JPS6291188A - Production of highly unsaturated fatty acid glyceride - Google Patents

Abstract

PURPOSE:To produce the titled glyceride having high quality with an extremely advantageous process, by reacting a highly unsaturated fatty acid or its lower alcohol ester with glycerol in the presence of heat-resistant lipase. CONSTITUTION:A long-chain highly unsaturated fatty acid having a carbon number of >=20 and an unsaturated bond number of >=3 (e.g. eicosatrienoic acid) or its lower alcohol ester and glycerol are put into a vessel, which is optionally added with a solvent and then added with water and a buffering solution. A heat-resistant lipase capable of keeping the activity at >=50 deg.C and originated from Pseudomonas genus, Penicillium genus, etc., or immobilized lipase is added to the reaction system and the reaction components are made to react with each other by raising the temperature to about 50-70 deg.C understirring. After the completion of reaction, the enzyme is inactivated and removed and the unreacted component is removed by fractionation to obtain the objective glyceride. The production of the objective compound may be carried out by reacting in a reaction column.

Description

DETAILED DESCRIPTION OF THE INVENTION FIELD OF INDUSTRIAL APPLICATION OF FAT The present invention relates to a method for producing highly unsaturated fatty acid glycerides using a thermostable lipase.

(b) Conventional technology Highly unsaturated fatty acids (hereinafter referred to as PUFA) are found in marine organisms such as fish and shellfish, algae, organs of higher animals, plants, and microorganisms, and are essential fatty acids for the species. In recent years, it has been attracting attention as it has been discovered to be effective in preventing arteriosclerosis in animals and humans, as well as having physiologically active effects such as thrombolytic and blood pressure lowering effects. R.E.P.
From this perspective, UFA glycerides are used in foods, medicines, and cosmetics.

It is useful in a wide range of fields as a raw material or base for agricultural chemicals, feedstuffs, diagnostic and analytical reagents, etc.

Up to now, attempts have been made to synthesize the glyceride of PUF A chemically, to convert it enzymatically, or to prepare it using a solvent.

That is, chemically, glycerin and the P[JFA are heated to 150 to 200''C in the presence of an acid or base catalyst to synthesize glyceride, but in this method, triglyceride of PUFA is the main product. However, due to the high temperature, the PUFA undergoes undesirable side reactions such as polymerization, isomerization, and oxidative discoloration, making it difficult to obtain high-quality target products.

In addition, several methods have been tried for enzymatic conversion, including lipase (Candida cylindrace
It has been described that the ester bond between PUFA and glycerin is hardly hydrolyzed even when using PUFA and glycerin, and therefore it can be seen that the reaction between PUFA and glycerin does not occur depending on such lipase. Furthermore, JP-A-59-14793 also describes that PUFA is difficult to react with conventional lipases.

As for a method using a solvent, JP-A-59-71396 discloses acetone, methyl ethyl ketone, and methanol.

A method of extracting fish oil with a 1-25 wt% water-containing solvent such as ethanol and concentrating and separating oils and fats containing PUFA has been disclosed, but the cost increase due to the use of such a solvent cannot be ignored, and it is also important among PUFAO. The eicosapen citric acid (Czo:s.omega.-3) content remains at about 30%.

As described above, several methods for producing PUFA glycerides have been attempted, but none of them have been satisfactory.

Problems to be Solved by the tC1 Invention Therefore, an object of the present invention is to polymerize PUFA glyceride using thermal history in a chemical synthesis method, as described above, in producing PUFA glyceride.

Isomerization 1 Changes such as coloration, i.e. instability mainly due to PUFA, low or non-reactivity of conventional lipases to PUFA in enzymatic conversion methods, low PUFA triglyceride content, production cost increase in solvent methods, etc. The object of the present invention is to solve various problems in the manufacturing process and to produce high quality PUFA glycerides in an extremely advantageous manner.

(Means for Solving Problem d1) As a result of intensive research, the present inventors discovered that the above object could be achieved by carrying out the reaction under specific conditions, and thus completed the present invention. That is, the present invention is a method for producing PUFA glyceride, which is characterized by reacting PUFA or its lower alcohol ester with glycerin using a heat-stable lipase.

The present invention will be explained in detail below. P[J used in the present invention
FA includes seafood, shrimp, squid, marine organisms such as chlorella seaweed, organs of higher animals such as cows and pigs, plant seeds,
It can be obtained from microorganisms such as mold, yeast, and bacteria, and generally refers to a long chain highly unsaturated fatty acid having 20 or more carbon atoms and 3 or more unsaturated bonds. Examples of these include eicosatrienoic acid (C2°, 3 ω-3
and ω-6), eicosatetraene M (C2°, 4
□ ω-3), arachidonic acid (C2014, ω-6),
Eicosapen citric acid (C2゜83.ω-3), docosatrienoic acid (Cz□,,ω-6), docosatetraenoic acid (C2□, 4.ω-6), docosahexaenoic acid (C2
□, 6. ω-3), tetracosatraenoic acid (C24:4
゜ω-6), etc., and the PUFA of the present invention contains these so-called ω-3 or ω-6 fatty acids having physiological activity as important components. In addition, in the present invention, these PUFAs can be used alone or in a mixed system, and can also be used as a mixed fatty acid containing these PUFAs.

Next, the lower alcohol of the lower alcohol ester of PUFA in the present invention refers to an alcohol having 6 or less carbon atoms,
Examples include methanol, ethanol, propatool, isopropanol, buta5ol, pentanol, hexanol, and the like. Esters of PUFA and these alcohols can be obtained by esterification by a conventional method, and can be used alone or as a mixture.

Conventional oil and fat hydrolases are generally used at 30 to 45°C and exhibit catalytic activity under mild reaction conditions near neutrality and normal pressure, but enzymes that act on PUFAs or their esters as described above have not been found. Not yet.

For this reason, an effective method for enzymatically producing PUFA glycerides has not been established. However, the inventors were able to make it possible to produce PUFA glycerides by applying thermostable lipases. The thermostable lipase may be one that can maintain its activity even at temperatures above 50°C, and such enzymes can be isolated and purified from living organisms or secretions of animals, plants, or microorganisms; The one is simple. An example of this is Pseudomonas spp.
Lipase from onasfluorescens, Penicillium cyclop
Lipase derived from alkaline earth metal microorganism strain No. 3783, Hu of Humicola sp.
lipase from micola lanuginose,
Lipase from Mucor genus l'lu (ormiehei, Rhizopus chinensi
Examples include lipase derived from S. Note that the present invention is not limited to these types of microorganisms,
Thermostable lipases from wild strains, mutant strains, auxotrophic strains, and drug-resistant strains belonging to these species may be used. Furthermore, the thermostable lipase can also be used as an immobilized product. For example, cellulose, dextran,
Adsorbs to enzyme immobilization carriers such as polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resin, porcelain, activated carbon, celite, alumina, photocrosslinkable or photocurable resin, alginate, carakeenan, etc.

Ionic, covalent or entrapped thermostable lipases can be used.

According to the method of the present invention, P [J l;' A or its lower alcohol ester and glycerin are reacted using a thermostable lipase to produce the PUFA glyceride as follows. That is, the reaction raw materials, PUFA or its lower alcohol ester, and glycerin are placed in a glass or stainless steel container, and if necessary, an inactive precipitate solvent such as n-hexane or n-hebutane is added, and then a binary starch is added. After adding water and a buffer solution as appropriate, and adding heat-stable lipase or its immobilized product, the pH is adjusted to pH 3 to 12, preferably pH 5 to I, while stirring or shaking and blowing an inert gas such as nitrogen gas.
The temperature is raised to 50°C or higher, preferably 50 to 70°C under O,
React for 5 to 120 hours. Here, although the enzyme used in the present invention can proceed at temperatures below 50°C, it takes a long time, and at temperatures above 70°C, even heat-stable lipases become less stable, and the enzyme may be deactivated. 50 ~
It is desirable to carry out the reaction at 70°C. The progress of the reaction can be monitored by TLC (thin layer chromatography), Ct, C (gas chromatography) or I (PLC (high speed? rf
This can be checked by measuring the decrease in the content of PUFA or its ester and the increase in the monoglyceride, diglyceride, and triglyceride content in the PUFA glyceride using t-isomer chromatography. After completion of the reaction,
The reaction product is heated to 80 to 90°C for a short time to inactivate the enzyme, the enzyme is removed by filtration or centrifugation, and unreacted products are removed using a fractionation method such as solvent fractionation or adsorption chromatography. A desired PUFA glyceride can be produced. In addition, in the present invention, it is also possible to pack the immobilized enzyme in a cylindrical container and pour the reaction solution from one side to cause the reaction to occur as a column-type glue actor, or as a dispersion-type glue actor in which the immobilized enzyme is suspended in the reaction solution. can also carry out the reaction.

(EL Examples Example 1 Marine chlorella, a type of green algae, is cultured in seawater, and the concentrated cultured cells are crushed, solvent extracted, and solvent fractionated to produce galactose and eicosaben citric acid (CZ).
A υ-moon lipid containing ω-3) as a main component was obtained. This was saponified and decomposed by a conventional method and extracted with a solvent to obtain PUFA. Fatty acid composition: C2°86. ω-3 (77%), C2°, 4. ω-6 (8%).

C18,2 (3%), Cls:+ (3%), Cl6
F. (5%).

75 g of this PUFA and 10 g of glycerin.

Transfer 100 mff of n-hexane to a 500 mff Mitsuro flask with a cooling prefix, add 0.3 M phosphate buffer (pl (7,
0) 0.8mff and thermostable lipase (Ilumic
olaIanug i nosa Yamaki, origin pharmaceutical (+@
0.1 g of Lipase CEJ, manufactured by Co., Ltd., trade name:
Stir at 60°C for 72 hours while blowing nitrogen gas (20
Orpm).

After the reaction was completed, the mixture was heated to 85"C for 5 minutes, the enzyme was removed by centrifugation, and unreacted PUFA was removed by silica gel column chromatography. The glyceride composition of the reaction product was determined by HPLC analysis. (86%), diglyceride (1%), and monoglyceride (3%).

Furthermore, as a result of analyzing the constituent fatty acids of the glyceride component by GLC, it was found that the composition was almost the same as that of the raw material PUFA.

Example 2 PUFA obtained in Example 1 was purified with ethanol using a conventional method.
Converted to FA ethyl ester. 50 g of the PUFA ethyl ester and 6.7 g of glycerin were placed in a 200 mA Erlenmeyer flask, and 0.5 M l-lith salt H1) street liquor (p
II 8.5) 0.5 ml and immobilized thermostable lipase (lipase derived from Mucor m1ehei immobilized on weakly basic ion exchange resin, manufactured by Novo Industries, Denmark, trade name rLipase3AJ) 0
．． 2 g was added thereto, and the mixture was shaken (200 rpm) at 65° C. for 90 hours in a nitrogen gas stream. After the reaction is complete, heat to 80°C for 5 minutes.
After heating for a minute, the immobilized substances were removed by filtration, and unreacted substances (PUFA) were removed by silica gel column chromatography. The composition of the product was nitriglyceride (25%), diglyceride (58%), and monoglyceride (17%), and its fatty acid composition was almost the same as that of the raw material.

Example 3 Immobilized thermostable lipase used in Example 2 (manufactured by Novo Industries, trade name “Lipage 3 A J”)
Filled with glass tube (2cm diameter x 3, Ocm length),
50 g of PUFA ethyl ester described in Example 2, 5 g of glycerin, 50 mA of n-heptane, 0.3 M! - 0.8 m of lithium-hydrochloric acid buffer (pH 7,5) and 0.05 g of polyvinyl alcohol (n = 1750) were treated in a separate container with a homomixer at 2000 rpm for 15 minutes to form a mixed solution. 10m1 of the lower part of the above column via the liquid feed pump! / hour flow rate,
A liquid circulation system was created to return the liquid flowing out from the top into the separate container mentioned above, and recycling was performed at 60°C for 80 hours.

When a portion of the reaction solution was extracted and its composition was analyzed by TLC, the main component was diglyceride.

Example 4 Fish oil derived from sardines was heated at 30°C using a non-heat-stable lipase (manufactured by Meito Sangyo Co., Ltd., [Lipase M Y J]) using a conventional method.
, and was allowed to react for 6 hours for hydrolysis. A saturated aqueous solution of calcium hydroxide is added to the reaction mixture, centrifuged to collect an oil layer, followed by saponification and decomposition using a conventional method and extraction with hexane to obtain PUF.
A concentrate of A was obtained. The main fatty acid composition according to GLC analysis is C2°86. n=3 (38%), C2□, 6. n=3
(25%), C2□81. n=3 (5%), C2°,
l (5%), C2□, I (3%).

C2°, 4. n=6 (2%), and others (22%). This puFA? 100 g of ficondensate, 10 g of glycerin, 0.5 m of water, thermostable lipase (Ps
derived from eudomonasf 1uorescens, manufactured by Amano Pharmaceutical 0@, trade name rLIpasePj) 0.15g
, Celite 012g was reacted in the same manner as in Example 1, and the composition of the reactant was determined. Triglyceride (80
%), diglycerides (13%), monoglycerides (7%)
), and their constituent fatty acids are the raw material PUFA? It was almost the same as the Q condensate.

(f) Effect of invention The effects of the present invention are as follows.

■ By using heat-stable lipase, it is now possible to react PUFAs or their esters, which were previously thought to be impossible to react with normal fat and oil hydrolases used at 30-45°C. It is now possible to produce UFA glyceride.

(2) Since this reaction is performed at a relatively low temperature of 50 to 70°C, PUFA, which has poor thermal stability, is polymerized.

High-quality PUFA glycerides can now be produced without adverse effects such as isomerization.

(2) PUFA glycerides obtained by conventional enzymatic methods had a limit in increasing the triglyceride content, but the method of the present invention can produce almost complete PUFA triglycerides.

■ Compared to natural fractionation or solvent fractionation methods, the reaction can be carried out at temperatures close to room temperature, and large amounts of solvent are not required.
Cost reduction in glyceride production can be expected.

Patent Applicant: Nisshin Oil Co., Ltd. Procedural Amendment (Spontaneous) Date: June 6, 1985 Director General of the Patent Office Uga Michi 6th Department ``(゛zuke 1, Case Indication 1985 Patent Application No. 232040 2, Title of the invention Process for producing highly unsaturated fatty acid glycerides 3, Relationship to the amended case Patent applicant Postal code 221 Address 1-3 Chiwaka-cho, Kanayō-ku, Yokohama-shi, Kanagawa Prefecture
Name Nisshin Oil Co., Ltd. Research call outage Call 0
45 (461)0181-1゛1) 7,゛...1(゛...8 f and...',・4゜Object of amendment (1) Detailed explanation of the invention in the specification column 5, Contents of amendment (1) On page 5-1 of the specification, line "Tetracosatraenoic acid" is corrected to "Tetracosatetraenoic acid."

(2) "As an example of this," is corrected from line 1 from the bottom of page 6 to page 7 of the specification as "these examples are."

(3) Same page 7 line 5 r 1anuginoseJ
lanuginosa”.

(4) Page 7, 3 lines from the bottom: “Carrageenan, etc.”
should be corrected as "carrageenan, etc."

(5) Specification page 13, lines 1) to 12 rrLIpase
Correct "P" to r r Lipase P J.

(6) On page 14 of the specification, line 7, "thermally stable" is corrected to "thermally stable."

Claims (2)

[Claims] (1) A method for producing a highly unsaturated fatty acid glyceride, which comprises reacting a highly unsaturated fatty acid or its lower alcohol ester with glycerin using a heat-stable lipase. (2) The highly unsaturated fatty acid is a fatty acid having 20 or more carbon atoms and 3 or more unsaturated bonds.
) The manufacturing method described in section 2.