U.S. patent application number 10/727292 was filed with the patent office on 2004-07-01 for method for producing composition containing soluble isoflavones.
Invention is credited to Araki, Hideo, Hashimoto, Yukio, Tsuzaki, Shinichi.
Application Number | 20040126443 10/727292 |
Document ID | / |
Family ID | 19027029 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126443 |
Kind Code |
A1 |
Tsuzaki, Shinichi ; et
al. |
July 1, 2004 |
Method for producing composition containing soluble isoflavones
Abstract
A method for producing a composition containing soluble
isoflavones is described, which uses soybean materials as raw
materials. The composition is obtained in a natural state without
addition of solubilizing agents and chemical modification, and has
high solubility under neutral to acidic conditions and good
long-term stability under refrigeration. By removing the insoluble
materials from the water-extract liquid of a soybean material
having a pH value of 2-7 and a temperature of 0-17.degree. C., a
composition containing isoflavones can be efficiently obtained with
high solubility under neutral to acidic conditions and good
stability under refrigeration.
Inventors: |
Tsuzaki, Shinichi; (Osaka,
JP) ; Araki, Hideo; (Osaka, JP) ; Hashimoto,
Yukio; (Osaka, JP) |
Correspondence
Address: |
J.C. PATENTS
SUITE 250
4 VENTURE
IRVINE
CA
92618
US
|
Family ID: |
19027029 |
Appl. No.: |
10/727292 |
Filed: |
December 2, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10727292 |
Dec 2, 2003 |
|
|
|
PCT/JP02/06252 |
Jun 21, 2002 |
|
|
|
Current U.S.
Class: |
424/757 |
Current CPC
Class: |
C07D 311/36 20130101;
C07H 17/07 20130101 |
Class at
Publication: |
424/757 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2001 |
JP |
2001-187783 |
Claims
What is claimed is:
1. A method for producing a composition containing soluble
isoflavones, comprising: preparing a soybean extract liquid that
has a pH value adjusted to 2-7 and a temperature adjusted to
0-17.degree. C.; and removing insoluble materials from the soybean
extract liquid.
2. The method of claim 1, wherein an amount of isoflavones is
0.2-20 wt %, a crude protein content is 30 wt % or less, and a
lipid content is 4 wt % or less in total solid content of the
soybean extract liquid.
3. The method of claim 1, wherein preparing the soybean extract
liquid comprises a process of adjusting the pH value of the soybean
extract liquid to 5.5-7 without a protease treatment.
4. The method of claim 2, wherein preparing the soybean extract
liquid comprises a process of adjusting the pH value of the soybean
extract liquid to 5.5-7 without a protease treatment.
5. The method of claim 1, wherein preparing the soybean extract
liquid comprises: a process of adjusting the pH value of the
soybean extract liquid to satisfy the equation
"2.ltoreq.pH<5.5"; and a process of treating the soybean extract
liquid with a protease.
6. The method of claim 2, wherein preparing the soybean extract
liquid comprises: a process of adjusting the pH value of the
soybean extract liquid to satisfy the equation
"2.ltoreq.pH<5.5"; and a process of treating the soybean extract
liquid with a protease.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation Application of an
international application, PCT/JP02/06252, filed on Jun. 21, 2002.
The international application also claims the priority benefit of
Japanese Basic Application No. 2001-187783, filed on Jun. 21,
2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method for producing a
composition containing soluble isoflavones.
[0004] 2. Description of the Related Art
[0005] Soybean isoflavones are a group of compounds having a
3-phenylchromone skeleton that exist in soybeans. Specifically,
soybean isoflavones are present in the forms of glucosides, malonyl
glucosides, acetyl glucosides or aglycones, etc. The glucosides
include daidzin, genistin and glycitin. The malonyl glucosides
include 6"-O-malonyldaidzin, 6"-O-malonylgenistin and
6"-O-malonylglycitin. The acetyl glucosides include
6"-O-acetyldaidzin, 6"-O-acetylgenistin and 6"-O-acetylglycitin.
The aglycones include daidzein, genistein and glycitein. The
general structures and the species of the above compounds can be
expressed by formulae (1)-(2) below:
1 (1) 1 The group of compounds having the general skeleton
expressed by formula (1) R1 R2 R3 daidzin H H H genistin OH H H
glycitin H OCH.sub.3 H 6"-O-malonyldaidzin H H COCH.sub.2COOH
6"-O-malonylgenistin OH H COCH.sub.2COOH 6"-O-malonylglycitin H
OCH.sub.3 COCH.sub.2COOH 6"-O-acetyldaidzin H H COCH.sub.3
6"-O-acetylgenistin OH H COCH.sub.3 6"-O-acetylglycitin H OCH.sub.3
COCH.sub.3 (2) 2 The group of compounds having the general skeleton
expressed by formula (2) R4 R5 daidzein H H genistein OH H
glycitein H OCH.sub.3
[0006] The above isoflavones are known to have estrogen-like
functions and antioxidative functions, etc., and therefore get
worldwide attentions as food components effective to prevent cancer
and osteoporosis or alleviate the menopausal syndrome. However,
isoflavone is difficult to dissolve in water, and its solubility in
water is 0.002-0.003 g under 25.degree. C. according to the
literature. Consequently, cloudiness or precipitation easily occurs
in or after the manufacturing process of food products, especially
drink products or dessert products, etc, and the applications of
isoflavones are restricted in these fields. Therefore, improvement
in the solubility of isoflavones is required.
[0007] To solve the above problem, for example, Japanese Patent
Application Laid Open No. 09-309902 or 10-298175 discloses a method
that includes isoflavone molecules in cyclodextrin to improve its
solubility in water. However, since it is necessary to previously
refine the isoflavones to certain purity in the method, the
operation steps are complicated. Moreover, since cyclodextrin is
used when isoflavones are added into drink products, the aroma
components like flavors are also included in cyclodextrin together
with the isoflavone molecules. Therefore, the balance of aroma is
easily broken down, and the commercial product design is difficult
accordingly. Moreover, isoflavones cannot be dissolved in water in
a high concentration due to the limitation of the solubility of
cyclodextrin. On the other hand, Japanese Patent Application Laid
Open No. 2000-325043 discloses another method that dissolves
isoflavones by heating the compounds together with a solubilizing
agent consisting of dehydrated or hydrated propylene glycol and/or
octenylsuccinic acid starch in the presence of water. Nevertheless,
the food additives like propylene glycol and octenylsuccinic acid
starch are not preferable to use in latest years. Moreover,
Japanese Patent Application Laid Open No. 2000-327692 discloses a
method for improving the solubility of isoflavones in water, which
forms .alpha.-glycosylisoflavone derivatives utilizing a
glycosyltransferase in the presence of an
.alpha.-glycosylsaccharide compound like dextrin. The
.alpha.-glycosylisoflavone derivative is formed by attaching a
glucose residue to a daidzin or genistin molecule with an
.alpha.-1,4 bond. However, since one or more equivalents of the
.alpha.-glycosylsaccharide compound must be used to bond with the
isoflavone molecules, the process is complicated. Moreover, the
percentage of isoflavones in total solid content is surely lowered
in the method, and there is no isoflavone molecule present in its
natural state. Furthermore, though the solubility and the long-term
stability under low temperature both are important properties when
a composition containing isoflavones is added in a liquid food
product like a drink product, there is no information about them.
Each of the aforementioned methods solubilizes isoflavones either
by adding a solubilizing agent or by chemically modifying
isoflavone molecules to form soluble derivatives, while there is no
easier method suitably used for industrial production that
solubilizes insoluble isoflavones in their natural form within a
wide pH range.
SUMMARY OF THE INVENTION
[0008] In view of the foregoing, one object of this invention is to
provide a method for producing a composition containing soluble
isoflavones, wherein soybean materials are used as raw materials
and isoflavones are extracted in their natural forms without
addition of solubilizing agents and chemical modification. The
composition containing soluble isoflavones has high solubility over
a wide pH range, and has good long-term stability under
refrigeratory preservation.
[0009] In the study of the aforementioned issue, the inventors
discovered that the isoflavones in soybeans can be easily extracted
with water even though their solubilities are low. Meanwhile, it is
absolutely surprising that the isoflavones can be solubilized
steadily even in a high-concentration state. After repeating the
outstanding study, it is confirmed that by adjusting the pH value
of the soybean extract liquid obtained from soybean materials to
5.5-7 and cooling the soybean extract liquid to 0-17.degree. C. and
then removing the insoluble materials thus produced, a composition
containing isoflavones that has a good solubility under neutral to
weak acidic conditions and good long-term stability under
refrigeratory preservation can be obtained with a higher recovery
ratio. Moreover, when the pH value of the soybean extract liquid is
adjusted smaller than 5.5, a composition containing isoflavones
that has a good solubility under acidic conditions can also be
obtained by removing the insoluble materials. However, since a lot
of the isoflavones precipitate together with proteins under the
condition, the recovery ratio of isoflavones is low. By treating
the soybean extract liquid with proteases and removing the
insoluble materials produced under an acidic low-temperature
condition, precipitation of isoflavone can be prevented, and a
composition containing isoflavones that has a good solubility under
acidic conditions and good long-term stability under refrigeratory
preservation can be obtained with a higher recovery ratio. The
present invention is completed based on the above discovery.
[0010] That is, this invention is directed to 1) a method for
producing a composition containing soluble isoflavones from a
soybean extract liquid, featuring with removal of insoluble
materials from the soybean extract liquid that has a pH value
adjusted to 2-7 and a temperature adjusted to 0-17.degree. C.; 2) a
method for producing a composition containing soluble isoflavones
according to 1), wherein the amount of isoflavones in total solid
content of the soybean extract liquid is 0.2-20 wt %, the crude
protein content is 30 wt % or less, and the amount of lipids is 4
wt % or less; 3) a method for producing a composition containing
soluble isoflavones according to 1) or 2), wherein the process of
preparing the soybean extract liquid includes adjusting the pH
value to 5.5-7 without a protease treatment; and 4) a method for
producing a composition containing soluble isoflavones according to
1) or 2), wherein the process of preparing the soybean extract
liquid includes a step of adjusting the pH value to satisfy the
equation "2.ltoreq.pH<5.5" and a protease treatment.
[0011] It is to be understood that both the foregoing general
description and the following detailed description are exemplary,
and are intended to provide further explanation of the invention as
claimed.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] The present invention is specifically described as follows.
The composition containing soluble isoflavones of this invention is
prepared from a soybean extract liquid, which is obtained by
treating soybean materials with an extracting solvent.
[0013] The soybean materials serving as the raw materials in this
invention include whole soybeans, dehulled soybeans, dehulled and
dehypocotyled soybeans, soybean hypocotyls, defatted soybeans and
defatted soybean hypocotyls, etc. Particularly, the soybean
hypocotyls contain more isoflavones than the other soybean
materials, approximately in an amount of 1-2 wt %, an is therefore
a suitable raw material for producing a composition containing a
high concentration of isoflavones. Generally, soybean materials can
be subjected to desired pre-treatments including physical
treatments like pulverization, flattening and puffing treatments,
dry-heating treatments like roasting treatment, wet-heating
treatments like steaming treatment, heating treatments like drying
treatment, and chemical treatments, etc. However, the physical
treatments like pulverization, flattening and puffing treatments
are preferably controlled not to destroy the soybean cells, or are
preferably not performed at all. When physically treated raw
material is subjected to water extraction, additional components
other than isoflavones contained in the raw material, such as
proteins or oils, easily exude in large amounts. Therefore, the
solubility of the composition containing isoflavones is lowered, or
the recovery ratio of isoflavones is lowered in the step of
removing the insoluble materials. However, by using the method
provided in this embodiment, exudation of proteins or lipids from
the raw material can be inhibited.
[0014] Moreover, when soybean hypocotyls are used as the raw
material, the unique grassy smell and astringent smell of unheated
soybean hypocotyls will remain if the extraction operation is
directly done to raw soybean hypocotyls. To reduce such smells, a
heating treatment is preferably conducted, while the heating
treatment should be controlled so that the soybean hypocotyls are
not scorched to cause a bitter taste. The heating method can be any
well-known method like a dry-heating method or a wet-heating
method. For example, a dry-heating step of soybean hypocotyls can
be conducted using a gas roaster (manufactured by Fuji Royal
Corporation), an electroheating roaster (by Nippon Glass
Corporation), a hot-blast roaster (by Buhler Corporation), a
microwave heater (by New Japan Radio Co., Ltd.), or an indirect
heating-type cooker, etc. The temperature of the heating treatment
is preferably 100.degree. C. or higher. The degree of heating can
be specified as the water content of the soybean hypocotyls, and
the heating treatment can be terminated when the water content is
reduced to 1-9.5 wt %, preferably 3-9 wt %. On the other hand, a
wet-heating process of soybean hypocotyls may utilize any method
that conducts heating under a moderate amount of water without
dissolving the isoflavones. Except steaming treatments, the methods
that conduct heating after a hydration treatment or similar methods
can also be used. When a steaming treatment is adopted, for
example, the steaming apparatuses generally used in food industry,
such as autoclaves, cookers or steam peelers, etc., can also be
used.
[0015] Moreover, before isoflavones are extracted from the
aforementioned soybean materials by water, the soybean materials
are preferably subjected to a pre-treatment that uses an aqueous
solvent of 4-80.degree. C., preferably 4-40.degree. C., to contact
and wash the soybean materials. The aqueous solvent can be water, a
solution of a salt or a buffer solution, etc., wherein the salt may
be a calcium salt, a sodium salt or a potassium salt, etc., and the
buffer solution may be a phosphoric acid buffer solution, a
carbonic acid buffer solution or a citric acid buffer solution,
etc. The pre-treatment is for removing the soluble components other
than isoflavones in advance, including soluble nitrogen-free
substances, nitrogen compounds and ash, etc., so as to increase the
concentration of isoflavones in the soybean extract liquid. In the
pre-treatment, it is not preferred to set the temperature under
4.degree. C. even though such temperatures can still make
sufficient cleaning effect, since the cost for the cooling
operation will be increased to lower the utility. In the washing
process, the weight ratio of the soybean material to water is not
particularly restricted, and is generally 1:3-1:30, preferably
1:5-1:15. The contact time with water is moderately adjusted so
that the extracted amount of the soluble components other than
isoflavones in the liquid part is maximized, and is generally 5-240
minutes. The methods for contacting with water include various
methods as the aforementioned water-extraction methods do, such as
batch-type methods, continuation-type methods, counterflow-type
methods and multistage-type methods. After the washing step is
performed as above, the liquid part can be removed with a
solid-liquid separation method like filtration or centrifugal
separation, and the residue, i.e., the soybean material, is
recovered.
[0016] Next, a method for obtaining a soybean extract liquid is
described, wherein isoflavones are extracted from soybean materials
or the pre-treated soybean materials mentioned above. The
extracting solvent can be an aqueous solvent like water or an
alcohol containing water. However, when the alcohol concentration
in the solvent is high, the bad-taste components in soybeans
including oils and phenol species, etc., are easily extracted
simultaneously. Therefore, to obtain a good-taste extract with a
required solubility, it is preferred to use water in the extraction
step. The extraction method can be the one disclosed in Japanese
Patent Application Laid Open No. 2000-14348. More specifically, the
extraction process is preferably conducted at a temperature that is
usually set for water-extraction of isoflavones. When the
temperature is overly low, the recovery ratio of isoflavones is
lowered, and the manufacturing process is inefficient. Therefore,
the extraction temperature is usually 80.degree. C. or higher, and
is preferably 80-150.degree. C. and more preferably 80-100.degree.
C. In the extraction process, the weight ratio of the raw material
to water is not particularly restricted, and is usually 1:3-1:30,
preferably 1:5-1:15. The contact time with water is moderately
adjusted so that the extracted amount of isoflavones is maximized,
and is generally 5-60 minutes. Moreover, sodium hydroxide,
potassium hydroxide, sodium bicarbonate or sodium carbonate, etc.,
can be added as required to make an alkaline condition having a pH
value of 8 or larger during the extraction process, so that the
efficiency of isoflavone extraction can be improved. However, since
some undesired components like proteins are also easily extracted
under the condition, the pH value is suitably adjusted to 6-8, more
preferably 6.5-7.5. Furthermore, a surfactant like glycerin fatty
acid ester or sorbitane fatty acid ester, etc., can also be added
in an amount of 0.01-1.0 w/v % relative to the water. The
extraction methods include various methods, such as batch-type
methods, continuation-type methods, counterflow-type methods and
multistage-type methods, and the extraction efficiency can be
improved with stirring. In addition, the extract liquid can be used
as the extracting solvent of a new batch of soybean material.
[0017] After the extraction process is performed as above, a
soybean extract liquid is obtained with a solid-liquid separation
method like filtration or centrifugal separation, etc. Moreover,
except the aforementioned soybean extract liquid, the
soybean-immersed waste solution produced during manufacture of tofu
or soybean milk or the soybean-boiled waste solution produced
during manufacture of boiled soybeans, etc., can also be used as a
soybean extract liquid. Moreover, it is preferably not to squeeze
the extraction residue during the solid-liquid separation to
prevent the proteins and the oils contained inside from
exuding.
[0018] In the soybean extract liquid obtained as above, the amount
of isoflavones in total solid content is usually 0.2-20 wt %, and
the crude protein content in total solid content is 30 wt % or
less, preferably 25 wt % or less. The lipid content in total solid
content is 4 wt % or less, preferably 2 wt % or less. In the
soybean milk obtained by pulverizing and extracting sovbeans, for
example, the crude protein content in total solid content is
usually 40-55 wt %, and the lipid content in total solid content is
25-35 wt %. Accordingly, it is confirmed that the exudation of
crude proteins and lipids is inhibited during the extraction
process for obtaining the soybean extract liquid of this
invention.
[0019] Thereafter, the pH value of the soybean extract liquid
obtained as above is adjusted to 2-7, and the soybean extract
liquid is cooled to 0-17.degree. C., preferably 0-10.degree. C.,
and kept at the same temperature for 10 minutes or more, preferably
30 minutes or more, whereby some low-temperature insoluble
materials are formed. If the pH value is adjusted smaller than 2,
the isoflavones may self-decompose. On the other hand, the
solubility of isoflavones gets low under a neutral to acidic
condition having a pH value larger than 7. The pH value can be
adjusted to any value within the range of 2-7, and may be adjusted
to be equal to or smaller than that of the product into which the
composition containing isoflavones will be added. When the cooling
temperature is higher than 17.degree. C., precipitation or
cloudiness occurs even if the pH value of the soybean extract
liquid is adjusted to be equal to or smaller than that of the
product. The acid for adjusting the pH value to 2-7 can be any one
among the inorganic acids and the organic acids that are usually
added in food. The applicable acids include hydrochloric acid,
sulfuric acid, phosphoric acid, acetic acid, malic acid, tartaric
acid, citric acid and ascorbic acid, etc., wherein hydrochloric
acid is particularly preferable. Subsequently, the insoluble
materials are removed with, for example, filtration or centrifugal
separation, and a composition containing soluble isoflavones is
obtained thereby. Generally, when proteins and other components get
insoluble and precipitate because of cooling effect, the
isoflavones contained in the soybean extract liquid easily
co-precipitate with them. However, since the raw material used in
this invention is not subjected to a physical treatment like
pulverization, exudation of proteins and other components from the
raw material can be inhibited to prevent co-precipitation.
Therefore, the recovery ratio of isoflavones is not lowered.
[0020] On the other hand, there is no problem that the pH value of
the soybean extract liquid is 5.5 or larger during removal of the
insoluble materials. However, in the cases where the soybean
extract liquid is made acidic with a pH value smaller than 5.5, the
isoflavones easily co-precipitate with the proteins contained in
the solution due to the interaction with the proteins when the
proteins precipitate at the isoelectric point. This
co-precipitation effect and the aforementioned co-precipitation
phenomenon caused by low temperature synergistically lower the
recovery ratio. As found by the inventors, when the pH value of the
extract liquid of the soybean material is adjusted under 5.5, the
amount of isoflavones in the soybean extract liquid is reduced by a
half after the produced insoluble materials are removed.
[0021] To prevent co-precipitation of isoflavones and to improve
the solubility of isoflavones under acidic condition in the
aforementioned cases, the soybean extract liquid is preferably
treated with proteases to decompose proteins into lower molecules.
Meanwhile, the pH value of the soybean extract liquid is adjusted
to satisfy the equation "2.ltoreq.pH<5.5", and the temperature
of the same is set to 0-17.degree. C. in the step of removing
insoluble materials.
[0022] The species of the protease and the treating process using
the same are not particularly restricted in the aforementioned
cases, and endo-proteases or exo-proteases can be used. Generally,
neutral proteases or alkaline proteases are preferably used to
treat the soybean extract liquid, since the pH value of the soybean
extract liquid is 6-7. Moreover, since .beta.-glucosidase is
present in proteases as an impurity, the .beta.-glucoside bond
between the aglycone moiety and the saccharide moiety of the
isoflavone glucoside is broken in the aforementioned process, and
the aglycones including daidzein, genistein and glycitein are
released. Since the aglycones generally have lower solubilities as
compared with glucosides, they are not desired. Therefore, the
source of proteases is preferably an animal or plant source that
has a low .beta.-glucosidase activity, while a microorganism source
is also feasible if only the .beta.-glucosidase activity thereof is
low. Moreover, the protease treatment is most preferably conducted
after the soybean extract liquid is prepared and before the pH
value of the same is adjusted to one of acidity. Though the
protease treatment can be performed simultaneously with the
pre-treatment wherein the soybean material is made contact with
low-temperature water before the extraction stage, or
simultaneously with the extraction stage using thermostable
proteases, the proteins are decomposed into lower molecules and are
extracted in a large amount. Moreover, it is also feasible to use
acidic proteases after the soybean extract liquid is made acidic.
However, since the proteins get insoluble because of precipitation
at the isoelectric point, the effect of the proteases is extremely
reduced.
[0023] The conditions of the protease treatment are not restricted
if only they can meet the requirement of decomposing the proteins
in the soybean extract liquid into lower molecules. The added
amount of proteases is moderately adjusted with a titer, and is
usually 0.01-10 wt % of the total solid content in the soybean
extract liquid. The reaction conditions are moderately adjusted
according to the optimum conditions of the used protease.
Generally, the pH value is preferably adjusted to 6-8 when a
neutral protease is used in the treatment, or is preferably
adjusted to 8-10 when an alkaline protease is used. The treating
temperature is preferably 40-60.degree. C., and the treating time
is preferably 0.5-5 hours. The reaction is terminated usually by
performing a heating treatment above 80.degree. C. to deactivate
the enzyme.
[0024] The composition containing soluble isoflavones obtained as
above can be directly used after the concentration thereof is
properly adjusted. The composition may also be used in the form of
condensed extract after being neutralized as required and
condensed, or in the form of dry matter after a further drying
process like a freeze-drying process or a spray-drying process,
etc. Moreover, if required, a further process like a purification
process using an adsorbent or a separation process using a solvent
can be performed to produce a composition containing soluble
isoflavones of higher purity. The adsorbent used in the
purification process can be of polystyrene type, methacryl type or
ODS type, etc, and the solvent used in the separation process can
be butanol or the like.
[0025] When the amount of isoflavones in total solid content is
0.2-10 wt % in the composition and the composition is used in a
product having a pH value larger than that on removing insoluble
materials, cloudiness or precipitation does not occur even if the
product is refrigerated under 10.degree. C. That is, the
composition shows high stability and a good solubility. The
solubility of pure isoflavone in water is merely 0.002-0.003 g,
while that of the isoflavones in the composition containing soluble
isoflavones of this invention is 0.3 g or more (0.3-100 g). That
is, it is possible to dissolve a large amount of isoflavones that
is 100 or more times the amount in the prior art. Therefore, all
neutral to weak acidic food products, such as neutral drinks like
tea drinks or weak acidic drinks like coffee as well as ice cream
and dessert, etc., can be applied with high concentrations of
isoflavones without loss of transparency. In addition, the
solubility in this invention is defined as the maximal amount of a
solute dissolved in a solvent of 100 g under 25.degree. C.
[0026] By using the method of this invention, the solubility issue
of isoflavones in the prior art can be completed solved. Therefore,
the scope of the applications of isoflavones can be significantly
expanded.
[0027] Moreover, in this invention, the quantification of
isoflavones is based on the standard analysis method of soybean
isoflavone food product specifications issued by Japan Health Food
& Nutrition Food Association, which is used to measure the
total amount of 12 kinds of isoflavone compounds as follows. The
amount of crude proteins is measured with the Kjeldahl method, and
the amount of lipids with an extraction method using a
chloroform-methanol mixed solvent.
[0028] <Method for Quantifying Isoflavones>
[0029] A sample containing approximately 1-10 mg of isoflavones is
pulverized as required and precisely weighed, and is added with 25
ml of 70% (v/v) ethanol. The extraction process is conducted with
stirring under room temperature for 30 minutes, and then an extract
liquid is obtained with centrifugal separation. The residue is
further subjected to the same extraction operation twice, and then
the extract liquid of the three times is added with 70% (v/v)
ethanol to prepare a sample solution of 100 ml. The sample solution
is filtered with a filter of 0.45 .mu.m and then analyzed with
high-performance liquid chromatography (HPLC) under the following
conditions:
2 Column: YMC-Pack ODS-AM303 (4.6 .times. 250 mm) Mobile Phase:
Acetonitrile:water:acetic acid = 15:85:0.1 - 35:65:0.1 (v/v/v) Flow
rate: 1.0 ml/min Temperature: 35.degree. C. Detection: UV (254 nm)
Injected amount: 10 .mu.L
[0030] A daizin standard is used to measure the daizin-converted
concentrations of 12 kinds of isoflavones, and each measured value
is multiplied with a corresponding quantification coefficient
listed below to calculate the correct concentration of the
corresponding isoflavone compound. The isoflavone quantification
coefficient is 1.000 for daizin, 0.814 for genistin, 1.090 for
glycitin, 1.444 for malonyldaizin, 1.095 for malonylgenistin, 1.351
for malonylglycitin, 1.094 for acetyldaizin, 1.064 for
acetylgenistin, 1.197 for acetylglycitin, 0.583 for daizein, 0.528
for genistein, and 0.740 for glycitein. The total amount of
isoflavones is then obtained by summing up the concentrations of
all isoflavone compounds.
EXAMPLES
[0031] Some examples of this invention are described as follows.
The examples are intended to exemplify the technical principles of
this invention, but not to restrict the scope of this
invention.
Experiment 1
[0032] 100 g of whole soybeans from the America is added with 500
ml of water in a pre-treatment, wherein the soybeans contact with
water under 20.degree. C. for 2 hours. The liquid part is then
removed with filtration. Next, the residue is added with 500 ml of
water to implement an extraction process under 98.degree. C. for 20
minutes, and then an extract liquid is obtained with filtration.
The residue is further added with 500 ml of water to implement
another extraction process under 98.degree. C. for 20 minutes, and
then another extract liquid is obtained with the same operation.
The two extract liquids are mixed into a soybean extract liquid. In
the soybean extract liquid, the amount of isoflavone in total solid
content is 1.3 wt %, the crude protein content is 24 wt %, and the
lipid content is 1.0 wt %. Subsequently, the soybean extract liquid
is cooled to 20-0.degree. C., as shown in Table 1, and then placed
still for 30 minutes, while the pH value is 6.5 at the moment.
Thereafter, the insoluble materials are removed with centrifugal
separation, and the soybean extract liquid is freeze-dried into
powder. The solubility of isoflavones in 100 g of water under
25.degree. C. is then measured using the composition containing
isoflavones. Moreover, a stability test is done by dissolving an
equivalent amount of powder containing 10 mg of isoflavones in 100
ml of water, adjusting the pH value of the solution to 7-5.5 with
sodium bicarbonate or citric acid, performing heat sterilization
under 95.degree. C. for 15 minutes and then preserving the solution
under 10.degree. C. for a month. The results are shown in Table
1.
3TABLE 1 Separation Solubility of Stability test temperature
(.degree. C.) isoflavones (g) pH = 7 pH = 6 pH = 5.5 20 >0.3 X X
X 15 >0.3 O O O 10 >0.3 O O O 0 >0.3 O O O Evaluation for
stability test: O: without precipitation; X: precipitation
occurs
[0033] Evaluation for stability test: O: without precipitation; X:
precipitation occurs
[0034] As shown in Table 1, when the insoluble materials are
removed from the soybean extract liquid at a temperature under
20.degree. C., the solubility of the composition containing
isoflavones in water is significantly increased. Moreover, the
results of the stability test indicate that excellent stability is
achieved under neutral (pH=7) to weak acidic (pH=5.5)
conditions.
Experiment 2
[0035] 100 g of whole soybeans from the America is added with 500
ml of water in a pre-treatment, wherein the soybeans contact with
water under 20.degree. C. for 2 hours. The liquid part is then
removed with filtration. Next, the residue is added with 500 ml of
water to implement an extraction process under 98.degree. C. for 20
minutes, and then an extract liquid is obtained with filtration.
The residue is further added with 500 ml of water to implement
another extraction process under 98.degree. C. for 20 minutes, and
then another extract liquid is obtained with the same operation.
The two extract liquids are mixed into a soybean extract liquid,
which has a pH value of 6.5 at the moment. In the soybean extract
liquid, the amount of isoflavone in total solid content is 1.3 wt
%, the crude protein content is 24 wt %, and the lipid content is
1.0 wt %. Next, a neutral protease from Bacillus subtilis
(Orientase 90N, produced by Hankyu Kyoei Bussan Inc.) is added in
an amount of 0.9 wt % relative to the total solid content, and the
reaction is carried out under 50.degree. C. for 1 hour. Next, the
soybean extract liquid is heated under 80.degree. C. for 30 minutes
to deactivate the enzyme, and the pH value and temperature of the
soybean are adjusted as in Table 2, wherein the pH value is
adjusted by adding HCl.sub.(aq). The soybean extract liquid is then
placed still for 30 minutes to complete the acid-precipitation
reaction. Thereafter, the insoluble materials are removed with
centrifugal separation, and the soybean extract liquid NaOH is
neutralized by adding NaOH to have a pH value of 6.5. The soybean
extract liquid is then freeze-dried into powder. The solubility of
isoflavones in 100 g of water under 25.degree. C. is then measured
using the composition containing isoflavones. Moreover, a stability
test is done by dissolving an equivalent amount of powder
containing 10 mg of isoflavones in 100 ml of water, adjusting the
pH value of the solution to 7-3.5 with sodium bicarbonate or citric
acid, performing heat sterilization under 95.degree. C. for 15
minutes and then preserving the solution under 10.degree. C. for a
month. The results are shown in Table 2.
4TABLE 2 pH value in pH value for Cooling Solubility of stability
test separation temperature (.degree. C.) isoflavones (g) 7 6 5.5
4.5 3.5 5.5 20 >0.3 X X X X X 5.5 15 >0.3 .largecircle.
.largecircle. .largecircle. X X 4.5 20 >0.3 .largecircle.
.largecircle. .largecircle. X X 4.5 15 >0.3 .largecircle.
.largecircle. .largecircle. .largecircle. X 3.5 20 >0.3
.largecircle. .largecircle. .largecircle. X X 3.5 15 >0.3
.largecircle. .largecircle. .largecircle. .largecircle.
.largecircle. 2.0 15 >0.3 .largecircle. .largecircle.
.largecircle. .largecircle. .largecircle. Evaluation for stability
test: .largecircle.: without precipitation; X: precipitation
occurs
[0036] As shown in Table 2, when the insoluble materials are
removed from the soybean extract liquid at a pH value smaller than
5.5 and a temperature under 20.degree. C., the solubility of the
composition containing isoflavones in water is significantly
increased as compared with pure isoflavones. Moreover, the results
of the stability test indicate that excellent stability is achieved
under neutral (pH=7) to acidic (pH=3.5) conditions.
Example 1
[0037] 100 g of whole soybeans from the America, in which the
amount of isoflavones is 0.2 wt %, is added with 500 ml of water in
a pre-treatment, wherein the soybeans contact with water under
20.degree. C. for 2 hours. The liquid part is then removed with
filtration. Next, the residue is added with 500 ml of water to
implement an extraction process under 98.degree. C. for 20 minutes,
and then an extract liquid is obtained with filtration. The residue
is further added with 500 ml of water to implement another
extraction process under 98.degree. C. for 20 minutes, and then
another extract liquid is obtained with the same operation. The two
extract liquids are mixed into a soybean extract liquid. In the
soybean extract liquid, the amount of isoflavone in total solid
content is 1.3 wt %, the crude protein content is 24 wt %, and the
lipid content is 1.0 wt %. The soybean extract liquid is cooled to
10.degree. C. and kept at the same temperature for 30 minutes,
while the pH value is 6.5 at the moment. Next, the insoluble
materials are removed with centrifugal separation, and the soybean
extract liquid is freeze-dried into powder. Thus, a composition of
9.4 g containing soluble isoflavones in an amount of 1.34 wt % is
obtained. In the example, the recovery ratio of isoflavones from
the whole soybeans is 63.0%.
Example 2
[0038] 100 g of soybean hypocotyls from the America, in which the
amount of isoflavones is 1.6 wt %, is dry-heated under a hot blast
of 140.degree. C. for 20 minutes using a gas roaster. After the
treatment, the soybean hypocotyls is added with 500 ml of water in
a pre-treatment, wherein the soybean hypocotyls contact with water
under 20.degree. C. for 2 hours. The liquid part is then removed
with filtration. Next, the residue is added with 500 ml of water to
implement an extraction process under 98.degree. C. for 20 minutes,
and then an extract liquid is obtained with filtration. The residue
is further added with 500 ml of water to implement another
extraction process under 98.degree. C. for 20 minutes, and then
another extract liquid is obtained with the same operation. The two
extract liquids are mixed into a soybean extract liquid. In the
soybean extract liquid, the amount of isoflavones in total solid
content is 6.0 wt %, the crude protein content is 22 wt %, and the
lipid content is 0.5 wt %. The soybean extract liquid is cooled to
10.degree. C. and kept at the same temperature for 30 minutes,
while the pH value is 6.5 at the moment. Next, the insoluble
materials are removed with centrifugal separation, and the soybean
extract liquid is freeze-dried into powder. Thus, a composition of
18.7 g containing soluble isoflavones in an amount of 6.06 wt % is
obtained. In the example, the recovery ratio of isoflavones from
the soybean hypocotyls is 70.8%.
Example 3
[0039] 100 g of whole soybeans from the America, in which the
amount of isoflavones is 0.2 wt %, is added with 500 ml of water in
a pre-treatment, wherein the soybeans contact with water under
20.degree. C. for 2 hours. The liquid part is then removed with
filtration. Next, the residue is added with 500 ml of water to
implement an extraction process under 98.degree. C. for 20 minutes,
and then an extract liquid is obtained with filtration. The residue
is further added with 500 ml of water to implement another
extraction process under 98.degree. C. for 20 minutes, and then
another extract liquid is obtained with the same operation. The two
extract liquids are mixed into a soybean extract liquid. In the
soybean extract liquid, the amount of isoflavone in total solid
content is 1.3 wt %, the crude protein content is 24 wt %, the
lipid content is 1.0 wt %, and the pH value is 6.5 at the moment.
Next, a neutral protease from Bacillus subtilis (Orientase 90N,
produced by Hankyu Kyoei Bussan Inc.) is added in an amount of 0.9
wt % relative to the total solid content, and the reaction is
carried out under 50.degree. C. for 1 hour. Next, the soybean
extract liquid is heated under 80.degree. C. for 30 minutes to
deactivate the enzyme, and the pH value is adjusted to 3.5 by
adding HCl.sub.(aq). The soybean extract liquid is cooled to
10.degree. C. and placed still for 30 minutes. Next, the insoluble
materials are removed with centrifugal separation, and the soybean
extract liquid is neutralized by adding NaOH to have a pH value of
6.5. The soybean extract liquid is then freeze-dried into powder,
and a composition of 8.8 g containing soluble isoflavones in an
amount of 1.40 wt % is obtained. In the example, the recovery ratio
of isoflavones from the whole soybeans is 61.6%.
Example 4
[0040] 100 g of soybean hypocotyls as obtained in Example 2, in
which the amount of isoflavones is 1.6 wt %, is added with 500 ml
of water in a pre-treatment, wherein the soybeans contact with
water under 20.degree. C. for 2 hours. The liquid part is then
removed with filtration. Next, the residue is added with 500 ml of
water to implement an extraction process under 98.degree. C. for 20
minutes, and then an extract liquid is obtained with filtration.
The residue is further added with 500 ml of water to implement
another extraction process under 98.degree. C. for 20 minutes, and
then another extract liquid is obtained with the same operation.
The two extract liquids are mixed into a soybean extract liquid. In
the soybean extract liquid, the amount of isoflavone in total solid
content is 6.0 wt %, the crude protein content is 22 wt %, the
lipid content is 0.5 wt %, and the pH value is 6.5 at the moment.
Next, a neutral protease from Bacillus subtilis (Orientase 90N,
produced by Hankyu Kyoei Bussan Inc.) is added in an amount of 0.9
wt % relative to the total solid content, and the reaction is
carried out under 50.degree. C. for 1 hour. Next, the soybean
extract liquid is heated under 80.degree. C. for 30 minutes to
deactivate the enzyme, and the pH value is adjusted to 3.5 by
adding HCl.sub.(aq). The soybean extract liquid is cooled to
10.degree. C. and placed still for 30 minutes. Next, the insoluble
materials are removed with centrifugal separation, and the soybean
extract liquid is neutralized by adding NaOH to have a pH value of
6.5. The soybean extract liquid is then freeze-dried into powder,
and a composition of 15.5 g containing soluble isoflavones in an
amount of 6.32 wt % is obtained. In the example, the recovery ratio
of isoflavones from the soybean hypocotyls is 61.2%.
Example 5
[0041] A soybean extract liquid as obtained in Example 4 is added
with HCl.sub.(aq) to have a pH value of 3.5 without the protease
treatment, and is cooled to 10.degree. C. and placed still for 30
minutes. After the insoluble materials are removed with centrifugal
separation, the soybean extract liquid is neutralized by adding
NaOH to have a pH value of 6.5. The soybean extract liquid is then
freeze-dried into powder, and a composition of 10.3 g containing
soluble isoflavones in an amount of 4.72 wt % is obtained. In the
example, the recovery ratio of isoflavones from the soybean
hypocotyls is 30.4%. Though the solubility of the composition in
water is relatively high, the recovery ratio of isoflavones gets
low since a part of the isoflavones are removed together with the
insoluble materials during the removal step under acidic
condition.
Example 6
[0042] 10 g of the composition containing soluble isoflavones as
obtained in Example 4 is dissolved in 100 ml of water, and then the
solution is conducted through a column (.phi. 2.5 cm.times.20 cm)
packed with 100 ml of an activated synthetic adsorbent of
styrenedivinylbenzene type (Diaion HP-20, produced by Mitsubishi
Chemical Corporation) in a flow rate of 100 ml/hr. Next, the
adsorbent is washed with 200 ml of water and 200 ml of 20% ethanol
in sequence to remove impurities, and then the target materials are
eluted with 300 ml of 70% ethanol. After being condensed under low
pressure to remove ethanol, the solution is freeze-dried into a
composition of 1.5 g containing soluble isoflavones in an amount of
26.2 wt %.
[0043] Utility in the Industry
[0044] By using the method of this invention, a composition
containing isoflavones can be obtained with a high solubility 100
or more times the solubility of pure isoflavone and good stability
preventing precipitation and cloudiness after long-term
preservation under refrigeration. The isoflavones can be easily
obtained in their natural states with a high recovery ratio using
soybean materials as raw materials without addition of solubilizing
agents or chemical modification. Therefore, the scope of the
applications of isoflavones in food-related fields can be
significantly expanded, and this invention should have great
significance to the industry.
[0045] It will be apparent to those skilled in the art that various
modifications and variations can be made to the structure of the
present invention without departing from the scope or spirit of the
invention. In view of the foregoing, it is intended that the
present invention covers modifications and variations of this
invention provided they fall within the scope of the following
claims and their equivalents.
* * * * *