JPS62262998A - Production of material containing lysophospholipid essentially free from residual enzymatic activity - Google Patents

Abstract

PURPOSE:To easily produce a lysophospholipid-containing material free from residual enzymatic activity on an industrial scale in high yield, by treating a natural phospholipid-containing substance with a phospholipase A2 preparation, etc., and drying and extracting the resultant lysophospholipid. CONSTITUTION:A natural phospholipid-containing substance such as yolk of chicken's egg is added with a phospholipase A2 preparation(-containing material) (e.g. purified phospholipase A2 preparation originated from animal pancreas) to effect enzymatic reaction. The phospholipid in the substance is decomposed by the reaction to obtain a lysophospholipid-containing material. The obtained lysophospholipid-containing material is dried at about <=80 deg.C by spray drying, etc., to powder having a water-content of <=10%. The powder is extracted with a polar solvent such as ethanol to extract lysophospholipid. The solvent is distilled off from the extract under reduced pressure to obtain the objective lysophospholipid-containing material composed of 68wt% neutral lipid and 32wt% phospholipid (30wt% thereof is lysophospholipid) and essentially free from residual enzymatic activity (provided that the enzymatic activity of residual phospholipase A2 is <=0.1 IU per 1g of the final product).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a lysophospholipid-containing product having substantially no residual enzymatic activity.

[Conventional technology]

Lysophospholipids are phospholipids in which one fatty acid is cleaved and replaced with water ME1, so they are more hydrophilic than phospholipids, and therefore form emulsions that are more stable against changes in pH and temperature than phospholipids. is a strong surfactant. For this reason, lysophospholipids are said to have a wider range of applications than phospholipids in the fields of foods, cosmetics, pharmaceuticals, and the like.

Lysophospholipids are conventionally produced from natural phospholipid-containing materials. For example, ``Novel method for producing natural lysolecithin'' disclosed in Japanese Patent Application Laid-Open No. 55-315 can be cited as a representative method. According to this conventional method, pancreatin is applied to animal and plant tissue homogenates that contain a large amount of lecithin, and after the reaction, the proteins are heat-denatured by heat treatment, and the lysolecithin adsorbed therein is separated from the coagulated proteins by filtration. The target natural lysolecithin is obtained by separating by a series of methods such as organic solvent extraction, and then, in the case of organic solvent extraction, the solvent used is distilled off from the extract.

[Problem that the invention seeks to solve]

However, it has been observed that the lysolecithin thus obtained retains the M speech properties of phospholipase A2, which is a constituent enzyme of vancreatin. Phospholipase A2
It is known that it contains many disulfide bonds in its molecule, and therefore exhibits extremely low stability against heat, organic solvents, etc., and is not easily deactivated by these agents (
Biochim, Biophys, Acta, vol. 159, p. 113, 1968). Therefore, lysolecithin in which the enzyme activity of phospholipase A2 remains is used in foods, cosmetics, pharmaceuticals, etc. When used for J construction, for example, if phospholipids, which are the substrate, are present, p
Unless l-1 is 4 or less, this phospholipid is hydrolyzed to generate new fatty acids, which reduces the stability of the product and significantly reduces its commercial value. Therefore, the use of such lysolecithin is limited.

In order to obtain a lipulin lipid-containing product that has substantially no residual enzyme activity of phospholipase A2, the manufacturing process is carried out in combination with solvent fractionation using various solvents, column treatment using silica gel, etc. Although the objective can be achieved to some extent, various problems are recognized, such as the operation takes too much time and cost, and the yield also decreases.

Under these circumstances, an object of the present invention is to provide a method for easily producing a lysophospholipid-containing material having substantially no residual enzymatic activity on an industrial scale and at a high yield. .

(Means for solving the problem) As a result of extensive research in accordance with the above purpose, the present inventor has found that
After subjecting a natural phospholipid-containing substance to an enzymatic reaction, if it is dried until its moisture content is 10% or less, residual enzymes can be easily removed by solvent extraction using a polar solvent and removal of the used solvent. The present inventors have discovered that a lysophospholipid-containing material having substantially no activity can be produced in a much higher yield than conventional methods, and have completed the present invention.

The present invention provides phospholipase A to natural phospholipid-containing substances.
2 preparation or an enzyme preparation containing 2 is applied to phospholipase to decompose the phospholipids in the above-mentioned substances into lysophospholipids, and then this substance is dried until the water content becomes 10% or less, and then the polar solvent is removed. The present invention provides a method for producing a lysophospholipid-containing product having substantially no residual enzymatic activity, the method comprising extracting lysophospholipids from this substance using be.

The natural phospholipid-containing substances used in the method of the present invention are substances containing phospholipids derived from living organisms such as animals, plants, and microorganisms. body, such as chicken egg yolk, cow brain,
Pig brain, soybean, rapeseed, chlorella cells, filamentous fungal cells (such as A. fungus cells); and crude phospholipid extracts extracted from these animal and plant tissues or microbial cells, such as commercially available soybean phospholipids (usually containing phospholipids). ff160% or more),
Examples include commercially available egg yolk phospholipids (usually phospholipid content of 30% or more). In addition, when using, for example, animal and plant tissues, microorganism bodies, etc., it is preferable to use these in crushed or liquid form in order to effectively carry out the enzyme action.

According to the method of the present invention, a phospholipid-containing substance as described above is first treated with a phospholipase A2 preparation or an enzyme preparation containing phospholipase A2 to decompose the phospholipid in the substance into lysophospholipid. put it here,
Phospholipase A2 preparations or enzyme preparations containing phospholipase A2 include, for example, a mixture of enzymes extracted from animal pancreas, namely vancreatin; vancreatin is subjected to heat treatment to inactivate protease and lipase to enrich phospholipase A2. (Japanese Unexamined Patent Publication No. 1983-8)
(Refer to Publication No. 8040); Purified phospholipase A2 preparation derived from animal pancreas: Phospholipase A derived from snake venom and venom
21J agent or the like may be used.

These commercially available products are preferably used.

Enzyme reactions using these enzyme preparations may be carried out under appropriately determined conditions according to conventional methods, and are not particularly limited in the present invention. Incidentally, in order to increase the conversion rate to lysophospholipids, the decomposition time by the enzyme may be lengthened.

According to the method of the present invention, the reaction product obtained by the enzymatic reaction is then dried until its water content is reduced to 10% or less. Setting the water content m to 10% or less is a critical condition for the method of the present invention. As is clear from the results of Test Example 1 described later, when the water content a exceeds 10%, a part of the enzyme used is likely to be transferred into the extract in the next solvent extraction step, so that the enzymatic activity of phospholipase A2 remains. This is because you will be able to get the final product that you are looking for.

At this time, the drying means is not particularly limited, and any conventional drying means may be used. However, the temperature of the reaction product should not exceed 80° C. during drying so as not to overheat the reaction product and cause thermal denaturation. When methods such as spray drying and freeze drying are used, the temperature of the reaction product itself is only about 60°C, so the proteins present therein are not denatured by heat at all or hardly.
Therefore, as is clear from the results of Test Example 2 described below, the extraction efficiency of lysophospholipids is good in the next solvent extraction step, and the yield of the final product can be increased.

Note that the reaction product dried to a moisture content of 10% or less is usually in the form of a powder.

According to the method of the invention, the powder obtained as described above is then subjected to solvent extraction with a polar solvent to extract lysophospholipids from this material. In this case, the polar solvent used is, but not particularly limited to, ethanol, methanol, a chloroform-methanol mixture, ethyl acetate, etc., and ethanol and methanol are particularly preferably used.

Note that non-polar solvents such as hexadiene and acetone have low extraction power for lysophospholipids, and are therefore not preferred in the present invention. Further, solvent extraction may be carried out according to a conventional method and is not particularly limited.

According to the method of the present invention, by distilling off the solvent used from the extract obtained in this way under reduced pressure, for example, a lysophospholipid-containing material having substantially no residual enzyme activity can be obtained.

This final product has a phospholipid content of 1% in the starting material.
It is converted into lysophospholipids at a conversion rate of 0 to 100%, and the lysophospholipids usually include lysophosphatidylcholine (LPG), lysophosphatidylethanolamine (LPE), and lysophosphatidic acid (L).
PA), lysophosphatidylinositol (LP), lysophosphatidylserine (LPS), etc. Therefore, the total composition of this final product is, for example, 68% neutral lipids and 32% phospholipids (of which If the proportion of lysophospholipids in the final product is less than 10%, the properties of lysophospholipids will deteriorate when used in the fields of food, cosmetics, pharmaceuticals, etc. This makes it difficult to obtain a product that retains its enzyme activity.Also, although this final product has virtually no residual enzyme activity,
Here, "having substantially no residual enzymatic activity" means that the enzymatic activity of residual phospholipase A2 is 0.11 u or less per 1 g of the final product. However, 11U
(1 international type position) indicates the level of enzyme activity that separates 1 μmol of fatty acid from the substrate T1 per minute. Residual enzyme activity is 0
．． If it exceeds 11 U/g, when this product is blended into other products as a raw material, enzymatic reactions will proceed during storage and free fatty acids will be produced, resulting in 1G of deterioration of the product.

Since the lysophospholipid-containing material obtained by the method of the present invention has substantially no residual enzymatic activity as described above, conventional phospholipids have thermal properties, such as When used as a surfactant, it can form stable emulsions even at high temperatures and in a wide DH range, and when used in the production of foods, cosmetics, pharmaceuticals, etc., it can improve the storage stability of these products.
Therefore, in these fields, we can expect the development of products for which conventional phospholipids or lysophospholipid S cargoes could not be used as raw materials.

[For production]

Conventional lysophospholipid-containing products! Compared to the FJ manufacturing method, typically the method shown in Japanese Patent Application Laid-Open No. 55-315, the method of the present invention involves subjecting a natural phospholipid-containing substance to an enzymatic reaction and then producing lysophospholipids. Prior to solvent extraction, instead of subjecting the enzymatic reaction product to heat treatment and filtration as conventionally done, the enzymatic reaction product was dried until the water content was 10% or less. Although it is not clear why a lysophospholipid-containing substance with virtually no residual enzymatic activity can be obtained, it is likely that the results of the test examples described below are present in coagulated proteins that have been simply filtered as in the conventional method. As is clearer, this is probably because about 28% of the water is still preserved.
Then, when subjected to 'FJ medium extraction, a part of the enzyme used, which was not inactivated even after heat treatment, was incorporated into the lysophospholipid micelles formed by the presence of water, and transferred directly into the extract solution. In contrast, according to the method of the present invention, the enzyme reaction product undergoes no or almost no thermal denaturation and its water content is reduced to 10% or less, so that the enzyme used during extraction is transferred to the extract solution. Therefore, it is presumed that a lysophospholipid-containing product having substantially no residual pre-M activity can be obtained in high yield.

〔Effect of the invention〕

The effects of the present invention will be explained with the results of Test Examples 1 and 2 below. In the present invention, all percentages are percentages by weight.

Test Example 1 This test example shows how the final product obtained by reducing the water content of the enzyme reaction product to 10% or less in the method of the present invention has substantially no residual enzyme activity. show.

10 chicken egg yolks (1g with bank creatin (Wako Jun'am>5
A solution of K9 dissolved in 10 ρ of fresh water was added, and an enzymatic reaction was carried out at 35 to 45° C. for 6 hours while stirring and maintaining p +−+ at 7.0 to 8.0 with a 1N aqueous sodium hydroxide solution. The obtained enzymatic reaction product was directly subjected to spray drying at an intake air temperature of 130 to 150°C.

Exhaust temperature: 50-75℃, temperature of reaction product itself: 50
~60°C), moisture content m4, 8% (D dry 1n
42 kg of yellow was obtained.

Next, 1,009 pieces of each of the dried egg yolks obtained in this manner were placed in a prepared 2p beaker, and after performing a humidifying operation, they were left to stand for one day to homogenize the whole, and the water content was 4.
8%, 9.1%, 13.6% and 21.0% powders were obtained.

Add 1 fJ of ethanol to each car containing the above-mentioned water-containing powder, perform an extraction operation with stirring at 40-45°C for 10 minutes, and then filter the resulting extract under vacuum. After concentration, yellow paste-like lysophospholipid-containing products were obtained.

Each content was then tested for yield and residual enzyme activity. The results are shown in Table 1 below. The water content was measured using a Kerato moisture meter (temperature: 105°C), and the residual enzyme activity of phospholipase A2 was measured using Biochim.
, Biophys, ACta, .

This was carried out according to the method described in Vol. 159, p. 105 (1968).

Table 1 From the results in Table 1 above, it can be seen that residual enzyme activity is difficult to recognize when the water content is 10% or less, whereas residual enzyme activity is considerably recognized when the water content exceeds 10%.

Test Example 2 In this test example, the method of the present invention was used in place of the conventional method (method disclosed in JP-A No. 55-315) in which the enzymatic reaction product was heated and then filtered. shows how the final product can be advantageously obtained in terms of yield and residual enzyme activity by drying the product to a moisture content of less than 10%.

Egg yolk liquid 2 KW obtained after the enzymatic reaction obtained according to the method of Test Example 1 above was heat denatured at 95 to 100°C for 1 hour, and then dehydrated by pressure filtration to form a solid with a water content of 28.0%. 1194 g of product (A) was obtained. At this time, it was not possible to further reduce the moisture level m by pressurizing or overpressuring.

500 g of the solid material (A) thus obtained was freeze-dried to yield 365 g of a dried material (B) with a moisture content of 1.6%.

On the other hand, 2 kg of the enzymatically reacted egg yolk liquid obtained according to the method of Test Example 1 above was subjected to spray drying without being subjected to heat denaturation treatment. Intake temperature: 130 to 150°C, exhaust temperature = 5
0-75°C, temperature of the reaction product itself = 50-60°C),
840 g of powder (C) with a moisture content of 4.3% was obtained.

Thus obtained 1llA, B and C each 200
9 and then add 21 ethanol to 40 to 45
After performing an extraction operation under stirring at .degree. C. for 10 minutes, the resulting extract was filtered and subjected to vacuum concentration to obtain a yellow paste-like lysophospholipid-containing material.

Each content was then tested for yield and residual enzyme activity. The results are shown in Table 2 below.

Table 2 From the results in Table 2 above, it can be seen that A has a high water content, and a portion of the enzyme used during solvent extraction migrates into the extract, resulting in a final product with considerable residual enzyme activity. The apparent yield is high because proteins, etc. are extracted in addition to lipids, and since B has a low water content, the final product has almost no residual enzyme activity. Because of this, lysophospholipids are difficult to extract during solvent extraction, and the yield of the final product is quite low.
Therefore, it can be seen that the yield of the final product is high and that residual enzyme activity is hardly noticeable.

〔Example〕

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

Example 1 100 kg of chicken egg yolk and 5 pieces of bank creatin (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
A solution prepared by dissolving 3 in 101 fresh water was added to the solution, and the enzyme reaction was carried out at 35 to 45° C. for 6 hours while stirring and maintaining the pH at 7.0 to 8.0 with a 1N aqueous sodium hydroxide solution. The obtained enzymatic reaction product was directly subjected to spray drying; intake air temperature = 130-150°C, exhaust temperature = 50-75°C, temperature of the reaction product itself: 50-60°C>, moisture content 5.2%.
42.5 kg of dried egg yolk was obtained.

Add 400g of ethanol to this dried egg yolk and add 40 to 45
The extract obtained by 110 filtration was subjected to an extraction operation under stirring for 10 minutes at 110 °C and was then concentrated in vacuum (with a product temperature of 30 °C or less).
A yellow paste-like lysophospholipid-containing material of 19.5 Ny (yield: 19.5%) was obtained.

Next, the lipid composition of this inclusion was determined by Iatroski1? NTH
When investigated using IO (manufactured by Fil Yatron) under the following measurement conditions, 67.9% of neutral lipids (mainly triglycerides and cholesterol) and 32.1% of phospholipids (
Behind this, LPC and LPE were 30.6%).

Measurement conditions Rod: 0 mA 5-II (silica gel) g1
g1 Solvent: Chloroform: Methanol: Water 80
:35 :3 (V/V/V) Development distance 11:10crR Further, the final product was examined for residual enzyme activity and found to be 0.
It was 11U/9 or less. In addition, residual activity of about 201 U/g was observed in the residue after separating the extract.

Example 2 Commercially available soybean phospholipid product (phospholipid content ff162%) 2
00 Kg commercially available purified phospholipase A,,! 1 drug (
A solution derived from primary pancreas: 1001tJ/119>709 dissolved in clear water 31 was added, and an enzymatic reaction was carried out at 50 to 55°C for 24 hours while stirring and maintaining the pH at 8.5 with a 4N aqueous calcium hydroxide solution. Next, 200 kg of fresh water
After adding 40 kg of Textrin powder as a bulking agent and emulsifying it with a homogenizer, it was spray-dried. ff13.8% (7)
Powder 228Kgel? Ta.

Add 2000M methanol to this powder and
After extraction under stirring for 20 minutes, the resulting extract was filtered and concentrated in vacuo to obtain 18ig of brown paste (yield: 91%).

Next, the lipid composition of this paste was examined in the same manner as in Example 1, and it was found that neutral lipids (mainly triglycerides and sterols) were 22.0% and phospholipids [78.0%].
% (34.2% of which was Lyso type).

Further, the residual M speech property was 0.11 U/9 or less.

A commercially available phospholipase A2 preparation (300 IU/9) of a commercially available phospholipase A2 preparation (snake venom-derived product, 300 IU/9) was dissolved in 51 liters of fresh water, and added to 150 kg of crushed bovine brain that had been breathed with 11 U. 40-50℃ while stirring
The enzymatic reaction was carried out for 6 hours. The obtained enzymatic reaction product was directly freeze-dried to obtain a dry product with a water content of 11.4%, 68.4 to 9.

Add ethanol 68ON to this dried product and heat at 40-45°C.
After extraction with stirring for 10 minutes, the extract obtained by filtration was concentrated in vacuo to obtain 10.2 g (yield: 6.8%) of a yellow base I-type lysophospholipid-containing substance. Obtained.

Next, the lipid composition of this content was investigated in the same manner as in Example 1, and it was found that 20% of neutral lipids (mainly cholesterol) and 46% of phospholipids (of which 21% were lyso-type).
) and glycolipids (mainly cerebrosides) were 34%.

Further, the residual enzyme activity was 0.11U157 or less.

Claims (1)

[Claims] 1. After treating a natural phospholipid-containing substance with a phospholipase A_2 preparation or an enzyme preparation containing phospholipase A_2 to decompose the phospholipid in the above-mentioned substance into lysophospholipid, the water content of this substance is reduced. Drying until the content is 10% or less, then extracting lysophospholipids from this substance using a polar solvent, and distilling off the polar solvent from this extract, substantially eliminating residual enzyme activity. A method for producing a lysophospholipid-containing product. 2. The method for producing a lysophospholipid-containing product having substantially no residual enzyme activity according to claim 1, wherein drying is carried out by spray drying or freeze drying. 3. A method for producing a lysophospholipid-containing material having substantially no residual enzyme activity according to claim 1, which uses ethanol or methanol as a polar solvent.