Phospholipid composition, fat and oil composition containing the same and process for producing phosphatidic acids

A phospholipid composition which satisfies the following requirements (i) and (ii): PA1 (i) a weight ratio of a nitrogen-containing phospholipid to the sum of a phospholipid, a glycolipid and a sterol derivative of less than 0.5; and PA1 (ii) a ratio of an area of a high-polar substance on a silica gel thin-layer chromatogram to the sum of areas of a phospholipid, a glycolipid and a sterol derivative on a silica gel thin-layer chromatogram of less than 500 area/.mu.g. A fat and oil composition containing from 0.001 to 30% by weight of the phospholipid composition is also disclosed. The present invention enables the blending of phospholipids with a frying oil, which has been considered difficult since it causes heat coloration. Thus a fat and oil composition, which is excellent in mold-release characteristics during cooking, has a good smell during heating, suffers from no coloration of oil after heating and shows a good flavor, can be obtained. A process for producing phosphatidic acids and lysophosphatidic acids is further disclosed.

FIELD OF THE INVENTION 
This invention relates to a phospholipid composition and a fat and oil 
composition containing the same. More particularly, it relates to an 
edible phospholipid composition which is excellent in preventing the 
adhesion of a material to a heating device during heating and shows a 
remarkably improved color even after heating. In particular, the 
phospholipid composition of the present invention is excellent in 
preventing the adhesion of a food material to a cooking device (for 
example, baking mold, baking plate) during heating and in suppressing 
sputtering during a cooking process, has a good flavor and does not suffer 
from any browning or coloration of phospholipids due to heating or 
generation of any offensive smell caused by the thermal decomposition or 
denaturation of phospholipids. 
This invention also relates to a process for producing phosphatidic acids 
(hereinafter sometimes referred to simply as PAs) and/or lysophosphatidic 
acids (hereinafter sometimes referred to simply as L-PAs) of a high 
purity. 
BACKGROUND OF THE INVENTION 
Phospholipids, which are basic substances constituting biomembranes, belong 
to lipids controlling the fundamental living activities, for example, 
protection of cell tissues, mediation of information, adjustment of 
material transfer. 
In recent years, it has attracted scientific and industrial attraction that 
various functional substances can be encapsulated in vesicles (or 
liposomes) consisting of phospholipids capable of forming bilayer 
membranes. In the field of pharmaceutical science and medicine, for 
example, it is expected that this phenomenon is applicable to a drug 
delivery system (DDS). 
The present inventors have tried to apply such high-functional lipids to 
the food industry and they previously succeeded in the development of a 
cooking oil suffering from no sputtering and having good mold release 
characteristics as disclosed in JP-A-1-27431 (the term "JP-A" as used 
herein means an "unexamined published Japanese patent application"). 
Examples of the industrial application of PA include, furthermore, 
improvement of dough properties in a baking process as disclosed in 
JP-A-58-51853, production of an emulsifier comprising PA and a zein 
complex as disclosed in JP-A-62-204838, application to drugs as disclosed 
in JP-A-54-105222, JP-A-55-11582, JP-A-56-127308 and JP-A-60-255728, 
application to cosmetics as disclosed in JP-A-59-27809 and application to 
chemical products as disclosed in JP-A-53-108503 and JP-A-60-243171. 
Namely, attempts have been made to use PA in various industrial fields. 
A known method for producing PA comprises treating lecithin with ground 
oilseeds or an oilseed extract. However, the product thus obtained is 
contaminated with impurities and thus should be purified. Phospholipids 
including PA and L-PA are commonly purified by column chromatography. In 
the purification by silicic acid column chromatography, in particular, 
each component can be eluted and fractionated by varying the polarity of 
the development solvent. A chloroform/methanol mixture is used as a 
development solvent for purifying PA and L-PA and these products are 
separated from each other by changing the polarity of the solvent by 
varying the mixing ratio. However, there arises a problem of the 
contamination with impurities and thus the product should be passed 
through the column several times in order to obtain a specimen of a high 
purity, which causes some disadvantages including an increase in the 
amount of the solvent to be used and a decrease in the yield. It is also 
proposed to separate PA and L-PA by thin layer chromatography and 
detecting each product with a non-decomposition reagent, followed by 
scratching and extracting the portion containing PA or L-PA from the 
plate. However, this method is seemingly ineffective from an industrial 
viewpoint, since the yield thus achieved is limited. 
On the other hand, there has been known that lecithin is specifically 
effective, compared with a number of other surfactants, in improving the 
mold-release characteristics of food materials which are liable to undergo 
heat adhesion (for example, sponge cake dough, egg roll). 
Typical examples of commonly employed lecithin, which comprises a 
phospholipid mixture comprising nitrogen-containing phospholipids (for 
example, phosphatidylcholine, phosphatidylethanolamine) as the main 
components, are obtained by extracting or purifying soybean, yolk or the 
like. 
These natural lecithins such as soybean lecithin and yolk lecithin are 
unstable and undergo browning when heated to 150.degree. C. or above, even 
in a fat-protected state, thus turning into a dark brown. 
The present inventors found out that a fat and oil composition containing 
0.1 % by weight or more of phospholipids, from which the 
nitrogen-containing phospholipids causing the heat coloration had been 
removed by, for example, an enzymatic treatment, would never undergo heat 
coloration when used in frizzling foods and have already applied the same 
for a patent (JP-A-2-27943). 
However, a fat and oil composition containing from 0.01 to 30 % by weight 
of such phospholipids free from any nitrogen-containing phospholipids 
still suffers from disadvantages that the oil per se is seriously colored 
when used at a higher temperature for a long period of time (for example, 
in frying). 
SUMMARY OF THE INVENTION 
The present inventors have conducted extensive studies in order to solve 
the above problems. As a result, they have found that there are some 
substances causing heat coloration other than the nitrogen-containing 
phospholipids. As the results of detailed examinations on the coloring 
substances other than the nitrogen-containing phospholipids, it has been 
found out that high-polar substances remaining around the original point 
in normal phase thin-layer chromatography (hereinafter sometimes referred 
to as TLC) would cause the heat coloration of the oil per se. 
Based on the above finding, a phospholipid composition, from which 
high-polar substances and nitrogen-containing phospholipids have been 
removed, is prepared by subjecting a starting material, which is a natural 
lecithin, preferably a lecithin treated with an enzyme (for example, 
phospholipase D, phospholipase A2) or fractionated so as to lower the 
contents of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and 
to elevate the contents of a phosphatidic acid (PA), a lysophosphatidic 
acid (L-PA) and a phosphatidylinositol (PI) to a definite level or above, 
to, for example, a column treatment. Then the present inventors have found 
that the fat and oil composition containing the phospholipid composition 
free from any high-polar substances or nitrogen-containing phospholipids 
would never undergo coloration of the oil per se, even when used as a 
frying oil at a high temperature, thus completing the present invention. 
Accordingly, the present invention provides a phospholipid composition 
which satisfies the following requirements (i) and (ii): 
(i) a weight ratio of a nitrogen-containing phospholipid to the sum of a 
phospholipid, a glycolipid and a sterol derivative of less than 0.5; and 
(ii) a ratio of an area of a high-polar substance in a silica gel 
thin-layer chromatogram to the sum of areas of a phospholipid, a 
glycolipid and a sterol derivative in a silica gel thin-layer chromatogram 
of less than 500 area/.mu.g. 
The present invention further provides a fat and oil composition containing 
from 0.001 to 30% by weight of the phospholipid composition as well as a 
fat and oil composition containing from 0.001 to 30% by weight of the 
phospholipid composition and from 5 to 99% by weight of a diglyceride 
component. 
The present invention furthermore provides a process for producing 
phosphatidic acids and/or lysophosphatidic acids which comprises purifying 
a phospholipid mixture containing a phosphatidic acid and/or a 
lysophosphatidic acid in a purification step comprising a step (a) and 
then washing a polyvalent metal salt of the phosphatidic acid and/or the 
lysophosphatidic acid obtained in the step (a) with an aqueous 
water-soluble solvent under an acidic condition, wherein the step (a) 
comprises: 
dispersing or dissolving the phospholipid mixture containing the 
phosphatidic acid and/or the lysophosphatidic acid in water or a nonpolar 
solvent and then 
reacting the phosphatidic acid and/or the lysophosphatidic acid with an 
aqueous solution of a polyvalent metal salt in the presence of a polar 
solvent under an alkaline condition to thereby give the polyvalent metal 
salt of the phosphatidic acid and/or the lysophosphatidic acid. 
The present invention still furthermore provides a process for producing 
phosphatidic acids and/or lysophosphatidic acids which comprises purifying 
a phospholipid mixture containing a phosphatidic acid and/or a 
lysophosphatidic acid in a purification step comprising the step (a) and a 
step (b) and then washing a polyvalent metal salt of the phosphatidic acid 
and/or the lysophosphatidic acid obtained in the step (b) with an aqueous 
water-soluble solvent under an acidic condition, wherein the step (b) 
comprises: 
adding a substance capable of precipitating a polyvalent metal salt of a 
phosphatidic acid and/or a lysophosphatidic acid into the polyvalent metal 
salt of the phosphatidic acid and/or the lysophosphatidic acid obtained in 
the step (a) in the presence of a nonpolar solvent to precipitate the 
polyvalent metal salt of the phosphatidic acid and/or the lysophosphatidic 
acid and then 
separating the polyvalent metal salt of the phosphatidic acid and/or the 
lysophosphatidic acid. 
The expression "phosphatidic acids and lysophosphatidic acids" as used 
herein means a metal salt of a phosphatidic acid and a lysophosphatidic 
acid and a free phosphatidic acid and lysophosphatidic acid which do not 
contain metal atom(s).

DETAILED DESCRIPTION OF THE INVENTION 
The term "high-polar substance" as used herein means those located around 
the original point (i.e., Rf value of from 0 to 0.1) in a silica gel thin 
layer chromatogram when a phospholipid is developed in silica gel 
thin-layer chromatography with chloroform/acetone/methanol/acetic 
acid/water (10:4:2:2:1). Details of technique, procedures and the like for 
thin-layer chromatography which may be used in the present invention is 
disclosed, for example, in "Shishitsu Bunseki-Ho Nyumon (Course of Method 
for LipidAnalysis)", Seibutsu Kagaku Jikken-Ho (Method for Biochemical 
Experiment), volume 9, published by Gakkai Shuppan Center (1985). These 
high-polar substances involve various substances such as those having 
primary amine groups such as amino acids, those formed through the 
decomposition of polar moieties of phospholipids, as disclosed, for 
example, in Tomioka and Kaneda, Kagaku to Seibutsu (Chemical and 
Organism), 14, 509 (1976) and it is highly difficult to determine each of 
these substances. The content of the high-polar substances may be 
conveniently determined by thin layer chromatography. Namely, a silica gel 
plate developed in the above-mentioned manner is allowed to undergo 
color-development with the use of sulfuric acid and the area of spots on 
the thin-layer chromatogram thus developed is measured with a TLC scanner. 
The high-polar substance content is expressed by the value of the area 
thus measured in the unit of area/.mu.g. 
The term "phospholipid" as used herein involves a nitrogen-containing 
phospholipid and a phospholipid which does not contain any nitrogen atoms. 
The term "phospholipid which does not contain any nitrogen atoms" as used 
herein means that containing a base which does not contain nitrogen atom 
and involves a phosphatidic acid (PA), a lysophosphatidic acid (L-PA), a 
phosphatidylinositol (PI) and a phosphatidylglycerol (PG). 
The term "nitrogen-containing phospholipid" as used herein involves 
nitrogen-containing glycerophospholipids such as a phosphatidylcholine 
(PC), a phosphatidylethanolamine (PE) and a phosphatidylserine (PS) as 
well as a sphingophospholipids such as a sphingomyelin (SM). 
The term glycolipid is used herein to mean a complex comprising 
sphingosines or similar amines, fatty acids and sugars (including amino 
sugars) and it can be classified roughly into (1) cerebrosides 
(galactolipides) (e.g., phrenosines), (2) sulfolipids (e.g., cerebron 
sulfuric acid) and (3) mucolipids (e.g., gangliosides and hematosides). 
Further, the term "sterol derivative" involves a sitosterol, a stigmasterol 
and a cholesterol. 
In the present invention, the phospholipids, glycolipids and sterol 
derivatives are separated from each other by chromatography and then 
determined. 
It is required that the weight ratio of the content of nitrogen-containing 
phospholipids of the phospholipid composition of the present invention to 
the total amount of phospholipids and glycolipids and sterol derivatives, 
if contained, in the phospholipid composition is less than 0.5, preferably 
less than 0.05, and more preferably less than 0.01. It is further required 
that the content of the high-polar substances is less than 500 area/.mu.g, 
preferably less than 200 area/.mu.g, in the area value as specified above 
based on the total area value of the phospholipids, glycolipids and sterol 
derivatives. A content of the nitrogen-containing phospholipids or the 
high-polar substances exceeding the level as specified above is 
undesirable from the viewpoints of, for example, odor and coloration. 
A phospholipid composition having the specific composition of the present 
invention can be obtained by, for example, a method in which a natural 
lecithin is used as a starting material, nitrogen-containing phospholipids 
(e.g., phosphatidylcholine, phosphatidylethanolamine) contained therein 
are decomposed with an enzyme (i.e., the formation of phosphatidic acid or 
lysophosphatidic acid), and then, the resulting products are fractionated 
with the use of a silicic acid column or an ion exchange column, as 
disclosed in "Seibutsu Kagaku [I]", Shin-Jikken Kagaku Koza (New Course of 
Experimental Chemistry), vol. 20, edited by The Chemical Society of Japan, 
page 457 (1985). 
One of the most convenient and effective methods therefor comprises 
selectively decomposing nitrogen-containing phospholipids (e.g., 
phosphatidylcholine, phosphatidylethanolamine) in soybean lecithin or yolk 
lecithin with the use of phospholipase D which is contained, for example, 
in cabbage leaves in a large amount, thus lowering the contents of these 
nitrogen-containing phospholipids and simultaneously elevating the 
contents of phosphatidic acid, lysophosphatidic acid and/or salts thereof 
(for example, sodium salt, calcium salt) and then removing high-polar 
substances from the resulting phospholipids, which is almost free from 
nitrogen-containing phospholipids, by using a silicic acid column, as 
disclosed in The Journal of Biological Chemistry, 239, (12), pages 
4066-4072 (1964). 
Further, a phospholipid composition of the present invention can more 
conveniently and efficiently be obtained by a process comprising purifying 
a phospholipid mixture containing a phosphatidic acid and/or a 
lysophosphatidic acid in a purification step comprising a step (a) and 
then washing a polyvalent meal salt of the phosphatidic acid and/or the 
lysophosphatidic acid obtained in the step (a) with an aqueous 
water-soluble solvent under an acidic condition, wherein the step (a) 
comprises: 
dispersing or dissolving the phospholipid mixture containing the 
phosphatidic acid and/or the lysophosphatidic acid in water or a nonpolar 
solvent and then 
reacting the phosphatidic acid and/or the lysophosphatidic acid with an 
aqueous solution of a polyvalent metal salt in the presence of a polar 
solvent under an alkaline condition to thereby give the polyvalent metal 
salt of the phosphatidic acid and/or the lysophosphatidic acid. 
In this process, the polyvalent metal salt of the phosphatidic acid and/or 
the lysophosphatidic acid obtained in the step (a) may further be 
subjected purification in a step (b), which comprises: 
adding a substance capable of precipitating a polyvalent metal salt of a 
phosphatidic acid and/or a lysophosphatidic acid into the polyvalent metal 
salt of the phosphatidic acid and/or the lysophosphatidic acid obtained in 
the step (a) in the presence of a nonpolar solvent to precipitate the 
polyvalent metal salt of the phosphatidic acid and/or the lysophosphatidic 
acid, and then 
separating the polyvalent metal salt of the phosphatidic acid and/or the 
lysophosphatidic acid. 
The phospholipid mixture containing PA or L-PA is available as a commercial 
product. Alternatively, it can be obtained by treating lecithin with 
ground oilseeds or an oilseed extract. The ground oilseeds may be obtained 
by grinding oilseeds, while the oilseed extract may be obtained by 
extracting the ground oilseeds and optionally followed by penetration 
purification by permeating through, for example, a membrane. These 
treatments may preferably be performed in the presence of an inert gas. 
Conventionally known treatment methods may be employed therefor. 
In the step (a), the phospholipid mixture containing the phosphatidic acid 
and/or the lysophosphatidic acid is dispersed or dissolved in water or a 
nonpolar solvent. The nonpolar solvent to be used in the present invention 
include a liquid organic compound containing 5 to 16 carbon atoms and 
having a dielectric constant of 10 or below. Examples of the liquid 
organic compound having a dielectric constant of 10 or below include 
hydrocarbons such as n-hexane and benzene, halogenated (e.g., a chlorine 
atom) hydrocarbons such as chloroform and hydrocarbons substituted with an 
aromatic hydrocarbon (e.g., phenyl) and the like, each containing 5 to 16 
carbon atoms. Among them, hydrocarbons having 5 to 16 carbon atoms are 
preferred and n-hexane is particularly preferred in view of safety. 
The dielectric constant as used herein is determined in accordance with the 
description on the dielectric constants of liquid organic compounds and 
liquids in Kagaku Binran Kiso-Hen, Kaitei 2-Hah (Chemical Handbook Basic 
Volume, the second revised edition), edited by Chemical Society of Japan 
(pages 1166-1168 and 1582-1583) (1975). 
Subsequently, a mixture of an aqueous solution of a polyvalent metal salt 
and a polar solvent is added to the dispersion or solution of the 
phospholipid mixture in an amount of 0.1 time by volume or more, 
preferably 0.4 to 0.8 times by volume, based on the water or the non-polar 
solvent. The volume ratio of the aqueous solution of the polyvalent metal 
salt to the polar solvent in this mixture may preferably be 0.1 or above, 
more preferably be from 0.5 to 2.0. The polar solvent to be used in this 
step includes a liquid organic compound containing 1 to 10 carbon atoms 
and having a dielectric constant exceeding 10. Examples of the liquid 
organic compound having a dielectric constant exceeding 10 include 
monohydric alcohols (e.g., methanol, ethanol), ketones (e.g., acetone) and 
polyhydric alcohols (e.g., ethylene glycol, glycerine) each containing 1 
to 10 carbon atoms. In view of safety, it is particularly preferable to 
use ethanol therefor. Examples of the polyvalent metal salt to be 
contained in the aqueous solution of the polyvalent metal salt include 
chlorides, sulfates, carbonates and phosphates of elements of the group 
IIA (for example, Mg, Ca, Sr, Ba) and those of the group IIIB (for 
example, Al). Among these polyvalent metal salts, chlorides are preferable 
and Ca salts are more preferable. In particular, calcium chloride is 
suitable therefor. The concentration of the polyvalent metal salt aqueous 
solution may be 1 time by mol or above, preferably 1 to 7 times by mol, 
based on the mole number of the phospholipid mixture. 
The resulting mixture is stirred under an alkaline condition at a pH value 
of 7.0 or above, preferably pH value of 10 to 12, at a temperature of 
0.degree. to 80.degree. C., preferably a temperature of 0.degree. to 
40.degree. C., for 0.5 hours or longer. 
Thus a mixture containing the polyvalent metal salt of PA and/or L-PA is 
obtained. 
In the purification step of the present invention, the polyvalent metal 
salt of PA and/or L-PA formed in the step (a) may further be subjected to 
the step (b) to selectively precipitate the polyvalent metal salt of PA 
and/or L-PA. 
In the case where the mixture containing the polyvalent metal salt of PA 
and/or L-PA obtained in the step (a) is in the form of a solution of a 
nonpolar solvent, the nonpolar solvent phase containing the polyvalent 
metal salt of PA and/or L-PA is separated from the mixture by a known 
procedure such as allowing to stand or centrifugation and the resulting 
nonpolar solvent phase is then subjected to the step (b). In the case 
where the mixture obtained in the step (a) is in the form of an aqueous 
dispersion, the mixture is preliminary dehydrated, desolvated and 
dissolved in a nonpolar solvent to give a nonpolar solvent solution of the 
polyvalent metal salt of PA and/or L-PA and then the resulting nonpolar 
solution is subjected to the step (b). Examples of the nonpolar solvent to 
be used in this step include those employed in the step (a). 
Next, a substance capable of precipitating polyvalent metal salt of PA 
and/or L-PA is added to the nonpolar solvent solution of the polyvalent 
metal salt of PA and/or L-PA in an amount of 0.1 time by volume or above, 
preferably from 0.1 to 2.0 times by volume, based on the nonpolar solvent 
solution to selectively precipitate the polyvalent metal salt of PA and/or 
L-PA. In this step, it is preferred that the nonpolar solvent containing 
the polyvalent metal salt of PA and/or L-PA is aged for 0.5 hours or 
longer under stirring. The polyvalent metal salt of PA and/or L-PA thus 
precipitated may be separated from the supernatant by known methods. When 
the amount of the added substance is less than 0.1 time by volume, the 
polyvalent metal salt of PA and/or L-PA are not precipitated. When the 
amount thereof exceeds 2.0 times by volume, on the other hand, impurities 
dissolved in the nonpolar solvent is extracted by the polar solvent, which 
lowers the purity of the obtained polyvalent metal salt of PA and/or L-PA. 
As the substance capable of selectively precipitating a polyvalent metal 
salt of PA and/or L-PA, a lipid and a polar solvent containing 1 to 10 
carbon atoms and having a polar group may be used. Examples of the lipids 
include monoglycerides, diglycerides, triglycerides and mixtures thereof. 
For instance, monoglycerides, diglycerides and triglycerides having 
saturated fatty acid residues or unsaturated fatty acid residues 
containing 8 to 24 carbon atoms may be used therefor. Among them, those 
having a slipping point, as defined in Kijun Yushi Bunseki Siken Ho 
(Standard Methods for the Analysis of Oils, Fats and Derivatives), Item 
2.3.4.2-90, published by The Japan Oil Chemists' Society (1991), of less 
than 20.degree. C. are preferably used, and, in view of safety, edible 
fats and oils, in particular, edible oils are more preferably used. 
Specific examples thereof include vegetable oils such as soybean oil, 
rapeseed oil, sunflower oil, corn oil, rice bran oil and cottonseed oil; 
and animal oils such as fish oil. As the polar solvent containing 1 to 10 
carbon atoms and having a polar group, liquid organic compounds having a 
dielectric constant exceeding 10 may be used. The liquid organic compounds 
having a dielectric constant exceeding 10 include monohydric alcohols, 
ketones, polyhydric alcohols and a mixture thereof. Examples of the 
monohydric alcohols include methanol, ethanol and isopropanol. Examples of 
the ketones include acetone and methyl ethyl ketone. Examples of the 
polyhydric alcohols include ethylene glycol and glycerol. Among them, a 
monohydric alcohol is particularly preferred. 
In order to elevate the purity of the polyvalent metal salt of PA or L-PA, 
it is preferable to repeat the step (b) twice or more. Namely, the 
obtained precipitate is further purified by dissolving again in a nonpolar 
solvent such as n-hexane and adding a substance capable of selectively 
precipitating the polyvalent metal salts of PA and/or L-PA, followed by 
separating the precipitated polyvalent metal salt of PA and/or L-PA. 
After the completion of the purification, the precipitate is washed with an 
aqueous water-soluble solvent under an acidic condition. This washing may 
preferably be performed at a pH value of 0.5 to 5 at a temperature of 
0.degree. to 40.degree. C. with the use of 5 times by weight or more, 
based on the solid matters, of the aqueous water-soluble solvent. Among 
the liquid organic compound containing 1 to 10 carbon atoms, which are 
exemplified above as the polar solvent, those having a dielectric constant 
exceeding 20 may be used as the water-soluble solvent to be used in this 
step. The liquid organic compounds having a dielectric constant exceeding 
20 include monohydric alcohols, ketones, polyhydric alcohols and mixtures 
thereof. Examples of the monohydric alcohols include methanol, ethanol and 
isopropanol. Examples of the ketones include acetone and methyl ethyl 
ketone. Examples of the polyhydric alcohols include ethylene glycol and 
glycerol. Ethanol is preferably selected therefor. The ratio of water to 
the water-soluble solvent in the aqueous water-soluble solvent may 
preferably range from 5 to 80% by weight. 
The polyvalent metal salt of PA and/or L-PA thus obtained may be formulated 
into various salt compounds such as sodium salts by a known salt-exchange 
procedure. Alternately, they may be desalted by a known method to thereby 
give free PA and/or L-PA which do not contain metal atoms. 
The fat and oil composition of the present invention contains from 0.001 to 
30% by weight of the phospholipid composition of the present invention. 
When the content of the phospholipid composition is less than 0.001% by 
weight, the effects of the present invention cannot be achieved in a fat 
and oil composition. On the other hand, a content of the phospholipid 
composition exceeding 30% by weight might cause some troubles in handling 
the fat and oil composition, for example, an increase in viscosity. 
It is reasonable to regard the content of acetone-insoluble matters as the 
content of the phospholipids content in the fat and oil composition of the 
present invention because the content of acetone-insoluble matters is 
conventionally regarded as the total phospholipids content and this 
accords with the definition of the lecithin content specified in Shokuhin 
Tenkabutsu Koteisyo (Food Additives Official Book). 
As the fat to be used in the fat and oil composition of the present 
invention, one or more materials may be selected from among, for example, 
vegetable oils such as soybean oil, rapeseed oil, palm oil, corn oil, 
cotton seed oil, coconut oil, palm kernel oil, rice bran oil, sesame oil, 
safflower oil, high-oleic safflower oil, sunflower oil and high-oleic 
sunflower oil; animal fats such as beef tallow, lard, fish oil, whale oil 
and milk fat; and those obtained by fractionating, hydrogenating or 
ester-exchanging these fats or oils. 
It is preferable that the fat and oil composition of the present invention 
further contains from 5 to 99% by weight of a diglyceride component. As 
the diglyceride component to be used in the present invention, a 
composition which satisfies the followings requirements is preferred: 
(1) a weight ratio of diglycerides to monoglycerides ranges from 5:1 to 
990:1; 
(2) diglycerides are contained so as to give a content, based on the total 
of the fats or oil composition, of from 5 to 99% by weight, preferably 
from 8 to 80% by weight; 
(3) fatty acid residues constituting diglycerides contain 8 to 24 carbon 
atoms and a content of unsaturated fatty acid residues is 70% by weight or 
more based on the fatty acid residues; and 
(4) diglycerides comprise 40% by weight or less of saturated/unsaturated 
fatty acid diglycerides, 5% by weight or less of saturated/saturated fatty 
acid diglycerides and the balance of unsaturated/unsaturated fatty acid 
diglycerides. 
When the diglyceride content in the fat and oil composition of the present 
invention is less than 5% by weight, dissolution of the phospholipid 
composition is poor in the resulting fat and oil composition. When the fat 
and oil composition contains a large amount of the diglyceride and the 
content of monoglycerides is also high, the fat and oil composition 
frequently shows fuming during heating. Thus it is particularly preferable 
that the diglyceride content ranges from 8 to 80% by weight. Similar to 
diglycerides, monoglycerides are effective in increasing the dissolution 
of phospholipids. However, differing from diglycerides, monoglycerides 
cause serious fuming during heating even contained in a small ratio (e.g., 
exceeding 10% by weight) in the fat and oil components. Therefore it is 
desirable to control the monoglyceride content in the fat and oil 
composition to 10% by weight or less, preferably 2% by weight or less. 
On the other hand, edible fats such as butter, shortening and lard have 
been widely used as an edible fat composition. In these fields, it has 
also been urgently required to develop a fat composition having the 
above-mentioned cooking properties (i.e., oxidation stability and heat 
resistance) and digestion properties (i.e., showing no oily feel). This 
requirement may be satisfied by providing the fat composition according to 
the present invention having a diglyceride component satisfying the 
following characteristics: 
(1) a weight ratio of diglycerides to monoglycerides ranges from 5:1 to 
990:1; 
(2) diglycerides are contained so as to give a content based on the total 
fat and oil content in the oil composition of from 5 to 99% by weight, 
preferably from 8 to 80% by weight; 
(3) fatty acid residues constituting diglycerides contain 8 to 24 carbon 
atoms; and 
(4) diglycerides comprise 40% by weight or more of saturated/unsaturated 
fatty acid diglycerides, 5% by weight or more of saturated/saturated fatty 
acid diglycerides and the balance of unsaturated/unsaturated fatty acid 
diglycerides. 
The content of the diglyceride component may preferably be elevated by 
adding fats rich in diglycerides which have been obtained by 
ester-exchanging a mixture of one or more fats or oil having a high 
unsaturated fatty acid residue content (for example, safflower oil, olive 
oil, cotton seed oil, rapeseed oil, corn oil, soybean oil, palm oil, rice 
bran oil, sunflower oil, sesame oil, lard, beef tallow, fish oil, butter 
or those obtained by fractionating, randomizing, hardening or 
ester-exchanging these fats or oils) with glycerol in a manner as 
disclosed, for example, in U.S. Pat. No. 4,976,984 or esterifying 
unsaturated fatty acids originating from these fats or oils with glycerol. 
In order to describe the present invention in greater detail, and not by 
way of limitation, the following Examples will be given, wherein all % are 
by weight unless otherwise noted. 
EXAMPLE 1 
Highly pure soybean lecithin (SLP-WSP, trade name, manufactured by Tsuru 
Lecithin Kogyo K.K.; content of acetone-insoluble matters: 95% or more) 
was treated with phospholipase D in accordance with the manner as 
disclosed in JP-A-58-51835 and thus a phospholipid composition, wherein 
the content of the total nitrogen-containing phospholipids (for example, 
phosphatidylcholine (PC), phosphatidylethanolamine (PE), 
phosphatidylserine (PS), sphingomyelin (SM)) based on the total amount of 
phospholipids, glycolipids and sterol derivatives is controlled to 0.01 or 
less, was obtained. The phospholipid composition was further subjected to 
silica gel column chromatography using WAKOGEL C-200 (manufactured by Wako 
Pure Chemical Industries, Ltd.) as a carrier and chloroform/methanol (2/3) 
as an eluent and the fraction of the final high-polar substance was 
removed while confirming with TLC. Table 1 shows the composition of the 
phospholipid composition thus obtained. 
Next, 0.2% of the phospholipid composition was blended with rapeseed oil 
containing 10% of rapeseed diglycerides so as to give an oil composition. 
The cooking properties of the obtained oil composition were evaluated. 
Table 2 shows the results. 
EXAMPLE 2 
The procedure of Example 1 was repeated except that the soybean lecithin 
was replaced by yolk lecithin (a purified yolk lecithin manufactured by 
Asahi Chemical Industry Co., Ltd.) to thereby give a phospholipid 
composition and an oil composition. Table 1 shows the composition of the 
phospholipid composition thus obtained, while Table 2 shows the results of 
the evaluation of the cooking properties of the obtained oil composition. 
COMATIVE EXAMPLES 1 AND 2 
The procedure of Example 1 was repeated except that the phospholipid 
composition was replaced by soybean lecithin (SLP-WSP, trade name, 
manufactured by Tsuru Lecithin Kogyo K.K.) (Comparative Example 1) or yolk 
lecithin (a purified yolk lecithin manufactured by Asahi Chemical Industry 
Co., Ltd.) (Comparative Example 2) to thereby give each an oil 
composition. Table 1 shows the composition of the soybean lecithin and 
that of the yolk lecithin, while Table 2 shows the results of the 
evaluation of the cooking properties of the obtained oil compositions. 
COMATIVE EXAMPLE 3 
Highly pure soybean lecithin (SLP-WSP, trade name, manufactured by Tsuru 
Lecithin Kogyo K.K.; content of acetone-insoluble matters: 95% or more) 
was treated with phospholipase D in accordance with the manner as 
disclosed in JP-A-58-51835 and thus a phospholipid composition, wherein 
the content of the total nitrogen-containing phospholipids (e.g., 
phosphatidylcholine (PC), phosphatidylethanolamine (PE), 
phosphatidylserine (PS), sphingomyelin (SM)) based on the total amount of 
phospholipids, glycolipids and sterol derivatives is controlled to 0.01 or 
less, was obtained. Table 1 shows the composition of the phospholipid 
composition thus obtained. 
By using the obtained phospholipid composition, an oil composition was 
prepared by the same method as in Example 1. Table 2 shows the results of 
the evaluation of the cooking properties of this oil composition. 
TABLE 1 
______________________________________ 
Comparative 
Example Example 
Sample 1 2 1 2 3 
______________________________________ 
Nitrogen-containing 
phospholipid: 
PC content Trace Trace 24 70 Trace 
(% by weight) 
PE content Trace Trace 18 15 Trace 
(% by weight) 
PS content Trace Trace 2 -- Trace 
(% by weight) 
SM content Trace Trace Trace 3 Trace 
(% by weight) 
PA + LPA content 
90 90 6 -- 90 
(% by weight) 
Glycolipid content 
3 1 12 -- 3 
(% by weight) 
Sterol derivative 
1 2 3 2 1 
content (% by 
weight) 
High-polar sub- 
20 100 2000 600 1000 
stance content*.sup.1 
(area/.mu.g) 
______________________________________ 
Note; *.sup.1 The highpolar substances given in Table 1 were determined b 
the following method. HPTLC Silica Gel 60 (trade name, a product 
manufactured by Merck & Co., Inc.) was used as a silica gel plate. Each 
sample was dissolved in chloroform/methanol (2:1) in such a manner as to 
give a concentration of about 20 mg/ml. 2 .mu.l of the sample solution wa 
accurately charged on the plate. Then it was developed with a solvent 
(chloroform/acetone/methanol/acetic acid/water = 10/4/2/2/1). Next, the 
plate was uniformly colordeveloped by immersing a color development 
solution (methanol/water/sulfuric acid = (90/90/6) for 5 seconds and then 
heating on a hot plate or in an oven at 120.degree. C. for 10 minutes. Th 
colordeveloped plate thus obtained was subjected to measurement with a TL 
scanner within one hour. The employed TLC scanner was a TLC Scanner CS900 
provided with a xenon lamp (a product manufactured by Shimadzu 
Corporation). The determination conditions as follows. 
Photo mode: Abs refraction 
Scan mode: ZigZag scan 
Determination wavelength: 540 nm 
Zero set mode: B.C. 
Swing Width: 8.0 mm 
The area of substances hardly shifting from the original point (Rf value of 
the peak: 0-0.1) per .mu.g of the sample charged on the plate was referred 
to as the amount of high-polar substances (area/.mu.g), 
TABLE 2 
______________________________________ 
Evaluation of cooking properties (in frying)*.sup.1 
Smell during Coloration of 
Example No. 
heating*.sup.2 
Oil after heating*.sup.3 
Flavor*.sup.4 
______________________________________ 
Example 1 
A A A 
Example 2 
A A A 
Comparative 
B B B 
Example 1 
Comparative 
B B B 
Example 2 
Comparative 
B B B 
Example 3 
______________________________________ 
Note; 
*.sup.1 The cooking properties (in frying) were evaluated 
in the following manner: 
Oil temperature: 180.degree. C. (gas caloric force: 0.50 1/min) 
Oil amount: 500 g 
Total cooking time: 20 minutes 
Cooking method: 
500 g of each oil composition obtained in the above Examples or 
Comparative Examples was introduced into a frying pan and the 
oil temperature was adjusted to 180.degree. C. Next, the following 
materials were coated with the composition as specified below and 
then fried in the oil. 
Materials: 
Green pepper: 4 pieces of 1/2-cut 
Egg plant: 3 pieces of 1/4-cut 
Sweet potato: 3 slices (each 5 mm in thickness) 
Prawn (medium-size): 3 
Coating composition: (mixed while cooling in an ice/water bath) 
Soft flour: 100 g 
Water: 140 g 
Whole egg: 25 g 
*.sup.2 Evaluation criteria for smell during heating: 
A: good 
B: poor 
*.sup.3 Evaluation criteria for coloration of oil: 
A: not colored 
B: colored 
*.sup.4 Evaluation criteria for flavor: 
A: good 
B: oily 
______________________________________ 
REFERENTIAL EXAMPLE 1 
10 g of the phospholipids containing PA and L-PA obtained in Comparative 
Example 3, which had been treated with an enzyme such as phospholipase D, 
were dissolved in 100 ml of n-hexane so as to give a concentration of 10% 
(wt/vol). To the hexane solution thus obtained, 20 ml of ethanol was 
added. Further, 30 ml of a 1.5M aqueous solution of calcium chloride was 
added thereto. Then 1N NaOH was added dropwise to the mixture under 
stirring until the pH value reached 10. After 4 hours, the stirring was 
ceased and the mixture was centrifuged in a 50 ml centrifugation tube at 
3,000 rpm for 10 minutes at 25.degree. C. The upper hexane layer was 
collected thereby 90 ml of a phospholipid calcium salt fraction was 
obtained. After adding 45 ml of ethanol, the mixture was stirred and then 
centrifuged at 3,000 rpm for 10 minutes at 25.degree. C. Thus calcium 
salts of PA and L-PA were obtained as a precipitate. The precipitate thus 
obtained was dissolved by adding 81 ml of n-hexane and then 40.5 ml of 
ethanol was added thereto. After stirring and centrifuging at 3,000 rpm 
for 10 minutes at 25.degree. C., purified calcium salts of PA and L-PA 
were obtained as a precipitate. 
The precipitate thus obtained was dissolved in 100 ml of n-hexane again and 
insoluble matters were removed. Further, the hexane was removed with an 
evaporator. Thus 6 g of calcium salts of PA and L-PA were obtained. 
REFERENTIAL EXAMPLE 2 
10 g of the phospholipids containing PA and L-PA obtained in Comparative 
Example 3 were dissolved in 100 ml of n-hexane so as to give a 
concentration of 10% (wt/vol). To the hexane solution thus obtained, 20 ml 
of ethanol was added. Further, 30 ml of a 1.5M aqueous solution of calcium 
chloride was added thereto. Then 1N NaOH was added dropwise to the mixture 
under stirring until the pH value reached 10. After 4 hours, the stirring 
was ceased and the mixture was centrifuged at 3,000 rpm for 10 minutes at 
25.degree. C. The upper hexane layer was collected thereby 90 ml of a 
phospholipid calcium salt fraction was obtained. Then 36 g of 
triglycerides derived from rapeseed oil were added thereto and the mixture 
was stirred and then centrifuged at 3,000 rpm for 10 minutes at 25.degree. 
C. Thus calcium salts of PA and L-PA were obtained as a precipitate. The 
precipitate thus obtained was dissolved by adding 81 ml of n-hexane and 
then 32.4 g of triglycerides derived from rapeseed oil were added thereto. 
After stirring and centrifuging at 3,000 rpm for 10 minutes at 25.degree. 
C., purified calcium salts of PA and L-PA were obtained as a precipitate. 
The precipitate thus obtained was dissolved in 100 ml of n-hexane again 
and insoluble matters were removed. Further, the hexane was removed with 
an evaporator. Thus 6 g of calcium salts of PA and L-PA were obtained. 
REFERENTIAL EXAMPLE 3 
The purification of PA with the use of a silica gel column is given as a 
referential example. 
73.5 g of the natural phospholipids containing PA obtained in Comparative 
Example 3, which had been treated with an enzyme such as phospholipase, 
were dissolved in 50 ml of n-hexane and then added dropwise to 250 ml of 
acetone which had been ice-cooled in an acetone/dry ice bath. After 
centrifuging in a 500 ml tube at 3,000 rpm for 10 minutes, 200 ml of 
ice-cooled acetone was added to the resulting pasty precipitate. Then the 
mixture was centrifuged again at 3,000 rpm for 10 minutes and the 
precipitate was desolvated with an evaporator. Thus 55.41 g of 
acetone-insoluble matters were obtained. 
The acetone-insoluble matters were dissolved in 70 ml of chloroform and 
divided into portions each weighing 52 g, followed by fractionating with 
the use of three columns. The amount of the solvent used for each column 
(WAKOGEL C-200; 800 g) is given below. PA was eluted by using a 
chloroform/methanol mixture as a solvent with varying the mixing ratio 
thereof. The mixing ratio and amounts of the solvents are as follows. 
______________________________________ 
1. Sample charge 52 g 
2. Chloroform/methanol = 5/1 
200 ml 
3. Chloroform/methanol = 2/1 
4500 ml 
4. Chloroform/methanol = 3/2 
4200 ml 
5. Chloroform/methanol = 1/1 
1800 ml 
6. Chloroform/methanol = 2/3 
600 ml 
______________________________________ 
The solvents passed through the columns were divided into portions of each 
450 ml and each portion was analyzed. As a result, it was found that PA 
was eluted in the second half of the fraction of the chloroform/methanol 
ratio of 2/1 to the first half of the fraction of said ratio of 1/1. 3,600 
ml of a fraction eluted with the chloroform/methanol mixture of 3/2 
showing a particularly high purity of PA was collected and desolvated with 
an evaporator to thereby give purified PA. Thus 19.67 g of purified PA was 
obtained in total by this method. The total amount required for the 
purification was 34,770 ml (20,698 ml of chloroform, 13,572 ml of 
methanol, 450 ml of acetone and 50 ml of n-hexane). 
The amounts of the solvents required for purifying 1 g of PA and/or L-PA in 
Referential Example 1 and Referential Example 3 were compared. Table 3 
shows the results. 
TABLE 3 
______________________________________ 
Referential 
Referential 
Example 1 
Example 3 
______________________________________ 
Amount of solvent required for 
59.7 1767.1 
purifying 1 g of PA and/or 
L-PA (ml/g) 
Purity of PA and/or L-PA 
99 99 
(%; determined by HPLC) 
______________________________________ 
As the above results show, the process of the present invention makes it 
possible to obtain PAs and/or L-PAs of high purity on an industrial scale. 
EXAMPLE 3 
Soybeans produced in China (Konan No. 2) were employed as the oilseed. 
These soybeans were ground in a Waring blender. 20 g of the ground matters 
thus obtained were packed in a glass column (50 ml) and then 120 ml of a 
0.1M sodium acetate/acetic acid buffer solution (pH 6.0) was passed 
through the column. The eluate was permeated through a membrane of a 
fractionation molecular weight of 13,000. Thus a clear extract was 
obtained. 
20 g of commercially available defatted lecithin (manufactured by Tsuru 
Lecithin Kogyo K.K.) was fed into a 300 ml four-neck flask provided with a 
stirrer. 120 ml of the above-obtained extract (pH 6.0) was added thereto. 
Then the mixture was continuously stirred for 24 hours while maintaining 
the reaction system at 30.degree. C. under a nitrogen gas stream (100 
ml/min.). After the completion of the reaction, the mixture was heated to 
70.degree. C. for 1 hour and cooled. Then 65 ml of ethanol was added 
thereto and the mixture was extracted with 150 ml of hexane. 
150 ml of the hexane extract obtained above was fed into a 300 ml four-neck 
flask provided with a stirrer. Then 25 ml of ethanol, 15 ml of water and 
1/2 times by volume of calcium chloride, based on the solid matters in the 
hexane extract, were added thereto. The pH value of the mixture was 
adjusted to 10.0 by adding a 1N solution of sodium hydroxide under 
stirring at room temperature. After aging by stirring for additional 4 
hours, the mixture was centrifuged to thereby give a hexane solution. 
To 150 ml of the aforesaid hexane solution, 75 ml of ethanol was added to 
thereby effect solvent-precipitation. Then the supernatant was removed by 
centrifuging. The precipitate was dissolved by adding hexane and thus the 
total volume was adjusted to 75 ml. The hexane solution was washed with a 
50% ethanol/water under an acidic (hydrochloric acid) condition of pH 
value of 1 at 15.degree. C. by stirring for 10 minutes. After separating, 
the obtained hexane phase was neutralized with an aqueous solution of 
sodium hydroxide and desolvated to thereby give PA. 
The precipitate formed in the aforesaid solvent-precipitation was weighed. 
Further, the heat coloration property of the obtained PA were evaluated by 
the method as specified below. Table 4 summarizes the results. 
Evaluation of heat coloration property 
0.025 g of PA was introduced into a test tube. 10 g of rapeseed oil 
containing 10% of diglyceride was added thereto and the PA was dissolved 
by stirring. The obtained solution was heated to 180.degree. C. for 70 
minutes on a block heater and then the color difference in comparison with 
hexane was measured with a color difference meter (SZ-.SIGMA.80, 
manufactured by Nippon Denshoku Kogyo K.K.). The .DELTA.E(H) value thus 
determined was referred to as the hue value. 
REFERENTIAL EXAMPLE 4 
150 ml of the hexane extract obtained in Example 3 was introduced into a 
300 ml four-neck flask provided with a stirrer and 25 ml of ethanol and 15 
ml of water were added thereto. Then the pH value of the mixture was 
adjusted to 10.0 by adding a 1N solution of sodium hydroxide under 
stirring at room temperature. After aging by stirring for additional 4 
hours, the mixture was centrifuged to thereby give a hexane solution. To 
150 ml of this hexane solution, 75 ml of ethanol was added. The 
precipitate formed at the addition of ethanol was weighed. Table 4 shows 
the results. 
REFERENTIAL EXAMPLE 5 
150 ml of the hexane extract obtained in Example 3 was introduced into a 
300 ml four-neck flask provided with a stirrer and 25 ml of ethanol, 15 ml 
of water and 1/2 times by volume of calcium chloride, based on the solid 
matters in the hexane extract, were added thereto. After aging by stirring 
for additional 4 hours, the mixture was centrifuged to thereby give a 
hexane solution. To 150 ml of this hexane solution, 75 ml of ethanol was 
added. The precipitate formed at the addition of ethanol was weighed. 
Table 4 shows the results. 
REFERENTIAL EXAMPLE 6 
75 ml of the hexane extract obtained in Example 3 was washed with a 50% 
ethanol/water by stirring under an acidic (hydrochloric acid) condition of 
pH value of 1 at 15.degree. C. for 10 minutes. After separation, the 
obtained hexane phase was neutralized with an aqueous solution of sodium 
hydroxide and desolvated to thereby give PA. The heat coloration 
properties of this PA were evaluated by the same method as in Example 3. 
Table 4 shows the results. 
TABLE 4 
______________________________________ 
Heat Coloration 
Precipitate 
Property 
(g) (hue after heating) 
______________________________________ 
Example 3 7.5 10 
Referential 0 -- 
Example 4 
Referential 0 -- 
Example 5 
Referential 0 30 
Example 6 
______________________________________ 
It is apparent that the present invention enables the blending of 
phospholipids with a frying oil, which has been considered difficult since 
it causes heat coloration. Thus, an oil composition, which is excellent in 
mold-release characteristics during cooking, has a good smell during 
heating, suffers from no coloration of oil after heating and shows a good 
flavor, can be obtained. 
Further, the process of the present invention gives PAs and L-PAs of high 
purity and contaminated with no impurities in a high yield through simple 
handling as compared to the conventional purification method using column 
chromatography, thus the process of the present invention is highly useful 
for industrial production of PAs and L-PAs. 
While the invention has been described in detail and with reference to 
specific examples thereof, it will be apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.