JPH02295489A - Production of highly unsaturated long chain fatty acid-containing triglyceride - Google Patents

Abstract

PURPOSE:To efficiently produce the subject triglyceride of high purity containing a high content of highly unsaturated long chain fatty acids by hydrolyzing highly unsaturated long chain fatty acid-containing fats and oils using lipase, subsequently carrying out deglicolination and then acting a specified enzyme on the resultant partially hydrolyzed material. CONSTITUTION:Highly unsaturated long chain fatty acid-containing fats and oils (preferably fish oil) are initially hydrolyzed using lipase and subsequently deglycerized to obtain a partially hydrolyzed material composed of a mixture of glycerides containing a high content of highly unsaturated long chain fatty acids and fatty acids. Chromobacterium genus-derived lipase is then acted thereon, preferably with stirring at 35-60 deg.C for 10-24hrs. to obtain the objective substance. In addition, an immobilized enzyme is preferably used as Chromobacterium genus-derived lipase.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing fats and oils, and more particularly to a method for producing triglycerides containing a large amount of long-chain highly unsaturated fatty acids.

In the present invention, long-chain highly unsaturated fatty acids have 20 or more carbon atoms per molecule, and 3 or more? refers to a fatty acid with a double bond of

(Prior Art) In recent years, the physiological activity of long-chain highly unsaturated fatty acids has attracted attention, and active studies have been conducted on their use. In other words, Western-style meat-based diets, including those of modern Japanese people, are associated with omega-6 long-chain highly unsaturated fatty acids (fatty acids with a double bond at the 6th C-C bond counting from the methyl end, e.g. Arachidonic acid (AA, cz..4), T-linolenic acid (GLA.c, Fatty acids with a double bond in the third C-C bond, such as Eicosapentae 71 (EPA, C2.
, ) and docosahexaenoic acid (D・HA, cz■.b), etc.), which can cause various adult diseases (cancer, high systemic pressure, heart disease, etc.). It is reported that there are. Therefore, the production of fats and oils containing long-chain polyunsaturated fatty acids using fish oil as a raw material has attracted attention for its usefulness as a supplement for omega-3 fatty acid intake.

(Problems to be Solved by the Invention) Naturally existing long-chain highly unsaturated fatty acids exist in the form of triglycerides. However, when fish oil is treated with a hydrolysis reaction using lipase, the long-chain polyunsaturated fatty acids are not completely concentrated into triglycerides, but some of them are concentrated into diglycerides or monoglycerides. The product is obtained as a mixture of monoglycerides. When only glycerides are separated from this mixture and the composition ratio of tri-, di-, and monoglycerides is examined, the content of triglycerides is usually 80% or less. From the standpoint of nutritional supplement products, the higher the triglyceride content, the more natural the fat will be accepted by the living body in terms of absorption and metabolism.

As a method for producing fats and oils containing high concentrations of long-chain polyunsaturated fatty acids, Candida
a) Method for producing fats and oils containing a high concentration of long-chain polyunsaturated fatty acids using selective hydrolysis of lipases
165796) is known. However, the fats and oils obtained by this production method have a triglyceride content of 75%.
%, G and monoglycerides were mixed, and there remained a problem in terms of quality as a food material.

An object of the present invention is to provide a production method that can efficiently obtain highly purified triglyceride containing a high concentration of long-chain highly unsaturated fatty acids.

(Means for Solving the Problems) The production method of the present invention is a partial hydrolyzate obtained by hydrolyzing fats and oils containing long-chain polyunsaturated fatty acids using lipase and then deglycerinizing the fats and oils containing long-chain polyunsaturated fatty acids. , using a mixture of glycerides and fatty acids containing a large amount of long-chain highly unsaturated fatty acids, and further using a mixture of Chromobacterium spp.
It is characterized by the action of lipase derived from P. terium.

As the long-chain highly unsaturated fatty acid-containing fat and oil raw material used in the present invention, marine animal oils, particularly fish oils obtained from sardines, mackerel, saury, horse mackerel, tuna, bonito, etc., are preferred because they contain large amounts of eicosapencitric acid and docosahexaenoic acid.

The lipase used for hydrolysis in the present invention is not particularly limited, and for example, lipases derived from microorganisms such as Candida, Pseudomonas, Chromobacterium, and Mucor can be used.

The method for producing fats and oils of the present invention involves using lipase to obtain fats and oils containing long-chain highly unsaturated fatty acids as constituent fatty acids with an acid value of 5.
0 to 150 and remove glycerin from the hydrolyzate to obtain a mixture of glyceride and fatty acid containing long chain highly unsaturated fatty acids as constituent fatty acids. In order to reduce the water content in this mixture to 1,000 ppm or less (preferably 5 to 100 ppm+), vacuum heating method, N
a. S.O. Dehydration is performed using dehydrating agents such as , molecular sieves, polymeric water absorbing agents, blowing dry nitrogen or carbon dioxide, or a combination thereof. Since the reaction for producing triglycerides from mono- and diglycerides is a dehydration reaction, the water content in the reaction system must always be kept within this range in order to obtain fats and oils with a high triglyceride content. As the enzyme for the reaction, lipase derived from Chromobacterium may be used as it is, but it is effective to use Celite, DE, etc.
It is better to use an immobilized enzyme immobilized on an immobilization carrier such as AE-Toyopearl (trade name, manufactured by Toyo Soda) or Sepabeads FP (trade name, manufactured by Mitsubishi Kasei). The amount of immobilized enzyme used is expressed in Unisoto (U), which represents fat hydrolysis activity, per 1 g of reacted fat! 00 to 5,000 units is preferred.

In order to carry out a more effective reaction, Chromobacterium-derived lipase should be used as an activity aid when preparing the immobilized enzyme by adding lecithin to the immobilized carrier at a level of 0.5 to 10%. That's good. The triglyceride production reaction may be carried out in the atmosphere using a dehydrating agent, but if the substance contains a large amount of long-chain polyunsaturated fatty acids, such as fish oil, dehydration by blowing dry nitrogen or carbon dioxide is recommended to avoid deterioration of the fatty acids. It is preferable not only from the viewpoint of preventing enzyme deactivation.

Further, tocopherol, ascorbic acid, TBHQ1 butylated hydroxyanisole, and butylated hydroxytoluene may be used in combination.

Preferably, the reaction is carried out at 20-70°C. If the temperature is lower than 20°C, the solidification of the reaction oil and the reaction rate will be slow, and if the temperature exceeds 70°C, the enzyme will be significantly deactivated.More preferably 35 to 6
Perform at 0°C.

Although it is preferable to stir the reaction, the reaction proceeds even if left standing.

Furthermore, it is also possible to utilize flow using the force of blowing dry inert gas, or to use an immobilized enzyme power ram to further make the reaction continuous.

After reacting in this manner for 1 to 48 hours (preferably 10 to 24 hours), glycerides are fractionated using known techniques such as chromatography, crystal fractionation, fractional distillation, liquid-liquid distribution, and deacidification methods, and triglycerides are Obtain fats and oils with a content of 90-100%.

(Effects of the Invention) According to the method of the present invention, since lipase derived from the genus Chromobacterium is activated, highly purified triglyceride containing a high concentration of long-chain polyunsaturated fatty acids can be easily obtained.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Fish oil (IV = 171.0, long chain highly unsaturated fatty acids in the constituent fatty acids 23.5% (EPA 13%, DHA 8
%)) The lipase obtained from Candida Cylindriac was weighed out at 100 units per gram of oil and fat, 50 g of distilled water was added, and the mixture was allowed to react at room temperature for 8 hours with stirring. After the reaction was completed, the aqueous layer containing lipase was removed to obtain a cracked oil. The acid value of this decomposed oil is 128
Met. A portion of this decomposed oil was taken, the fatty acid portion was removed by alkaline deoxidation, and the glycerides were separated.The triglyceride, di-, and monoglyceride ratios were analyzed by TLC-chromatography. It was 2%. Moreover, the content of long chain highly unsaturated fatty acids in its constituent fatty acids was 47.3%.

This decomposed mixture was washed three times with hot water at 40°C to deglycerinate, and then heated to 50°C and adjusted to a water content of 1100 ppm or less while blowing dry nitrogen. Raw, unimmobilized Chromobacterium lipase was added to the fish oil decomposition product at a concentration of 1,000 units per gram of oil and fat, and the mixture was shaken while blowing dry nitrogen and reacted at 45° C. for 24 hours.

After the reaction, the fatty acid part of the oil and fat was removed by alkaline deoxidation to separate the glycerides, and the triglyceride and dimonoglyceride ratios were analyzed by TLC-chromatography. Ta. In addition, the content of long chain highly unsaturated fatty acids in its constituent fatty acids is 45.3% (EPAIO%, DHA28
%)Met.

Example 2 10% by weight of Na. Add SOa to add moisture to 300pp
Chromobacterium lipase, which had been dehydrated to m and immobilized on Celite 535, was added at 1,000 units per gram of oil, and the mixture was shaken and stirred while blowing dry nitrogen, and reacted at 50° C. for 24 hours. After the reaction, the fatty acid part of the oil and fat is removed by alkaline deoxidation method, and the glyceride is fractionated.
As a result of analyzing the monoglyceride ratio by TLC-chromatography, it was found to be 98% triglyceride, 2% diglyceride, and 0% monoglyceride. In addition, the content of long-chain highly unsaturated fatty acids in its constituent fatty acids is 42.3% (E
PAII%, DHA 26%).

Example 3 Weigh out the immobilized enzyme obtained by immobilizing Tacomobacterium lipase on aminated polyacrylonitrile resin to 100 g of fish oil so that it becomes 50 units per 1 g of oil, add 30 g of distilled water, and heat at 37° C. for 12 hours with stirring. Allowed time to react. After the reaction is complete, remove the aqueous layer containing lipase,
Obtained cracked oil. The acid value of this cracked oil was 125.

A portion of this decomposed oil was taken, and the fatty acid portion was removed by alkaline deoxidation to separate the glycerides.The ratio of tri-, di-, and monoglycerides was analyzed by TLC-chromatography, and the results showed that triglyceride was 70%, diglyceride was 25%, The monoglyceride content was 5%. In addition, the content of Changzhen polyunsaturated fatty acids among its constituent fatty acids was 46.2%.

This decomposition mixture was washed three times with hot water at 40°C to deglycerinate it, and the temperature was raised to 50°C and the water content was adjusted to 100 ppm or less while blowing dry nitrogen. DEAE-1-
2,000 units of Chromobacterium lipase immobilized on Yopal was added to 1 g of oil, shaken and stirred while blowing dry nitrogen, and reacted at 60° C. for 15 hours. After the reaction, the fatty acid part of the oil and fat was removed by alkaline deoxidation method, the glyceride was fractionated, and the tri-, g-, and monoglyceride ratios were analyzed by TLC-chromatography scanner method.
The content was 97% triglyceride, 3% diglyceride, and 0% monoglyceride. In addition, the content of long chain highly unsaturated fatty acids in its constituent fatty acids is 40.4% (E P A 9%, D
HA27%).

Example 4 A fish oil decomposition product that had been decomposed and deglycerinated with immobilized lipase in the same manner as in Example 3 was heated to 60° C. and dehydrated by suction to a water content of 60 pplll. 500 units of Chromobacterium lipase immobilized on Sephabeads FP was added to 1 g of oil, and 10% by weight of Molecular Sieve 3A was added as a continuous dehydrating agent to the oil and fat, shaken and stirred, and reacted at 50°C for 12 hours. .

After the reaction, the fatty acid part of the oil and fat was removed by alkaline deoxidation method to separate the glyceride, and the triglyceride and dimonoglyceride ratio was analyzed by TLC-chromatography scanner method. As a result, triglyceride 98%, diglyceride 2%, monoglyceride O
%Met. In addition, the content of long chain highly unsaturated fatty acids in its constituent fatty acids is 42.4% (EPAIO%, DMA28%
)Met.

Comparative Example 1 In a fish oil decomposition product that had been decomposed, deglycerinated, and dehydrated in the same manner as in Example 1, lipase derived from Candida cylindricae and immobilized on Celite 535 was added at a concentration of 1,000 g per 1 g of fat.
The mixture was stirred and shaken at 45°C, and reacted for 24 hours while blowing dry nitrogen for continuous dehydration.

After the reaction, the fatty acid portion of the oil and fat was removed by alkaline deoxidation to separate the glycerides, and the triglyceride and di-monoglyceride ratios were analyzed by TLC-chromatography. Ta. In addition, the content of long chain highly unsaturated fatty acids in its constituent fatty acids is 45.3% (EPA II%, DHA2
9%), and no increase in the triglyceride ratio could be expected.

Claims (1)

[Claims] It is a partial hydrolyzate obtained by hydrolyzing fats and oils containing long-chain polyunsaturated fatty acids using lipase and then deglycerinizing them. A method for producing a long-chain polyunsaturated fatty acid-containing triglyceride, which comprises using a mixture and further acting on a lipase derived from Chromobacterium.