefore, purification of triglycerides in which unsaturated fatty acids are bonded at the 1st and / or 3rd positions and saturated fatty acids having 16 to 18 carbon atoms are bonded at the 2nd position is performed by alkaline deoxidation, steam distillation, molecular distillation, flow down Any one of membrane distillation, vacuum precision distillation, column chromatography, solvent extraction, membrane separation, or a combination thereof may be used to remove the transesterified fatty acid and the unreacted unsaturated fatty acid.

  The triglyceride obtained by the present invention in which palmitic acid is bonded to the 2-position and arachidonic acid and / or docosahexaenoic acid is bonded to the 1- and 3-positions is considered to be a structure of triglyceride in human breast milk. It can be effectively used for infant formula, infant formula, follow-up milk, formula for pregnant women and lactating women, and the like. That is, one of the triglycerides of the present invention in which palmitic acid is bonded at the 2-position and arachidonic acid and / or docosahexaenoic acid is bonded at the 1- and 3-positions is used for premature infant formula, infant formula, follow-up milk, etc. By adding to the product in the production process of the formula or to the product, a formula closer to human breast milk can be obtained.

  In the present invention, triglycerides in which the same unsaturated fatty acids of ω6 system, ω9 system or ω3 system are bonded to the 1st and 3rd positions are obtained, and these are considered to be the structures of triglycerides in human breast milk. Although it is sufficiently useful as a source of unsaturated fatty acids of ω6, ω9 and ω3, respectively, in the present invention, different unsaturated fatty acids of ω6, ω9 or ω3 are present in the 1st and 3rd positions. The usefulness is further increased by obtaining a bound triglyceride, for example, a triglyceride having an ω6 arachidonic acid bonded to the 1-position and an ω3-docosahexaenoic acid bonded to the 3-position. That is, if it is a triglyceride in which an ω6 arachidonic acid is bonded to the 1st position and an ω3 docosahexaenoic acid is bonded to the 3rd position, two triglycerides, arachidonic acid (ω6) and docosahexaenoic acid (ω3), are used. It becomes possible to simultaneously supply various types of unsaturated fatty acids.

  As the utilization of the triglyceride of the present invention, addition to formulas intended for premature babies and infants, for example, addition to dairy products such as milk and soy milk, and foods using fats and oils can be considered. Examples of foods using fats and oils include natural foods containing fats and oils such as meat, fish and nuts, foods to which fats and oils are added when cooking Chinese dishes, ramen, soups, tempura, fries, fried chicken, fried rice, donuts, carin sugar Foods using fats and oils as a heating medium such as butter, margarine, mayonnaise, dressing, chocolate, instant ramen, caramel, biscuits, ice cream, etc. or foods with added fats during processing, pork, hard biscuits, bread The food etc. which sprayed or apply | coated the fats and oils at the time of processing finishing etc. can be mention | raise | lifted.

  In addition, for example, agricultural foods such as bread, noodles, rice, confectionery, tofu and processed foods thereof, fermented foods such as sake, medicinal liquor, mirin, vinegar, soy sauce, miso, dressing, yogurt, ham, bacon, sausage, Examples include livestock foods such as mayonnaise, marine foods such as kamaboko, fried tempura, hampen, fruit juice drinks, soft drinks, sports drinks, alcoholic drinks, tea drinks, and the like.

  When used as health foods or functional foods, the form may be the following pharmaceutical preparations or the above-mentioned food and drink products. For example, protein (a protein source is a highly nutritious milk protein with balanced amino acid, soybean Proteins such as protein and egg albumin are the most widely used, but in addition to these degradation products, egg white oligopeptides, soybean hydrolysates, etc., mixtures of amino acids alone are also used), sugars, fats, trace elements, Processing forms such as natural liquid foods, semi-digested nutritional foods and component nutritional foods, drinks, and the like containing vitamins, emulsifiers, fragrances and the like may be used.

  The food / beverage products of this invention can be processed and manufactured by a general manufacturing method, adding the required amount of the triglyceride of this invention. The blending amount differs depending on the dosage form, food form, and properties, and is generally preferably 0.001 to 50%, but is not particularly limited. Ingestion as a health food or functional food is based on the physiological function and titer of the highly unsaturated fatty acid bound to positions 1 and 3 of the triglyceride of the present invention. When cooking lunches, the novel triglycerides of the present invention can be added and given to patients in the form of functional foods prepared on site.

  When the triglyceride of the present invention is used as a pharmaceutical, the administration form may be any form that can be conveniently administered orally or parenterally, for example, injection solution, infusion solution, powder, granule, tablet , Capsule, enteric solvent, troche, liquid for internal use, suspension, emulsion, syrup, liquid for external use, poultice, nasal drop, ear drops, eye drops, inhalant, ointment, lotion, suppository These can be used alone or in combination depending on the symptoms.

These various preparations are known to be commonly used in the field of pharmaceutical formulation technologies such as prison agents, binders, preservatives, stabilizers, disintegrants, lubricants, and corrigents depending on the purpose in accordance with conventional methods. It can be formulated using adjuvants. The dose varies depending on the purpose of administration, fatty acids (physiological activity, titer, etc.) bound to positions 1 and 3 of triglyceride and the situation of the subject (gender, age, weight, etc.). In the case of oral administration to adults, the total amount of the structured lipid of the present invention is 0.01 mg to 10 g per day, preferably 0.1 mg to 2 g, more preferably 1 mg to 200 mg, and parenteral administration. The total amount of the structured lipid of the present invention can be appropriately adjusted and administered in the range of 0.001 mg to 1 g, preferably 0.01 mg to 200 mg, more preferably 0.1 mg to 100 mg per day.
Furthermore, the triglyceride of the present invention is a triglyceride that has not been isolated or synthesized so far and can be used as a standard substance for analysis.

Next, the present invention will be described more specifically with reference to examples.
In this example, fatty acids and triglycerides are abbreviated as follows for convenience. First, the following one-letter abbreviations representing fatty acids are used. 8: caprylic acid, P: palmitic acid, A: arachidonic acid, M: mead acid, D: docosahexaenoic acid. Next, triglyceride is expressed in three letters by a one-letter abbreviation representing a fatty acid bonded to the first position, a one-letter abbreviation representing a fatty acid bonded to the second position, and a one-letter abbreviation representing a fatty acid bonded to the third position. . Therefore, the structure of triglyceride is expressed as in the following example, for example. Example: 8P8 (caprylic acid at position 1, palmitic acid at position 2, triglyceride with caprylic acid bound at position 3)

Example 1.
Using a mixture of tripalmitin (PPP) and caprylic acid 1: 2 (wt / wt) as a substrate material, 10.5 g of the substrate mixture and immobilized Rhizomucor miehei lipase (manufactured by Novo Nordisk); A reaction solution consisting of 1.2 g of ribozyme IM60) was placed in a vial with a screw cap and incubated at 50 ° C. for 48 hours with shaking (140 times / min). After the reaction, the reaction solution was replaced with a new substrate mixture leaving only the immobilized enzyme, and the reaction was performed under the same conditions. The reaction was performed 4 times while repeatedly using the immobilized enzyme, and each reaction solution was recovered.

  70 ml of 0.5N KOH solution (20% ethanol solution) was added to each reaction solution (10.5 g), and the glyceride fraction was extracted with 100 ml of hexane, and then the solvent was removed by an evaporator to recover glycerides. As a result of examining the glyceride composition with Iatroscan (manufactured by Yatron Co., Ltd.), 8% diglyceride was contained in the first glyceride, but the partial glyceride content in the second and subsequent glycerides was 1% It was the following. The fatty acid composition (mol%) of the 2nd to 4th glyceride fractions was 45.1% caprylic acid and 54.9% palmitic acid.

  In order to increase the exchange rate of caprylic acid, the transesterification was carried out again using the second to fourth glyceride fractions as raw materials. The ribozyme IM60 (1.2 g) used in the above reaction was added with 3.5 g of the prepared glyceride and 7 g of caprylic acid, and reacted at 30 ° C. for 48 hours with shaking (fifth time). After the reaction, the reaction solution was replaced with a new substrate, and the reaction was performed under the same conditions (sixth time). The glyceride fraction was recovered from the 5th and 6th reaction liquids by hexane extraction (total 4.8 g). The fatty acid composition (mol%) of the obtained glyceride was 64.2% caprylic acid and 35.8% palmitic acid. The partial glyceride contained in this glyceride is 1% or less, and the result of analysis with an ODS column (Wakosil-II 3C18, 4.6 x 150 mm, 2 bottles) using acetone / acetonitrile (1: 1, vol / vol) as an elution solvent. The purity of 8P8 was 93%.

  Using the obtained 8P8 (3.5 g) and 7 g of arachidonic acid (purity 90%) as raw materials, transesterification was performed at 30 ° C for 48 hours with the ribozyme IM60 used in the above reaction (the seventh time). Was extracted with hexane under alkaline conditions to obtain a glyceride fraction (4.8 g). Analysis of the fatty acid composition of glycerides revealed that caprylic acid, palmitic acid, γ-linolenic acid and arachidonic acid were 38.5, 23.1, 2.4 and 34.0 mol%, respectively. Results of fractionation of this glyceride by high performance liquid chromatography using an ODS column (SH-345-5, 20 x 500mm, manufactured by YMC) with acetone / acetonitrile (1: 1, vol / vol) as the elution solvent 8PA and APA were obtained in 0.72 and 0.44 g, respectively.

Example 2
Reaction was performed on a scale 100 times that described in Example 1 to prepare 8P8, which was used as a raw material.
Rhizopus delemar (Rhizopus delemar) of lipase (Tanabe Seiyaku Co., Ltd.; Talipase).. The J. Ferment Bioeng, 81, a ceramic carrier SM-10 (manufactured by NGK Insulators, Ltd.) in accordance with 299-303 (1996) Immobilized. After 10 g of immobilized enzyme (31,000 U / g) is packed in the column, 100 ml of water-saturated soybean oil: caprylic acid 1: 2 (wt / wt) mixture is flowed at 30 ° C. at a flow rate of 3 ml / hr to immobilize the enzyme. Activated.

  Next, after removing excess water by flowing 50 ml of soybean oil with no added water, transesterification was performed while flowing a mixture of 8P8 and arachidonic acid ethyl ester (purity 90%) under the same conditions. went. After 100 g of the reaction solution was distilled under high vacuum and the glyceride fraction was recovered as a residue, it was extracted with hexane under alkaline conditions according to Example 1. The solvent was removed by an evaporator to obtain 35.7 g of a hexane extract. The composition ratio of triglyceride and fatty acid ester contained in this hexane extract was analyzed by eartroscan to be 91: 9. As a result of analyzing the fatty acid composition, caprylic acid, palmitic acid, γ-linolenic acid, dihomo-γ-linolenic acid and arachidonic acid were 24.4, 34.5, 1.5, 2.6 and 37.0 mol%, respectively.

Example 3
In order to remove excess water contained in the immobilized Rhizomucor miehei lipase (manufactured by Novo Nordisk Co., Ltd .; ribozyme IM60) used in Example 1, 12 g of the immobilized enzyme, SUNTGA A reaction mixture consisting of 60 g of -25 (manufactured by Suntory Ltd.) was placed in a 100 ml vial with a screw cap and reacted at 30 ° C. for 48 hours with shaking (first time). Leave only the immobilized enzyme in the reactor, add 8P8 (12 g) prepared in Example 2 and 48 g of Mead acid ethyl ester (purity 90%) and replace with nitrogen, and then transesterify with shaking at 30 ° C for 72 hours. The reaction was carried out (a few times).

  After the reaction, the second and third reaction mixtures were combined, 100 g of which was distilled under high vacuum in the same manner as in Example 2 to recover the glyceride fraction as a residue. Subsequently, hexane was extracted under alkaline conditions according to Example 1, and then hexane was removed by an evaporator to obtain 24.1 g of a glyceride fraction. The composition ratio of triglyceride and fatty acid ester contained therein was analyzed by eartroscan to be 92: 8. When the high performance liquid chromatography was performed according to Example 1 and the fatty acid ester and each triglyceride component were quantified from the peak area of the differential refractometer, the MPM was 12.0%.

The fatty acid composition of this fraction was 31.2, 35.7, and 33.1 mol% for caprylic acid, palmitic acid, and mead acid, respectively.
In order to increase the transesterification rate, the obtained transesterified triglyceride was transesterified again with medeic acid ethyl ester. 12 g of transesterified triglyceride and 48 g of medic acid ethyl ester were added to the above immobilized enzyme, and the reaction was carried out at 30 ° C. with shaking for 72 hours (fourth time). After the reaction, 55 g of the reaction solution was distilled by the method described above to obtain 12.3 g of a glyceride fraction. The fatty acid composition of this fraction was 5.2, 38.6 and 56.1 mol% caprylic acid, palmitic acid and mead acid, respectively.

Example 4
In order to remove excess water contained in the immobilized Rhizomucor miehei lipase (manufactured by Novo Nordisk Co., Ltd .; ribozyme IM60) used in Example 1, 2 g of the immobilized enzyme, SUNTGA The reaction mixture consisting of 10 g of -25 (manufactured by Suntory Ltd.) was placed in a 20 ml vial with a screw cap and reacted at 30 ° C. with shaking for 48 hours (first time). Only the immobilized enzyme is left in the reactor, 8P8 (12 g) prepared in Example 2 and 8 g of fatty acid mixture obtained by hydrolysis of SUNTGA-25 are added, and the mixture is sufficiently purged with nitrogen, and then shaken at 30 ° C. for 48 hours. The transesterification reaction was carried out (2 to 5 times). After the reaction, glycerides extracted with hexane from the second to fifth reaction mixture were combined and used as a substrate for another transesterification reaction.

  2 g of transesterified triglyceride and 10 g of a fatty acid mixture derived from SUNTGA-25 were added to the reactor containing the immobilized enzyme and reacted at 30 ° C. for 48 hours with shaking (6th and 7th times). The glyceride fraction was extracted from the 6th and 7th reaction mixture, and the reaction was carried out in the same manner as the substrate for repeated transesterification reactions (8th time). The fatty acid composition constituting the triglyceride obtained by repeating the transesterification reaction three times and the fatty acid compositions at the 1,3-position and 2-position of the triglyceride were analyzed by gas chromatography. The results are shown in Table 1.

Example 5 FIG.
Using the APA obtained in Example 1 as a standard substance, the proportion of APA in the total triglycerides in human breast milk was analyzed by high performance liquid chromatography. An evaporative light scattering detector (DDL31, manufactured by EUROSEP Instruments) was used as the detector, an ODS column (COSMOSIL, 4.6 x 250 mm, manufactured by Nacalai Tesque) was used, and acetone / acetonitrile (1: 1, 1: 1) was used as the eluent. A gradient from vol / vol) to 100% acetone was used. As a result, it was confirmed that APA was present in human breast milk at a ratio of 0.1 to 0.6% by weight based on the total triglycerides. From the content of arachidonic acid in human breast milk (about 0.5 to 1.0% by weight ratio in fats and oils in breast milk), it was considered that 10 to 50% of arachidonic acid in breast milk was present as APA.

Example 6
A human milk type triglyceride-containing formula was prepared by mixing 100 g of powdered milk with 0.3 g of the novel structured lipid (APA or 8PA) obtained in Example 1. The ratio of arachidonic acid to the total fatty acids in this formula was 0.8% when APA was mixed and 0.4% when 8PA was mixed.

Example 7
400 g of a new structured lipid prepared in a large amount by the same method as in Example 4 and refined oil and fat, 48 g of purified egg yolk lecithin, 20 g of oleic acid, 100 g of concentrated glycerin and 40 ml of 0.1N caustic soda were dispersed in a homogenizer. Add distilled water for injection to a total volume of 4 liters. This was emulsified with a high-pressure spray type emulsifier to prepare a lipid emulsion. After 200 ml of the lipid emulsion is dispensed into a plastic bag, it is autoclaved at 121 ° C. for 20 minutes to obtain a fat infusion.

Example 8 FIG.
The novel structured lipid obtained in Example 3 was prepared as an emulsion injection according to a conventional method. The content of newly structured lipid in the emulsion injection solution is 10% (W / V), 1.2% (W / V) egg yolk lecithin is added as an emulsifier, and it is further infiltrated with glycerin so as to be isotonic with blood. The pressure was adjusted.

Example 9
Newborn boars (body weight> 1 kg) were randomly divided into 4 groups with 6 animals per group (the litters were in a separate group). All groups are fed with formula milk, and arachidonic acid-containing triglycerides are not added to the formula (prepared milk group), and SUNTGA-25 (Suntory Co., Ltd.) is added to the formula as arachidonic acid-containing triglycerides. A group added at a concentration (SUN group), a group obtained by adding APA obtained by the method of Example 1 at a concentration of 0.4 g / L to the formula milk (APA group), and 8PA obtained by the method of Example 1 There were four groups (8PA group) added to the formula milk at a concentration of 0.82 g / L. The SUN group, the APA group, and the 8PA group were prepared so that the amount of arachidonic acid in the prepared milk was almost the same. Table 2 shows the composition of all fatty acids of SUNTGA-25 (abbreviated as SUN in the table), APA, and 8PA, and the fatty acid composition at the 2-position of triglyceride.

  On day 18 of administration, the animals were fasted for 10 to 12 hours, blood was collected, and the liver and lungs were removed (stored at -80 ° C until analysis). The fatty acid composition in plasma and liver was affected by fatty acids present in the formula, and the arachidonic acid content in each of the SUN, APA, and 8PA groups was higher compared to the formula group However, there was no significant difference between the SUN group, APA group, and 8PA group. This is thought to be because the tissue is directly affected by dietary fatty acids. Therefore, next, the fatty acid composition of phospholipids in the lung was analyzed. The results are shown in Table 3.

  There was a significant difference in the proportion of arachidonic acid in the phospholipid fatty acid composition. It can be expected that the proportion of arachidonic acid in the pulmonary phospholipids of the SUN group will be higher than in the formula milk group. However, despite the inclusion of the same amount of arachidonic acid, the proportion of arachidonic acid in the pulmonary phospholipids of the APA group and the 8PA group was significantly higher than that of the SUN group. This result is considered to be due to the positional characteristics of the triglyceride of the structured lipid of the present invention.

Claims (7)

The following general formula (I):

(Where R ¹ and R ² are 6,9,12-octadecatrienoic acid (γ-linolenic acid) 18: 3, ω6, 8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid) ) 20: 3, ω6,5,8,11,14-eicosatetraenoic acid (arachidonic acid) 20: 4, ω6, 7,10,13,16-docosatetraenoic acid 22: 4, ω6, 4, 7,10,13,16-docosapentaenoic acid 22: 5, ω6,6,9-octadecadienoic acid 18: 2, ω9,8,11-eicosadienoic acid 20: 2, ω9, 5,8,11- Eicosatrienoic acid (medeic acid) 20: 3, ω9, 6,9,12,15-octadecatetraenoic acid (stearidonic acid) 18: 4, ω3, 11,14,17-eicosatrienoic acid (dihomo- α-linolenic acid) 20: 3, ω3, 8,11,14,17-eicosatetraenoic acid 20: 4, ω3, and 7,10,13,16,19-docosapentaenoic acid 22: 5, ω3 Represents an acyl group of an unsaturated fatty acid selected from the group consisting of: even if this acyl group is oxidized by hydroxylation, epoxidation or hydroxyepoxidation Well, and n represents an integer from 14 to 16)
And at least one of R ^{1 and} R ² is a ω6, ω9, or ω3 unsaturated fatty acid. One of the following triglycerides:
1,3-diarachidonyl-2-palmitoyl triglyceride,
1-arachidonyl-3-docosahexanoyl-2-palmitoyl triglyceride,
1- (dihomo-γ-linolenoyl) -3-docosahexanoyl-2-palmitoyl triglyceride,
1-arachidonyl-3- (dihomo-γ-linolenoyl) -2-palmitoyl triglyceride,
1,3-bis (dihomo-γ-linolenoyl) -2-palmitoyl triglyceride,
1-arachidonyl-3- (5,8,11-eicosatrienoyl) -2-palmitoyl triglyceride or 1-docosahexanoyl-3- (5,8,11-eicosatrienoyl) -2-palmitoyl triglyceride. The food composition formed by mix | blending the triglyceride of Claim 1 or 2 according to special nutrition demand. Said food composition, functional food, dietary supplements, premature infant modified milk, food composition according to claim 3, characterized in that the infant formula, infant food, maternal food or geriatric foods object. The animal feed formed by mix | blending the triglyceride of Claim 1 or 2 . A therapeutic nutritional product containing at least one triglyceride according to claim 1 or 2 and optionally mixed with a neutral carrier suitable for oral, enteral or parenteral administration. The analytical standard reagent which consists of a triglyceride of Claim 1 or 2 .