U.S. patent application number 12/227951 was filed with the patent office on 2009-07-23 for composition for preventing and improving metabolic syndrome.
This patent application is currently assigned to J-OIL MILLS, INC.. Invention is credited to Yousuke Isobe, Syuichi Kamo, Sanshirou Saito, Toshiro Sato.
Application Number | 20090186145 12/227951 |
Document ID | / |
Family ID | 38344671 |
Filed Date | 2009-07-23 |
United States Patent
Application |
20090186145 |
Kind Code |
A1 |
Saito; Sanshirou ; et
al. |
July 23, 2009 |
Composition for Preventing and Improving Metabolic Syndrome
Abstract
The present invention is intended to provide a composition for
preventing or improving metabolic syndrome, which is safe and
efficiently ingestible. Provided is a composition for reducing
blood cholesterol level, improving blood HDL/LDL cholesterol ratio,
reducing blood triglyceride level, reducing blood sugar level,
controlling increase in liver weight, and/or reducing body weight,
the composition containing soybean germ protein as an active
ingredient. The composition according to the present invention
improves blood cholesterol level, triglyceride, and glucose levels
thereby contributing to health promotion.
Inventors: |
Saito; Sanshirou; (Tokyo,
JP) ; Sato; Toshiro; (Tokyo, JP) ; Kamo;
Syuichi; (Tokyo, JP) ; Isobe; Yousuke; (Tokyo,
JP) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 320850
ALEXANDRIA
VA
22320-4850
US
|
Assignee: |
J-OIL MILLS, INC.
TOKYO
JP
|
Family ID: |
38344671 |
Appl. No.: |
12/227951 |
Filed: |
June 19, 2007 |
PCT Filed: |
June 19, 2007 |
PCT NO: |
PCT/JP2007/062333 |
371 Date: |
December 3, 2008 |
Current U.S.
Class: |
426/656 ;
426/590; 426/648 |
Current CPC
Class: |
A61K 36/185 20130101;
A61K 36/48 20130101; A61P 1/16 20180101; A61P 3/04 20180101; A23V
2002/00 20130101; A61P 3/06 20180101; A61P 3/10 20180101; A21D
2/266 20130101; A23L 33/185 20160801; A21D 13/80 20170101; A61P
3/08 20180101; A23V 2002/00 20130101; A23V 2200/326 20130101; A23V
2200/3262 20130101; A23V 2250/5488 20130101; A23V 2002/00 20130101;
A23V 2250/5488 20130101; A23V 2250/0616 20130101; A23V 2250/5118
20130101; A23V 2250/5108 20130101; A23V 2200/3262 20130101 |
Class at
Publication: |
426/656 ;
426/648; 426/590 |
International
Class: |
A23J 3/16 20060101
A23J003/16; A23L 1/307 20060101 A23L001/307; A23L 2/38 20060101
A23L002/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2006 |
JP |
2006-208949 |
Claims
1. A composition for improving metabolic syndrome, comprising
soybean germ protein as an active ingredient.
2. The composition according to claim 1, wherein metabolic syndrome
is improved through improvement of lipid metabolism.
3. The composition according to claim 1, wherein lipid metabolism
is improved through improvement of HDL/LDL cholesterol ratio and/or
reduction of blood triglyceride level.
4. The composition according to claim 1, wherein metabolic syndrome
is improved through reduction of blood sugar level and/or control
of increase the liver weight.
5. A composition for reducing body weight, comprising soybean germ
protein as an active ingredient.
6. The composition according to claim 1, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
7. An agent for improving blood HDL/LDL cholesterol ratio, reducing
blood triglyceride level, reducing blood sugar level, and/or
reducing body weight, the agent comprising soybean germ protein as
an active ingredient.
8. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 1.
9. The composition according to claim 2, wherein lipid metabolism
is improved through improvement of HDL/LDL cholesterol ratio and/or
reduction of blood triglyceride level.
10. The composition according to claim 2, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
11. The composition according to claim 3, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
12. The composition according to claim 9, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
13. The composition according to claim 4, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
14. The composition according to claim 5, wherein the soybean germ
protein is a substance obtained by treating soybean germ through a
process including solvent treatment and heat treatment.
15. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 2.
16. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 3.
17. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 9.
18. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 4.
19. Foods, beverages, functional foods, dietary supplements, and
diets comprising the composition according to claim 5.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
preventing and improving metabolic syndrome utilizing soy germ
protein, more specifically to a composition for improving the blood
lipid condition and the blood sugar level, controlling the increase
in liver weight, and reducing the body weight.
BACKGROUND ART
[0002] In Japan, from ancient times, soybeans have been eaten in
various forms, such as bean curd, soybean milk, deep-fat fried bean
curd, boiled beans, fermented soybeans, soybean flour, miso
(fermented soybean paste), and soy sauce. In recent years, various
physiological components are receiving attention, so that soybean
is expected to serve as food useful for health maintenance. Soybean
protein (defatted soybean), which is prepared by removing oil from
soybean, contains about 40 to 50% protein, and has relatively good
amino acid balance of a vegetable protein. Therefore, soybean
protein is called "field meat", and widely used for food and feed
applications. Functional components contained in soybeans, such as
isoflavone, lecithin, and vitamin E are also receiving attention,
and these useful components have been used as supplements for, for
example, prevention of various diseases. However, soybeans also
contains a trypsin inhibitor which inhibits the activity of
proteolytic enzymes, an antigenic protein as an allergen which
causes an allergy, phytic acid which inhibits absorption of
minerals and the like. Effective use of soybean as a nutritional
functional food is based on two main points of efficient use of
useful components and reduction of antinutrient components.
[0003] On the other hands, in recent years, metabolic syndrome has
become a significant public health issue particularly among people
of middle and advanced age, and food and drugs for improving the
syndrome have been demanded. It is well known that soybeans reduce
the blood cholesterol level. The effect is likely due to that
soybean protein specifically binds to cholesterol precursors such
as bile acid, and is excreted as feces.
[0004] In order to investigate the physiological functions such as
the cholesterol reducing effect, identification of functional
fractions of peptide components obtained from decomposed soybean
protein has been studied. For example, it is confirmed that
undigested fractions obtained by enzymatic digestion of soybean
protein lower the cholesterol level (Patent Document 1, Non-patent
Documents 1 and 2). As other soybean components which improve the
blood neutral lipid, cholesterol and blood sugar levels, 11S
globulin called glycinin (Patent Document 2), 7S globulin called
.beta.-conglycinin, isoflavone, and others are said to be
effective, but no clear conclusion has been provided. Further,
proteins of soybean germ have little been studied.
[0005] Regarding the substances derived from soybean germ, it is
reported that isoflavone aglycon, which is contained in a substance
prepared by treating soybean germ with aspergillus oryzae,
hydrolyzing the product, and extracting and concentrating the
product with a solvent, is effective as an active ingredient for
promoting bioactivity (Patent Document 3). However, there still are
different opinions among researchers about the efficacy of
isoflavone inclusive of its metabolites, so that no clear
conclusion has been provided.
[0006] The protein composition of soybean germ is markedly
different from that of soybean. Soybean germ has a low nutritional
value because it contains little storage protein such as
conglycinin and .beta.-conglycinin, and its total protein content
is low. In addition, soybean germ abundantly contains antinutrients
such as saponin and allergens, so that its excessive ingestion
causes an allergy, growth inhibition or the like. Soybean germ
further has a bad flavor. Therefore, it is believed to be
unsuitable for uses in foods, beverages, and medicines. The
inventors have applied for a patent on a soybean germ protein
composition which resolves the problem (Japanese Patent Application
No. 2005-36942). The composition is prepared by removing
antinutrient components from soybean germ through solvent treatment
in combination with heat treatment. The process increases the
content of soybean germ protein while decreasing the content of
allergens derived from the protein, and improves the flavor.
However, functionality of soybean germ protein, more specifically,
processing characteristics and physiological effects caused by the
ingestion of the protein have not known at all until the completion
of the present invention. [0007] Patent Document 1: Japanese Patent
Publication No. 07-025796 [0008] Patent Document 2: Japanese Patent
Application Laid-Open No. 2003-88334 [0009] Patent Document 3: WO
2003/077904 [0010] Non-patent Document 1: Atherosclerosis, 72, 115,
1988 [0011] Non-patent Document 2: J. Nutr., 120, 977, 1990
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0012] The present invention is intended to identify a functional
component of soybean having the effect of improving the blood lipid
and blood sugar levels, which has not been revealed, and utilize
the component in food, beverages, drugs or a material thereof.
Means for Solving the Problems
[0013] An aspect of the present invention is a composition for
improving blood lipid level and blood sugar level, an active
ingredient of the composition being a fraction containing soybean
germ protein obtained through fractionation carried out such that
the fraction contains germ protein, which is a minor element of
soybeans, at a ratio of 20% or more, more preferably 40% or more.
Further, when the obtained soybean germ protein is subjected to,
for example, solvent treatment, chemical treatment, or physical
treatment thereby removing antinutrient components, and in
combination with heat treatment, the protein content in the
resultant soybean germ protein increases while the content of
allergens derived from the protein decreases, and the flavor of the
protein is improved.
[0014] More specifically, the present invention is directed to a
composition for improving blood HDL/LDL cholesterol ratio, reducing
blood triglyceride level, reducing blood sugar level, and reducing
body weight, the composition containing soybean germ protein as an
active ingredient.
ADVANTAGEOUS EFFECT OF THE INVENTION
[0015] The composition according to the present invention improves
blood cholesterol level, triglyceride and blood sugar level,
thereby contributing to health promotion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a view showing electrophoretic patterns of
proteins.
[0017] FIG. 2 is a view showing electrophoretic patterns of
defatted proteins.
[0018] FIG. 3 is a view showing electrophoretic patterns of a
concentrated soybean germ protein.
[0019] FIG. 4 is a graph showing the change in total blood
cholesterol concentration after administration of germ protein and
SPC.
[0020] FIG. 5 is a graph showing the change in blood LDL
cholesterol concentration after administration of germ protein and
SPC.
[0021] FIG. 6 is a graph showing the change in blood HDL
cholesterol concentration after administration of germ protein and
SPC.
[0022] FIG. 7 is a graph showing the change in blood HDL
cholesterol/blood LDL cholesterol ratio after administration of
germ protein and SPC.
[0023] FIG. 8 is a graph showing the change in blood triglyceride
concentration after administration of germ protein and SPC.
[0024] FIG. 9 is a graph showing the change in blood sugar level
after administration of germ protein and SPC.
[0025] FIG. 10 is a graph showing the change in liver weight per
body weight after administration of germ protein and SPC.
[0026] FIG. 11 is a graph showing the change in total blood
cholesterol concentration after administration of germ protein and
SPC.
BEST MODE FOR CARRYING OUT THE INVENTION
[0027] Common soybean contains about 0.8% soybean germ protein,
though the content varies among species. Isolation of soybean germ
protein from soybean may employ any method. For example, soybean
may be half-crushed, and sieved to obtain soybean germ. Under the
method, the soybean germ protein is concentrated to about 50-fold.
The concentrate may be further defatted thereby removing the lipid
component to increase the concentration of the soybean germ
protein. Alternatively, the protein may be extracted with water or
any organic solvent, preferably hydrous alcohol at 0.degree. C. to
100.degree. C. When the extracted soybean germ protein is heated at
50 to 300.degree. C. for 1 to 180 minutes, soybean germ protein
having an excellent flavor and a high nutritional value is
obtained. If the heat treatment is carried out at a temperature
lower than 50.degree. C. and/or for a period of less than 1 minute,
modification of the protein is insufficient, and if the temperature
is higher than 300.degree. C. and/or the period is more than 180
minutes, the protein is burned and the amino acids decrease.
According to a method of removing the antinutrient components from
soybean germ through extraction with an aqueous solvent and then
collecting specific proteins by isoelectric precipitation under pH
control with an acid or alkali, extraction and collection can be
carried out with simple equipment. Alternatively, according to a
method of extracting and removing low polarity substances using
supercritical carbon dioxide, carbon dioxide is vaporized in a
state that it is returned to normal temperature and normal pressure
conditions, which advantageously dispenses with subsequent drying
treatment.
[0028] In the present invention, the solvent treatment and heat
treatment may be performed sequentially or simultaneously, or
either one treatment may be dispensed with. They may be combined
with other treatment processes such as pulverization, separation,
and chromatography. The soybean germ protein of the present
invention may be produced from common soybean protein such as
defatted soybean, soybean protein concentrate, soybean protein
isolate, and soybean milk through removal of storage protein
specifically abundant in the cotyledon of soybean, and fibers
abundant in seed coat. The germ portion of soybean contains
isoflavone and saponin at relatively high concentrations, so that
excess intake of the germ needs to be careful of. However,
combination of the above methods provides soybean germ protein
which contains less isoflavone and saponin, and can safely be
taken.
[0029] For example, when soybean germ is subjected to solvent
treatment with hexane and/or hydrous alcohol, and heat treatment at
50 to 300.degree. C. for 1 to 80 minutes, the isoflavone content is
decreased to 0.5% or less, and the saponin content is decreased to
1% or less. More specifically, when soybean germ is subjected to
solvent treatment with 70% ethanol at 70.degree. C. for 30 minutes,
and heat treatment at 100.degree. C. for 40 minutes, the isoflavone
content is decreased to about 0.1%, and the saponin content is
decreased to about 0.2%.
[0030] The obtained soybean germ protein is markedly different from
the protein composition of ordinary soybean, and thus can be
readily identified by SDS-PAGE analysis.
[0031] The soybean germ protein obtained according to the present
invention has the following effects.
[0032] Firstly, the soybean germ protein of the present invention
is superior to casein and soybean protein in the effects of
reducing the blood LDL cholesterol and increasing the blood HDL
cholesterol, and in the effect of improving the blood HDL/LDL
cholesterol ratio. In addition, the soybean germ protein prevents
fat accumulation in organs thereby preventing metabolic syndrome.
The effects are intrinsic to the soybean germ protein and are not
found in soybean protein concentrate.
[0033] Secondly, the soybean germ protein of the present invention
has the effect of reducing the blood triglyceride.
[0034] Thirdly, the soybean germ protein of the present invention
has the effect of reducing the blood sugar level.
[0035] Fourthly, the soybean germ protein of the present invention
has the effect of suppressing accumulation of body fat such as
visceral fat, and reducing the body weight.
[0036] In the form of known soy products, the content of germ
protein is so low that the above-described effects achieved by the
present invention cannot be obtained. However, physiological action
can be provided by increasing the concentration of the germ protein
according to the present invention. More specifically, the effects
are achieved by an ingestion of about 0.05 g to 50 g of the soybean
germ protein of the present invention per day, so that the protein
can be added to various food or beverages.
[0037] The soybean germ protein according to the present invention
may be used alone as an active ingredient, or in the form of, for
example, a solid, liquid, sol, gel, or plastic composition
containing a cereal powder such as starch, oils and fats,
emulsifying agent, fragrant material, or thickener according to the
requirements of the end product (e.g., drug, functional food,
dietary supplement, food, or drink).
[0038] Examples of the starch include corn starch, waxy corn
starch, high amylose corn starch, potato starch, wheat starch,
tapioca starch, green bean starch, sago starch, rice starch, pea
starch, and processed products of these starches prepared by
subjecting a starch to single or combined physical or chemical
treatment such as esterification treatment, etherification
treatment, crosslinking treatment, acid treatment, oxidation
treatment, heat-moisture treatment, and gelatinization
treatment.
[0039] Examples of the oils and fats include soy oil, soybean germ
oil, rapeseed oil, high oleic rapeseed oil, corn oil, sesame oil,
sesame salad oil, perilla oil, linseed oil, peanut oil, safflower
oil, high oleic safflower oil, sunflower oil, high oleic sunflower
oil, high linolic sunflower oil, mid oleic sunflower oil,
cottonseed oil, grape seed oil, macadamia nut oil, hazelnut oil,
walnut oil, pumpkin seed oil, camellia oil, tea seed oil, olive
oil, rice bran oil, wheat germ oil, palm oil, palm olein, palm
kernel oil, coconut oil, cacao butter, algae oil, and hydrogenated
oil, interesterified oil, and fractionated oil of these oils. These
oils may be used alone or in combination of two or more of
them.
[0040] The emulsifying agent may be one commonly used for food.
Examples of the emulsifying agent include glycerin fatty acid
ester, glycerin organic acid ester, sorbitan fatty acid ester,
propylene glycol fatty acid ester, polyglycerin fatty acid ester,
polyglycerin-condensed ricinolate, sucrose fatty acid ester,
calcium stearoyl lactate, alkyl glycosides, erythritol fatty acid
esters, polyoxyethylene sorbitan fatty acid esters, lecithin,
enzymatically decomposed lecithin, and enzymatically modified
lecithin. These emulsifying agents may be used alone or in
combination of two or more of them.
[0041] The fragrant material may be a natural or synthetic fragrant
material commonly used in foods.
[0042] The thickener increases the viscosity of the aqueous
solution, and examples thereof include polysaccharides such as gum
arabic, arabino galactan, guar gum, xanthan gum, psyllium seed gum,
gellan gum, Tara gum, locust bean gum, tamarind seed gum,
water-soluble soy polysaccharide (hemicellulose), sodium alginate,
pullulan, pectin, karaya gum, ghatti gum, gum tragacanth, curdlan,
glucomannan, chitin, chitosan, microfibrillar cellulose, and
microcrystalline cellulose.
[0043] Examples of other additives include protein-derived
substances such as collagen peptide, lactoprotein peptide, casein
peptide, oligopeptide, whey protein concentrate, pea protein and
gelatin, fibers such as soy fiber and pea fiber, and dextrin such
as highly branched cyclic dextrin.
[0044] In addition, a pH adjuster and a saccharide may be added to
the soybean germ protein.
[0045] Examples of the pH adjuster include lactic acid, gluconic
acid, succinic acid, fumaric acid, citric acid, L-malic acid,
DL-malic acid, glacial acetic acid, glucono delta lactone,
L-tartaric acid, and DL-tartaric acid.
[0046] Examples of the saccharide include glucose (grape sugar),
maltose, fructose (fruit sugar), galactose, trehalose,
oligosaccharide, sucrose, and sorbit.
[0047] When the soybean germ protein of the present invention is
contained in a baked confectionery such as cookie with a content of
1 to 50%, the baked confectionery has crispness which cannot be
achieved with a common soybean protein, and gives a good smell and
a good flavor.
[0048] The soybean germ protein of the present invention
complements the flavor of food when it is mixed with a grain such
as barley, wheat, rice, Japanese millet, foxtail millet, brown
rice, black bean, red rice, green rice, red bean, black bean, red
kidney bean, chickpea, white kidney bean, or the germ portion of
these beans, or a seed such as peanut, walnut, sesame, pine nut, or
chestnut. In addition, the soybean germ protein is suitable for,
for example, cereals, baked confectionery, and energy bars owing to
its physiological functionality.
EXAMPLES
[0049] The present invention is described in detail with reference
to the following examples, but the scope of the present invention
is not limited to the examples.
Test Example 1
Preparation 1 of Soybean Germ Protein
[0050] Soybeans were mechanically crushed, and sieved to obtain a
concentrated fraction of soybean germ protein containing 70% or
more of soybean germ. The concentrated fraction obtained by the
method was subjected to total nitrogen analysis, and confirmed to
have a total protein content of 40%. 70% or more of the protein was
derived from soybean germ, so that the content of the soybean germ
protein in the product was 28% or more, and the concentration
factor was 35-fold or more. The protein composition of the sample
was analyzed by SDS-PAGE. In the SDS-PAGE analysis, 1 g of the
obtained sample was suspended in 5 ml of a 10 mM phosphate buffered
saline (PBS: 8.2 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 137
mM NaCl, 3 mM KCl), and subjected to ultrasonic treatment for 30
minutes to conduct extraction. 50 .mu.l of the extract was
dissolved in 250 .mu.l of 50 mM Tris-HCl buffer containing 35 mM
sodium dodecyl sulfate and 1% .beta.-mercaptoethanol, and filtered
through a MILLEX-HV 0.22 .mu.m disposable filter to obtain a
soluble protein sample. The sample was mixed with 20 .mu.l of a 10%
glycerol solution containing 1% bromophenol blue, and subjected to
boiling treatment for 5 minutes in a boiling water bath to obtain a
sample for electrophoresis. In the above treatment, the addition of
.beta.-mercaptoethanol is an important step, because the
electrophoretic patterns markedly differ according to whether
.beta.-mercaptoethanol is added or not.
[0051] Electrophoresis was carried out using PhastSystem (Pharmacia
LKB Biotechnology). The electrophoresis gel was PhastGel, Gradient
10-15, and staining was carried out with CBB. After the
electrophoresis, the stained polypeptide bands isolated by SDS-PAGE
were analyzed with a chromatoscanner CS9300 (manufactured by
Shimadzu Corporation) thereby measuring the absorption at 560 nm.
Ordinary soybeans show a strong band in the vicinity of 33 kD,
while the soybean germ protein shows a strong band in the vicinity
of 30 kD. Similarly, ordinary soybeans show a strong band in the
vicinity of 48 kD, while the soybean germ protein shows a
characteristic strong band in the vicinity of 52 kD, and shows
bands in the vicinity of 21 kD and 35 kD, which are not shown by
the ordinary soybean protein. The image was graphed by the
chromatoscanner, and the peak areas in the graph indicated that the
bands of the soybean germ protein were stronger than those of the
ordinary soybean protein.
[0052] The electrophoretic patterns of the proteins are shown in
FIG. 1.
Test Example 2
Preparation 2 of Soybean Germ Protein
[0053] Soybeans were mechanically crushed, and sieved to obtain a
fraction containing 70% or more of soybean germ. The fraction was
defatted through hexane extraction at low and high temperatures,
which is a common procedure for an oil pressing process, to obtain
a soybean germ protein fraction. The soybean germ protein obtained
by the above method was subjected to total nitrogen analysis, and
found to have a total protein content of 43%, a soybean germ
protein content of 30% or more, and a concentration factor of
37-fold or more. The protein composition of the sample was analyzed
by SDS-PAGE. In the SDS-PAGE analysis, 1 mg of the obtained sample
was suspended in 5 ml of a 10 mM phosphate buffered saline (PBS:
8.2 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 137 mMNaCl, 3 mM
KCl), and subjected to ultrasonic treatment for 30 minutes to
conduct extraction. 50 .mu.l of the extract was dissolved in 250
.mu.l of 50 mM Tris-HCl buffer containing 35 mM sodium dodecyl
sulfate and 1% .beta.-mercaptoethanol, and filtered through a
MILLEX-HV 0.22 .mu.m disposable filter to obtain a soluble protein
sample. The sample was mixed with 20 .mu.l of a 10% glycerol
solution containing 1% bromophenol blue, and subjected to boiling
treatment for 5 minutes in a boiling water bath to obtain a sample
for electrophoresis.
[0054] Electrophoresis was carried out using PhastSystem (Pharmacia
LKB Biotechnology). The electrophoresis gel was PhastGel, Gradient
10-15, and staining was carried out with CBB. After the
electrophoresis, the stained polypeptide bands isolated by SDS-PAGE
were analyzed with a chromatoscanner CS9300 (manufactured by
Shimadzu Corporation) thereby measuring the absorption at 560 nm.
Ordinary soybeans show a strong band in the vicinity of 33 kD,
while the soybean germ protein shows a strong band in the vicinity
of 30 kD. Similarly, ordinary soybeans show a strong band in the
vicinity of 48 kD, while the soybean germ protein shows a
characteristic strong band in the vicinity of 52 kD, and show bands
in the vicinity of 21 kD and 35 kD, which are not shown by the
ordinary soybean protein. The image was graphed by the
chromatoscanner, and the peak areas in the graph indicated that the
bands of the soybean germ protein were stronger than those of the
ordinary soybean protein.
[0055] The electrophoretic patterns of the defatted proteins are
shown in FIG. 2.
Test Example 3
Preparation 3 of Soybean Germ Protein
[0056] Soybeans were mechanically crushed, and sieved to obtain a
fraction containing 90% or more of soybean germ. The fraction was
subjected to hexane extraction in a common procedure for an oil
pressing process. Subsequently, 70% hydrous ethanol was added to
the fraction, the mixture was stirred at 70.degree. C. for 30
minutes, and then filtered to collect the residue. The residue was
subjected to heat treatment at 100.degree. C. for 40 minutes under
reduced pressure to obtain a soybean germ protein fraction. The
soybean germ protein obtained by the method was subjected to total
nitrogen analysis, and found to have a total protein content of
62%, a soybean germ protein content of 56% or more, and a
concentration factor of 70-fold or more. The sample was analyzed by
SDS-PAGE in the same manner as Test Example 1, and found to have a
characteristic composition of soybean germ protein in which the
band in the vicinity of 30 kD was stronger than that in the
vicinity of 33 kD, the band in the vicinity of 52 kD was stronger
than that in the vicinity of 48 kD, and other bands were shown in
the vicinity of 21 kD and 35 kD.
[0057] Further, antigenicity to bovine antiserum which was obtained
using soybean protein as an antigen was measured by ELISA, and
found to be 40 U/10 mg or less. The isoflavone content and saponin
content were 0.5% or less, which are one-fifth or less of those in
an ordinary soybean germ, and lower than those in whole soybeans.
Therefore, high intake of the soybean germ protein will not cause
bitterness or a problem of excess intake of these trace components.
FIG. 3 shows an electrophoretic pattern of the concentrated soybean
germ protein.
[Analysis of Isoflavone and Saponin Contents]
[0058] The isoflavone and saponin contents in the soybean germ
proteins obtained in Test Examples 1 to 3 were measured.
[0059] Isoflavone content was analyzed according to a method based
on the soy isoflavone food standard established by Japan Health
Food & Nutrition Food Association, and the isoflavone content
was determined in terms of aglycon.
[0060] The saponin content was analyzed by the following method. A
sample containing about 10 mg of soy saponin was weighed, and
refluxed at 80.degree. C. for 2 hours in 5 ml of a 10% hydrogen
chloride-methanol test solution. Insoluble matter was removed from
the solution by centrifugation and filtration, and then the
solution was analyzed by HPLC. As standard substances for
quantitation, 10 mg each of soyasapogenol A and soyasapogenol B
manufactured by Koshiro Company Limited were dissolved respectively
in 100 ml of ethanol, and the solutions were used. HPLC used ODS
columns, and the absorption at UV 210 nm was measured under the
conditions of
mobile phase: acetonitrile/water/methanol (6:3:1); flow rate: 0.7
ml/min; and temperature: 45.degree. C. The integrated value of the
sample was compared with that of the standard substances, and the
saponin content was determined in terms of aglycon. The results are
shown in Table 1.
TABLE-US-00001 TABLE 1 Isoflavone and saponin contents in soybean
germ protein Protein Isoflavone Saponin content content content
Test Example 1 40.0% 1.8% 1.7% Test Example 2 43.2% 2.2% 2.1% Test
Example 3 62.2% 0.18% 0.33%
Example 1
[0061] High fat diets were fed to rats. The concentrations of
various physiological substances in the blood were compared among
rats fed with casein, with soybean protein concentrate (hereinafter
abbreviated as SPC) as other common soybean protein or with the
soybean germ protein of Test Example 3 (hereinafter abbreviated as
germ protein).
[0062] Three diets prepared were: 1. casein diet, 2. SPC diet and
3. germ protein diet. The compositions of these diets are
summarized in Table 1.
[0063] The germ protein was in the form of powder.
[0064] CD (SD) IGS male rats aged nine weeks were placed in
individual cages, and preliminarily bred with a purified diet
(AIN93G) for one week. Before starting the main test, the rats were
grouped into three divisions each containing eight rats such that
the divisions have an equal average body weight. The rats of the
respective divisions were fed with 1. casein diet (control), 2. SPC
diet containing SPC in place of casein, or 3. germ protein diet
containing germ protein in place of casein. The test period was 28
days, and water and the diets were freely ingested. The test was
carried out in an animal room under the following conditions:
temperature, 22.+-.3.degree. C.; humidity, 60.+-.15%; frequency of
ventilation, 12 to 15 times/hour; and photoperiod, 12 hours (7 to
19 o'clock).
[0065] The rats were fasted for 18 hours from the evening of the
last day of administration, and the blood was collected from the
aorta abdominal is under ether anesthesia. The blood was taken in
blood-collecting vessels containing a serum separating medium, and
the vessels were centrifuged at 3000 rpm for 15 minutes to separate
the serum. The serum was assayed for the levels of LDL cholesterol
(FIG. 4), HDL cholesterol (FIG. 5), triglyceride (FIG. 7), and
blood sugar level (FIG. 8). Further, the HDL cholesterol/LDL
cholesterol ratio was calculated from the LDL cholesterol and HDL
cholesterol levels (FIG. 6). After blood collection, the rats were
exsanguinated to death, their livers were harvested and weighed.
Further, mesenteric fat, retroperitoneal fat, perinephric fat and
epididymal fat were isolated from the rats of the casein diet group
and germ protein diet group to study the accumulation of fats. The
measurement values were expressed by average .+-.standard
deviation. In the statistical analysis, Tukey multiple comparison
test was carried out as necessary, and the significant differences
between the measurement values were examined.
TABLE-US-00002 TABLE 2 Composition of rat diets (unit: %)
Ingredients Casein diet SPC diet Germ protein diet Casein 20.0000
-- -- SPC -- 25.6716 -- Germ protein -- -- 28.1967 L-cystine 0.30
0.30 0.30 Lard 35 35 35 Corn starch 19.9486 14.2770 11.7519 Sucrose
15 15 15 Cellulose powder 5 5 5 AIN-93G mineral mixture 3.50 3.50
3.50 AIN-93G vitamin mixture 1.00 1.00 1.00 Choline bitartrate 0.25
0.25 0.25 Tertiary butyl 0.0014 0.0014 0.0014 hydroquinone
[0066] FIGS. 1 to 11 indicate the followings.
[0067] 1. FIG. 4 indicates that the germ protein diet decreases the
total blood cholesterol.
[0068] 2. FIG. 5 indicates that the germ protein diet decreases the
blood LDL cholesterol.
[0069] 3. FIG. 6 indicates that the germ protein diet increases the
blood HDL cholesterol.
[0070] 4. FIG. 7 indicates that the germ protein diet increases the
blood HDL/LDL cholesterol ratio more than the casein diet, and that
the germ protein diet significantly increased the HDL/LDL ratio in
comparison with the SPC diet (P<0.05). The germ protein diet
also significantly increased the cholesterol ratio in terms of
HDL/total cholesterol in comparison with the SPC diet.
[0071] 5. FIG. 8 indicates that the SPC and germ protein diets
significantly decreased the blood triglyceride in comparison with
the casein diet (P<0.05).
[0072] 6. FIG. 9 indicates that the SPC and germ protein diets
significantly decreased the blood sugar level in comparison with
the casein diet (P<0.01 and P<0.05 for the SPC and germ
protein diets, respectively).
[0073] 7. FIG. 10 indicates that only the germ protein diet
significantly decreased the liver weight per body weight in
comparison with the casein diet (P<0.05), which demonstrates
that the germ protein diet is effective in prevention of fatty
liver.
[0074] 8. FIG. 11 indicates that the accumulation of the mesenteric
fat, retroperitoneal fat, perinephric fat and epididymal fat was
smaller in weight in the division fed with the germ protein diet
than the division fed with the casein diet, which shows that the
germ protein diet suppresses accumulation of fats.
[0075] The above facts indicate that the germ protein diet
decreased the blood triglyceride and blood sugar level in
comparison with the casein diet. In addition, the germ protein diet
improved the HDL/LDL cholesterol ratio and HDL/total cholesterol
ratio in comparison with SPC, showing that it prevents lipid
accumulation in the liver. Accordingly, it was proved that the germ
protein of the present invention has physiological function
different from that of ordinary soybean protein.
Example 2
[0076] Baked confectionery was made using the soybean germ proteins
of Test Examples 1, 2, and 3 as soybean protein materials. As a
comparative example, commercial separated soybean protein was used
in place of the soybean germ proteins. The ingredients are as
follows.
TABLE-US-00003 Weak flour 130 g Butter 100 g Soybean protein
material 100 g Sugar 80 g Egg 1 Vanilla essence few drops
[0077] An appropriate amount of milk was added to and mixed with
the above ingredients. The dough was preserved at 4.degree. C. for
2 hours, shaped into disks having a diameter of about 3 cm, and
baked in an oven at 170.degree. C. for about 12 minutes to make
cookies. The cookies were tasted, and evaluated by sensory test by
ten panelists. As a result of the sensory test, the cookies made of
the soybean germ protein were more fragrant and crisp than those
made of the separated soybean protein, and thus achieved higher
scores in terms of flavor, texture, and taste.
TABLE-US-00004 TABLE 3 Result of sensory test (fractional
components are rounded off) Fragrance Crispness Taste Test Examples
1 3 4 3 Test Examples 2 4 5 4 Test Examples 3 5 5 5 Comparative
Example 2 2 1 5: very good; 4: good; 3: normal; 2: not good; 1:
bad
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