U.S. patent application number 13/549568 was filed with the patent office on 2012-11-01 for equol-containing fermentation product of soybean embryonic axis, and method for production thereof.
This patent application is currently assigned to OTSUKA PHARMACEUTICAL CO., LTD.. Invention is credited to Hiroyuki Kimura, Masatoshi Mizuno, Masahiro Oono, Ikutaro Sato, Toshimi Suzuki, Kentaro Tadano, Shigeto Uchiyama, Tomomi Ueno, Takeshi Yamauchi.
Application Number | 20120277302 13/549568 |
Document ID | / |
Family ID | 38122801 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120277302 |
Kind Code |
A1 |
Kimura; Hiroyuki ; et
al. |
November 1, 2012 |
EQUOL-CONTAINING FERMENTATION PRODUCT OF SOYBEAN EMBRYONIC AXIS,
AND METHOD FOR PRODUCTION THEREOF
Abstract
An object of the invention is to provide an equol-containing
fermented soybean hypocotyl material that is useful for foods,
pharmaceutical preparations, cosmetic products, etc. The
equol-containing fermented soybean hypocotyl material of the
invention is obtained by fermenting soybean hypocotyls using at
least one microorganism having an equol-producing ability by
utilizing at least one daidzein compound selected from the group
consisting of diadzein glycosides, daidzein, and
dihydrodaidzein.
Inventors: |
Kimura; Hiroyuki; (Saga,
JP) ; Yamauchi; Takeshi; (Shiga, JP) ; Ueno;
Tomomi; (Saga, JP) ; Suzuki; Toshimi; (Saga,
JP) ; Tadano; Kentaro; (Saga, JP) ; Sato;
Ikutaro; (Saga, JP) ; Uchiyama; Shigeto;
(Saga, JP) ; Oono; Masahiro; (Saitama, JP)
; Mizuno; Masatoshi; (Shiga, JP) |
Assignee: |
OTSUKA PHARMACEUTICAL CO.,
LTD.
Tokyo
JP
|
Family ID: |
38122801 |
Appl. No.: |
13/549568 |
Filed: |
July 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12095828 |
Jun 24, 2009 |
|
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PCT/JP2006/324255 |
Dec 5, 2006 |
|
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13549568 |
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Current U.S.
Class: |
514/456 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
25/24 20180101; A61P 19/10 20180101; A61K 8/498 20130101; A61P 3/06
20180101; A61P 7/10 20180101; A23V 2002/00 20130101; A61P 15/12
20180101; A61K 2800/85 20130101; A61P 5/30 20180101; A61P 13/08
20180101; A61P 5/32 20180101; A61P 15/00 20180101; A61Q 19/10
20130101; A61K 8/9789 20170801; A23L 11/09 20160801; A61P 3/00
20180101; A23L 33/105 20160801; A61P 35/00 20180101; A61P 3/04
20180101; A61Q 19/00 20130101; A61Q 19/02 20130101; A23V 2002/00
20130101; A23V 2250/21172 20130101; A23V 2002/00 20130101; A23V
2250/5488 20130101; A23V 2200/3262 20130101 |
Class at
Publication: |
514/456 |
International
Class: |
A61K 31/352 20060101
A61K031/352; A61K 8/49 20060101 A61K008/49 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 2005 |
JP |
2005-352337 |
Oct 11, 2006 |
JP |
2006-277934 |
Claims
1. A fermented soybean hypocotyl material comprising 1 to 20 mg of
equol and 0.1 to 1.5 mg of at least one daidzein compound selected
from the group consisting of daidzin, malonyldaidzin,
acetyldaidzin, daidzein and dihydrodaidzein per 1 g of the
fermented soybean hypocotyl material on a dry weight basis.
2. The fermented soybean hypocotyl material according to claim 1,
which further comprises 0.05 to 2.5 mg of at least one genistein
compound selected from the group consisting of genistin,
malonylgenistin, acetylgenistin, genistein and dihydrogenistein per
1 g of the fermented soybean hypocotyl material on a dry weight
basis.
3. The fermented soybean hypocotyl material according to claim 1,
which is fermented by an enteric bacterium.
4. The fermented soybean hypocotyl material according to claim 3,
wherein the enteric bacterium is a lactic acid bacterium.
5. The fermented soybean hypocotyl material according to claim 1,
wherein the content of allergen in the soybean hypocotyl is
reduced.
6. A food, pharmaceutical or cosmetic product comprising the
fermented soybean hypocotyl material according to claim 1.
Description
[0001] This is a Continuation Application of U.S. application Ser.
No. 12/095,828 filed Jun. 24, 2009, which is a U.S. National Stage
Entry of PCT/JP2006/324255 filed Dec. 5, 2006, which claims
priority from JP Patent Appln. No. 2005-352337 filed Dec. 6, 2005
and JP Patent Appln. No. 2006-277934 filed Oct. 11, 2006, the
contents of all of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to fermented soybean hypocotyl
containing equol and a method of producing the same.
BACKGROUND ART
[0003] Isoflavones (soybean isoflavones: daidzein, genistein,
glycitein) contained in soybeans have structures similar to
estradiol, and have anti-estrogen actions associated with binding
to estrogen receptors (hereinafter referred to as ER) and
estrogen-like actions. The epidemiological studies and intervention
studies of soybean isoflavones that have been done before suggest
their preventive effects due to their anti-estrogen actions on
breast cancer, prostate cancer and other hormone-dependent cancers
and improving effects due to estrogen-like actions on menopausal
disorders, postmenopausal osteoporosis and hyperlipidemia.
[0004] Recently, it has been pointed out that the active principle
of the physiological effects of these soybean isoflavones may be a
metabolite of daidzein, equol. More specifically, it has been
reported that equol has an ability to bind to ER (especially to
ER.beta.) greater than soybean isoflavones and that it has
remarkably high transition capability to target organs such as
breast and prostate tissues. Moreover, a case-control study reports
that there are significantly less patients who produce equol in the
patients of breast cancer and prostate cancer. The effects of
soybean isoflavones to improve the bone density and lipid
metabolism were examined regarding postmenopausal women categorized
into two groups: those who produce equol and those who do not. A
significant improvement in those who produce equol was
observed.
[0005] Equol is produced by metabolism of daidzein by enteric
bacteria. The abilities to produce equol vary between individuals,
and the percentage of Japanese who produce equol production is
reportedly about 50%. That is, about 50% of Japanese are not able
to produce equol (non-equol-producing individuals). Such an
individual cannot enjoy useful physiological benefits based on the
action of equol even if they ingest soybeans and processed soybean
foods. Therefore, in order to attain useful physiological benefits
based on the action of equol in a non-equol-producing individual,
ingesting equol itself is thought to be effective.
[0006] A known method of producing equol is subjecting a raw
material containing daidzein compounds to fermentation treatment by
microorganisms (hereinafter referred to as equol-producing
bacteria) which metabolize daidzein to produce equol. Known
starting materials containing daidzein for use in this production
method include soybeans, Ge Gen Tang (Chinese traditional medicine,
also known as Kakkonto), red globe grapes, alfalfa or others.
Moreover, equol-producing bacteria are already known. For example,
bacteroides E-23-15 (FERM BP-6435), streptococcus E-23-17 (FERM
BP-6436), streptococcus A6G225 (FERM BP-6437) and lactococcus 20-92
(FERM BP-10036) have been isolated from human excrement by the
inventors of the present invention (refer to patent documents 1 and
2).
[0007] However, simply subjecting the above-mentioned starting
materials containing daidzein compounds to fermentation treatment
by using equol-producing bacteria cannot yield sufficient amount of
equol in the fermented product, and there has been the problem that
desired useful benefits based on the action of equol cannot be
sufficiently expected by ingesting the fermented product as it
is.
[0008] In contrast, the hypocotyl portions of soybeans have been
known to contain isoflavones, saponins and other useful components
in a proportion higher than in the cotyledon portions which are
used as processed soybean foods, and various uses have been
developed for its extract (e.g., patent document 3). However,
soybean hypocotyl extract itself is disadvantageously expensive.
Moreover, when the soybean hypocotyl extract is used as a starting
material for producing equol, addition of other nutrients are
necessary to allow fermentation by equol-producing bacteria, which
can be another problem. For these reasons, the soybean hypocotyl
extract cannot be currently used as a starting material for
industrially producing equol.
[0009] Meanwhile, since the soybean hypocotyl itself has a
characteristic bitterness, there is a trend to avoid using the
substance itself as it is, and much of the soybean hypocotyl is
currently disposed. Furthermore, likewise soybean cotyledon
portions, soybean hypocotyls contain allergens, therefore the
soybean hypocotyl could not be taken by or administer to people
suffering from soybean allergy. Therefore, to effectively utilize
the soybean hypocotyl itself, it is important to impart added
values to increase its usefulness.
[0010] [Patent document 1] International Publication
WO99/007392
[0011] [Patent document 2] International Publication
WO2005/000042
[0012] [Patent document 3] Japanese Unexamined Patent Publication
No. 2002-234844
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0013] An object of the invention is to provide an equol-containing
fermented soybean hypocotyl material that is useful for foods,
pharmaceutical preparations, cosmetic products, etc. Another object
of the invention is to provide a method for producing an
equol-containing fermented soybean hypocotyl material.
Means for Solving the Problem
[0014] The present inventors conducted intensive research to
achieve the above objects and found that an equol-containing
fermented soybean hypocotyl material can be obtained very
efficiently by fermenting soybean hypocotyls using at least one
microorganism having an equol-producing ability by utilizing at
least one daidzein compound. The present inventors also found that
the thus-obtained equol-containing fermented soybean hypocotyl
material is useful as an allergen-reduced material, because the
allergens contained in the soy bean hypocotyl are reduced. The
present invention has been accomplished based on these finding.
[0015] In other words, the present invention provides
equol-containing fermented soybean hypocotyl materials and uses
thereof as below:
[0016] Item 1: An equol-containing fermented soybean hypocotyl
material obtained by fermenting soybean hypocotyls using at least
one microorganism having an equol-producing ability by utilizing at
least one daidzein compound selected from the group consisting of
diadzein glycosides, daidzein, and dihydrodaidzein.
[0017] Item 2: A fermented soybean hypocotyl material according to
Item 1, wherein said at least one microorganism is a lactic acid
bacterium of the genus Lactococcus.
[0018] Item 3: A fermented soybean hypocotyl material according to
Item 1, wherein said at least one microorganism belongs to
Lactococcus garvieae.
[0019] Item 4: A fermented soybean hypocotyl material according to
Item 1, which contains 0.1 to 20 wt % of equol per total weight of
dry fermented soybean hypocotyl material.
[0020] Item 5: A fermented soybean hypocotyl material according to
Item 1, which further contains daidzin compounds, genistin
compounds, genistei compounds, glyciti compounds and glycitei
compounds.
[0021] Item 6: A fermented soybean hypocotyl material according to
Item 1, which further contains ornithine.
[0022] Item 7: A food containing a fermented soybean hypocotyl
material of Item 1.
[0023] Item 8: A food according to Item 7, which is a dietary
supplement.
[0024] Item 9: A food according to Item 7, which contains 0.1 to 90
g of the fermented soybean hypocotyl material per 100 g of the
food.
[0025] Item 10: A pharmaceutical preparation containing a fermented
soybean hypocotyl material of Item 1.
[0026] Item 11: A pharmaceutical preparation according to Item 10,
which is used for preventing or treating menopausal disorders,
osteoporosis, prostatic hypertrophy, or metabolic syndrome.
[0027] Item 12: A pharmaceutical preparation according to Item 10,
which is used for lowering the blood cholesterol level.
[0028] Item 13: Use of a fermented soybean hypocotyl material of
Item 1 for producing preparations for preventing or treating
menopausal disorders, osteoporosis, prostatic hypertrophy, or
metabolic syndrome.
[0029] Item 14: Use of a fermented soybean hypocotyl material of
Item 1 for producing preparations for lowering the blood
cholesterol level.
[0030] Item 15: A method for treating menopausal disorders
comprising the step of administering an effective amount of a
fermented soybean hypocotyl material of Item 1 to a patient
suffering from menopausal disorders.
[0031] Item 16: A method for lowering the blood cholesterol level
comprising the step of administering an effective amount of a
fermented soybean hypocotyl material of Item 1 to a patient who is
in need of lowering the blood cholesterol level.
[0032] Item 17: A cosmetic product containing the fermented soybean
hypocotyl material of Item 1.
[0033] Item 18: A cosmetic product according to Item 17, which
contains 0.1 to 10 g of the fermented soybean hypocotyl material
per 100 g of the cosmetic product.
[0034] The present invention also provides a method for producing
an equol-containing fermented soybean hypocotyl material as
below:
[0035] Item 19: A method for producing an equol-containing
fermented soybean hypocotyl material comprising the step of
fermenting soybean hypocotyls using at least one microorganism
having an equol-producing ability by utilizing at least one
daidzein compound selected from the group consisting of diadzein
glycosides, daidzein, and dihydrodaidzein.
Effects of the Invention
[0036] The fermented soybean hypocotyl material of the invention
contains equol and other active ingredients, such as isoflavones
and saponins, and can thereby find a variety of applications in the
food, pharmaceutical, cosmetic and other fields. The fermented
soybean hypocotyl material of the invention, in particular, has a
significantly higher equol content than those of the materials
obtained by fermenting a raw material containing daidzein compounds
such as soybean, Ge Gen Tang (Chinese traditional medicine, also
known as Kakkonto), red globe grapes, alfalfa, and the like, and
can thereby attain much superior equol-derived active physiological
effects.
[0037] Furthermore, because the thus-obtained fermented soybean
hypocotyl material is reduced in the amount of the allergens
inherently contained in soy bean hypocotyls, it can be safely taken
by or administered to people suffering from soybean allergy.
Moreover, the fermented soybean hypocotyl material of the invention
is made from soybean hypocotyls that are discarded during soybean
food processing, and therefore has high industrial potential in
terms of effective use of resources.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows the equol concentrations of the fermented
liquids obtained in Examples 1 to 3.
[0039] FIG. 2 shows the detection results of total proteins
contained in the fermented soybean hypocotyl material of Example 1,
soybean cotyledons, and soybean hypocotyls
(electrophoretogram).
[0040] FIG. 3 shows the detection results of major allergens (Gym4,
Gm30K, and Gm28K) contained in the fermented soybean hypocotyl
material of Example 1, soybean cotyledons, and soybean hypocotyls
(electrophoretogram).
[0041] FIG. 4 shows the detection results of major allergens (7S
globulin mix, oleocine, and trypsin inhibitor) contained in the
fermented soybean hypocotyl material of Example 1, soybean
cotyledons, and soybean hypocotyls (electrophoretogram).
BEST MODE FOR CARRYING OUT THE INVENTION
[0042] Embodiments of the present invention are described below in
detail.
[0043] Microorganisms having an ability to produce equol (metabolic
activity) by utilizing at least one daidzein compound selected from
the group consisting of diadzein glycosides, daidzein, and
dihydrodaidzein are used as equol-producing bacteria in the present
invention. Specific examples of diadzein glycosides include
daidzin, malonyldaidzin, acetyldaidzin, etc.
[0044] Such microorganisms (equol-producing bacteria) are not
particularly limited as long as they have equol-producing ability
and are acceptable for foods, pharmaceuticals, or cosmetics. It has
been revealed that microorganisms having the above-mentioned
include, for example, bacteria of the genus Lactococcus, such as
Lactococcus garvieae, etc.; bacteria of the genus Streptococcus,
such as Streptococcus intermedius, Streptococcus constellatus,
etc.; and bacteria of the genus Bacteroides such as Bacteroides
ovatus. Amongst equol-producing bacteria, lactic acid bacteria
belonging to the genera Lactococcus, Streptococcus, etc. are
preferable; lactic acid bacteria of the genus Lactococcus are
particularly preferable; and Lactococcus garvieae is particularly
preferable. Bacteria having equol-producing ability can be isolated
from human feces using the presence or absence of equol production
as an indicator. Equol-producing bacteria isolated from human feces
and identified by the present inventors, as well as others, i.e.,
Lactococcus 20-92 (FERM BP-10036), Streptococcus E-23-17 (FERM
BP-6436), Streptococcus A6G225 (FERM BP-6437), and Bacteroides
E-23-15 (FERM BP-6435) have been deposited. Such deposited bacteria
can be used in the present invention. Among these, Lactococcus
20-92 is preferable.
[0045] In the present invention, soybean hypocotyls are used as a
fermentation material. Soybean hypocotyls are portions that develop
into plumules and radicles when the soybeans germinate, and are
known to contain large amounts of daidzein compounds such as
daidzein glycosides, daidzein, etc. Soybean hypocotyls for use in
the present invention are not limited in origin, and may be
processed or unprocessed, as long as their daidzein compound
contents are not lost. Usable examples include raw soybean
hypocotyls; hypocotyls separated from heated, dried, steamed or
otherwise treated soybeans; materials obtained by heating, drying,
steaming or otherwise treating hypocotyls separated from
unprocessed soybeans. Soybean hypocotyls that have been subjected
to removal of fat and/or protein are also usable in the present
invention. The form of the soybean hypocotyls for use in the
present invention is also not limited, and may be powdered, ground
or crushed. From the viewpoint of efficient production of equol, it
is preferable to use powdered soybean hypocotyls.
[0046] Such soybean hypocotyls are fermented by adding a suitable
amount of water to the soybean hypocotyls to adjust the water
content, and inoculating equol-producing bacteria as mentioned
above.
[0047] The amount of water added to the soybean hypocotyls can be
suitably selected according to the type of equol-producing
bacteria, the type of fermentation tank, etc. It is usually
appropriate that, at the start of fermentation, water be present
with soybean hypocotyls in a proportion of 400 to 4000 parts by
weight, preferably 500 to 2000 parts by weight, and more preferably
600 to 1000 parts by weight, per 100 parts by weight of soybean
hypocotyls (on a dry weight basis).
[0048] In order to improve the fermentation efficiency, flavor and
taste of the fermented product, etc., nutritional ingredients can
be added, as necessary, to the soybean hypocotyls used as a
fermentation material. Examples of usable nutritional ingredients
include yeast extracts, polypeptones, meat extracts, and other
nitrogen sources; glucose, sucrose, and other carbon sources;
phosphate, carbonate, sulfate, and other mineral salts; vitamins;
amino acids; etc. In particular, when using a microorganism having
an ability to convert arginine to ornithine (hereinafter referred
to as "ornithine/equol-producing microorganism") as an
equol-producing microorganism, it is possible to obtain a fermented
material containing ornithine by adding arginine to soybean
hypocotyls and then performing fermentation. In such a case, the
amount of added arginine may be, for example, about 0.5 to about 3
parts by weight per 100 parts by weight of soybean hypocotyls (on a
dry weight basis). Equol-producing microorganisms having an ability
to convert arginine to ornithine can be selected from Lactococcus
garvieae strains, and specific examples include Lactococcus 20-92
(FERM BP-10036).
[0049] The pH of the fermentation material (starting material which
is subjected to fermentation) is not limited as long as the
equol-producing bacteria are viable. From the viewpoint of good
proliferation of equol-producing microorganisms, it is desirable to
adjust the pH of the fermentation material to about 6 to about 7,
and preferably about 6.3 to about 6.8.
[0050] Isoflavones (including daidzein compounds as mentioned
above) can be added to the fermentation material. Addition of
isoflavone(s) to the fermentation material makes it possible to
increase the equol content of the resulting fermented soybean
hypocotyl material, improving the utility of the fermented soybean
hypocotyl material.
[0051] The fermentation of soybean hypocotyls is performed under
environmental conditions that are suitable for the growth
characteristics of the equol-producing bacteria used. For example,
when using the equol-producing bacteria specifically listed above,
the fermentation (cultivation) is performed under anaerobic
conditions.
[0052] The fermentation temperature is not limited as long as it is
suitable for the growth of the equol-producing bacteria, and may
be, for example, 20 to 40.degree. C., preferably 35 to 40.degree.
C., and more preferably 36 to 38.degree. C.
[0053] The fermentation time can be suitably selected according to
the amount of equol produced, the residual amount of daidzein
compounds, the type of equol-producing microorganism, etc., and it
is usually 1 to 10 days, preferably 2 to 7 days, and more
preferably 3 to 5 days.
[0054] Equol is produced and accumulated in the fermented soybean
hypocotyl material produced by a fermentation treatment under the
conditions mentioned above, and thus the fermented soybean
hypocotyl material is capable of exhibiting the useful
physiological activity of equol. The equol content of such a
fermented soybean hypocotyl material varies depending on the
equol-producing bacteria used, fermentation conditions, etc.; and
equol is usually contained in an amount of 0.1 to 1 g, preferably
0.2 to 1 g, and more preferably 0.5 to 0.8 g, per 100 g, on a dry
weight basis, of fermented soybean hypocotyl material.
[0055] The fermented soybean hypocotyl material contains various
isoflavones other than equol, such as daidzin, malonyldaidzin,
acetylmalonyldaidzin, daidzein, dihydrodaidzein, and other daidzein
compounds (these components may be referred to as "daidzein
compounds"); genistin, malonylgenistin, acetylgenistin, genistein,
dihydrogenistein, and other genistein compounds (these components
may be referred to as "genistein compounds"); glycitin,
malonylglycitin, acetylglycitin, glycitein, dihydroglycitein, and
other glycitein compounds (these components may be referred to as
"glycitein compounds"); etc. Thus, the fermented material also
exhibits the useful physiological activities of such isoflavones.
The isoflavone (including equol) content of the fermented soybean
hypocotyl material may be, for example, on a dry weight basis,
about 0.5 to about 2 g, preferably about 0.5 to about 1.5 g, and
more preferably about 0.8 to about 1.5 g, per 100 g of the
fermented soybean hypocotyl material.
[0056] The proportions of isoflavones other than equol in the
fermented soybean hypocotyl material are different from those in
unfermented soybean hypocotyls. In particular, in the fermented
soybean hypocotyl material, the total content of genistein
compounds, whose activities as endocrine disrupters raise concerns,
is as low as 14 wt. % or less, and preferably 12 wt. % or less.
Therefore, the fermented soybean hypocotyl material is also more
advantageous than unfermented soybean hypocotyls from the viewpoint
of isoflavone proportions.
[0057] Specific examples of proportions of isoflavones in the
fermented soybean hypocotyl material include the following, in
which "mg" indicates the total content of each isoflavone per 1 g
of fermented soybean hypocotyl material on a dry weight basis.
[0058] Equol: 1 to 20 mg, and preferably 2 to 10 mg;
[0059] Daidzein compounds: 0.1 to 3 mg, and preferably 0.1 to 1.5
mg;
[0060] Genistein compounds: 0.05 to 2.5 mg, and preferably 0.05 to
2 mg;
[0061] Glycitein compounds: 0.1 to 4 mg, and preferably 2 to 3.5
mg.
[0062] The proportions of these isoflavones contained in the
fermented soybean hypocotyl material are, for example, as below, in
which "wt. %" indicates the percentage relative to the total amount
of isoflavones contained in the fermented soybean hypocotyl
material.
[0063] Equol: 30 to 75 wt. %, preferably 40 to 70 wt. %, and more
preferably 45 to 65 wt. %;
[0064] Daidzein compounds: 1 to 20 wt. %, preferably 2 to 15 wt. %,
and more preferably 4 to 8 wt. %;
[0065] Genistein compounds: 0.1 to 20 wt. %, preferably 1 to 15 wt.
%, and more preferably 1 to 10 wt. %;
[0066] Glycitein compounds: 10 to 50 wt. %, preferably 15 to 35 wt.
%, and more preferably 25 to 30 wt. %;
[0067] The fermented soybean hypocotyl material of the present
invention contains isoflavones in such proportions that cannot be
achieved by known methods. Therefore, the fermented soybean
hypocotyl material of the present invention may be referred to as
an isoflavone-containing material comprising the isoflavones in the
above proportions.
[0068] For production of a fermented soybean hypocotyl material
having an isoflavone proportions as above, Lactococcus 20-92 (FERM
BP-10036) can be used particularly advantageously.
[0069] Further, since the fermented soybean hypocotyl material also
contains saponins derived from soybean hypocotyls, it is also
possible to achieve the useful physiological activity based on such
saponins (e.g., antiviral activity). The saponin content of the
fermented soybean hypocotyl material is usually 1 to 8 g,
preferably 2 to 5 g, and more preferably 3 to 4 g, per 100 g, on a
dry weight basis, of the fermented soybean hypocotyl material.
[0070] Further, as mentioned above, ornithine is contained in a
fermented soybean hypocotyl material obtained by adding arginine to
soybean hypocotyls and performing fermentation using an
ornithine/equol-producing microorganism. Specifically, ornithine
may be contained in such a fermented soybean hypocotyl material in
an amount of, for example, about 0.5 to about 2.0 g, preferably
about 0.8 to about 1.5 g, and more preferably about 0.9 to about
1.2 g, per 100 g, on a dry weight basis, of the fermented soybean
hypocotyl material.
[0071] The fermented soybean hypocotyl material obtained by
fermentation under the conditions described above may be in its
post-fermentation state without additional treatment, or may be
dried as necessary to form a dry solid product, for use as an
ingredient for foods, pharmaceuticals, cosmetics, etc. In order to
improve its storage stability, the fermented soybean hypocotyl
material is preferably dried to solid form. The heated and dried
fermented soybean hypocotyl material may be powdered, as
necessary.
[0072] As previously described, because the fermented soybean
hypocotyl material of the invention contains a variety of effective
physiologically active substances such as equol, it expresses a
variety of physiological and pharmacological activities. For
example, the fermented soybean hypocotyl material of the invention
is useful for the prevention or alleviation of symptoms or diseases
such as, e.g., menopausal disorders, osteoporosis, prostatic
hypertrophy, metabolic syndrome, and for lowering of the blood
cholesterol level, skin whitening, acne treatment, treatment of
intestinal disorders, obesity, urinary disorders, etc. Among such
uses, the fermented soybean hypocotyl material of the invention is
especially useful for the prevention or alleviation of general
malaise-complaints and menopausal symptoms (for example,
osteoporosis, menopausal disorders, etc.) in middle-aged women.
When a fermented soybean hypocotyl material is produced by
fermenting an arginine-containing fermentation material using an
ornithine/equol-producing bacteria, ornithine is also formed and
stored therein. Such a fermented soybean hypocotyl material can
also exhibit active physiological effects derived from ornithine,
such as improved liver function, improved growth hormone secretion,
increased immunostimulation, increased amount of muscles, increased
basal metabolism, etc.
[0073] When the fermented soybean hypocotyl material of the
invention is used as a food ingredient, it may be prepared in the
form of, e.g., a drink, granules, fine grains, capsules, tablets, a
powder, dairy product, gum, gum drop, pudding, bar, or other solid
food. A food containing the fermented soybean hypocotyl material
exhibits not only the equol-derived effective physiological
activities, but also physiological activities derived from
isoflavones, saponins, and other like materials. Such foods,
therefore, provide excellent health-keeping effects, and are highly
useful. When a fermented soybean hypocotyl material produced by
fermenting an arginine-containing fermentation material using
ornithine/equol-producing bacteria is used in a food, such a food
also contains ornithine, and therefore has further enhanced
usefulness.
[0074] Food containing the fermented soybean hypocotyl material of
the invention are usable, not only as general foods, but also as
foods for specified health uses, dietary supplements, functional
foods, foods for invalids, etc. Foods containing the fermented
soybean hypocotyl material of the invention are particularly usable
as dietary supplements.
[0075] The proportion of the fermented soybean hypocotyl material
of the invention in a food can be suitably determined according to
the type of food, equol content, age and sex of the subject,
expected effects, and other factors. For example, the total amount
of the fermented soybean hypocotyl material per 100 g of a food may
be generally 0.1 to 90 g, preferably 0.1 to 10 g, and more
preferably 0.5 to 2 g on a dry weight basis.
[0076] The daily dosage of a food containing the fermented soybean
hypocotyl material depends upon the equol content of the fermented
soybean hypocotyl material, age and body weight of the subject,
daily number of doses, and other factors; but, for example, an
adult may take a daily dosage of 0.1 to 10 g of the fermented
soybean hypocotyl material.
[0077] When the fermented soybean hypocotyl material of the
invention is used as a pharmaceutical ingredient, the fermented
soybean hypocotyl material is prepared in the form of, e.g.,
tablets, pills, a powder, a liquid medicine, a suspension, an
emulsion, granules, capsules, a suppository, or the like. A
pharmaceutical preparation containing the fermented soybean
hypocotyl material of the invention is useful for the prevention or
alleviation of symptoms or diseases, e.g., menopausal disorders
(including menopausal complaints, osteoporosis, and
hyperlipidemia), osteoporosis, prostatic hypertrophy, metabolic
syndrome, and for the reduction of the blood cholesterol level,
treatment of intestinal disorders, obesity, urinary disorders, etc.
Such a pharmaceutical preparation is especially suitable for use in
the prevention or treatment of general malaise-complaints and
menopausal symptoms (e.g., osteoporosis, menopausal disorders,
etc.) in middle-aged women.
[0078] The dosage of a pharmaceutical preparation containing the
fermented soybean hypocotyl material of the invention depends upon
the equol content of the fermented soybean hypocotyl material, age
and body weight of the subject, symptoms, number of doses per day,
and other factors; but, for example, an adult may take a daily
dosage of 0.5 to 6 g of the fermented soybean hypocotyl material on
a dry weight basis.
[0079] When the fermented soybean hypocotyl material of the
invention is used as a cosmetic ingredient, the fermented soybean
hypocotyl material may be prepared in any desired form, such as,
e.g., paste-like, mousse-like, gel-like, liquid, emulsion,
suspension, cream, ointment, sheet-like, or like form. Such
cosmetic products can be used in a wide various usages: e.g., basic
skin care products such as emulsions, creams, lotions, oils, and
packs; cleansing products such as facial washes, cleansers, and
body cleansers; cleansing wipes; purifying agents; etc. Cosmetic
products containing the fermented soybean hypocotyl material of the
invention are used for skin whitening and clearing acne.
[0080] The proportion of the fermented soybean hypocotyl material
of the invention in a cosmetic product can be suitably determined
according to the type of the cosmetic product, equol content, and
the like. For example, the total amount of the fermented soybean
hypocotyl material per 100 g of a food may be 0.1 to 10 g, and
preferably 0.5 to 5 g on a dry weight basis.
EXAMPLES
[0081] The present invention is described in detail with reference
to Test Examples, Examples, etc. below, but is not limited to these
examples.
Examples 1 to 3
[0082] Powdered soybean hypocotyls, arginine, and water were mixed
in such a manner that the compositions of the mixture was as shown
in Table 1 to prepare soybean hypocotyl solutions. Into 5 ml
samples of the soybean hypocotyl solution was inoculated
Lactococcus 20-92 (FERM BP-10036; Lactococcus garvieae), and
subjected to static cultivation at 37.degree. C. for 96 hours under
anaerobic conditions. After cultivation, the resulting culture
(fermented liquids) were sterilized by heating at 100.degree. C.
for one minute, subsequently dried at 80.degree. C., and further
powdered using a homogenizer, thereby obtaining powdered fermented
soybean hypocotyl materials.
[0083] Table 1 shows equol concentrations in culture 96 hours after
cultivation. Table 1 also shows viable bacterial counts and pH of
culture media 96 hours after cultivation, yields of the powdered
fermented soybean hypocotyl materials, and equol concentrations in
the powdered fermented soybean hypocotyl materials. The results
established that fermentation of the powdered soybean hypocotyls
using an equol-producing bacterium can produce equol very
efficiently.
TABLE-US-00001 TABLE 1 Ex. 1 Ex. 2 Ex. 3 Composition Powdered
soybean 0.25 g 0.5 g 0.75 g of soybean hypocotyls (dried hypocotyl
wt.) solution Arginine 0.005 g 0.005 g 0.005 g Water Appropriate
Appropriate Appropriate quantity quantity quantity Total amount 5
ml 5 ml 5 ml pH 6.75 .+-. 0.03 6.54 .+-. 0.02 6.39 .+-. 0.03
Analytical Viable bacterial 7.9 .+-. 0.1 8.2 .+-. 0.1 8.3 .+-. 0.2
result of counts of fermented fermented liquid (log cfu/ml) liquid
pH of fermented 7.00 .+-. 0.03 6.88 .+-. 0.01 6.76 .+-. 0.02 liquid
Analytical Equol concentration 385.6 .+-. 101.5 344.6 .+-. 62.1
417.5 .+-. 68.0 result of in powdered soybean powered hypocotyl
material fermented (mg/100 g) soybean hypocotyls Note: Each example
was carried out using powdered soybean hypocotyls of three
different lots (N = 3). The analytical results in the table are
average .+-. SD values.
Example 4
[0084] Lactococcus 20-92 (FERM BP-10036; Lactococcus garvieae) was
inoculated into 5 ml of a soybean hypocotyl solution containing 10
wt. % of powered soybean hypocotyls and 0.1 wt. % of L-arginine,
and subjected to static cultivation at 37.degree. C. for 96 hours
under anaerobic conditions. After cultivation, the resulting
culture (fermented liquid) was sterilized by heating at 100.degree.
C. for one minute, then dried at 80.degree. C., and further
powdered using a homogenizer, thereby obtaining a powered fermented
soybean hypocotyl material.
[0085] The powdered soybean hypocotyls used as starting materials
(referred to as "pre-fermentation" in Tables 2 and 3) and the
obtained powdered fermented soybean hypocotyl material (referred to
as "post-fermentation" in Tables 2 and 3) were analyzed for
compositional components. Table 2 shows the analytical results for
soybean isoflavones, and Table 3 shows the analytical results for
nutritional components. These results also established that
fermented soybean hypocotyl materials containing high levels of
equol can be produced by fermenting soybean hypocotyls with
equol-producing bacterium. The results further revealed that the
contents of oligosaccharides such as raffinose, stachyose and the
like after the fermentation remain almost the same as before,
indicating that they are hardly influenced by fermentation.
However, it was found that arginine is converted to ornithine by
fermentation. Consequently, it was established that when
arginine-added soybean hypocotyls are fermented with Lactococcus
20-92, not only equol but also ornithine can be produced.
TABLE-US-00002 TABLE 2 Soybean isoflavones Per 100 g Component
Pre-fermentation Post-fermentation Equol N.D. 632.0 mg Daidzin
566.4 mg 29.7 mg Malonyldaidzin 124.9 mg N.D. Acetyldaidzin 364.8
mg 25.4 mg Daidzein 7.1 mg 24.4 mg Dihydrodaidzein N.D. 49.4 mg
Genistin 111.7 mg 3.2 mg Malonylgenistin 35.1 mg N.D.
Acetylgenistin 146.1 mg 3.7 mg Genistein 0.9 mg 22.5 mg
Dihydrogenistein N.D. 112.0 mg Glycitin 331.7 mg 53.6 mg
Malonylglycitin 65.0 mg N.D. Acetylglycitin 169.2 mg 34.8 mg
Glycitein 19.1 mg 292.3 mg Dihydroglycitein N.D. 8.2 mg Total
isoflavones 1942.0 mg 1291.2 mg N.D. refers to "Not Detected"
TABLE-US-00003 TABLE 3 Nutritional component Per 100 g Component
Pre-fermentation Post-fermentation Moisture 3.2 g 6.2 g Protein
38.1 g 38.3 g Fat 13.0 g 14.5 g Ash 4.3 g 4.0 g Saccharide 30.9 g
26.8 g Dietary fiber 10. g 10.2 g Energy 414 kcal 411 kcal Sucrose
7.95 g 7.42 g Raffinose 1.37 g 1.34 g Stachyose 9.04 g 8.38 g Trans
fatty acids N.D. N.D. Phospholipids(as stear-, 3.33 g 2.92 g ole-,
and lecitin) Free arginine 881 mg 12 mg Free ornithine N.D. 1.06 g
Syasapogenol A N.D. N.D. Soyasapogenol B N.D. N.D. Soybean saponine
3.6 g 3.8 g N.D. refers to "Not Detected"
Examples 5-11
[0086] Powdered fermented soybean hypocotyls (Examples 5-11) were
produced under the same conditions as in Example 3, except that
powdered soybean hypocotyls of seven different lots from that in
Example 3 were used. Proportions of isoflavones contained in the
thus-obtained fermented soybean hypocotyl materials were evaluated.
As is clear from the results shown in Table 4, the fermented
soybean hypocotyl materials of Examples 5-11 have a high equol
content and contain isoflavones in such proportions that cannot be
achieved by known methods.
TABLE-US-00004 TABLE 4 Isoflavone Proportions Daidzein Genistein
Glycitein Equol Compounds Compounds Compounds Example 5 6.51 mg
0.71 mg 0.53 mg 2.71 mg (62.2 wt. %) (6.8 wt. %) (5.1 wt. %) (25.9
wt. %) Example 6 6.25 mg 0.48 mg 0.35 mg 3.12 mg (61.3 wt. %) (4.7
wt. %) (3.4 wt. %) (30.6 wt. %) Example 7 5.38 mg 1.18 mg 1.45 mg
3.00 mg (48.9 wt. %) (10.7 wt. %) (13.2 wt. %) (27.2 wt. %) Example
8 6.43 mg 0.61 mg 0.48 mg 2.62 mg (63.4 wt. %) (6.0 wt. %) (4.7 wt.
%) (25.8 wt. %) Example 9 6.05 mg 0.51 mg 0.30 mg 2.57 mg (64.2 wt.
%) (5.4 wt. %) (3.2 wt. %) (27.3 wt. %) Example 10 6.11 mg 0.37 mg
0.10 mg 2.74 mg (65.6 wt. %) (4.0 wt. %) (1.1 wt. %) (29.4 wt. %)
Example 11 6.3 mg 0.49 mg 0.37 mg 3.19 mg (60.9 wt. %) (4.73 wt. %)
(3.6 wt. %) (30.8 wt. %)
In Table 4, the upper figures indicate the amount (mg) of each
isoflavone per 1 g of fermented soybean hypocotyl material, and the
lower figures indicate the percentage (wt %) of each isoflavone per
total weight (100% wt) of isoflavones contained in each fermented
soybean hypocotyl material.
Example 12
[0087] Powdered fermented soybean hypocotyls were produced under
the same conditions as in Example 3 above, except that powdered
soybean hypocotyls of a different lot from that in Example 3 above
were used. The obtained fermented soybean hypocotyl material
contained 6.5 mg of equol, 0.6 mg of daidzein compounds, 0.6 mg of
genistein compounds, and 3.2 mg of glycitein compounds, per g.
Aglycone accounted for 90 wt. % or more in the total isoflavone
content in the fermented soybean hypocotyl material.
[0088] Tablets having the following formula (weight 2.51 g and 10.9
mg equol content per tablet) were prepared using the thus obtained
fermented soybean hypocotyls.
TABLE-US-00005 Fermented soybean hypocotyl material 66.7 wt. %
Erythritol 33.2 wt. % Total 100.0 wt. %
Example 13
[0089] Granules having the following formula were prepared using
the fermented soybean hypocotyl material used in Example 5
above.
TABLE-US-00006 Fermented soybean hypocotyl material 66.7 wt. %
Erythritol 33.2 wt. % Total 100.0 wt. %
Example 14
[0090] Cosmetic product having the following formula were prepared
using the fermented soybean hypocotyl material of Example 1
above.
TABLE-US-00007 Fermented soybean hypocotyl material 10 g Paraffin
oil 60 ml Olive oil 40 ml Glycerol monostearic acid ester 50 ml
Lanolin 10 ml propylene glycol 30 ml Water balance Total 1000 g
Example 15
[0091] Cosmetic product having the following formula were prepared
using the fermented soybean hypocotyl material of Example 1
above.
TABLE-US-00008 Fermented soybean hypocotyl material 10 g Paraffin
oil 30 ml Olive oil 30 ml Glycerol monostearic acid ester 60 ml
Lanolin 20 ml propylene glycol 40 ml Water balance Total 1000 g
Test Example 1
[0092] It is known that soybean hypocotyls inherently contain Gym4,
Gm30K, Gm28K, 7S globulin mix (.beta.-conglycine), oleocine,
trypsin inhibitor and like allergens. The presence or absence of
allergens in the fermented soybean hypocotyl material prepared in
Example 1 was detected by the following method.
[0093] First, an adequate amount of the fermented soybean hypocotyl
material prepared in Example 1 was added to an extraction buffer
(Tris HCl pH 7.5, containing 1 M EDTA and an adequate amount of
protease inhibitor), followed by sufficient agitation to extract
water-soluble components from the fermented soybean hypocotyl
material. Subsequently, solid matter was removed therefrom by
filtration, giving an extract. Total protein in the thus-obtained
extract was assayed using a Bio-Rad Protein Assay. Major allergens
(Gym4, Gm30K, Gm28K, 7S globulin mix, oleocine, and trypsin
inhibitor) contained in the thus-obtained extract were then
detected by western blotting. For comparison, detections of total
protein and major allergens were conducted in the same manner as
described above using soybean cotyledon and soybean hypocotyl
powders instead of the fermented soybean hypocotyl material.
[0094] FIGS. 2 to 4 show the results. FIG. 2 shows the results of
total protein detection. FIG. 3 shows the results of Gym4, Gm30K,
and Gm28K detections. FIG. 4 shows the results of 7S globulin mix,
oleocine, and trypsin inhibitor detections.
[0095] From these results, it was confirmed that major allergens
inherently contained in soybeans or soybean hypocotyls are
eliminated in the fermented soybean hypocotyl material.
* * * * *