U.S. patent application number 09/768595 was filed with the patent office on 2001-08-02 for process for producing isoflavone aglycone-containing composition.
This patent application is currently assigned to Kikkoman Corporation. Invention is credited to Izumi, Toru, Kikuchi, Mamoru, Manaka, Tatuo, Obata, Akio, Saito, Makoto, Tobe, Koichiro.
Application Number | 20010010930 09/768595 |
Document ID | / |
Family ID | 18546961 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010930 |
Kind Code |
A1 |
Obata, Akio ; et
al. |
August 2, 2001 |
Process for producing isoflavone aglycone-containing
composition
Abstract
An isoflavone aglycone-containing composition having genistein
as a main aglycone is produced by a process comprising allowing a
protease and .beta.-glucosidase to act on a soy protein raw
material, an extract of a soy protein raw material or a by-product
of a soy protein raw material to water-solubilize the protein of
soybean origin and to convert isoflavone glycosides to the
corresponding aglycones, separating water-soluble components from
the enzymatic reaction mixture, and recovering water-insoluble
matter.
Inventors: |
Obata, Akio; (Chiba Pref.,
JP) ; Manaka, Tatuo; (Chiba Pref., JP) ; Tobe,
Koichiro; (Chiba Pref., JP) ; Izumi, Toru;
(Chiba Pref., JP) ; Saito, Makoto; (Chiba Pref.,
JP) ; Kikuchi, Mamoru; (Chiba Pref., JP) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Assignee: |
Kikkoman Corporation
|
Family ID: |
18546961 |
Appl. No.: |
09/768595 |
Filed: |
January 25, 2001 |
Current U.S.
Class: |
435/125 |
Current CPC
Class: |
C12P 17/06 20130101 |
Class at
Publication: |
435/125 |
International
Class: |
C12P 017/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2000 |
JP |
2000-20393 |
Claims
What is claimed is:
1. A process for producing an isoflavone aglycone-containing
composition comprising allowing a protease and .beta.-glucosidase
to act on a soy protein raw material, an extract of a soy protein
raw material or a by-product of a soy protein raw material to
water-solubilize the protein of soybean origin and to convert
isoflavone glycosides to the corresponding aglycones, separating
water-soluble components from the enzymatic reaction mixture, and
recovering water-insoluble matter.
2. The process according to claim 1, wherein said protease and said
.beta.-glucosidase are in the form of an enzyme preparation having
both protease activity and .beta.-glucosidase activity.
3. The process according to claim 1, wherein said soy protein raw
material is at least one of whole soybeans, dehulled soybeans and
defatted soybeans.
4. The process according to claim 1, wherein said extract of a soy
protein raw material is at least one of a soy protein isolate, a
soy protein concentrate, soybean milk, and a soybean extract
containing isoflavone glycosides.
5. The process according to claim 1, wherein said by-product of a
soy protein raw material is at least one of soybean molasses,
soybean whey, and soybean curd waste.
6. The process according to claim 1, wherein the majority of said
isoflavone aglycones is genistein.
7. The process according to claim 1, wherein the enzyme reaction
mixture contains 1 to 50% by weight of an organic solvent.
8. The process according to claim 1 or 2, wherein said protease and
said .beta.-glucosidase are acid enzymes the optimum pH of which is
from 2.0 to 6.0.
9. The process according to claim 1 or 2, wherein said protease and
said .beta.-glucosidase are thermostable enzymes the optimum
temperature of which is 50.degree. C. or higher.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a process for obtaining a
composition containing soybean isoflavone aglycones mainly
comprising genistein.
[0003] 2. Description of the Related Art
[0004] Isoflavone compounds, such as malonyldaidzin,
malonylglycitin, malonylgenistin, daidzin, glycitin, genistin,
daidzein, glycitein, and genistein, are known to have estrogenic
activity, antioxidative activity, antibacterial activity,
antilipemia activity, anticholesterol activity, and the like. In
recent years, cancer cell differentiation and induction activity,
oncogene inhibitory activity, and prophylactic activity on cancers
have been confirmed. Thus, the usefulness of these isoflavone
compounds has been attracting attention. Many researches have
revealed that the pharmacological effects, such a cancer
prophylactic effect, of isoflavone compounds are primarily
attributed not to the glycosides themselves but their aglycones,
such as daidzein or genistein. Of the soybean isoflavone aglycones
genistein has recently been proved particularly excellent in
physiological activities, including antiosteoporosis activity,
antiarteriosclerotic activity, and anticancer activities in the
breast, the stomach and the prostate (see M. Numoto, Cancer
Research, vol. 53, p. 5815 (1993) and S. Barnes, Biochem. Biophys.
Res. Commun., vol. 179, p. 661 (1997)).
[0005] Methods of obtaining isoflavone compounds include the
methods described in Japanese Patent Laid-Open Nos. 62-126186 and
11-89589, for example. Since 95% or more of isoflavone compounds in
soybeans are present in the form of glycosides, however, the
isoflavone compounds obtained by the former method mainly comprise
glycosides with little amount of aglycones. Further, the isoflavone
aglycones obtained by the latter method, which uses soybean
hypocotyl tissue as a raw material, mainly comprise daidzein with
the genistein content being about 1/6 of daidzein.
[0006] The inventors of the present invention previously applied
for a patent based on their findings that soy sauce cake and soy
sauce oil, which are by-produced in soy sauce production, contain
isoflavone compounds substantially comprising aglycones such as
daidzein and genistein and that these aglycones can be obtained
efficiently by organic solvent extraction (see Japanese Patent
Laid-Open No. 6-170756). The problems associated with this method,
however, are that the yield of aglycones is low, the raw materials
are uncommon, and a special extraction operation is needed.
SUMMARY OF THE INVENTION
[0007] The present inventors have conducted extensive
investigations on production of isoflavone aglycones from soybeans
and found as a result that a composition containing isoflavone
aglycones the majority of which is genistein can easily be obtained
by allowing an enzyme preparation having protease activity and
.beta.-glucosidase activity to thoroughly act on a soybean raw
material that is easily available in the market and recovering
water-insoluble matter.
[0008] Having been completed based on the above finding, the
present invention provides a process for producing an isoflavone
aglycone-containing composition comprising allowing a protease and
.beta.-glucosidase to act on a soy protein raw material, an extract
of a soy protein raw material or a by-product of a soy protein raw
material to water-solubilize the protein of soybean origin and to
convert isoflavone glycosides to the corresponding aglycones,
separating water-soluble components from the enzymatic reaction
mixture, and recovering water-insoluble matter. The majority of the
aglycones present in the resulting composition is genistein.
[0009] According to the present invention, a composition containing
isoflavone aglycones mainly comprising genistein can be obtained
from an easily commercially available raw material through an easy
and simple operation.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The raw material used in the present invention is a soy
protein raw material, an extract of a soy protein raw material or a
by-product of a soy protein raw material. The soy protein raw
material includes whole soybeans, dehulled soybeans, and defatted
soybeans. The extract of a soy protein raw material includes a soy
protein isolate, a soy protein concentrate, soybean milk, and a
soybean extract containing isoflavone glycosides. The by-product of
a soy protein raw material includes soybean molasses, soybean whey,
and soybean curd waste. To help a protease enzyme act, it is
preferred that these raw materials be previously heat treated to
denature the protein and deactivate the trypsin inhibitor. The heat
treatment is preferably carried out at 80 to 200.degree. C. for
about 10 to 60 minutes.
[0011] The enzymes which can be used in the present invention,
i.e., a protease and .beta.-glucosidase, can be crude enzymes as
produced by culturing microorganisms or commercially available
crude enzymes, such as protease preparations. The crude enzymes may
be used as concentrated or purified by ultrafiltration or column
chromatography. While not limiting, the amounts of the enzymes
(protease and .beta.-glucosidase) preferably range from 0.1 to 10
parts by weight in terms of the solid content per 100 parts by
weight of the soybean raw material.
[0012] The enzymes (protease and .beta.-glucosidase) are preferably
of acid species whose optimum pH is in a range of from 2.0 to 6.0
and of thermostable species whose optimum temperature is 50.degree.
C. or higher.
[0013] Any commercially available crude enzymes can be used as long
as they have protease activity and .beta.-glucosidase activity.
Enzyme preparations of the genus Penicillium or Aspergillus origin
are preferred. Analysis of .beta.-glucosidase activity on various
commercially available protease preparations by the method of
Ebata, et al. (Nippon Nogeikagaku Kaishi, vol. 46, p. 323 (1972))
revealed that those of Penicillium or Aspergillus origin,
especially those having an optimum pH in an acidic region (acid
protease preparations) exhibit high activity. Specific examples of
preferred commercial enzyme preparations having both protease
activity (PA) and .beta.-glucosidase activity (GA) are Molsin F
(PA: 38,000 U/g; GA: 260 U/g; available from Kikkoman Corp.),
Sumizyme FP (PA: 50,000 U/g; GA: 172 U/g; available from Shin-nihon
Kagaku Kogyo K.K.), Protease M Amano (PA: 5,500 U/g; GA: 132 U/g;
available from Amano Pharmaceutical Co., Ltd.), Miso Koso Amano A1
(PA: 3,000 U/g; GA: 28 U/g; available from Amano Pharmaceutical
Co., Ltd.), AO Protease (PA: 24,000 U/g; GA: 20 U/g; available from
Kikkoman Corp.), and IP Koso (PA: 34,000 U/g; GA: 4 U/g; available
from Kikkoman Corp.). These enzyme preparations are sold as enzymes
for food so that their safety as food has been established.
[0014] Of these enzyme preparations those having a protease
activity of 5,000 U/g or more (as determined by the method
described in P. E. Wilcox, Methods Enzymol., vol. 19, pp. 64-80
(1970)) and a .beta.-glucosidase activity of 100 U/g or more (as
determined by the method described in J. Ebata et al, Nippon
Nogeikagaku Kaishi, pp. 323-329 (1972)) are preferred.
[0015] The manner of carrying out the enzymatic reaction is not
particularly restricted. For example, the soybean raw material is
added to a previously prepared aqueous dispersion of the enzyme
preparation followed by stirring, or the aqueous enzyme dispersion
is sprayed directly on the soybean raw material, or the enzyme
preparation is added to a mixture of the soybean raw material and
water, followed by stirring, or enzymes (protease and
.beta.-glucosidase) are brought into contact with the soybean raw
material to impregnate. While the amount of water added is not
particularly limited, too much water requires of necessity larger
equipment to increase the cost and to reduce the productivity. A
preferred amount of water is about 50 to 10,000 parts by weight per
100 parts by weight of the soybean raw material.
[0016] The enzyme reaction conditions, while not particularly
limited unless abrupt deactivation of the enzymes or putrefaction
by bacteria occurs, are preferably 40 to 70.degree. C. and 1 to 24
hours. Since isoflavone glycosides are sparingly soluble in water,
it is preferred to add an organic solvent to the reaction system in
an amount of 1 to 50% by weight or to use thermostable enzymes to
allow the reaction to be conducted at an increased temperature of
60 to 70.degree. C. By such manipulations, the solubility of the
isoflavone glycosides increases, and contamination with bacteria
can be suppressed, thereby improving the reaction efficiency. Any
organic solvent can be added in an arbitrary amount as far as is
consistent with the enzymatic actions and effective to improve the
solubility of isoflavone glycosides. Ethanol, methanol, acetone or
dimethyl sulfoxide can be used for example. The pH of the reaction
system is not limited either, unless the enzymes are inactivated. A
preferred pH range is from 2 to 9. In using an acid protease
preparation, a pH range of 3 to 5 is preferred.
[0017] The enzymatic reaction can be regarded completed when a
sampled aliquot of the reaction mixture shows conversion of 90% or
more of the isoflavone glycosides into the corresponding aglycones.
On completion of the reaction, the reaction mixture is adjusted to
pH 2 to 5, and water-soluble components are removed by
ultrafiltration, centrifugal separation or a like technique to
recover water-insoluble matter. If desired, the recovered
water-insoluble matter is washed with water at pH 2 to 5. The
separated water-soluble components (the soluble matter of the
reaction mixture plus the washing) include water-solubilized amino
acids and peptides resulting from decomposition by the protease and
the sugar moieties resulting from hydrolysis by the
.beta.-glucosidase. Accordingly, the above operation removes
components other than isoflavone aglycones and provides the
insoluble matter enriched in aglycones.
[0018] A general process according to the present invention will be
described hereunder for illustrative purposes only but not for
limitation.
[0019] The above-described soybean raw material can be treated as
such or, for facilitating the enzymes' reacting, previously reduced
to powder in a grinder, etc. It is recommended to previously
subject the raw material to heat treatment at 80 to 200.degree. C.
for about 10 to 60 minutes to deactivate nutrition inhibitory
factors such as soybean trypsin inhibitor thereby facilitating the
protease reaction.
[0020] An enzyme preparation having protease activity and
.beta.-glucosidase activity is then made to act on the soybean raw
material. The enzyme preparation is used as dispersed in water
beforehand. An organic solvent, such as ethanol, may be added to
the enzyme dispersion in an amount of 1 to 50% by weight so as to
improve the solubility of isoflavone glycosides. The soybean raw
material is added to the enzyme dispersion prepared in a reactor
whose temperature can be maintained constant, and the mixture is
stirred to carry out enzyme reaction. Alternatively, water is added
to the soybean raw material, and the enzyme preparation is added
thereto, followed by stirring to conduct the reaction.
[0021] The pH and the temperature of the enzyme reaction system are
adjusted to the optimum to ensure efficient progress of the enzyme
reaction.
[0022] After confirming that 90% or more of the isoflavone
glycosides in the reaction mixture has converted to the
corresponding aglycones, the reaction mixture is adjusted to pH 2
to 5, and water-soluble components are removed by ultrafiltration,
centrifugal separation or a like technique to recover
water-insoluble matter. If desired, the recovered water-insoluble
matter is washed with water at pH 2 to 5. The resulting wet solid
is dried by means of, for example, a vacuum drier and pulverized to
obtain an isoflavone aglycone-containing composition. Rich in
isoflavone aglycones, the resulting powder can be utilized as
health foods and general beverages and foodstuffs. The resulting
powder or an intermediate product obtained in the course of the
above-mentioned preparation process may be purified by organic
solvent extraction or by use of a resin to provide high purity
isoflavone aglycone preparations, which can be used as health
foods, cosmetics or ingredients of pharmaceutical preparations.
[0023] The present invention will further be illustrated in greater
detail with reference to Examples, but it should be understood that
the invention is not construed as being limited thereto. Unless
otherwise noted, all the percents are by weight.
EXAMPLE 1
[0024] Defatted soybeans (200 g) were ground and extracted with
1000 ml of 80% ethanol to obtain 23 g of an extract containing
isoflavone glycosides. To the extract was added 500 ml of water to
dissolve the extract. The solution was adjusted to pH 4.5, 2 g of
Sumizyme FP (available from Shin-nihon Kagaku Kogyo K.K.) was added
thereto, and the mixture was stirred at 55.degree. C. overnight. It
was found that 90% or more of the isoflavone glycosides had been
converted to aglycones. The reaction mixture was adjusted to pH 4
with hydrochloric acid to precipitate an isoflavone fraction, which
was collected by filtration. The filter cake was dissolved in 1000
ml of 0.1N NaOH, adjusted to pH 8, and passed through an activated
carbon column. After washing the column with water, the adsorbed
isoflavone aglycones were eluted with 2000 ml of 0.1N NaOH. The
eluate was adjusted to pH 4.5 with hydrochloric acid to precipitate
the aglycones, which were collected by filtration and dried in a
vacuum drier to give 300 mg of an isoflavone aglycone powder having
a purity of 70%. The isoflavone aglycone content of the resulting
powder was found made up of 58% of genistein, 38% of daidzein, and
4% of glycitein, proving to be a composition mainly comprising
genistein. The analysis of the aglycone composition was in
accordance with H. Wang et al., J Agric. Food Chem., vol. 42, p.
666 (1994).
EXAMPLE 2
[0025] Three liters of water was added to 100 g of a commercially
available soy protein isolate Fuji Pro F (available from Fuji Oil
Co., Ltd.), and the mixture was heated in a boiling water bath for
10 minutes. After cooling, 10 g of Molsin F (available from
Kikkoman Corp.) and 300 ml of ethanol were added thereto at a
temperature kept at 50.degree. C., followed by stirring at pH 5
overnight (16 hours). After the reaction, 95% or more of the
isoflavones in the reaction mixture were found to have been
converted to the corresponding aglycones. The reaction mixture was
adjusted to a pH of 4.5. The precipitate was collected by
centrifugation, washed with water at pH 4.5, and dried in a
freeze-drier to obtain a product weighing 34 g. The product was
found to have an isoflavone aglycone content of about 0.61%, which
is about three times that of the raw material. Analysis by
high-performance liquid chromatography revealed that the isoflavone
aglycone composition of the product was made up of 56% of
genistein, 39% of daidzein, and 5% of glycitein, proving to
comprise genistein as a main aglycone.
EXAMPLE 3
[0026] To 1 kg of a commercially available isoflavone glycoside
Novasoy (available from Archer Daniels Midland Co.) was added 251
of distilled water. After the mixture was adjusted to pH 4.5 with
hydrochloric acid, 150 g of Molsin F (Kikkoman Corp.) was added
thereto, followed by stirring at 60.degree. C. overnight (16
hours). After the reaction, 95% or more of the isoflavones in the
reaction mixture were found converted to aglycones. The reaction
mixture was adjusted to a pH of 2.5 with hydrochloric acid to
precipitate an isoflavone fraction, which was washed with water at
pH 2.5 and collected by filtration. The filter cake was stirred in
25 l of 90% ethanol for 2 hours to extract the isoflavones. The
insoluble enzyme component was removed, the separated ethanol
extract was concentrated, and the concentrate was spray-dried to
obtain 480 g of an isoflavone aglycone powder. The resulting powder
had an isoflavone aglycone content of 52%, which was found to be
made up of 54% of genistein, 40% of daidzein, and 6% of glycitein,
proving to comprise genistein as a main aglycone.
* * * * *