JPH0582197B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a method for producing highly unsaturated fatty acid glycerides using a thermostable lipase.

(b) Conventional technology Polyunsaturated 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. At the same time, in recent years it has attracted attention as it has been discovered that it is effective in preventing arteriosclerosis in animals and humans, and has physiologically active effects such as thrombolytic and blood pressure lowering effects. From this viewpoint, the PUFA glycerides are useful in a wide range of fields as raw materials or bases for foods, pharmaceuticals, cosmetics, agricultural chemicals, feeds, diagnostic and analytical reagents, and the like.

Up to now, attempts have been made to synthesize glycerides of PUFA by chemically synthesizing them, enzymatically converting them, or preparing them using solvents.

That is, chemically, glycerin and the PUFA are heated to 150 to 200°C in the presence of an acid or base catalyst,
This method synthesizes glycerides, but although triglyceride of PUFA is the main product, due to the high temperature, the PUFA undergoes undesirable side reactions such as polymerization, isomerization, oxidation, and coloring, resulting in a high-quality target product. There are drawbacks that make it difficult to obtain.

In addition, several methods have been tried for enzymatic conversion, such as JP-A-58-
No. 165796 states that even when lipase (derived from Candida cylindracea) is used, the ester bond between PUFA and glycerin is hardly hydrolyzed.
It can be seen that the reaction between and glycerin also does not occur. Furthermore, JP-A-59-14793 also describes that PUFA is difficult to react with conventional lipases.

A method using a solvent is disclosed in JP-A-59-71396.
No. 1 discloses a method for extracting fish oil with a solvent containing 1 to 25 wt% water such as acetone, methyl ethyl ketone, methanol, or ethanol, and concentrating and separating fats and oils containing PUFA, but the cost increase due to the use of such solvents cannot be ignored. Also, PUFA
Eicosapentaenoic acid (C ₂₀ : ₅ ,
ω-3) The content remains at around 30%.

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

(c) Problems to be Solved by the Invention Therefore, the purpose of the present invention is to solve the above-mentioned problems such as polymerization, isomerization, and coloring of PUFA glyceride due to thermal history in the chemical synthesis method when producing PUFA glyceride. , i.e. instability mainly due to PUFA, low or non-reactivity of conventional lipases towards PUFA in enzymatic conversion methods, PUFA
The object of the present invention is to solve the problems of conventional methods such as low triglyceride content and increased production costs in solvent methods, and to produce high-quality PUFA glycerides in an extremely advantageous manner.

(d) Means for solving the problem As a result of intensive research, the present inventors discovered that the above object can be achieved by carrying out the reaction under specific conditions, which led to the completion of the present invention. It is. 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 at 65° C. or higher using a heat-stable lipase.

The present invention will be explained in detail below. The PUFA used in the present invention is seafood, shrimp, squid, marine chlorella,
It can be obtained from marine organisms such as seaweed, organs of higher animals such as cows and pigs, plant seeds, and microorganisms such as mold, yeast, and bacteria, and generally has 20 or more carbon atoms and 3 or more unsaturated bonds. Refers to long-chain highly unsaturated fatty acids. Examples of these include eicosatrienoic acids (C ₂₀ : ₃ ω-3 and ω
-6), eicosatetraenoic acid (C ₂₀ : ₄ , ω-
3), arachidonic acid (C ₂₀ : ₄ , ω-6), eicosapentaenoic acid (C ₂₀ : ₅ , ω-3), docosatrienoic acid (C ₂₂ : ₃ , ω-6), docosatetraenoic acid (C ₂₂ : ₄ , ω-6), docosahexaenoic acid (C ₂₂ : ₆ , ω-3), and tetracosatetraenoic acid (C ₂₄ : ₄ , ω-6).
PUFA has these so-called physiological activities ω
-3 or ω-6 fatty acids are important components. In addition, in the present invention, these PUFA
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 a monohydric alcohol having 6 or less carbon atoms, and examples thereof include methanol, ethanol, propanol, isopropanol, butanol, 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 PUFA or their esters as described above have not been found. Not yet. Therefore, 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 a thermostable lipase. The thermostable lipase may be one that can maintain its activity even at 50°C or higher, and such enzymes can be used in living organisms such as animals, plants, or microorganisms.
Although it can be isolated and purified from secretions, it is convenient to use microorganism-derived products. Examples of these include Pseudomonas spp.
Lipase derived from Penicillium cyclopium of the genus Penicillium, lipase derived from the Alcaligenes strain No. 3783, Humicola lanuginosa of the genus Humicola
Lipase from Mucor miehei, Rhizopus chinensis
Examples include lipase derived from Note that the present invention is not limited to these types of microorganisms, and 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, polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resin, magnetic material, activated carbon, celite, alumina, photocrosslinkable or photocurable resin,
It is possible to use a thermostable lipase that is adsorbed, ionically bonded, covalently bonded, or encapsulated in an enzyme immobilization carrier such as alginate or carrageenan.

According to the method of the present invention, PUFA or its lower alcohol ester and glycerin are reacted using a heat-stable lipase to produce PUFA glyceride as follows. That is, it is a reaction raw material.
PUFA or its lower alcohol ester and glycerin are placed in a glass or stainless steel container, and if necessary, an inert organic solvent such as n-
After adding hexane, n-heptane, etc., further adding an appropriate amount of water and a buffer solution, and adding heat-resistant lipase or its immobilized product, while stirring or shaking while blowing an inert gas such as nitrogen gas, Under PH3-12, preferably PH5-10, the temperature is raised to 65°C or higher, preferably 65-70°C, and
Incubate for 120 hours. Although the enzyme used in the present invention can proceed at temperatures below 65°C, it takes a long time, and at temperatures above 70°C, even if it is a thermostable lipase, its stability decreases, leading to deactivation of the enzyme. It is desirable to conduct the reaction at a temperature of 65 to 70°C due to the risk of deterioration of unsaturated groups due to high temperatures.
The progress of the reaction can be monitored by TLC (thin layer chromatography), GLC (gas chromatography) or
HPLC (high performance liquid chromatography) etc.
Reduction of PUFA or its ester content, said PUFA
It can be checked by measuring the increase in monoglyceride, diglyceride and triglyceride content in glycerides. After the reaction is completed, the reactants are heated to 80~
The enzyme is inactivated by heating to 90℃ for a short time, the enzyme is removed by filtration or centrifugation, and unreacted substances are removed using fractionation methods such as solvent fractionation and adsorption chromatography to obtain the desired PUFA glyceride. can be manufactured. In addition, in the present invention, the immobilized enzyme can be packed in a cylindrical container, and the reaction solution can be poured in from one side to perform the reaction as a column-type reactor, or it can be reacted as a dispersion-type reactor in which the immobilized enzyme is suspended in the reaction solution. It can be performed.

(e) 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 eicosapentaenoic acid (C ₂₀ : ₅ , ω- A glycolipid containing 3) as the main component was obtained. This is saponified and decomposed by the usual method,
PUFA was obtained by solvent extraction. Fatty acid composition:
C ₂₀ : ₅ , ω-3 (77%), C ₂₀ : ₄ , ω-6 (8%),
C ₁₈ : ₂ (3%), C ₁₈ : ₁ (3%), and C ₁₆ : ₀ (5%). This PUFA is mixed with ethanol using a conventional method.
Converted to PUFA ethyl ester. Add 50g of the PUFA ethyl ester and 6.7g of glycerin to a 200ml Erlenmeyer flask, and add 0.5M Tris-HCl buffer (PH8.5).
0.5ml and immobilized thermostable lipase (Mucor
miehei-derived lipase immobilized on a weakly basic ion-exchange resin, manufactured by Novo Industries, Denmark, product name ``Lipase3A'') was added, and the mixture was shaken at 65°C for 90 hours (200 rpm) in a nitrogen gas stream. )did. After the reaction was completed, the mixture was heated to 80°C for 5 minutes, the immobilized substances were removed by filtration, and unreacted substances (PUFA) were removed by silica gel column chromatography. The composition of the product was triglyceride (25%), diglyceride (58%), and monoglyceride (17%), and its fatty acid composition was almost the same as that of the raw material.

Example 2 75g of PUFA obtained in Example 1 and 10g of glycerin
Transfer 100ml of g, n-hexane to a 500ml three-necked flask with a condenser tube, and add 0.8ml of 0.3M phosphate buffer (PH7.0).
and thermostable lipase (Humicola lanuginosa
Origin, manufactured by Amano Pharmaceutical Co., Ltd., product name "Lipase CE") 0.1
72g at 66°C while blowing nitrogen gas.
The mixture was stirred (200 rpm) for hours. After the reaction was completed, the enzyme was heated to 85℃ for 5 minutes, the enzyme was removed by centrifugation, and the unreacted material was removed using silica gel column chromatography.
After removing PUFA, the glyceride composition of the reaction product was determined by HPLC analysis, and it was found to be triglyceride (86%), diglyceride (12%), and monoglyceride (2%). In addition, as a result of GLC analysis of the constituent fatty acids of the glyceride component, it was found that
It had almost the same composition as PUFA.

Example 3 Fish oil derived from sardines was treated with a non-heat-stable lipase (“Lipase MY” manufactured by Meito Sangyo Co., Ltd.) using a conventional method for 30 minutes.
℃ for 6 hours to perform hydrolysis. A saturated aqueous calcium hydroxide solution was added to the reaction mixture, and the mixture was centrifuged to recover an oil layer, followed by saponification and decomposition using a conventional method and extraction with hexane to obtain a PUFA concentrate. The main fatty acid composition according to GLC analysis is C ₂₀ : ₅ , ω-3 (38%),
C ₂₂ : ₆ , ω-3 (25%), C ₂₂ : ₅ , ω-3 (5%),
C ₂₀ : ₁ (5%), C ₂₂ : ₁ (3%), C ₂₀ : ₄ , ω-6 (2
%) and other (22%). 100g of this PUFA concentrate, 10g of glycerin, 0.5ml of water, heat-stable lipase (derived from Pseudomonas fluorescens, Amano Pharmaceutical)
Co., Ltd., product name "Lipase P") 0.15g, Celite
Using 0.2g, the reaction was carried out at 66°C in the same manner as in Example 2, and the glyceride composition of the reaction product was determined. Triglyceride (81%), diglyceride (13%)
%) and monoglycerides (6%), and their constituent fatty acids were almost the same as the raw PUFA concentrate.

Example 4 Immobilized thermostable lipase used in Example 1 (manufactured by Novo Industries, trade name "Lipase 3A")
was packed in a glass tube (2 cm diameter x 30 cm length) and prepared in Example 1.
50 g of PUFA ethyl ester, 5 g of glycerin, 50 ml of n-heptane, 0.8 ml of 0.3 M Tris-HCl buffer (PH7.5) and 0.05 g of polyvinyl alcohol (n = 1750) were mixed in a separate container with a homomixer.
The mixed solution was treated at 2000 rpm for 15 minutes, and the mixed solution was pumped from the bottom of the above column via a liquid feed pump.
A liquid circulation system was created in which the liquid was fed at a flow rate of 10 ml/hour and the liquid flowing out from the top was returned to the separate container mentioned above, and recycling was performed at 65°C for 80 hours. When a portion of the reaction solution was extracted and its composition analyzed by TLC, the main component was diglyceride.

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

Conventionally, it was thought that the reaction could not be carried out using normal fat and oil hydrolase enzymes used at 30-45℃.
It has become possible to react PUFA or its ester using a thermostable lipase, and it has become possible to produce glycerides of the PUFA.

Since this reaction is carried out at a relatively low temperature of 65 to 70°C, high-quality PUFA glycerides can be produced without adverse effects such as polymerization and isomerization on PUFA, which has poor thermal stability.

PUFA glycerides obtained by conventional enzymatic methods had a limit in increasing the triglyceride content, but the method of the present invention almost completely increases the triglyceride content.
Can produce PUFA triglycerides.

Compared to natural fractionation or solvent fractionation methods, this method allows the reaction to occur at temperatures close to room temperature, and does not require large amounts of solvent, which can lead to cost reductions in the production of PUFA glycerides.

Claims (1)

[Claims] 1. A method for producing a polyunsaturated fatty acid glyceride, which comprises reacting a polyunsaturated fatty acid or its lower alcohol ester with glycerin at 65°C or higher using a heat-stable lipase. 2. The production method according to claim 1, wherein the highly unsaturated fatty acid is a fatty acid having 20 or more carbon atoms and 3 or more unsaturated bonds.