JPH11263750A - Long-chain unsaturated fatty acid menthol ester and its production by enzyme method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject ester under mild conditions readily and efficiently by reacting a menthol with a specific long-chain unsaturated fatty acid (alkyl ester) in the presence of a lipase. SOLUTION: (A) A menthol (1-menthol) is reacted with (B) a >=16C long-chain unsaturated fatty acid (alkyl ester) (e.g. γ-linolenic acid) in the molar ratio of the component A to the component B of preferably 1:0.5 to 1:10, more preferably 1:1 to about 1:10 in the presence of (C) preferably about 1-50,000 units of a lipase based on 1 g of a reaction mixture solution in 0-500 wt.% of water at 15-60 deg.C for 30 minutes to 144 hours to give the objective long-chain unsaturated fatty acid menthol ester of the formula (R1 is the acyl group part of γ- linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid). A lipase derived from a bacterium of the genus Pseudomonas or a fungus of the genus Candida, etc., is preferably used as the component C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an ester of menthol with a long-chain unsaturated fatty acid by an enzymatic method. Specifically, the present invention relates to a method for producing a long-chain unsaturated fatty acid menthol ester by lipase and a long-chain unsaturated fatty acid menthol ester obtained by the method.

[0002]

2. Description of the Related Art In recent years, attention has been paid to the physiologically active action of highly unsaturated fatty acids. In particular, eicosapentaenoic acid (hereinafter referred to as "EPA") and docosahexaenoic acid (hereinafter referred to as "DHA") have a preventive effect on adult diseases such as arteriosclerosis and thrombosis, an anticancer effect, and an enhancement of learning ability. It is known to have many bioactive effects such as action. Further, γ-linolenic acid (hereinafter, referred to as “GLA”) has a healing action and an immunostimulatory action against atopic dermatitis, and arachidonic acid (hereinafter, referred to as “AA”) is prostaglandin, thromboxane. It is also an important fatty acid as a precursor of local hormones such as leukotriene, and amides of AA and monoethanolamine and 2-AA monoglycerides are known to have neuroactive effects. Various studies have been made on how to use it.

The present inventors have proposed an ester form with menthol as one measure for effectively exerting the physiologically active action of a polyunsaturated fatty acid, and have been studying to establish a synthesis method thereof. . In particular, l-menthol is a substance that gives a refreshing feeling as a mint component obtained from mint oil or other mint oils, and is used as a soft drink, confectionery, fragrance, tobacco fragrance, painkiller, antipruritic, antiseptic, etc. It is a highly safe substance widely used.

Heretofore, a chemical synthesis method using an inorganic catalyst has been employed as a method for synthesizing a useful ester of a long-chain unsaturated fatty acid.
There were problems such as complicated purification. Further, in order to improve this point, for example, a method using an enzyme for producing a sterol ester or a wax ester has been proposed (JP-A-60-45128, JP-A-62-262997).
However, there is no production example of ester synthesis of a highly unsaturated fatty acid or its alkyl ester and menthol either chemically or enzymatically.

[0005]

SUMMARY OF THE INVENTION The present inventors have conducted repeated studies on the synthesis of menthol esters of highly unsaturated fatty acids such as GLA, AA, EPA, and DHA, which are said to have poor reactivity, and menthol, The inventors have found that menthol ester can be efficiently synthesized when reacting with a lipase enzyme as a catalyst, and have completed the present invention.

[0006] That is, an object of the present invention is to provide a method for producing a long-chain unsaturated fatty acid menthol ester of a long-chain unsaturated fatty acid having 16 or more carbon atoms or an alkyl ester thereof and menthol. Another object of the present invention is to provide a highly unsaturated fatty acid menthol ester obtainable by the above-mentioned method or the like.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides an enzyme reaction using a lipase which can be easily and easily performed under extremely mild conditions at ordinary temperature and pressure without the need for complicated operations or special reaction equipment. It is based on the finding that the desired highly unsaturated fatty acid menthol ester can be efficiently produced, and that the menthol ester can be easily separated and purified from the reaction system. Therefore, the present invention consists of the following.

(1) A method comprising reacting menthol with a long-chain unsaturated fatty acid having 16 or more carbon atoms or an alkyl ester thereof in the presence of lipase, and recovering the produced long-chain unsaturated fatty acid menthol ester. A method for producing a chain unsaturated fatty acid menthol ester. (2) The method according to (1), wherein the menthol is l-menthol.

(3) In the above (1) or (2),
A method wherein the long-chain unsaturated fatty acid is selected from the group consisting of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. (4) In the above (1) or (2), the long-chain unsaturated fatty acid alkyl ester is selected from the group consisting of γ-linolenic acid ester, arachidonic acid ester, eicosapentaenoic acid ester, and docosahexaenoic acid ester. how to.

(5) The method according to (1) to (4), wherein the lipase is a lipase derived from Pseudomonas. (6) The method according to (1) to (4), wherein the lipase is a lipase derived from the genus Candida. (7) General formula (I):

[0011]

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Wherein R ₁ is an acyl group moiety of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid, and a long-chain unsaturated fatty acid menthol ester represented by the formula: (8) General formula (II):

[0013]

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A long-chain unsaturated fatty acid menthol ester represented by the formula: wherein R ₁ is the acyl moiety of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The lipase used in the present invention is
The origin is not particularly limited, and any of various microorganisms, animals, and plants may be used as long as it has the enzyme reactivity in the present invention. As lipase of microbial origin,
For example, those derived from microorganisms belonging to the genus Staphylococcus, Pseudomonas, Candida, Rhizopus, Chromobacterium, etc., especially lipase derived from Pseudomonas sp. And lipases derived therefrom. As lipases of animal origin,
Examples thereof include pancreatic lipase, digestive tract lipase and the like, and examples of lipase of plant origin include lipase of rice bran, rapeseed seed, palm pulp, castor seed and the like. As these enzymes, commercially available ones may be used as they are, but it is not particularly necessary to use a purified commercial product, and the microorganism itself having the ability to produce the enzyme of interest, its culture solution, and the culture solution may be used. A crude enzyme solution or a composition containing an enzyme obtained by the treatment can also be used. The use form of the lipase may be as it is, but if the lipase is immobilized on a fixing agent (for example, an acrylic resin, an ion exchange resin, a ceramics carrier, celite, etc.) and used, the lipase is stabilized,
Enzyme activity can be maintained with good reproducibility even after repeated reactions or long-term continuous use. However, the use form of the lipase is not particularly limited. The amount of the lipase to be used varies depending on its activity and the composition of the reaction mixture, and may be appropriately determined, but is not particularly limited, and is usually about 1 to about 50,000 units, preferably about 1 to about 50,000 units per gram of the reaction mixture. Ten to about 10,000 units are used. Here, one unit (U) is a unit of the amount of an enzyme that releases 1 μmol of fatty acid per minute from olive oil.

As the menthol used in the present invention, both optically active and optically inactive can be used.
-Or l-menthol, a racemate, and a diastereomer are included, and a 1R, 3R, 4S-form of 1-menthol is preferable. Menthol is a monocyclic monoterpene with 8 optically active forms and 4 inactive forms.

The long-chain unsaturated fatty acid having 16 or more carbon atoms used in the present invention is a natural or non-natural fatty acid having at least one double bond. Its derivatives (eg, substituted derivatives, addition derivatives, etc.) can be preferably used. As the polyunsaturated fatty acids, in the ω-3 series, fish oils, for example, those extracted from the head of tuna or bonito, or sardines,
Mackerel, Pacific saury or horse mackerel extracted from whole fish,
Alternatively, squid or cod liver is used as a raw material, which is purified to a high concentration as a free fatty acid.Examples of the ω-6 system include oils and fats or eggs produced by Mortierella spp. Extracted from egg yolk oil,
In the case of botanical origin, borage oil and evening primrose oil are used as raw materials.
The fatty acid in a free state may be purified to a high concentration. The ω-3 polyunsaturated fatty acids in the polyunsaturated fatty acids are fatty acids having at least 4 to 6 double bonds, and a double bond is formed from the third position from the methyl group terminal of the chain structure. Beginning fatty acids, for example EP
A, DHA and the like. On the other hand, ω-6 highly unsaturated fatty acids are fatty acids having at least 3 to 5 double bonds, and the double bond starts from the sixth position from the methyl group end of the chain structure. Fatty acid, for example, GL
A, dihomo-γ-linolenic acid, AA, docosapentaenoic acid and the like. As used herein, a "polyunsaturated fatty acid" refers to at least three fatty acids on a straight or branched alkyl chain.
It means a fatty acid having 18 or more carbon atoms having two double bonds.

The oil-and-fat raw material containing a polyunsaturated fatty acid (hereinafter sometimes referred to as "PUFA") used in the present invention includes marine animal oils such as tuna, skipjack, sardine, mackerel, saury, horse mackerel, squid or Fish oil obtained from cod is preferred because it contains a lot of EPA and DHA. In addition, the oils and fats produced by Mortierella spp.
It is preferable as an oil or fat raw material containing a lot of AA, and borage or evening primrose seed oil is preferable because it contains a lot of GLA. PUFA-containing fats and oils are extracted by methods such as boiling extraction, solvent extraction, and press extraction. In order to hydrolyze the extracted PUFA-containing fats and oils as free fatty acids, from the problem of oxidative stability of PUFA,
Among the chemical hydrolysis methods, a saponification decomposition method having relatively mild reaction conditions must be selected. Also, as a method for suppressing the deterioration of fats and oils, free fatty acids can be obtained by using a method using lipase. The obtained fatty acid contains the desired highly unsaturated fatty acid by a combination of a method using urea addition, a distillation method such as molecular distillation and precision distillation, a method using chromatography, and a method using the fatty acid specificity of lipase. It can be roughly concentrated to an amount.

In the present invention, an alkyl ester thereof may be used in place of the long-chain unsaturated fatty acid. The alkyl group on the alcohol side constituting this ester is not particularly limited as long as the reaction of the present invention can be performed, but usually has 1 to 1 carbon atoms.
24, preferably 1 to 4, and preferably a linear one.

According to the present invention, long-chain unsaturated fatty acids having 16 or more carbon atoms, in particular, highly unsaturated fatty acids such as GLA, AA, EPA, DHA, and the like, which can be expected to have physiological effects and whose synthesis methods have not been reported so far, have been reported. The ester and menthol can be reacted in the presence of lipase to produce, for example, a fatty acid ester represented by Formula (I) or Formula (II). The reaction proceeds by contacting menthol with a fatty acid as a substrate in the presence of lipase. In the conventional ester synthesis reaction, in order to increase the frequency of contact between the enzyme and the substrate, a reaction system in which a hydrocarbon solvent such as n-hexane, n-heptane, or n-octane is mixed is well known. Since a large amount of solvent corresponding to 10 to 20 times the volume (volume) of the solvent is used, the tank capacity in actual production becomes enormous, and the amount of solvent used is greatly reduced. The disadvantage of high cost has been pointed out. On the other hand, the method of the present invention can easily and efficiently produce the desired highly unsaturated fatty acid menthol under ordinary conditions and under ordinary temperature and pressure without requiring complicated operations and special reaction equipment. In addition to the advantage that the ester can be produced and that the menthol ester can be easily separated and purified from the reaction system, it has the advantage that the reaction can be performed even in a reaction system in which no hydrocarbon solvent is present. ing. That is, the reaction can be carried out in a reaction system composed of a long-chain unsaturated fatty acid or its alkyl ester, menthol, water, an enzyme and the like.

The raw material molar ratio of menthol to fatty acid or its alkyl ester in the reaction mixture for carrying out the ester synthesis reaction is usually 1: 0.5 to 1:10, preferably 1: 1 to 1:10.
However, other molar ratios may be used depending on the type of the raw material and the degree of reactivity. As other reaction conditions, the reaction conditions of lipase which are usually performed can be used. That is, using the lipase, 0 to 500% by weight
The ester synthesis reaction can be carried out by allowing the mixture to stand at a temperature of 15 to 60 ° C. for 30 minutes to 144 hours, shaking or stirring under a water content of 30 ° C. If the temperature is lower than 15 ° C., the reaction rate of the lipase becomes slow.
However, in order to sufficiently express the activity of the lipase used in the present invention, the water content should be 5 to 400% by weight, 25 to 5% by weight.
0 ° C temperature condition, stirring and reacting,
In order to prevent the oxidative deterioration of the polyunsaturated fatty acid, it is preferable that the reaction is performed in a relatively short time of about 1 to 96 hours under a nitrogen stream. Although the esterification reaction proceeds even when it is allowed to stand, the reaction efficiency is greatly increased by stirring. In the present invention, the synthesis rate of fatty acid menthol ester by lipase is expressed by the following formula: ester synthesis rate (%) = [(free fatty acid amount in reaction mixture at start of reaction−amount of free fatty acid in reaction mixture at end of reaction) {(Amount of free fatty acid in reaction mixture at start of reaction)] × 100. As a method for fractionating the obtained menthol ester, a hexane-alkaline partitioning method which is usually performed (that is, a method in which an unreacted fatty acid is converted to a fatty acid salt by an alkali reaction and removed to the aqueous layer side), a solvent liquid- Examples thereof include a method by liquid distribution, a method by low-temperature crystallization fractionation, a method by molecular distillation or precision distillation, and a method by chromatography.
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

[0022]

EXAMPLES Example 1 Various long-chain unsaturated fatty acids and l-ment
Long-chain lipase from Pseudomonas sp.
Manufacture of saturated fatty acid menthol ester Various long-chain unsaturated fatty acids (purity 95% or more) and l-menthol
(Tokyo Chemical Industry; purity: 95%) 24 g of reaction mixture
(Molar ratio 3: 1), 6 g of distilled water in which 24,000 U (800 U per 1 g of reaction mixture) of lipase produced by Pseudomonas SP (KWI-56, KWI-56) was added, and added under a nitrogen stream. The esterification reaction was performed at 35 ° C. for 50 hours with stirring. A 0.5N sodium hydroxide solution was added to the reaction mixture to completely convert unreacted fatty acids into fatty acid salts. To this was added n-hexane, the resulting menthol ester was extracted with n-hexane, and the solvent was distilled off using a rotary evaporator to obtain a menthol ester. The ester synthesis rate was calculated from the acid value consumed by the reaction, and the results are shown in Table 1.

[0023]

[Table 1]

From Table 1, it can be seen that each of the polyunsaturated fatty acids was 6
Ester synthesis rate exceeding 0%, especially α-
Linolenic acid and docosahexaenoic acid had a high ester synthesis rate, and showed the same value as oleic acid and linoleic acid.

Example 2 Various long-chain unsaturated fatty acids and l-men
Long chain lipase from Candida spp.
Manufacture of saturated fatty acid menthol ester Various long-chain unsaturated fatty acids (purity 95% or more) and l-menthol
(Tokyo Chemical Industry; purity: 95%) 24 g of reaction mixture
(Molar ratio 3: 1), 12,000 U of lipase (Lipase OF, manufactured by Meito Sangyo Co., Ltd.) produced by Candida Sylindrache
6 g of distilled water in which (400 U per 1 g of the reaction mixture) was dissolved was added, and the esterification reaction was carried out at 40 ° C. for 24 hours while stirring under a nitrogen stream. A 0.5N sodium hydroxide solution was added to the reaction mixture to completely convert unreacted fatty acids into fatty acid salts. N-Hexane was added to this and the resulting menthol ester was converted to n-hexane.
Extraction with hexane and distilling off the solvent with a rotary evaporator gave menthol ester.
The ester synthesis rate was calculated from the acid value consumed by the reaction, and the results are shown in Table 2.

[0026]

[Table 2]

From Table 2, among the highly unsaturated fatty acids, α-linolenic acid has a high ester synthesizing rate, and shows the same value as oleic acid and linoleic acid. Example 3 Various long-chain unsaturated fatty acid ethyls and l-menthol
Chain unsaturation by lipase from Pseudomonas spp.
Production of fatty acid menthol ester Pseudomonas was added to 24 g (molar ratio 3: 1) of a reaction mixture of various long-chain unsaturated fatty acid ethyl (purity 95% or more) and l-menthol (Tokyo Kasei Kogyo Co., Ltd .; purity 95%).・ 6 g of distilled water containing 24,000 U (800 U / g of reaction mixture) of lipase produced by SP (Kurita Kogyo Co., Ltd., KWI-56) was added, and the esterification reaction was carried out at 35 ° C. for 50 hours while stirring under a nitrogen stream. Was done. A 0.5N sodium hydroxide solution was added to the reaction mixture to completely convert unreacted fatty acids into fatty acid salts. To this was added n-hexane, the mixture other than the fatty acid was extracted with n-hexane, and the solvent was distilled off with a rotary evaporator, whereby the produced fatty acid menthol ester, unreacted fatty acid ethyl and menthol mixed fractions. Obtained. From this fraction, Iatroscan (manufactured by Jatron Co., Ltd.,
Each peak area was determined using a model MK-5 and chromarod S-III), the molar ratio was calculated based on this value, and the menthol ester synthesis rate was determined from the following equation. Synthesis rate (%) = [(amount of fatty acid menthol ester) / (amount of fatty acid ethyl ester + amount of menthol)] × 100 The results are shown in Table 3.

[0028]

[Table 3]

From Table 3, it can be seen that the conversion of the long-chain unsaturated fatty acid menthol ester by the transesterification reaction was lower than that of the corresponding esterification reaction of Example 1; The synthesis rate is shown.

[0030]

According to the present invention, a method for producing a long-chain unsaturated fatty acid menthol ester is provided. In particular, since highly unsaturated fatty acid menthol ester can be efficiently produced at a high synthesis rate, it can be effectively used for cosmetic bases, pharmaceuticals, biochemical reagents, and the like.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:72) (72) Inventor Kazuki Maruyama 1-12-17 Ninomiya, Tsukuba, Ibaraki Prefecture Oceanic Residential 201 (72) Inventor Moriyama Shigeru Shiba 3-20-11 Maihama, Urayasu-shi, Chiba (72) Inventor Takashi Baba 19-5, Higashi-Manabe-cho, Tsuchiura-shi, Ibaraki Prefecture (72) Inventor Tomoaki Oguri 2-9-1, Koboku, Tsuchiura-shi, Ibaraki 607

Claims (8)

[Claims] 1. A method comprising reacting menthol with a long-chain unsaturated fatty acid having 16 or more carbon atoms or an alkyl ester thereof in the presence of a lipase, and recovering the long-chain unsaturated fatty acid menthol ester produced. A method for producing an unsaturated fatty acid menthol ester. 2. The method according to claim 1, wherein the menthol is l-menthol. 3. The method according to claim 1, wherein the long-chain unsaturated fatty acid is selected from the group consisting of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. 4. The long-chain unsaturated fatty acid alkyl ester is γ
3. The method according to claim 1, wherein the method is selected from the group consisting of linolenic acid esters, arachidonic acid esters, eicosapentaenoic acid esters, and docosahexaenoic acid esters. 5. The method according to claim 1, wherein the lipase is a lipase derived from Pseudomonas. 6. The method according to claim 1, wherein the lipase is a lipase derived from the genus Candida. 7. A compound of the general formula (I): (Wherein R ₁ is an acyl group moiety of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid). 8. A compound of the general formula (II): (Wherein R ₁ is an acyl group moiety of γ-linolenic acid, arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid).