JPH0529433B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a method for producing a long-chain highly unsaturated fatty acid alcohol ester. In the present invention, long-chain highly unsaturated fatty acids mean fatty acids having 20 or more carbon atoms and 3 or more double bonds per molecule, and hereinafter referred to as PUFA.
It is abbreviated as [Prior Art] PUFA has recently attracted attention for its physiological activity and pharmacological effects on humans, and active studies have been conducted on the use of PUFA. [Problems to be solved by the invention] However, since PUFA has a high boiling point, high vacuum and high temperatures are required to recover it, which increases energy costs, and because it is exposed to high temperatures, distillation residues due to polymerization etc. It has been pointed out that there is a problem in that the yield increases, the yield decreases, and double bond isomerization occurs, producing a large amount of undesirable isomers. In view of the above-mentioned problems, an object of the present invention is to provide a process by which alcohol esters of PUFA can be effectively obtained. [Means for solving the problem] The method for producing PUFA alcohol ester of the present invention involves hydrolyzing fats and oils containing PUFA as a constituent fatty acid with lipase, and adding the fatty acid obtained from the hydrolyzate to threads. It is characterized by being esterified with a monohydric or polyhydric alcohol or transesterified with an alcohol ester using lipase in the presence of water. [Function] Examples of PUFA-containing oils and fats used in the present invention include fish oil, whale oil, krill oil, marine chlorella oil, etc., but mixed oils, conjugated isomerized oils, partially hydrogenated oils, etc. containing these oils and fats are also suitable. Can be used. In the method of the present invention, when hydrolyzing these fats and oils, it is preferable to use a lipase having triglyceride regioselectivity and a lipase not having triglyceride regioselectivity in combination for the purpose of increasing the hydrolysis rate. In the present invention, the lipases used as lipolytic enzymes with triglyceride regioselectivity include lipases obtained from microbial sources such as Aspergillus and Rhizopus, and animal sources such as Pancreatin. , Aspergillus niger, Phizopus delemar and Pancreatin are preferred. In addition, oil-degrading enzymes without triglyceride regioselectivity include Penicillium and Geotricum, both of microbial origin.
In particular, lipases obtained from Geotrichum candidum and Candida cylindracea are preferably used. The hydrolysis conditions may be carried out according to known methods, but the amount of the lipolytic enzyme used is expressed as U (unit), which represents the fat hydrolyzing activity. for
60 to 1000 U, and 200 to 3000 U of a lipolytic enzyme without regioselectivity, preferably in a ratio of 1:1 to 30. More preferably, the former enzyme is 100 to 500 U, the latter enzyme is 300 to 1500 U, and the ratio is 1:3 to 15.
It is. The amount of water added is preferably 5 to 500% (by weight, same hereinafter), more preferably 10 to 200%, based on the fat or oil. The pH of water is preferably in the range of 4.5 to 8.5, and it is more effective to use a buffer to adjust the pH, with a particularly desirable pH range of 5.5 to 8.0. Furthermore, for a more effective reaction,
Emulsifiers such as polyvinyl alcohol and fatty acid esters can also be used, and addition of bile salts is also effective in increasing hydrolysis activity. The hydrolysis reaction may be carried out in the atmosphere, but if it contains a large amount of long-chain highly unsaturated acids, such as fish oil or whale oil, it may be carried out under an inert gas atmosphere, such as nitrogen gas or carbon dioxide atmosphere. Not only can this prevent deterioration of fatty acids, but also the deactivation of enzymes. Also, antioxidants, such as tocopherols,
TBHQ, BHA, and BHT may be used together. The hydrolysis reaction is preferably carried out at 20-60°C. Below 20°C, the reaction is slow, and above 60°C, the enzyme is inactivated. It is more preferable to carry out the reaction at a temperature of 30 to 45°C. Although it is preferable to stir the reaction, the reaction can also be carried out in an emulsified state and allowed to stand still. Further, the reaction may be a single-stage reaction, but may also be a multi-stage reaction in order to proceed more efficiently and quickly. An immobilized enzyme column can also be used for continuous reaction. When fats and oils containing long-chain unsaturated fatty acids having 20 or more carbon atoms are hydrolyzed as described above, the decomposition rate usually reaches 70 to 99%. In addition, in the hydrolysis reaction of the present invention, a monohydric alcohol and/or an inert solvent can be added in order to improve the hydrolysis rate. The monohydric alcohol mentioned above is an aliphatic alcohol having 1 to 28 carbon atoms, such as methanol, ethanol, propanol, butanol, hexanol,
Examples include octanol, decanol, dodecanol, hexadecanol, octadecanol, tetracosanol, hexacosanol, etc., and particularly preferred are butanol, octanol, decanol, dodecanol, hexadecanol, octadecanol, etc. In addition, the amount added is 0.1 to 20 per mole of oil or fat.
It is a mole. As the inert solvent, hexane, petroleum ether, etc. are used, and the amount used is at most 10 times, preferably at most 3 times, the amount of oil and fat. The fatty acid obtained by hydrolysis can be transferred as it is to the next esterification or transesterification step, but it is more preferable to perform an operation to concentrate the PUFA. Concentration of such PUFA can be performed using known chromatography methods, urea addition methods, fractional crystallization methods, molecular distillation methods,
This can be carried out by a liquid-liquid distribution method, a double bond addition method, or the like. Among these, in the fractional crystallization method, a solvent such as acetone, hexane, petroleum ether, etc. is used, and the ratio of lipid to solvent is preferably about 1:1 to 1:10, and the temperature is preferably 10 to -60°C. In addition, in the urea addition method, the methanol:urea:fatty acid ratio is 5:3:1 to
Preferably, the ratio is 10:5:1, and in the molecular distillation method, it is preferably carried out at a temperature of 100 to 260°C and a temperature and pressure of several mmTorr. Such concentration results in a higher concentration of PUFA than the raw material, but EPA (eicosapentaenoic acid) accounts for 20% or more (preferably 25% or more) and DHA of all fatty acids.
(Docosahexaenoic acid) It is preferable to concentrate to 10% or more. Next, the monohydric or polyhydric alcohol used in the present invention is preferably a monohydric or polyhydric alcohol having 1 to 28 carbon atoms, such as methanol, ethanol, propanol, butanol, propylene glycol, ethylene glycol, glycerin, pentaerythritol, etc. can be mentioned. Further, as the alcohol ester, any fatty acid ester of the above-mentioned monohydric or polyhydric alcohol can be used. Although esterification and transesterification can be carried out using acid or alkali catalysts, it is highly preferable to use lipase for the purposes of the present invention. As the lipase used for esterification and transesterification, lipases of the genus Rhizopus, Aspergillus, Mucor, Candida, Geotrigram, etc. can be used alone or in combination. The amount of lipase used is 50 to 1 g of substrate (fatty acid + alcohol + alcohol ester).
3000U, preferably 100-1000U. Esterification and transesterification reactions are preferably carried out using water and an organic solvent that does not inactivate enzymes, and in an inert gas atmosphere such as N ₂ or CO ₂ to prevent oxidation of fatty acids. Water is 0.1-10% relative to the substrate, organic solvents such as hexane and petroleum ether are relative to the substrate.
It is recommended to use 0.3 to 10 times, preferably 0.5 to 5 times. The amount of alcohol or alcohol ester used may be in the range of 0.1 to 20 moles per mole of fatty acid, and a dehydrating agent or the like may be present in the esterification or transesterification reaction system to allow the reaction to proceed while dehydrating. is preferred. The reaction is preferably continued at a reaction temperature of 20 to 60°C for 5 to 80 hours, usually 5 to 40 hours. The enzyme used in the present invention can be immobilized or adsorbed on a suitable inert support for convenience such as reuse or recovery. Such inert supports include celite, alumina, chitosan, and the like. [Effect of the invention] As described above, according to the method of the present invention, PUFA
alcohol ester can be obtained efficiently. [Examples] The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples. Example 1 A mixed lipase of 450,000 U of Candida lipase and 450,000 U of Rhizopus lipase was added to 500 g of fish oil (iodine value 182.1) together with 500 ml of distilled water, and hydrolyzed to produce a hydrolyzate with a decomposition rate of 93%. Obtained. The obtained decomposition product is centrifuged to separate the oil layer, and then concentrated by fractional distillation, solvent fractionation, and urea addition.
Fatty acids of 45% EPA and 20% DHA were obtained. 50 g of this fatty acid and 5 g of glycerin were added to 100 g of hexane, and a mixed lipase of 10,000 U of Candeida layer lipase and 10,000 U of Rhizopus lipase was added together with 2 g of water, and an esterification reaction was carried out for 24 hours while dehydrating. . The composition of the obtained reaction product was analyzed using a thin layer chromatography FID (synchrograph) analyzer, and the results showed that it contained 30% free fatty acids and 6 monoglycerides.
%, 40% diglyceride, and 24% triglyceride. Example 2 50 g of concentrated long-chain unsaturated fatty acids obtained in Example 1 and 50 g of fish oil (IV = 181.2, EPA 14.2, DHA 11.1) were added to 100 g of hexane, and 30,000 U of Rhizopus lipase was added to this. It was added together with distilled water, and the transesterification reaction was carried out for 24 hours while dehydrating. After the reaction was completed, triglycerides were fractionated using column chromatography, methyl esterification was performed, and the fatty acid composition was analyzed using gas chromatography.
It was DHA (15.1%). The fatty acid composition is shown in Table 1.
It looked like this. Example 3 50 g of the concentrated long chain unsaturated fatty acid obtained in Example 4, 50 g of fish oil (IV = 182.1, EPA 14.2, DHA 11.1) and 10 g of butyl alcohol were added to 100 g of hexane, and 30,000 U of Aspergillus lipase was added to this. 5g of
After adding the mixture with water, the transesterification reaction was carried out for 10 hours while dehydrating. After the reaction was completed, the triglycerides were fractionated using column chromatography, and the fatty acid composition was analyzed using gas chromatography, which revealed that EPA was 29.2%.
DHA was 16.8%. The fatty acid composition is shown in Table-1.

[Table] ω (omega) is a number that indicates where the first double bond is located, counting from the terminal methyl group. Therefore, for example, C20:5ω3 indicates a fatty acid with 20 carbon atoms and five double bonds, of which the first double bond is the third fatty acid counting from the terminal methyl group.

Claims (1)

[Claims] 1 Oils and fats containing long-chain highly unsaturated fatty acids are hydrolyzed with lipase, and the resulting fatty acids are esterified with monohydric or polyhydric alcohol using lipase in the presence of water added to the system, or alcohol A method for producing a long-chain highly unsaturated fatty acid alcohol ester, which comprises transesterification with an ester.