Food additive and use thereof

An additive for cholesterol-containing food, containing a rice bran component and/or a derivative thereof as an active ingredient, a method of using same, and food treated thereby; and a process for producing mayonnaise controlled in the rise of a blood cholesterol level, or the absorption of cholesterol. A method of decreasing the effect of cholesterol contained in food on a living organism, for example, controlling the rise of a blood cholesterol level by adding to cholesterol-containing food a food additive capable of forming a complex with cholesterol, such as an extract of a rice bran component, .gamma.-orizanol (.gamma.-OZ) which is a mixture of esters of ferulic acid with various alcohols prepared by refining said extract and/or a derivative of a rice bran component, thereby forming a cholesterol complex of the food additive, whereby the cholesterol contained in food becomes nonabsorbable by a living organism or can be removed therefrom.

TECHNICAL FIELD 
This invention relates to an additive for a cholesterol-containing food 
which comprises rice bran components and/or derivatives thereof as an 
active component, use of the same and a food obtained by this method. 
More particularly, it relates to a food additive which comprises a rice 
bran component extract (hereinafter referred to simply as the extract), 
ferulates of various alcohols obtained by purifying the extract, 
.gamma.-oryzanol (hereinafter referred to simply as .gamma.-OZ), i.e., a 
mixture of these esters, and/or derivatives of the rice bran components 
capable of forming a cholesterol complex; use of the same; and a food 
which has been prepared by this method which forms a cholesterol complex. 
BACKGROUND ART 
In recent years, an increase in cholesterol intake accompanying the 
diversification in our eating habits has become a serious social problem 
in connection with diseases. Namely, we frequently eat foods of a high 
cholesterol content these days. As a result, cholesterol contained in 
these foods elevates the blood cholesterol level, which is the major cause 
of, for example, arteriosclerosis. 
To lower the cholesterol content in these foods, there have been reported 
some methods for decomposing cholesterol with the use of cholesterol 
oxidase. 
Rice bran contains a number of components. For example, .gamma.-OZ, which 
is prepared by purifying these components, is a mixture of ferulates of 
various vegetable sterols and triterpene alcohol. For example, .gamma.-OZ 
consists of 14% of campesterol ferulate, 1% of stigmasterol ferulate, 4% 
of .beta.-sitosterol ferulate, 2% of cycloartanol ferulate, 35% of 
cycloartenol ferulate and 44% of 24-methylene cycloartanol ferulate. 
As examples of the application of .gamma.-OZ to medical purposes, there has 
been reported that it has pharmacological effects of promoting growth, 
controlling diencephalic function, lowering serum cholesterol level, 
increasing amine-like compounds in amygdaloid nucleus and stimulating 
gonad, and clinical effects of treating menopausal disorders and autonomic 
imbalance. 
Regarding the above-mentioned effect of .gamma.-OZ of lowering serum 
cholesterol level, its effects on catabolism/excretion of cholesterol in 
rat has been reported Geriatric Medicine, vol. 18, 519-524 (1980); 
Domyakukoka (arteriosclerosis), vol. 11 (2), 411-416 (1983)!. 
In the field of food, .gamma.-OZ has been employed as an antioxidant. 
However, since .gamma.-OZ exhibits only a very weak antioxidant effect, it 
is scarcely used alone but mixed with tocopherol so as to enhance its 
effect. 
Also, it is reported that organic acid esters of triterpene alcohol, 
namely, derivatives of the main constituent of .gamma.-OZ, are applicable 
to drugs for treating hyperlipemia (JP-A-61-243022 and JP-A-61-243099; the 
term "JP-A" as used herein means an "unexamined published Japanese patent 
application"). Although these drugs aim at treating this disease by 
preventing an increase in serum cholesterol level or lowering the same, 
their function mechanisms have not been clarified in detail so far. 
As discussed above, .gamma.-OZ or rice bran component derivatives have been 
studied and applied as drugs. However, no attempt has been made so far to 
clarify their function per se of directly binding to cholesterol to form a 
cholesterol complex and they have not been used as a food additive by 
taking advantage of this function. 
Today, mayonnaise is a seasoning which is essentially needed in our 
westernized eating habits in Japan. In general, mayonnaises are produced 
by adding edible oils, vinegar, seasonings etc. to egg white and yolk to 
form an oil-in-water emulsion. 
With the recent tendency toward health foods such as low-cholesterol or 
low-salt foods and a variety of tastes, there have been proposed 
mayonnaises free from cholesterol or containing cholesterol in a reduced 
amount which are produced by using no or little egg yolk. For example, 
JP-A-50-64466 has proposed an egg yolk-free mayonnaise produced by using 
starch paste and egg white, while JP-A-63-275 has proposed a mayonnaise 
produced by using soybean flour. Further, JP-A-60-43347 has proposed a 
process for producing a mayonnaise having a function of lowering blood 
cholesterol level or suppressing an increase of the blood cholesterol 
level, in which monolinolein is added to an edible oil to be used. 
Not only chemical seasonings and spices but also egg components, in 
particular, egg yolk largely contribute to the flavor of mayonnaise. In 
addition, egg yolk plays an important role in the shape retention when 
mayonnaise is used in salad dressing placed on salad. 
Namely, since mayonnaise has a high acidity due to vinegar which is used in 
a large amount, a stable emulsion of mayonnaise can hardly be achieved by 
using common edible emulsifiers and it is highly important to use the 
emulsifying power of egg yolk. 
Thus, it is considered that the use of egg yolk is an essential requirement 
in the production of mayonnaise. 
The conventional mayonnaises being free from egg yolk or containing egg 
yolk in a reduced amount have no or only deteriorated body taste of egg 
yolk. Further, they are not satisfactory in shape retention properties as 
a mayonnaise to be used as a seasoning. 
On the other hand, the method of adding monolinolein to fat and oil suffers 
from a problem that monolinolein is hardly available. In addition, only an 
unsatisfactory effect of lowering blood cholesterol level is achieved by 
this method. 
DISCLOSURE OF THE INVENTION 
The present inventors have conducted extensive studies to solve these 
problems. As a result, they have found out that rice bran components 
and/or derivatives thereof have an ability to form a complex with 
cholesterol and that the complex formed by adding the rice bran components 
and/or derivatives thereof to a food is not absorbed by a living body, 
thus suppressing an increase in serum cholesterol level. 
The present inventors have further found out that a mayonnaise capable of 
remarkably suppressing an increase in blood cholesterol level can be 
produced by using a fat and oil containing rice bran components and/or 
derivatives thereof in the production of a mayonnaise having an 
oil-in-water emulsion structure comprising fats and oils, souring agents, 
whole egg and/or egg yolk, etc. containing cholesterol, and other 
seasonings and additives required. 
They have furthermore found out for the first time that similar effects can 
be achieved by using whole egg and/or egg yolk treated with rice bran 
components and/or derivatives thereof and that a mayonnaise, which is 
produced by using whole egg and/or egg yolk treated with rice bran 
components and/or derivatives thereof together with fats and oils 
containing rice bran components and/or derivatives, has a function of 
further remarkably suppressing an increase in blood cholesterol level. The 
present invention has been completed based on these findings. 
The present invention provides a method for relieving the effects of 
cholesterol contained in various foods on a living body by converting the 
cholesterol into another form which is not absorbed by a living body or 
eliminating the cholesterol from the foods after converting it into 
another form which is not absorbed. 
The present invention further provides a method for producing a mayonnaise 
which can suppress an increase in the blood cholesterol level, i.e., the 
absorption of cholesterol, though egg yolk is used in the production of 
the mayonnaise. 
According to the present invention, the absorption of cholesterol by a 
living body can be prevented and thus an increase in the blood cholesterol 
level can be prevented by adding an additive, which comprises rice bran 
components and/or derivatives thereof as an active component and which has 
an ability to form a cholesterol complex, to a food and thus forming a 
cholesterol complex together with cholesterol contained in the food, or 
removing the thus formed cholesterol complex after the addition. 
A cholesterol complex means a state where cholesterol has bound to the rice 
bran component and/or a derivative thereof added to the food, its 
solubility is largely lowered, and thus the absorption of cholesterol by a 
living body is prevented. Since such a complex is hardly soluble in 
solvents, it can be removed as a precipitate from the food by a 
conventional means. Thus, a food of a reduced cholesterol content can be 
obtained. 
The rice bran components and/or derivatives' thereof to be used in the 
additive of the present invention also include an extract prepared by 
extracting the components of rice bran from rice bran by a conventional 
means. Such an extract can be obtained by, for example, extracting oily 
components from rice bran with a solvent, degumming, dewaxing and 
alkali-hydrolyzing these oily components, neutralizing the hydrolyzate, 
subjecting it to solid/liquid separation and distilling the residue, 
followed by solvent-extraction and column treatment. As a particular 
example thereof, the following extraction process may be exemplified. 
##STR1## 
Needless to say, any materials such as an extract which is obtained in each 
intermediate step of the above method and contains various rice bran 
components may be used in the present invention, so long as it can form a 
complex with cholesterol. Furthermore, extracts obtained by any methods 
other than the above-mentioned one may be used and the degree of 
purification of the extract may be at an arbitrary level, so long as they 
can form a cholesterol complex. 
In addition, .gamma.-OZ obtained by further purifying the above-mentioned 
extract as well as its single component thereof obtained by furthermore 
purifying the same, for example, cycloartenol ferulate (hereinafter 
referred to simply as CAFE) or 24-methylene cycloartenol ferulate 
(hereinafter referred to simply as 24-MFE) are also regarded as the rice 
bran components. 
As examples of the derivatives of the rice bran components, esters of 
triterpene alcohols with .alpha.-alkyl(C.sub.1 to C.sub.4)-cinnamic acid 
having one or two substituents binding to the benzene ring, and esters of 
triterpene alcohols with cinnamic acid or benzoic acid having two 
substituents selected from combinations of a C.sub.1 -C.sub.4 alkoxy group 
with a nitro group, a C.sub.1 -C.sub.4 alkoxy group and an amino group and 
a C.sub.1 -C.sub.4 alkoxy group with a C.sub.2 -C.sub.5 acylamino group 
binding to the benzene ring may be cited. 
As examples of triterpene alcohol, cycloartenol, cyclobranol and 
24-methylene cycloartenol may be cited. 
Among these additives, the extract, .gamma.-OZ and CAFE can be preferably 
used as the rice bran component, while an ester of cycloartenol with 
3-methoxy-4-hydroxy-.alpha.-methylcinnamic acid, i.e., 
cycloartenol-.alpha.-methylferulate (hereinafter referred to simply as 
CAMFE) can be preferably used as a rice bran component derivative. 
As a matter of course, as an additive, each of the above-mentioned extract, 
.gamma.-OZ, various components of rice bran and the rice bran component 
derivatives can be used singly or a mixture of two or more of them can be 
used. 
As particular examples of the food to be used as the subject of the present 
invention, those containing cholesterol at a higher level compared with 
other foods and those containing a large amount of cholesterol being 
condensed in the processing step, though the cholesterol content of the 
starting material is not relatively high, may be exemplified. In the case 
of diet foods of limited cholesterol intake, the present invention is 
applicable to any foods. 
As examples of foods of a high cholesterol content, meats, eggs and fish 
may be cited. Examples of the meats include pork, beef, chicken and their 
internals. Examples of the eggs include chicken egg, salmon roe, cod roe 
and herring roe. Examples of the fish include squid, corbicula, eel and 
shishamo smelt. Further, primary and secondary processed products of these 
foods, for example, egg yolk powder, mayonnaise, hamburger and cake, are 
included. As examples of the foods containing a large amount of 
cholesterol being condensed in the processing step, dairy products such as 
butter, cheese, cream and milk powder may be cited. The cholesterol 
content in these final products can be lowered by treating the starting 
material thereof, for example, cow's milk with the food additive of the 
present invention. 
The term "mayonnaise" to be used herein means a so-called mayonnaise or 
dressing, and the mayonnaise includes everything produced by conventional 
methods. That is to say, the mayonnaise means a seasoning which has an 
acidic pH value and an oil-in-water emulsion structure formed by using egg 
white and yolk as an emulsifier. 
A dressing means a seasoning other than mayonnaises which has an 
oil-in-water emulsion structure containing egg yolk. 
As the fats and oils to be used in the production of the mayonnaise, 
various edible oils containing, either inherently or artificially, the 
rice bran components and/or derivatives thereof can be used. As an example 
of an edible oil inherently containing rice bran components and/or 
derivatives thereof originating in the starting material, rice oil 
(unpolished rice germ oil) obtained from rice bran and the like may be 
exemplified. 
Rice oil can be prepared from rice bran, for example, in the following 
manner. The feedstock rice bran is sieved and ground rice grains are 
separated. After a pre-extraction treatment, cold extraction and miscella 
distillation are effected. The crude rice bran oil thus obtained is then 
centrifuged, degummed, dewaxed, deacidified, decolored and winterized. 
Thus, a refined rice oil can be obtained. 
Now, a concrete example of the preparation process will be given. 
##STR2## 
Alternately, fats and oils obtained by adding the rice bran components 
and/or derivatives thereof to various edible oils may be used. 
The edible oil, to which the rice bran components and/or derivatives 
thereof are to be added, may be arbitrarily selected, so long as it is 
usable in the production of mayonnaise. For example, corn oil, soybean 
oil, sunflower oil, rapeseed oil and cottonseed oil may be exemplified. 
To produce mayonnaise, the rice bran components and/or derivatives thereof 
may be preferably used at a ratio of from 0.01 to 10%. 
In the method for the production of a mayonnaise in accordance with the 
present invention, whole egg and/or egg yolk which have been treated with 
the rice bran components and/or derivatives thereof can be used as the 
whole egg and/or egg yolk component. Either the whole fresh egg or the 
yolk alone may be used and a dry egg yolk can be used as the egg yolk. 
Alternately, an egg yolk powder prepared by treating egg yolk with the 
rice bran components and/or derivatives thereof followed by freeze-drying 
may be used. 
The addition method varies depending on the form of the food to which the 
rice bran components and/or derivatives thereof are to be added. Namely, 
the rice bran components and/or derivatives thereof may be added in the 
form of a powder or, alternately, in the form of a liquid prepared by 
using an appropriate medium which has been commonly employed in the 
production of foods. In order to achieve the direct contact with 
cholesterol contained in the food, it is preferable to add the rice bran 
components and/or derivatives thereof in the form of a liquid. 
To use as a food additive, the rice bran components and/or derivatives 
thereof are preferably added in a step during the course of processing the 
food. After adding the rice bran components and/or derivatives thereof, 
the food is stirred by an appropriate means. It is enough to stir the food 
until the formation of the complex is completed. In general, 10 minutes to 
24 hours are needed therefor. 
To facilitate the contact of a liquid food with the rice bran components 
and/or derivatives thereof, it is effective to add a step of drying. In 
this drying step, commonly employed drying means, for example, heat drying 
or freeze-drying may be employed. Freeze-drying is a preferable method 
from the viewpoint of maintenance of the qualities of the food. 
The rice bran components and/or derivatives thereof are added at a ratio of 
from 1 to 10 parts by weight, preferably from 2 to 6 parts by weight, per 
part by weight of cholesterol contained in the food. Although this 
addition level varies depending on the purity of the additive, it is 
generally recommended to add the rice bran components and/or derivatives 
thereof in an amount required for the formation of the complex with 
cholesterol which is to be eliminated in order to control the cholesterol 
content in the food to the desired level. Cholesterol and the active 
component usually undergo the reaction of forming the cholesterol complex 
at a molar ratio of 1:1. 
The rice bran components and/or derivatives thereof capable of forming a 
cholesterol complex thus added form the cholesterol complex in the food to 
thereby lower the absorption ratio of cholesterol in a living body. 
Therefore, it is unnecessary to remove the cholesterol complex. However, 
the cholesterol complex can be removed from the food by an appropriate 
method by taking advantage of a difference in solubility between 
cholesterol and the cholesterol complex. A rice bran extract is preferably 
used as a food additive from the viewpoint of safety, since it is a 
natural material which can remain in a food.

BEST MODE FOR CARRYING OUT THE INVENTION 
To further illustrate the present invention in greater detail, and not by 
way of limitation, the following Test Examples and Examples will be given. 
Unless otherwise noted, all % in these Test Examples and Examples are by 
weight. 
The extract, .gamma.-OZ, CAFE, 24-MFE and CAMFE employed in Test Examples 1 
to 4 and Examples 1 to 9 were prepared each in the following manner. The 
extract was prepared by subjecting rice bran to extracting with hexane; 
degumming, dewaxing and deacidifying the oily components thus obtained to 
thereby separate an edible oil; neutralizing the foots thus obtained, 
followed by distillation of the insoluble matters, washing with a solvent 
and treatment with an alumina column. As the .gamma.-OZ, a commercially 
available product was used. CAFE and 24-MFE were prepared by repeatedly 
recrystallizing .gamma.-OZ from acetone/methanol, acetone and ethyl 
acetate by reference to the method of Endo, Misu and Inaba et al. 
Yukagaku (Oil Chemistry), 18, 63-67 (1969)!. CAMFE was prepared in 
accordance with the method described in JP-A-61-243099. 
Test Example 1 
Comparison of various additives in ability to form cholesterol complex: 
The extract, CAFE, 24-MFE and CAMFE were used as test samples. First, 0.33 
mmol (201 mg) of CAFE and 0.33 mmol (129 mg) of cholesterol were dissolved 
in 100 ml of tetrahydrofuran. After adding 100 ml of water, the mixture 
was evaporated to dryness under reduced pressure. The dry product thus 
obtained was suspended in 100 ml of water, filtered and dried under 
reduced pressure (85.degree. C., 2 hours) and the infrared absorption 
spectrum was measured. Other test samples were similarly treated and the 
infrared absorption spectra were measured. Table 1 shows the results. 
The ability to form cholesterol complex was evaluated based on the results 
of the infrared absorption spectrometry. In Table 1, .circleincircle. 
means that a very strong ability to form cholesterol complex was observed, 
.largecircle. means that a somewhat weaker ability to form cholesterol 
complex was observed. 
TABLE 1 
__________________________________________________________________________ 
IR of IR of Difference in 
Sample 
pure material 
preparation 
wave number 
Evaluation 
__________________________________________________________________________ 
Extract 
1700 cm.sup.-1 (CO) 
1700 cm.sup.-1 (CO) 
0 impossible 
1680 cm.sup.-1 (CO) 
1680 cm.sup.-1 (CO) 
0 
CAFE 1675 cm.sup.-1 (CO) 
1690 cm.sup.-1 (CO) 
+15 cm.sup.-1 
.circleincircle. 
change in OH 
absorption 
24-MFE 
1695 cm.sup.-1 (CO) 
1690 cm.sup.-1 (CO) 
+5 cm.sup.-1 
.largecircle. 
change in OH 
absorption 
CAMFE 1700 cm.sup.-1 (CO) 
1700 cm.sup.-1 (CO) 
0 .circleincircle. 
1668 cm.sup.-1 (CO) 
1673 cm.sup.-1 (CO) 
+5 cm.sup.-1 
change in OH 
absorption 
__________________________________________________________________________ 
As a result, it was suggested that CAFE, 24-MFE and CAMFE were able to form 
cholesterol complex, though it was not judged by the infrared absorption 
spectrometry whether the extract was able to form cholesterol complex or 
not. However, the extract is assumed to be able to form cholesterol 
complex, since it is a mixture of CAFE, 24-MFE, campesterol, stigmasterol, 
etc. and CAFE and 24-MFE constituting the same form cholesterol complex. 
Further, the results of animal experiments, which will be described 
hereinafter, also support this assumption. 
Test Example 2 
Relationship between the degree of freeze-drying of CAMFE-treated egg yolk 
and migration of CAMFE into egg yolk layer: 
(1) Preparation of Sample 
To each of 5 egg yolk samples weighing 10 g, 260 mg of CAMFE was added and 
the mixture was stirred at 200 rpm with a mechanical stirrer for 1 hour at 
25.degree. C. Then, these samples were freeze-dried in such a manner as to 
give water losses of 50%, 40%, 30%, 20% and 10%. Table 2 shows the drying 
ratio of each sample. 
TABLE 2 
______________________________________ 
Sample Loss in egg yolk (%) 
______________________________________ 
No. 1 51.0 
No. 2 41.6 
No. 3 29.6 
No. 4 18.8 
No. 5 10.7 
______________________________________ 
(2) Procedure 
Each sample thus dried was ground and sieved (22-mesh) to give a dry egg 
yolk powder. Then, water was added in an amount corresponding to the loss 
due to the freeze-drying (about 5, 4, 3, 2 and 1 g). The obtained mixture 
was stirred at 200 rpm with a mechanical stirrer for 1 hour. After adding 
about 8 g of water, it was further stirred at 500 rpm for 2 hours. A 
portion (9 g) of this mixture was centrifuged at 10,000 rpm for 30 
minutes. 
The egg yolk layer was extracted with chloroform (20 ml.times.1, 10 
ml.times.2) and the extract was dried over magnesium sulfate (2 g) and 
then concentrated. The concentrate was dissolved in chloroform to give a 
volume of 10 ml. A 5 .mu.l portion thereof was analyzed by HPLC to thereby 
determine the CAMFE content. 
Table 3 shows the results wherein a content of CAMFE is expressed in a 
chart area (.mu.g.sec). 
Analytical conditions for HPLC were as follows. 
Detector: UV absorptiometer (detection wavelength: 293 nm). 
Column: Asahipak ODP-50, 4.6 mm (i.d.).times.150 mm. 
Column temp.: 45.degree. C. 
Mobile phase: acetonitrile/water=95/5. 
TABLE 3 
______________________________________ 
Sample Egg yolk layer 
______________________________________ 
No. 1 7,680,942 
No. 2 7,623,906 
No. 3 2,386,327 
No. 4 1,265,665 
No. 5 905,997 
______________________________________ 
With an increase in the degree of freeze-drying, CAMFE became liable to 
migrate into the egg yolk layer. Namely, it is considered that CAMFE would 
come in contact with oily components in the egg yolk with a decrease of 
water, thus being absorbed by the egg yolk. The migration ratio at a water 
loss of 40% or above was particularly high. 
Test Example 3 
Degree of migration of the extract, CAFE or CAMFE contained in freeze-dried 
egg yolk into egg yolk layer: 
(1) Preparation of Sample 
To 10 g of egg yolk, 260 mg of the extract, CAFE or CAMFE was added, 
respectively. Next, the mixture was stirred at 200 rpm with a mechanical 
stirrer at 25.degree. C. for 1 hour and then freeze-dried (50% loss) to 
thereby give a sample. 
(2) Procedure 
Each sample was ground and sieved (22-mesh) to thereby give a dry egg yolk 
powder. Then water was added in an amount corresponding to the loss due to 
the freeze-drying (about 5 g in each case). The obtained mixture was 
stirred at 200 rpm with a mechanical stirrer for 1 hour. After adding 
about 8 g of water, it was further stirred at 500 rpm for 2 hours. A 
portion (9 g) of this mixture was centrifuged at 10,000 rpm for 30 
minutes. 
The precipitate layer was separated from the egg yolk layer by decantation 
and the egg yolk layer and the precipitate layer were extracted with 
chloroform (20 ml.times.1, 10 ml.times.2) and each extract was dried over 
magnesium sulfate (2 g) and then concentrated. Each concentrate was 
dissolved in chloroform to give a volume of 10 ml. A 5 .mu.l portion 
thereof was analyzed by HPLC to thereby determine each additive. Table 4 
shows the results wherein a content of an additive is expressed in a chart 
area (.mu.g.sec). 
Analytical conditions for HPLC were the same as those used in Test Example 
2. 
TABLE 4 
______________________________________ 
Egg yolk Precipitate 
Sample layer (A) layer (B) A/B 
______________________________________ 
extract-treated 
7,384,067 3,819,789 1.933 
CAFE-treated 9,033,489 6,578,706 1.373 
CAMFE-treated 
7,680,942 7,868,723 0.976 
______________________________________ 
It is considered that the extract and CAFE has strong affinities for the 
egg yolk layer in comparison with CAMFE. 
Test Example 4 
Degree of migration of the extract, CAFE or CAMFE contained in freeze-dried 
cow's milk into cow's milk layer: 
(1) Preparation of Sample 
To 100 g of cow's milk, 22 mg of the extract, CAFE or CAMFE was added, 
respectively. Next, the mixture was stirred at 200 rpm with a mechanical 
stirrer at 25.degree. C. for 1 hour and then freeze-dried to give a 
sample. 
(2) Procedure 
To each sample, water was added in an amount corresponding to the loss due 
to the freeze-drying (about 88 g in each case). The obtained mixture was 
stirred at 500 rpm with a mechanical stirrer for 1 hour at 25.degree. C. 
and centrifuged at 6,000 rpm for 30 minutes. The cow's milk layer and the 
precipitate layer were separated by decantation. The cow's milk layer was 
freeze-dried and, after adding 50 ml of chloroform, heated under reflux 
for 30 minutes. Then, it was dried over magnesium sulfate, filtered and 
concentrated. On the other hand, the precipitate layer as such was 
extracted with chloroform (20 ml.times.1, 10 ml.times.2). After filtering 
off the magnesium sulfate, the extract was concentrated. Each of these 
extracts was adjusted to a volume of 10 ml with chloroform. A 5 .mu.l 
portion of each extract was analyzed by HPLC to thereby determine the 
amount of each additive. Table 5 shows the results wherein a content of an 
additive is expressed in a chart area (.mu.g.sec). 
Analytical conditions for HPLC were the same as those used in Test Example 
2. 
TABLE 5 
______________________________________ 
Cow's milk Precipitate 
Sample layer (A) layer (B) A/B 
______________________________________ 
extract-treated 
299,511 1,104,309 0.2712 
CAFE-treated 166,548 1,100,244 0.1514 
CAMFE-treated 
24,517 1,057,800 0.0232 
______________________________________ 
It is considered that CAMFE has a weak affinity for the cow's milk layer in 
comparison with CAFE. 
Example 1 
Evaluation of ability to form cholesterol complex in food: 
CAFE and CAMFE were used as samples. 
To 10 g of commercially available egg yolk, 200 mg of CAFE was added and 
the mixture was stirred at room temperature for 1 hour and freeze-dried to 
give a sample for infrared absorption spectrometry. Further, a sample 
containing CAMFE (210 mg) and a control sample were prepared in the same 
manner. Table 6 shows the results. 
TABLE 6 
______________________________________ 
Differential 
IR of spectrum of 
Sample complex preparation Evaluation 
______________________________________ 
CAFE 1690 cm.sup.-1 (CO) 
1710 cm.sup.-1 (CO) 
.largecircle. 
CAMFE 1673 cm.sup.-1 (CO) 
1675 cm.sup.-1 (CO) 
.largecircle. 
______________________________________ 
Based on these results, it is considered that CAFE and CAMFE form a complex 
with cholesterol contained in egg yolk. In the case of CAFE, the 
absorption of the CAFE complex was involved in the absorption of the 
background (1710 cm.sup.-1), since the background could not be completely 
eliminated due to the inhomogeneities of the carrier, i.e., egg yolk. 
However, the peak assignable to the pure CAFE (1675 cm.sup.-1) was not 
observed and thus it was judged that CAFE is able to form a cholesterol 
complex. 
Example 2 
Formation of cholesterol complex in egg yolk: 
To 10 g of egg yolk, 260 mg of the extract, CAFE or CAMFE was added and the 
mixture was stirred at 200 rpm with the use of a mechanical stirrer at 
room temperature for 1, 5 or 20 hours, followed by freeze-drying. In each 
case, about 5 g of a freeze-dried egg yolk powder containing a cholesterol 
complex was obtained. 
Example 3 
Formation of cholesterol complex in cow's milk: 
To 100 g of egg yolk, 10 or 500 mg of the extract, CAFE or CAMFE was added 
and the mixture was stirred at 200 rpm at room temperature for 1, 5 or 20 
hours, followed by freeze-drying. In each case, about 12 g of a 
freeze-dried milk powder containing a cholesterol complex was obtained. 
Example 4 
Method for removing cholesterol from egg yolk combined with freeze-drying 
step: 
To 10 g of egg yolk, 260 mg of the extract, CAFE or CAMFE was added, 
respectively, and the mixture was stirred at 200 rpm with the use of a 
mechanical stirrer at room temperature for 1, 10 or 20 hours, followed by 
freeze-drying. 
The freeze-dried egg yolk thus obtained was ground and sieved (22-mesh) to 
give a dry egg yolk powder. Next, water was added in an amount 
corresponding to the loss due to the freeze-drying (about 5 g) and the 
resulting mixture was further stirred at 200 rpm with a mechanical stirrer 
for 2 hours. After adding about 3 or 8 g of water, it was stirred at 500 
rpm for additional 30 minutes or 2 hours. A 6.5 g or 9 g portion of this 
water-reconstituted egg yolk was centrifuged. 
Then, the precipitate layer was separated from the egg yolk layer by 
decantation and the egg yolk layer was freeze-dried. Thus, 1.94 to 2.37 g 
(77.6 to 94.8%) of a low-cholesterol egg yolk powder was obtained. 
Table 7 shows the results of the enzymatic determination of cholesterol 
contained in the egg yolk layer and the precipitate layer prepared by 
stirring for 1 hour, adding 8 g of water and centrifuging 9 g of the 
water-reconstituted egg yolk. 
TABLE 7 
______________________________________ 
Sample Cholesterol conc. (mg/dl) 
______________________________________ 
untreated yolk 
(yolk layer) 171.23 (100%) 
(precipitate layer) 
23.15 
Ext.-treated 
(yolk layer) 145.35 (84.9%) 
(precipitate layer) 
47.11 
CAFE-treated 
(yolk layer) 113.64 (66.4%) 
(precipitate layer) 
77.36 
CAMFE-treated 
(yolk layer) 102.76 (60.0%) 
(precipitate layer) 
86.39 
______________________________________ 
By this method, cholesterol contained in the egg yolk was reduced by 20 to 
40%. 
Example 5 
Method for removing cholesterol from cow's milk combined with freeze-drying 
step: 
To 100 g of cow's milk, 10 or 500 mg of the extract, CAFE or CAMFE was 
added and the mixture was stirred at 200 rpm at room temperature for 1, 5 
or 20 hours, followed by freeze-drying. Next, water was added in an amount 
corresponding to the loss due to freeze-drying (about 88 g) and the 
resulting mixture was further stirred at 200 rpm at room temperature for 
1, 5 or 20 hours and then centrifuged. 
Then cow's milk layer was separated from the precipitate layer by 
decantation and freeze-dried. Thus, 10.5 to 11.8 g (87.5 to 98.3%) of a 
low-cholesterol cow's milk powder was obtained. 
When determined by the enzymatic method, it was found that cholesterol was 
reduced by 75 to 95%. 
Example 6 
Administration test of cholesterol complex-containing food by using rat: 
As test samples, the extract, .gamma.-OZ, CAFE, 24-MFE and CAMFE were 
employed. 
&lt;Test animal&gt; 
Species and strain: Slc:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Classification and groups 
Food: a powdery food containing 1% of cholesterol (hereinafter sometimes 
referred to simply as CHL) and 0.5% sodium cholate (CE-2, a product of 
Clea Japan Inc.). 
(2) Test method and blood collection 
Each test sample was added to the food at a ratio as specified in Table 8 
and the animals were allowed to take it ad libitum. The body weight change 
and food intake were measured. After 7 days, the blood of each animal was 
taken from the abdominal aorta and the total cholesterol level in the 
serum was measured. Tables 9 to 11 show the results. 
TABLE 8 
______________________________________ 
No. of 
Sample 
No. Group rats g/100 g CE-2 
______________________________________ 
1 Normal 8 
2 Control (CHL) 8 1 
3 Ext./CHL (0.5:1) 8 1.713 
4 Ext./CHL (1:1) 8 2.562 
5 Ext./CHL (2:1) 8 4.186 
6 .gamma.-OZ/CHL 
(0.5:1) 8 1.788 
7 .gamma.-OZ/CHL 
(1:1) 8 2.576 
8 .gamma.-OZ/CHL 
(2:1) 8 4.152 
9 CAFE/CHL (0.5:1) 8 1.780 
10 CAFE/CHL (1:1) 8 2.558 
11 CAFE/CHL (2:1) 8 4.166 
12 24-MFE/CHL (0.5:1) 8 1.798 
13 24-MFE/CHL (1:1) 8 2.594 
14 24-MFE/CHL (2:1) 8 4.188 
15 CAMFE/CHL (1:1) 8 2.594 
______________________________________ 
(3) Body weight is expressed by the mean .+-. standard deviation (g). 
TABLE 9 
__________________________________________________________________________ 
Administration time (day) 
No. 
Group 0 3 7 9 
__________________________________________________________________________ 
1 Normal 269 .+-. 12 
278 .+-. 13 
296 .+-. 13 
308 .+-. 15 
2 Control (CHL) 
268 .+-. 10 
272 .+-. 8 
290 .+-. 11 
306 .+-. 10 
3 Ext./CHL (0.5:1) 
269 .+-. 08 
271 .+-. 10 
290 .+-. 21 
298 .+-. 18 
4 Ext./CHL (1:1) 
270 .+-. 09 
270 .+-. 10 
293 .+-. 18 
305 .+-. 23 
5 Ext./CHL (2:1) 
268 .+-. 10 
273 .+-. 9 
296 .+-. 19 
302 .+-. 8 
6 .gamma.-OZ/CHL 
(0.5:1) 
268 .+-. 9 
295 .+-. 41 
301 .+-. 9 
313 .+-. 6 
7 .gamma.-OZ/CHL 
(1:1) 
268 .+-. 10 
274 .+-. 14 
294 .+-. 10 
311 .+-. 14 
8 .gamma.-OZ/CHL 
(2:1) 
267 .+-. 10 
277 .+-. 15 
295 .+-. 10 
307 .+-. 9 
9 CAFE/CHL (0.5:1) 
268 .+-. 9 
270 .+-. 13 
293 .+-. 14 
307 .+-. 14 
10 CAFE/CHL (1:1) 
270 .+-. 8 
278 .+-. 5 
299 .+-. 7 
314 .+-. 8 
11 CAFE/CHL (2:1) 
268 .+-. 11 
267 .+-. 18 
283 .+-. 20 
299 .+-. 20 
12 24-MFE/CHL 
(0.5:1) 
268 .+-. 9 
273 .+-. 17 
291 .+-. 21 
305 .+-. 16 
13 24-MFE/CHL 
(1:1) 
268 .+-. 9 
273 .+-. 11 
296 .+-. 5 
312 .+-. 5 
14 24-MFE/CHL 
(2:1) 
270 .+-. 8 
263 .+-. 14 
280 .+-. 18 
297 .+-. 21 
15 CAMFE/CHL 
(1:1) 
267 .+-. 8 
278 .+-. 11 
298 .+-. 15 
311 .+-. 15 
__________________________________________________________________________ 
(4) Food intake is expressed by the means .+-. standard deviation 
(g/animal/day). 
TABLE 10 
__________________________________________________________________________ 
Administration time (day) 
No. Group 0-3 4-7 8-9 
__________________________________________________________________________ 
1 Normal 19.4 .+-. 2.9 
21.8 .+-. 3.2 
25.3 .+-. 5.4 
2 Control (CHL) 
16.0 .+-. 1.2 
22.5 .+-. 2.9 
27.3 .+-. 1.7 
3 CAFE/CHL (0.5:1) 
18.5 .+-. 2.2 
23.0 .+-. 1.5 
27.6 .+-. 2.0 
4 CAFE/CHL (1:1) 
17.2 .+-. 1.8 
23.5 .+-. 1.9 
26.4 .+-. 2.1 
5 ACFE/CHL (2:1) 
15.6 .+-. 3.2 
21.7 .+-. 1.5 
28.6 .+-. 1.7 
6 24-MFE/CHL 
(0.5:1) 
17.4 .+-. 3.9 
22.5 .+-. 2.1 
26.6 .+-. 5.4 
7 24-MFE/CHL 
(1:1) 
17.0 .+-. 2.7 
22.5 .+-. 2.7 
28.5 .+-. 2.6 
8 24-MFE/CHL 
(2:1) 
13.3 .+-. 3.3 
21.5 .+-. 3.1 
26.4 .+-. 4.1 
9 Ext./CHL (0.5:1) 
18.8 .+-. 4.5 
23.5 .+-. 3.6 
28.9 .+-. 5.0 
10 Ext./CHL (1:1) 
19.5 .+-. 2.6 
23.5 .+-. 2.0 
26.4 .+-. 6.2 
11 Ext./CHL (2:1) 
19.1 .+-. 1.8 
21.9 .+-. 1.9 
28.8 .+-. 3.8 
12 .gamma.-OZ/CHL 
(0.5:1) 
19.9 .+-. 1.5 
22.6 .+-. 2.6 
29.0 .+-. 2.5 
13 .gamma.-OZ/CHL 
(1:1) 
17.8 .+-. 3.0 
23.2 .+-. 1.3 
29.8 .+-. 4.9 
14 .gamma.-OZ/CHL 
(2:1) 
19.2 .+-. 2.4 
22.3 .+-. 2.5 
28.9 .+-. 4.1 
15 CAMFE/CHL 
(1:1) 
17.8 .+-. 2.2 
23.2 .+-. 2.2 
27.1 .+-. 2.1 
__________________________________________________________________________ 
(5) Total cholesterol level in the serum is expressed by the mean .+-. 
standard deviation (mg/dl). 
TABLE 11 
______________________________________ 
No. Group Mean .+-. standard deviation 
______________________________________ 
1 Normal 83.25 .+-. 15.39*** 
2 Control (CHL) 176.13 .+-. 25.40 
3 Ext./CHL (0.5:1) 168.25 .+-. 28.36 
4 Ext./CHL (1:1) 128.51 .+-. 20.56*** 
5 Ext./CHL (2:1) 110.82 .+-. 26.49*** 
6 .gamma.-OZ/CHL 
(0.5:1) 170.75 .+-. 38.04 
7 .gamma.-OZ/CHL 
(1:1) 136.13 .+-. 28.56* 
8 .gamma.-OZ/CHL 
(2:1) 112.38 .+-. 21.68*** 
9 CAFE/CHL (0.5:1) 174.13 .+-. 32.99 
10 CAFE/CHL (1:1) 122.88 .+-. 23.05*** 
11 CAFE/CHL (2:1) 140.38 .+-. 29.96* 
12 24-MFE/CHL (0.5:1) 165.63 .+-. 33.75 
13 24-MFE/CHL (1:1) 135.25 .+-. 28.09* 
14 24-MFE/CHL (2:1) 123.75 .+-. 23.37*** 
15 CAMFE/CHL (1:1) 100.75 .+-. 8.01*** 
______________________________________ 
*P &lt; 0.05; ***: P &lt; 0.001. 
As described above, increases in the total cholesterol level in the serum 
were significantly suppressed in the groups of CAFE/CHL (1:1), CAFE/CHL 
(2:1), 24-MFE/CHL (1:1), 24-MFE/CHL (2:1), extract/CHL (1:1), extract/CHL 
(2:1), .gamma.-OZ/CHL (1:1), .gamma.-OZ/CHL (2:1) and CAMFE/CHL (1:1). 
None of these additives caused any significant change in body weight or 
food intake. Thus, it can be concluded that they are applicable to foods 
without any problem. 
Example 7 
Administration test of egg yolk powder-containing food by using rat: 
As test samples, the extract, .gamma.-OZ, CAFE and CAMFE were employed. 
&lt;Test animal&gt; 
Species and strain: Slc:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Preparation of egg yolk powder 
To 2500 g of egg yolk, no additive or 50 g of each additive was added. 
After stirring with a mechanical stirrer for 1 hour, the mixture was 
freeze-dried. Thus, about 1250 g of a egg yolk powder with cholesterol 
complex formation was obtained. 
(2) Food and group 
Each egg yolk powder obtained in the above (1) was mixed with a 0.5% sodium 
cholate-containing food (CE-2) at a ratio of 1:1 and employed in a test 
wherein groups were classified as in Table 12. 
TABLE 12 
______________________________________ 
No. Powdery Sodium 
Yolk Yolk powder 
of food cholate 
pow- with 
No. Group rats CE-2 (%) 
(%) der complex 
______________________________________ 
1 Normal 8 99.5 0.5 -- -- 
2 Control 8 49.5 0.5 50.0 -- 
3 Extract 8 49.5 0.5 -- 50.0 
4 .gamma.-OZ 
8 49.5 0.5 -- 50.0 
5 CAFE 8 49.5 0.5 -- 50.0 
6 CAMFE 8 49.5 0.5 -- 50.0 
______________________________________ 
(3) Test method and blood collection 
(3) Test method and blood collection 
The rats were allowed to take each food as listed in Table 12 ad libitum 
for 3 days. On the days 2 and 3, the blood of each animal was collected 
from the tail vein. On the day 4 of the administration, the blood was 
collected from the abdominal aorta and the total cholesterol level in the 
serum was measured. 
Further, the liver was taken out and the cholesterol level in the liver was 
measured. Tables 13 to 15 show the body weight gain, food intake, total 
cholesterol level in the serum and cholesterol level in the liver of the 
rats. 
(4) Body weight and food intake are expressed by the mean .+-. standard 
deviation (g). 
TABLE 13 
______________________________________ 
Intake 
Body weight (g) (g/animal/day) 
No. Group day 0 day 3 days 0-3 
______________________________________ 
1 Normal 275.3 .+-. 11.6 
283.4 .+-. 9.9 
19.9 .+-. 1.3 
2 Control 274.6 .+-. 10.9 
289.2 .+-. 14.4 
16.5 .+-. 0.8 
3 Extract 275.1 .+-. 10.2 
291.6 .+-. 12.5 
18.2 .+-. 1.3 
4 .gamma.-OZ 
275.9 .+-. 9.6 
292.2 .+-. 10.5 
17.9 .+-. 1.8 
5 CAFE 274.8 .+-. 9.9 
287.2 .+-. 11.7 
17.1 .+-. 0.2 
6 CAMFE 274.7 .+-. 13.1 
288.4 .+-. 13.2 
17.3 .+-. 0.5 
______________________________________ 
(5) The total cholesterol level in the serum is expressed by the mean .+- 
standard deviation (mg/dl). 
TABLE 14 
______________________________________ 
Total CHL level in serum (mg/dl) 
No. Group day 0 day 2 day 3 
______________________________________ 
1 Normal 65.3 .+-. 7.9*** 
66.6 .+-. 9.5*** 
63.8 .+-. 10.7 
2 Control 90.0 .+-. 8.2 
95.6 .+-. 9.0 
68.8 .+-. 11.0 
3 Extract 70.6 .+-. 9.2** 
70.8 .+-. 10.3*** 
55.7 .+-. 6.8* 
4 .gamma.-OZ 
71.3 .+-. 10.3** 
72.8 .+-. 8.8*** 
57.4 .+-. 7.5* 
5 CAFE 81.1 .+-. 11.7 
81.4 .+-. 16.3* 
66.8 .+-. 16.0 
6 CAMFE 76.6 .+-. 10.7* 
74.5 .+-. 10.7*** 
56.4 .+-. 8.1* 
______________________________________ 
*: P &lt; 0.05; **: P &lt; 0.01; ***: P &lt; 0.001. 
(6) Liver weight (g) and the total cholesterol level in the liver are 
expressed by the mean .+-. standard deviation (mg/g, mg/total). 
TABLE 15 
______________________________________ 
Liver weight 
CHL 
No. Group (g) (mg/g liver) 
(mg/liver) 
______________________________________ 
1 Normal 11.4 .+-. 1.1 
4.8 .+-. 0.8*** 
55.1 .+-. 11.3*** 
2 Control 12.7 .+-. 1.3 
16.7 .+-. 1.2 
213.1 .+-. 27.4 
3 Extract 11.9 .+-. 0.6 
8.1 .+-. 0.6*** 
98.5 .+-. 21.8*** 
4 .gamma.-OZ 
12.6 .+-. 0.6 
8.2 .+-. 1.1*** 
104.0 .+-. 18.2*** 
5 CAFE 12.7 .+-. 0.9 
14.1 .+-. 0.9*** 
179.2 .+-. 15.0** 
6 CAMFE 12.4 .+-. 0.7 
10.1 .+-. 2.8*** 
124.9 .+-. 37.4*** 
______________________________________ 
*: P &lt; 0.05; **: P &lt; 0.01; ***: P &lt; 0.001. 
Example 8 
Forced oral administration of egg yolk powder by using rat: 
As test samples, the extract, .gamma.-OZ, CAFE and CAMFE were employed. 
&lt;Test animal&gt; 
Species and strain: Slc:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Food and group 
The egg yolk powder and egg yolk powder with cholesterol complex formation, 
prepared in the same manner as the one described in Example 7, were each 
adjusted to a dose of 3 g/6 ml/animal with the use of purified water for 
injection and forcibly administered per os to the rats three times a day 
for 3 days. 
On the days 2 and 3, the blood of each animal was collected from the tail 
vein. On the day 4 of the administration, the blood was collected from the 
abdominal aorta and the total cholesterol level in the serum was measured. 
Further, the liver was taken out and the total cholesterol level in the 
liver was measured. Tables 16 to 18 show the body weight gain, food 
intake, total cholesterol level in the serum and cholesterol level in the 
liver of the rats. 
(2) Body weight is expressed by the mean .+-. standard deviation (g). 
TABLE 16 
______________________________________ 
No. Group Day 0 Day 3 
______________________________________ 
1 Normal 265.4 .+-. 8.1 
277.6 .+-. 7.4 
2 Control 264.3 .+-. 13.1 
275.5 .+-. 13.1 
3 Extract 263.5 .+-. 11.8 
276.2 .+-. 12.8 
4 .gamma.-OZ 265.2 .+-. 13.3 
271.5 .+-. 16.6 
5 CAFE 266.5 .+-. 13.0 
279.4 .+-. 14.2 
6 CAMFE 262.3 .+-. 10.2 
271.7 .+-. 14.2 
______________________________________ 
(3) The total cholesterol level in the serum is expressed by the mean .+- 
standard deviation (mg/dl). 
TABLE 17 
______________________________________ 
Total CHL level in serum (mg/dl) 
No. Group day 0 day 2 day 3 
______________________________________ 
1 Normal 52.3 .+-. 6.4*** 
55.9 .+-. 5.0*** 
55.5 .+-. 5.2*** 
2 Control 67.6 .+-. 4.0 
86.4 .+-. 10.5 
70.4 .+-. 5.9 
3 Extract 53.2 .+-. 6.4*** 
61.6 .+-. 5.6*** 
52.8 .+-. 6.9*** 
4 .gamma.-OZ 
54.4 .+-. 4.6*** 
62.1 .+-. 4.1*** 
47.4 .+-. 3.2*** 
5 CAFE 61.1 .+-. 7.0* 
78.9 .+-. 10.1 
62.3 .+-. 6.5* 
6 CAMFE 67.6 .+-. 11.8 
74.0 .+-. 8.1* 
59.6 .+-. 10.6* 
______________________________________ 
*: P &lt; 0.05; **: P &lt; 0.01; ***: P &lt; 0.001. 
(4) Liver weight (g) and the total cholesterol level in the liver are 
expressed by the mean .+-. standard deviation (mg/g, mg/total). 
TABLE 18 
______________________________________ 
Liver weight 
CHL 
No. Group (g) (mg/g liver) 
(mg/liver) 
______________________________________ 
1 Normal 8.7 .+-. 0.4*** 
5.2 .+-. 0.4*** 
45.2 .+-. 4.4*** 
2 Control 10.3 .+-. 0.8 
11.2 .+-. 1.8 
116.5 .+-. 23.9 
3 Extract 9.8 .+-. 0.5 
5.1 .+-. 0.6*** 
49.7 .+-. 8.2*** 
4 .gamma.-OZ 
9.6 .+-. 0.8 
5.3 .+-. 0.4*** 
50.4 .+-. 5.3*** 
5 CAFE 10.3 .+-. 0.7 
9.7 .+-. 0.7* 
99.8 .+-. 9.2 
6 CAMFE 9.6 .+-. 0.7 
7.0 .+-. 1.2 
67.5 .+-. 15.2*** 
______________________________________ 
*: P &lt; 0.05; **: P &lt; 0.01; ***: P &lt; 0.001. 
As described above, all of the test samples significantly suppressed 
increase in the total cholesterol level in the serum and the total 
cholesterol level in the liver in Examples 6 to 8. The extract, .gamma.-OZ 
and CAMFE showed particularly remarkable significant differences. None of 
these additives caused any significant difference in body weight gain and 
food intake. 
Example 9 
Forced administration test of cholesterol complex by using normal and 
diabetic rats: 
As test samples, CAFE, the extract and CAMFE were employed. 
&lt;Test animal&gt; 
Species and strain: Slc:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Preparation of diabetic rat 
42 to 44 mg/kg of alloxan was intravenously administered to male Slc:SD 
rats aged 8 weeks to thereby give diabetic rats. 
In the test, diabetic rats 13 weeks after the administration of alloxan 
were used. 
(2) Labeled compound 
.sup.3 H-cholesterol (hereinafter referred to as .sup.3 H-CHL) purchased 
from Japan Isotope Association was used. 
(3) Formation of liquid preparation to be administered 
.sup.3 H-CHL was mixed with the extract, CAFE or CAMFE each at a ratio of 
1:2 and dissolved in chloroform. After evaporating to dryness under 
reduced pressure, each complex was obtained. This complex was suspended in 
5% acacia and thus a liquid preparation to be administered was formed. In 
the liquid preparation, the concentration of CHL was adjusted to 20 mg/ml. 
(4) Administration method 
The .sup.3 H-CHL liquid preparation, extract/.sup.3 H-CHL liquid 
preparation, CAFE/.sup.3 H-CHL liquid preparation or CAMFE/.sup.3 H-CHL 
liquid preparation was orally administered to normal and diabetic rats in 
a dose of 5 ml/kg by using a sound. The administration radioactivity was 
adjusted to 3.7 MBq/kg. The animals were fasted for about 18 hours before 
the administration and, immediately after the administration, allowed to 
take food. 
(5) Measurement of serum level 
Two, four, and six hours after the administration, the blood of each rat 
was collected from the tail vein. Twenty-four hours after the 
administration, the blood was collected from the abdominal aorta. The 
collected blood was centrifuged (3,000 rpm, 15 min) at 4.degree. C. to 
give the serum. 
To 100 .mu.l of the serum, 1 ml of a tissue solubilizer SOLUENE-350 
(KRAD) was added. After the completion of the dissolution, 9 ml of a 
scintillator ECONOFLUOR (Du Pont NEN Research Products) was added and the 
radioactivity was measured with a liquid scintillation counter (TRI-CARB 
4530, PCAKRAD). Tables 19 and 20 show the results of the measurement of 
the serum .sup.3 H-CHL level. 
TABLE 19 
______________________________________ 
Normal rat 
No. Serum .sup.3 H-CHL level (.mu.g/ml) 
No. Group of rats 2 hrs 
4 hrs 6 hrs 
24 hrs 
______________________________________ 
1 CHL 3 34.2 26.6 16.6 11.6 
2 Ext./CHL 3 20.3 16.7 14.4 10.3 
3 CAFE/CHL 3 17.5 16.2 16.2 11.6 
4 CAMFE/CHL 3 11.0 12.1 6.1 2.8 
______________________________________ 
TABLE 20 
______________________________________ 
Diabetic rat 
No. Serum .sup.3 H-CHL level (.mu.g/ml) 
No. Group of rats 2 hrs 
4 hrs 6 hrs 
24 hrs 
______________________________________ 
1 CHL 3 12.5 12.7 12.2 10.5 
2 Ext./CHL 3 8.8 11.1 9.0 8.2 
3 CAFE/CHL 3 10.9 11.1 11.6 6.8 
4 CAMFE/CHL 3 4.6 4.1 2.4 2.8 
______________________________________ 
As shown above, the serum CHL level was suppressed by administering the 
complexes of CHL with the test samples in Example 9. In particular, CAMFE 
showed a remarkable effect. 
CAFE employed in the following Examples 10 to 19 was prepared by repeatedly 
recrystallizing, similar to the procedure as in Example 1. As the extract, 
an extract obtained by degumming and dewaxing oily components extracted 
from rice bran with a solvent, followed by treatment with an alkali, 
neutralization and solid/liquid separation, and distilling the residue 
followed by solvent-extraction and column treatment was employed. 
Example 10 
Production of mayonnaise by using rice bran component-containing fat and 
oil: 
After stirring 6 g of egg yolk by using an eggbeater, 4.5 ml of rice 
vinegar was added thereto and mixed. Next, 30 ml of salad oil (a product 
of The Nissin Oil Mills, Ltd. containing 2% of the extract was slowly 
added to give a mayonnaise. 
Example 11 
Production of mayonnaise by using whole egg and/or egg yolk treated with 
rice bran component: 
(1) Production of mayonnaise by using dry egg yolk 
To 6 g of egg yolk, 312 mg of the extract.sup.1) was added and mixed by 
stirring with an eggbeater, followed by freeze-drying. A 3 g portion of 
the extract-containing dry egg yolk thus obtained was weighed and put into 
a mortar, and 3 ml of water, 4.5 ml of rice vinegar and 30 ml of salad oil 
were slowly added with stirring to give a mayonnaise. 
1): The amount of the added extract, i.e. 312 mg was determined by 
referring that 78 mg of cholesterol was contained in 6 g of egg yolk (13 
mg/1 g of egg yolk). 
The amount of the extract to be added was set to about twice as much on a 
molar basis. 
(2) Production of mayonnaise by using egg yolk 
To 6 g of egg yolk, 312 mg of the extract.sup.1) was added and mixed by 
using an eggbeater. After adding and mixing 4.5 ml of rice vinegar, 30 ml 
of salad oil was slowly added to give a mayonnaise. 
Example 12 
Production of mayonnaise by using whole egg and/or egg yolk treated with 
rice bran component and rice bran component-containing fat and oil: 
(1) Production of mayonnaise by using extract-containing dry egg yolk 
A 3 g portion of the extract-containing dry egg yolk obtained in the same 
manner as in Example 11 (1) was weighed and put into a mortar, and 3 ml of 
water and 4.5 ml of rice vinegar was added thereto. Next, 30 ml of the 
extract-containing salad oil obtained in the same manner as in Example 10 
was slowly added to give a mayonnaise. 
(2) Production of mayonnaise by using egg yolk 
To 6 g of egg yolk, 312 mg of the extract.sup.1) was added and mixed by 
using an eggbeater. After adding and mixing 4.5 ml of rice vinegar, 30 ml 
of the extract-containing salad oil obtained in the same manner as in 
Example 10 was slowly added to give a mayonnaise. 
Example 13 
Production of mayonnaise by using CAFE-containing fat and oil: 
The procedures of Examples 10, 11 and 12 were repeated but substituting the 
extract with CAFE to produce mayonnaises. 
Example 14 
Forced oral administration test of mayonnaise produced by using rice bran 
component-containing fat and oil: 
&lt;Test animal&gt; 
Species and strain: Jcl:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Preparation of diabetic rat 
To male Jcl:SD rats aged 8 weeks, 42 to 44 mg/kg of alloxan was 
intravenously administered to give diabetic rats. 
In the test, diabetic rats 13 weeks after the administration of alloxan 
were used. 
(2) Production of mayonnaise 
To 6 g of egg yolk, 1,050 mg of cholesterol was added and mixed by using an 
eggbeater and 4.5 ml of rice vinegar was added thereto. Then, 30 ml of 
salad oil containing 7% of the extract was slowly added to give a 
mayonnaise. The mayonnaise of the control group was produced by the 
similar method. 
(3) Administration method and blood collection 
The control mayonnaise prepared above was forcibly administered per os to 
the rats in a dose of 9 g/kg twice a day for 2 days. At 9:00 a.m. on the 
day 3, each mayonnaise was forcibly administered per os to the rats in the 
same dose. After 4 and 8 hours, the blood of each animal was collected 
from the tail vein. After 24 hours, the blood was collected from the 
abdominal aorta and the total cholesterol level in the serum was measured. 
Example 15 
Forced oral administration test of mayonnaise produced by using 
extract-containing egg yolk: 
&lt;Test animal&gt; 
Species and strain: Crj:SD rat (SPF). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Labeled compound 
.sup.3 H-cholesterol (hereinafter referred to as .sup.3 H-CHL) and .sup.14 
C-cholesterol (hereinafter referred to as .sup.14 C-CHL) purchased from 
Japan Isotope Association were used. 
(2) Preparation of dry egg yolk 
1.5 ml of .sup.14 C-CHL (9.25 MBq/6.25 ml ethanol solution) and 0.5 ml of 
distilled water were introduced into a 50 ml round-bottom flask and the 
ethanol was evaporated under reduced pressure. Next, 1 ml of distilled 
water and 4 g of egg yolk were added thereto. In the case of the 
extract/.sup.14 C-CHL group, 209.2 mg of the extract.sup.1) was further 
added thereto. After stirring, each mixture was frozen with dry 
ice/ethanol and freeze-dried for about 8 hours. 
1): The amount of the added extract, i.e. 209.2 mg was determined by 
referring that 52.3 mg of cholesterol was contained in 4 g of egg yolk 
(13.08 mg/1 g of egg yolk). The amount of the extract to be added was set 
to about twice as much on a molar basis. 
(3) Production of mayonnaise 
A 1 g portion of the .sup.14 C-CHL or extract/.sup.14 C-CHL-containing dry 
egg yolk was weighed and put into a mortar. Then, 1 ml of water, 9 ml of 
salad oil and 1.5 ml of rice vinegar were slowly added and stirred to give 
a mayonnaise. The radioactivity (Bq/g) of the mayonnaise was measured with 
a liquid scintillator by sampling part of the mayonnaise. 
(4) Preparation of cholesterol suspension for tail vein administration 
A 35 .mu.l portion of .sup.3 H-CHL 1,2,6,7-3H-(N)-! was pipetted into a 10 
ml test tube with a glass stopper and evaporated to dryness under a 
nitrogen atmosphere. Then, it was dissolved in 200 .mu.l of 95% ethanol. 
Then, it was suspended in physiological saline and the total volume was 
adjusted to 4 ml. 
(5) Administration method 
From the day of the administration, the rats were transferred into an RI 
feeding room and individually placed in stainless metabolism cages. At 
10:00 a.m., the .sup.3 H-CHL suspension was intravenously administered 
through the tail vein in a dose of 2.5 ml/kg. Immediately thereafter, the 
.sup.14 C-CHL or the extract/.sup.14 C-CHL containing mayonnaise was 
orally administered in a dose of 10 g/kg by using a sound. The food was 
taken away from the cage at 4:00 p.m. on the day before the administration 
(water was given without restriction). Feeding was started again 6 hours 
after the administration. Then, 2, 4, 6, 8, 10, 24, 48 and 72 hours after 
the administration, the blood of each animal was collected from the tail 
vein. Further, 96 hours after the administration, the blood was taken from 
the abdominal aorta under etherization. After collecting, the blood was 
centrifuged to give the serum. 
(6) Measurement of serum cholesterol level 
To 100 mg of the serum, 1 ml of a tissue dissolution agent SOLUENE-350 
(KRAD) was added. After the completion of the dissolution, 9 ml of a 
scintillator ECONOFLUOR (Du Pont NEN Research Products) was added and the 
radioactivity was measured with a liquid scintillation counter (TRI-CARB 
4530, KRAD). 
(7) Measurement of cholesterol absorption ratio 
To 100 mg of the serum, 1 ml of the tissue dissolution agent SOLUENE-350 
was added. Next, 9 ml of scintillator was further added thereto and the 
.sup.14 C and .sup.3 H radioactivities were measured with a liquid 
scintillation counter. Then, the absorption ratio of cholesterol (.sup.14 
C/.sup.3 H.times.100) was determined. 
Example 16 
Forced oral administration test of mayonnaise by using extract-containing 
egg yolk: 
The procedure of Example 14 was repeated but varying Example 14 &lt;Test 
method&gt; (2) and (3) as follows. 
(2) Production of mayonnaise 
To 6 g of egg yolk, 1,050 mg of cholesterol and 2,100 mg of the extract 
were added and mixed by using an eggbeater. Then, 4.5 ml of rice vinegar 
was added and 30 ml of salad oil was further added slowly to give a 
mayonnaise. The mayonnaise of the control group was produced in the 
similar manner. 
(3) Administration method and blood collection 
The mayonnaise of the control group (9 g/kg) or the extract-treated 
mayonnaise (10 g/kg) prepared above was orally administered to the animals 
twice a day for 2 days. 
Example 17 
Forced oral administration test of mayonnaise produced by using rice bran 
component-treated whole egg and/or egg yolk and rice bran 
component-containing fat and oil: 
The procedure of Example 15 was repeated but varying Example 15 &lt;Test 
method&gt; (3) as follows. 
(3) Production of mayonnaise 
A 1 g portion of the .sup.14 C-CHL or .sup.14 C-CHL-added dry egg yolk was 
weighed and put into a mortar. Then, 1 ml of water, 9 ml of salad oil 
containing 2% of the extract and 1.5 ml of rice vinegar were slowly added 
with stirring to give a mayonnaise. The radioactivity (Bq/g) of the 
mayonnaise was measured with a liquid scintillator by sampling part of the 
prepared mayonnaise. 
Example 18 
Forced oral administration test of mayonnaise produced by using rice bran 
component-treated whole egg and/or egg yolk and rice bran 
component-containing fat and oil: 
The procedure of Example 16 was repeated but varying Example 16 &lt;Test 
method&gt; (2) as follows. 
(2) Production of mayonnaise 
To 6 g of egg yolk, 1,050 mg of cholesterol and 2,100 mg of the extract 
were added and mixed by using an eggbeater. After adding 4.5 ml of rice 
vinegar, 30 ml of salad oil containing 7% of the extract was further added 
slowly to give a mayonnaise. The mayonnaise of the control group was 
produced in the same manner. 
Example 19 
Forced oral administration test of mayonnaise produced by using rice bran 
component-treated whole egg and/or egg yolk and rice bran 
component-containing fat and oil: 
The procedure of Example 15 was repeated but varying Example 15 &lt;Test 
method&gt; (3) as follows. 
(3) Production of mayonnaise 
A 1 g portion of .sup.14 C-CHL or the extract/.sup.14 C-CHL-added dry egg 
yolk was weighed and put into a mortar. Then, 1 ml of water, 9 ml of salad 
oil containing 2% of the extract and 1.5 ml of rice vinegar were slowly 
added with stirring to thereby give a mayonnaise. The radioactivity (Bq/g) 
of the mayonnaise was measured with a liquid scintillator by sampling part 
of the prepared mayonnaise. 
Table 21 shows the results of the measurement of the total cholesterol 
level in the serum in Examples 14, 16 and 18. 
TABLE 21 
__________________________________________________________________________ 
No. Content Total CHL in blood (mg/dl) 
of (%) Dose 
before 
after (hrs) 
Group 
rats 
CHL 
Ext. 
(g/kg) 
0 4 8 24 
__________________________________________________________________________ 
Control 
8 2.7 
-- 9 218 .+-. 70 
679 .+-. 204 
680 .+-. 162 
550 .+-. 284 
Ex. 14 
8 2.7 
5.0 
9 219 .+-. 69 
556 .+-. 286 
520 .+-. 113* 
458 .+-. 170 
Ex. 16 
8 2.7 
5.0 
10 222 .+-. 66 
540 .+-. 135 
515 .+-. 100* 
444 .+-. 230 
Ex. 18 
8 2.7 
10.0 
10 220 .+-. 60 
390 .+-. 103** 
321 .+-. 115*** 
263 .+-. 141* 
__________________________________________________________________________ 
*: P &lt; 0.05; **: P &lt; 0.01; ***: P &lt; 0.001. 
The total cholesterol level in the serum is expressed by the mean .+-. 
standard deviation (mg/dl). 
The data obtained 8 hours after the final administration as given in Table 
21 indicate that in the mayonnaise produced by adding the 
extract-containing salad oil to egg yolk (corresponding to the mayonnaise 
described in Example 10) and the mayonnaise produced by adding the extract 
to egg yolk and then adding salad oil thereto corresponding to the 
mayonnaise described in Example 11 (2)!, the total cholesterol level in 
the serum of the extract-treated egg yolk/salad oil was obviously lowered 
compared with the control case with the use of egg yolk/salad oil. 
Compared with the control case with the use of egg yolk/salad oil, the 
total cholesterol level in the serum was remarkably lowered in the case of 
the mayonnaise obtained from the extract-treated egg 
yolk/extract-containing salad oil corresponding to the mayonnaise 
described in Example 12 (2)!. 
Table 22 shows the area under curve of the serum total cholesterol level 
curves (0-96 hours) of Examples 15, 17 and 19 expressed in the mean 
+standard deviation (.mu.g.hr.ml.sup.-1). 
TABLE 22 
______________________________________ 
Area Under Curve 
No. Content (0-96 hrs) 
of (%) Dose calcd. ratio 
Group rats CHL Ext. (g/kg) 
(.mu.g .multidot. hr .multidot. ml.sup.-1) 
2 (%) 
______________________________________ 
Control 
3 0.26 -- 10 1472.3 .+-. 207.0 
100 
Ex. 15 3 0.26 1.04 10 1274.3 .+-. 234.0 
86.6 
Ex. 17 3 0.26 1.80 10 1261.8 .+-. 212.6 
85.7 
Ex. 19 3 0.26 2.84 10 729.3 .+-. 117.7** 
49.5 
______________________________________ 
**: P &lt; 0.01. 
The data given in Table 22 indicate that the use of the extract-treated, 
freeze-dried egg yolk/salad oil suppressed the absorption of cholesterol 
by 13.4% compared with the case wherein the freeze-dried egg yolk/salad 
oil was used, and the use of the extract-containing salad oil suppressed 
the absorption of cholesterol by 14.3% compared with the case where the 
freeze-dried egg yolk/salad oil was used. 
When the above-mentioned methods were employed in combination, that is to 
say, the extract-treated, freeze-dried egg yolk/extract-containing oil was 
used, the absorption of cholesterol was suppressed by 50.5% compared with 
the case where the freeze-dried egg yolk/salad oil was used, showing a 
remarkable synergistic effect. 
FIG. 15 shows the serum cholesterol level within a period of 2 to 72 hours 
after the administration, while FIG. 16 shows the cholesterol absorption 
within a period of 2 to 96 hours after the administration. 
These FIGS. 15 and 16 indicate that the serum cholesterol level and 
cholesterol absorption ratio after 24 hours from the administration were 
in the following order. In particular, the case with the use of the 
extract-treated, freeze-dried egg yolk/extract-containing salad oil showed 
extremely low values. 
1. Use of extract-treated, freeze-dried egg yolk/extract-containing salad 
oil. 
2. Use of extract-treated, freeze-dried egg yolk/salad oil. 
3. Use of freeze-dried egg yolk/extract-containing salad oil. 
4. Use of freeze-dried egg yolk/salad oil (control). 
When the extract was substituted with CAFE and the total serum cholesterol 
level and the area under curve thereof (0-96 hours) were measured in the 
same manner as those of Examples 14 to 19, the obtained results were 
similar to the data obtained in Examples 14 to 19. 
Example 20 
Method for producing cake: 
To 72 g of egg yolk or the extract-containing egg yolk, 36 g of wheat 
flour, 15 g of sugar and 1 g of baking powder were added at once and 
mixed. The obtained mixture was baked in an oven at 160.degree. C. for 40 
minutes. This cake contained 60 g of egg yolk per 100 g. 
Example 21 
Effect of the intake of cake produced by using extract-containing egg yolk 
on serum cholesterol: 
&lt;Test animal&gt; 
Species and strain: Slc:SD rat (SPE). 
Sex: Male. 
Age at purchase: 7 weeks. 
&lt;Test method&gt; 
(1) Group 
______________________________________ 
No. of Composition of food (g) 
Group rats Cake CE-2 
______________________________________ 
Treated control 
10 20 10 
Control 10 freely taking solid CE-2 
Ext.-containing 
10 20 10 
______________________________________ 
(2) Test method and blood collection 
To the male Slc:SD rats (SPF, aged 8 weeks), 42 to 44 mg/kg of alloxan was 
intravenously administered. From 11 weeks thereafter, 20 g of the egg 
yolk-containing cake and 4 pellets (about 10 g) of a solid food CE-2 were 
given to the animals to thereby prepare hypercholesterolemia rats. 
The animals were fed for 10 days by giving 20 g of the cake produced by 
adding 2% (based on the egg yolk weight) of the extract and 4 pellets 
(about 10 g) of the solid food CE-2. On the days 5 and 10, the blood of 
each animal was collected from the tail vein and the total CHL in the 
serum was measured. 
The total cholesterol level in the serum is expressed by the mean .+-. 
standard deviation (mg/dl). 
TABLE 23 
__________________________________________________________________________ 
Total CHL in serum of rat after administering cake 
produced by using extract-containing egg yolk 
No. of Total CHL level in serum (mg/dl) 
Group rats 
Day 0 Day 5 Day 10 
__________________________________________________________________________ 
Treated 
10 563.3 .+-. 402.2 
659.6 .+-. 433.5 
(0) 760.3 .+-. 443.0 
(0) 
control 
Control 
10 547.5 .+-. 355.9 
202.4 .+-. 58.9 
(100)** 
227.9 .+-. 100.5 
(100)** 
Ext.- 10 568.8 .+-. 360.6 
254.9 .+-. 162.1 
(88)* 
267.2 .+-. 192.7 
(93)** 
containing 
__________________________________________________________________________ 
Mean .+-. standard deviation; *: P &lt; 0.05; **: P &lt; 0.01. 
(): Suppression ratio. 
Industrial Applicability! 
According to the present invention, cholesterol contained in a food can be 
reduced and the absorption thereof by a living body can be prevented. 
Thus, a food additive and a food, which are useful in inhibiting the 
accumulation of cholesterol in the living body and in preventing 
arteriosclerosis, can be obtained. According to the present invention, 
mayonnaises having an effect of lowering blood cholesterol level or 
suppressing an increase of blood cholesterol level can be obtained.