JPH03108489A - Production of long-chain highly unsaturated fatty acid monoglyceride - Google Patents

Abstract

PURPOSE:To obtain the title monoglyceride suitable for drug, healthy food, etc., having excellent digestion and absorption by reacting specific fats and oils with alkali lipase and then hydrolyzing. CONSTITUTION:(A) Fats and oils (e.g. fish oil of sardine or mackerel) containing a long-chain highly unsaturated fatty acid (e.g. arachidonic acid) is reacted with (B) 1-1,000 units based on 1 g of the component A of an alkali lipase (e.g. pancreatic lipase) having 1,3-position specificity by adding 2-200% based on the component A of water and (C) preferably 0.1-30mol based on 1mol of the component A of an alkali salt (e.g. potassium hydrogencarbonate) at 20-40 deg.C and hydrolyzed preferably in 50-90% hydrolysis ratio to give the objective monoglyceride.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing long-chain highly unsaturated fatty acid monoglycerides.

In the present invention, long chain highly unsaturated fatty acids (hereinafter referred to as PUF
A) means a carbon atom with 2 or more double bonds and 3 or more double bonds.
0 or more fatty acids, and long-chain highly unsaturated fatty acid monoglyceride (hereinafter referred to as PUFMC) means a monoglyceride having PUFA as the main constituent fatty acid.

PUFA is arachidonic acid (C20
:4), eicosapentaenoic acid (C20:5), and docosahexaenoic acid (C22:6), which are important essential fatty acids involved in biological regulatory functions.

PUFMG produced by the present invention is useful as a pharmaceutical, health food, or functional food.

According to the present invention, there is provided an industrially advantageous method for producing PUFMG, which is characterized in that oils and fats containing PUFA as constituent fatty acids are hydrolyzed by an alkaline lipase having 1.3 regiospecificity in the presence of an alkali salt. Ru.

[Prior Art] PUFA has many double bonds and is an unstable substance that is easily subjected to thermal polymerization and oxidation. Conventionally known methods for concentrating PUFA include chromatography using a mixed fatty acid decomposition product, a method using a urea adduct, a low-temperature solvent fractional crystallization method, and a method using molecular distillation. However, these methods are not economical or involve denaturation of PUFA, and there has been a desire for a concentration method that does not cause denaturation at room temperature and pressure.

PUF is created by hydrolyzing fats and oils with lipase produced by specific microorganisms (Candida cylindracie or Alcaligenes).
A method for concentrating A in the form of glyceride is disclosed in JP-A-58-1.
No. 65796 and No. 61-15692. The methods described in these publications all involve the discovery of a group of lipases with substrate specificity that hydrolyze the glyceride bonds of ordinary fatty acids, but little or no hydrolysis of the glyceride bonds of PUFA. The gist of the invention is to hydrolyze fats and oils under acidic to neutral conditions (no alkaline substances are added) using these specific lipases, and by decomposing and removing only normal fatty acids from fats and oils, PtJFA can be produced. Although it is concentrated in the form of glyceride, the concentration of PUFA is insufficient and is not necessarily satisfactory as an industrial method. In addition, a method of decomposing fats and oils with alkaline lipase in the presence of a hydroxide compound of a divalent or higher metal is disclosed in Japanese Patent Publication No. 61-20275 and No. 60-19999. A method for producing monoglyceride by hydrolyzing is disclosed in JP-A-60-102192.

[Problem to be solved by the invention]

The purpose of the present invention is to establish an industrially advantageous method for producing long-chain polyunsaturated fatty acid monoglycerides (PUFMG) useful as pharmaceuticals, health foods, functional foods, or materials for producing them. .

The present inventors have discovered that the method of producing monoglycerides by hydrolyzing fats and oils with alkaline lipase under alkaline conditions (Japanese Unexamined Patent Publication No. 102192/1982) is industrially excellent and that PUFA in fats and oils are mainly glycerides. 2
Focusing on the fact that PUFA is bound to the 2nd position of glycerides, we hydrolyze fats and oils containing PUFA as a constituent fatty acid in the presence of alkaline salts using alkaline lipase with 1,3 position specificity, and mainly bind to the 2nd position of glycerides. If the fatty acids bonded to the 1st and 3rd positions could be decomposed and removed while leaving the PUFAs intact, it was possible to produce monoglycerides (PUFMG) containing PUFAs as the main constituent fatty acids, and we have developed an industrial approach to this. The present invention was completed as a result of various research aimed at establishing an advantageous manufacturing method.

[Means to solve the problem]

The present invention is characterized by hydrolyzing fats and oils containing rl, long-chain polyunsaturated fatty acids as constituent fatty acids using an alkaline lipase with 1,3 position specificity in the presence of an alkaline salt. Method for producing monoglycerides.

2. The method for producing long-chain highly unsaturated fatty acid monoglycerides as described in 1 above, which comprises hydrolyzing fats and oils until the decomposition rate reaches 50 to 90%.

3. The method for producing a long-chain highly unsaturated fatty acid monoglyceride as described in 1 or 2 above, which comprises extracting the long-chain highly unsaturated fatty acid monoglyceride from the hydrolysis reaction mixture with an organic solvent and separating it from the fatty acid salt. ”.

In the present invention, the oil and fat used as a starting material must contain PUFA as a constituent fatty acid, and examples of such oil and fat include fish oils such as sardine, mackerel, saury, cod, squid, horse mackerel, etc. Krill, algae, and fungal fats and oils can be mentioned, and these fats and oils contain about 1% to 40%
% PUFA is mainly contained in the 2nd position.

In the present invention, an alkaline lipase with 1,3 position specificity specifically acts on the glyceride bond at the 1.3 position of fats and oils, but has weak or almost no effect on the glyceride bond at the 2 position. Any alkaline lipase with specific properties may be used. Examples of such alkaline lipase include pancreatic lipase, and the lipase produced by the genus Achromobacter, known as iJ! AL-86
No. 5 (Feikoken Bibori No. 1213), famous as a lipase that produces alkaline earth metals! Bacterium PL-266 (Feikoken Bacterium No. 3187) or PL-679 (Feiken Bacterial No. 3783), Lipase B (Sara Polo Beer Co., Ltd.) as a lipase produced by Pseudomonas;
Other examples include alkaline lipase 5P-398 manufactured by Novo.

These alkaline lipases may be used in an amount of 1 to 1000 units, preferably 5 to 100 units per gram of fat or oil. The enzyme unit was determined by the method of King et al.
Biol, Chem, 45(5), 1159. (1
982) J.

Examples of alkali salts include Ca (011) z ,
Mg(Of()z.

Hydroxide such as Ba(Oll)z, hydrogen carbonate or hydrogen phosphate of sodium or potassium at 0.00% per mole of fat or oil.
It is preferable to add about 1 to 30 moles.

When performing a hydrolysis reaction, water is 2 to 200% based on fats and oils.
(W/W)), react at room temperature and pressure, and hydrolyze at a decomposition rate of about 50 to 90% depending on the type of fat or oil.

The reaction temperature may be around 20 to 40°C; too high reaction temperature is not preferred because it induces spontaneous transition of the 2-position fatty acid.

After completion of the reaction, any known method can be used as appropriate to separate and collect the target PUFMG from the reaction mixture, but an extraction method using an organic solvent is recommended as the industrially simplest and most advantageous method.

That is, if the reaction mixture is treated using an organic solvent such as acetone, hexane, or ether as an extractant, PUFMG is extracted, but fatty acid salts are not extracted, so that the target product PUFMG can be separated and collected very easily.

The decomposition rate of fats and oils in the present invention was determined using the following formula.

However, in the formula, SV indicates the saponification value of the fat and oil, and AV is the acid value of the oil obtained by making the hydrolysis reaction mixture of fat and oil acidic, extracting the oil with diethyl ether, washing with water, removing the ether after dehydration. shows.

〔Effect of the invention〕

The effect or advantage of the present invention is to hydrolyze approximately 50% or more of fats and oils containing PUFA in the presence of an alkaline salt using an alkaline lipase with 1,3 position specificity, and to convert PUFA into phosphorus.
This provides an industrial and economical method for accumulating and extracting UFMG.

In addition, in the method of the present invention, the rate of oil and fat decomposition is increased by adding an alkali salt to the reaction system, and the accumulation of PUFA is facilitated. Furthermore, after the reaction, PUFMG can be easily separated from soap at room temperature and pressure using an organic solvent. We can provide an advantageous method that does not impair product quality.
Furthermore, Takagi et al. reported that the autoxidation rate of PUFA is the slowest and most stable when it exists as a monoglyceride among mono-, di-, and triglycerides (JA
OC5, Vol, 65 No, 7 p-1156 (19
88)), and the use of PUFA in the form of PUFMG in medicines and foods is also superior in terms of digestion and absorption.

PUFMC; can also be applied as a functional food emulsifier as a functional monoglyceride.

Next, the present invention will be explained in more detail by showing comparative examples and examples.

Example 1 Sardine oil (manufactured by Riken Vitamin Co., Ltd.) 50g, calcium hydroxide 6g and lipase PL-266 (20,000 units/g)
250 mg of distilled water dissolved in 12+d was added to the baffled 5
The mixture was collected in a 00 mfl flask and reacted at 35°C for 24 hours.

Collect 2 g of the reaction product, add 2 mf/5N hydrochloric acid and 20 ml of petroleum ether, shake thoroughly, and extract the oil.
The petroleum ether layer was recovered by centrifugation at 1,000 g. After washing the petroleum ether layer with 201 nIl of distilled water, the petroleum ether was distilled off using a rotary evaporator to obtain an oil component.

Regarding this oil content, Standard Oil and Fat Analysis Methods 2 and 4 edited by Japan Oil Chemists' Association
- The acid value was measured according to 1-83. In addition, the saponification value of sardine oil was measured according to the same analytical method 2.4.3-71. The decomposition rate of sardine oil is calculated by dividing the acid value by the saponification value.
When calculated by multiplying by 100, it was 74%. In addition, 5 mg of this oil was added to a silica gel plate (manufactured by Merck & Co., Ltd., N
0, 13895) at a position 1 cm from the lower end, and petroleum ether/ether/acetic acid (70:30:1. v/v)
After developing 10 cm with a mixed solvent, a spot of monoglyceride (abbreviated as MG) was detected under ultraviolet irradiation with a wavelength of 254 nm, scraped into a test tube with a stopper, and analyzed using the above analysis method 2.
Fatty acid methyl ester was prepared according to 4.20.2.77. A glass column filled with 10% DEGS (manufactured by Gas Kuro Kogyo Co., Ltd., inner diameter 3 mm, length 2 m, No. AA45
G
The composition of fatty acid methyl esters was analyzed using the method (abbreviated as C). Similarly, fatty acids contained in the oil were separated by TLC, and the composition was analyzed by GC as methyl esters. These results are shown in Table 1 together with the fatty acid composition of sardine oil.

(Margins below this page) PA, SA, AA, and DPA are palmitic acid,
Indicates stearic acid, arachidonic acid, and docosapene citric acid. Furthermore, AA is a value that also includes erucic acid.

As can be seen from Table 2, compared to the raw material sardine oil, MG
contains 2.2 times more PUFAs such as EPA and DHA, 68%
and highly concentrated.

Furthermore, 5 μg of the recovered oil was added to Chromaro Nodo S-■ (
10 cm was developed using a mixed solvent of benzene, chloroform, and acetic acid (50:30:1, v/v), and the MC content in the oil was analyzed using Iatroscan T H-10 (manufactured by Yatron). When we measured the rate, it was 19.5% (
area%).

Example 2゜Example 1. 200 d of acetone was added to 40 g of the reaction product obtained in , and after stirring until uniformly dispersed, the acetone layer was collected by centrifugation at 2.500 g. Acetone is distilled off from this acetone layer using a rotary evaporator and PUF is created.
6.7 g of MG was obtained. As analyzed by the above GC, E
It contained 43.5% PA and 30.1% DHA. MG82.4 as measured by the above IATROScan
%, DC 16.2%. This shows that highly pure PUFMG can be prepared by preliminary extraction and scraping.

Comparative Example 1 50g of sardine oil (manufactured by Riken Vitamin Co., Ltd.) and distilled water containing 250mg of lipase PL-266 dissolved in 5001+11 with a baffle! Collect in a flask and incubate at 35°C for 2
The reaction was carried out with shaking for 4 hours. Example 1. The decomposition rate measured in the same manner as above was 38%. The content of PUFA in MC measured in the same manner is shown in Table 2.

Table 2 Decomposition reaction without using alkali salt From the results in Table 2, when no alkali salt is used, PLJFA is M
It can be seen that it is not concentrated in G at all.

Comparative Example 2 Comparative Example 1. Lipase PL-679 (100,000 units/g) 42 ml was used. A shaking reaction was carried out in the same manner as above for 7 days, and the decomposition rate and PUFA content in MG were measured. The decomposition rate is 1
7.0%, EPA in MG 15.6%, DHAll, 0
%, decomposition does not progress much, and PUF to MG
No concentration of A was observed.

Example 3゜Sardine oil (manufactured by Yoneyama Pharmaceutical Co., Ltd.) 50g, hydrogen carbonate) IJ
12 ml of distilled water in which 12 g of Lipase P L-266 and 125 mg of Lipase PL-266 were dissolved was collected in a 500 ml flask with a baffle, and reacted with shaking at 35°C for 24 hours.

The decomposition rate measured by the above method was 58%, and the MG content was 3.
It was 2.3%. The PUFA content of MG is shown in Table 3.

Table 3 PUFA in MG The results in Table 3 show that PUFA was concentrated 2.1 times in MG compared to sardine oil.

Example 4゜Example 3. Add 200 mff of ether to 40 g of the reaction product obtained in
1 was added and stirred for 1 hour, and then filtered through Nα1 filter to remove insoluble matter. After washing the collected ether layer with 100 ml of distilled water, the ether was distilled off using a rotary evaporator to obtain 7.4 g of PUFMG.

As measured by the above method, MG86.0%, DC8
, 5%, and as measured by CC, it has an EP A22.
8%, DMA 19.0%.

Example 5 8Inf distilled water in which 50 g of cod oil (manufactured by Riken Vitamin Co., Ltd.), 0.5 g of calcium hydroxide, and 35 mg of lipase PL-679 were dissolved! , as Example 1. The reaction was carried out in the same manner as above for 8 hours. When the decomposition rate was measured using the above method, it was 52% and contained 9.8% MG. Table 4 shows the PUFA content of MG.

Table 4 Concentration of PUFA using Lipase PL-679 Table 5 Concentration of PUFA using magnesium hydroxide The results in Table 4 show that PUFA can be effectively concentrated even from fats and oils with low PUFA content.

Example 6゜Sardine oil 30g, magnesium hydroxide 2.5g and 15
50 ml of distilled water with mg of lipase PL-266 dissolved in it
Example 1. The reaction was carried out in the same manner as above for 48 hours.

The decomposition rate measured by the above method was 71%, MG18.2
%. Table 5 also shows the PUFA content of MG.

(Margins below this page) As can be seen from the results in Table 5, even an alkali salt such as magnesium hydroxide can effectively concentrate PUFA into MG.

Example 7 8 d of distilled water in which 30 g of sardine oil (manufactured by Riken Vitamin Co., Ltd.), 50 n+g of Banqueretic lipase (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) were dissolved, 65.0 g of calcium hydroxide, and 8 μm of sodium cholate were collected, Example 1. as well as 7
It reacted for 2 hours.

When the decomposition rate was measured using the above method, it was 62% and the MO was 2.
It contained 0.4%.

Table 6 shows the results of analyzing PUFA contained in MG using the GC described above.

Table 6 PUFAI reduction by pancreatic lipase As can be seen from Table 6, vancreatin also reduces PUFAI.
UFA can be effectively concentrated to 53.6% in MG.

Comparative Example 3 30 g of cod oil (manufactured by Shiga Pharmaceutical Co., Ltd.), 8 ml of distilled water in which 75 μg of lipase PL-266 had been dissolved, and 5 g of sodium bicarbonate were collected, and the same amount as that of Example 1. The reaction was carried out in the same manner as above for 7 hours. The decomposition rate measured by the above method was 38%.
Baka TG containing 2.2% remained in an amount of 20.1%.

150 mf of acetone was added to 20 g of this reaction product, and extraction was performed with stirring for 1 hour. After removing insoluble materials using N011 filter paper, the acetone phase was concentrated to dryness using a rotary evaporator to obtain 10.4 g of oil. The PUFA content of this oil is G
When measured at C, EPA was 13.3% and DHA was 8%.
．． It was 0%. The EPA and DHA contents of the cod oil used are 11.1% and 7.3%, so the decomposition rate is 50%.
It can be seen that if the concentration is lower, PUFA cannot be sufficiently concentrated.

Example date.

Sardine oil (manufactured by Riken Vitamin Co., Ltd.) 30g, 7.5μl
Lipase 5P-398 (manufactured by Novo), 3.0 g of calcium hydroxide, and 8 g of distilled water were collected and reacted for 24 hours in the same manner as in Example 1. When the decomposition rate was measured by the method described above, it was 64% and the production of MG was 9.8%. MG
Table 7 shows the results of measuring the PUFA contained therein by GC.

Table 7 Concentration of PUFA with Lipase 5P-398 As can be seen from Table 7, the use of Lipase 5P-398 allows particularly effective concentration of DMA to MG.

Example 9 30 g of sardine oil (manufactured by Riken Vitamin Co., Ltd.), 8 ml of distilled water in which 50 mg of Lipase B (manufactured by Sarapolo Beer Co., Ltd.) were dissolved, and 3.0 g of calcium hydroxide were collected, and 3.0 g of calcium hydroxide were collected. The reaction was carried out in the same manner as above for 24 hours.

When the decomposition rate was measured by the above method, it was 88% and contained 17.2% MG. Table 8 shows the results of measuring PUFA in MG by GC.

Example 10 30g of squid oil (manufactured by Riken Vitamin Co., Ltd.), 60ml of distilled water in which 10mg of lipase PL-266 was dissolved! and 140 g of dipotassium hydrogen phosphate were collected, and Example 1. The reaction was carried out in the same manner as above for 12 hours.

The decomposition rate was measured by the above method and found to be 76%, containing 15.5% MG. Table 9 also shows the results of measuring the PUFA content of MG.

Table 9 Concentration of PUFA using hydrogen phosphate Table 8 Concentration of PUFA using Lipase B As can be seen from the results in Table 8, PUFA can be concentrated using Lipase B, but EPA is particularly effective. It can be concentrated into

As can be seen from Table 9, PUFA can be concentrated even if a large amount of alkali salt or water is used.

Comparative example 4゜Sardine oil 30g (manufactured by Riken Vitamin Co., Ltd.), Pseudomonas lipase (manufactured by Sigma Co., Ltd.) 10■, Hydroxylation power Luciram 2
Example 1. Similarly, 12
Time reacted. The decomposition rate measured by the above method was 72%, M
Contained 6.7% of G. PUFA in MG is E
P AlB, 8%, DHA 15.6%, and 1 of sardine oil
It was almost the same compared to 7.0% and 14.4%.

This result shows that PUFA cannot be concentrated in MG when a lipase without position specificity is used.

Reference Example 1.3 When TG is hydrolyzed by a position-specific lipase, the DC generated is 1.2 DC, and 1,3 DG is not generated, and with a lipase without position specificity, 1.2 DC is 1.3 DC. It is thought that it will generate about twice as much.

0.2g of olive oil, 0.2g of 2.2% calcium chloride.
6 composition, 0.1% sodium cholate for 1.5 d, 50
mM Tris-f (CI buffer (pH 8,0) 5
ml and 50μ of each type of lipase were collected into a 18D test tube with a stopper, and a shaking reaction was performed at 40°C for 3 minutes. 6N
- After terminating the reaction by adding 2In1 of hydrochloric acid, 10d of petroleum ether and 1ml of ethanol were added, and extraction was performed with thorough stirring.

The petroleum ether layer was collected and concentrated under a nitrogen stream, and the ratio of 1.3 DC to 1,2 DC was measured using IATROScan.

Note that lipase O is an example of a lipase without position specificity.
Regarding F? Uke? The same procedure was carried out except that the pH of IJt was changed to 7.0, and the results are also shown in Table 10.

Table 10

Claims (1)

[Claims] 1. A long-chain highly unsaturated fatty acid characterized by hydrolyzing an oil or fat containing a long-chain highly unsaturated fatty acid as a constituent fatty acid using an alkaline lipase having 1 or 3 position specificity in the presence of an alkaline salt. Method for producing saturated fatty acid monoglycerides. 2. The method for producing long-chain highly unsaturated fatty acid monoglyceride according to claim 1, which comprises hydrolyzing the fat or oil until the decomposition rate reaches 50 to 90%. 3. The method for producing a long-chain highly unsaturated fatty acid monoglyceride according to claim 1 or 2, which comprises extracting the long-chain highly unsaturated fatty acid monoglyceride from the hydrolysis reaction mixture with an organic solvent and separating it from the fatty acid salt.