HK1139014B - Equol-containing extract, method for production thereof, method for extraction of equol, and equol-containing food - Google Patents

Abstract

Disclosed is an extract produced by extracting an equol-containing valuable component from an equol-containing fermentation product of a soybean embryonic axis. Also disclosed is a method for producing the extract. An equol-containing fermentation product of a soybean embryonic axis is extracted by using an aqueous ethanol solution as an extraction solvent to extract an equol-containing valuable component from the fermentation product with a high degree of efficiency. Alternatively, an equol-containing fermentation product of a soybean embryonic axis is successively subjected to the extraction with an aqueous ethanol solution and the extract with ethanol to extract equol and glycitein with a high degree of efficiency, thereby reducing the content of a saponin that causes an unpleasant taste.

Description

Equol-containing extract, method for producing same, method for extracting equol, and equol-containing food

Technical Field

The present invention relates to an equol-containing extract obtained by extracting an effective ingredient from an equol-containing fermented soybean hypocotyl, and a method for producing the same. The present invention also relates to a method for efficiently purifying an equol-containing material to obtain high-purity equol. The present invention further relates to an equol-containing food material and an equol-containing food.

Background

Isoflavones contained in soybeans (soybean isoflavones: daidzein, genistein, glycitein) have a structure similar to estradiol, and have an antiestrogenic action and an estrogen-like action associated with binding to an estrogen receptor (hereinafter referred to as ER). Epidemiological studies and intervention studies of soybean isoflavone have been conducted to show that they have a preventive effect on breast cancer, prostate cancer and other hormone-dependent cancers due to their antiestrogenic action and have an improving effect on menopausal disorders, postmenopausal osteoporosis and hyperlipidemia due to their estrogen-like action.

Recently, it has been pointed out that the active ingredient of these physiological effects of soy isoflavones may be a metabolite of soy isoflavones, i.e. equol. More specifically, equol has been reported to have a higher ability to bind to ER (particularly ER β) than soybean isoflavone and to have a remarkably high transitive ability to target organs such as breast and prostate tissues (non-patent documents 1 to 4). In addition, case-control studies report that significantly fewer patients produce equol in breast and prostate cancer patients. The effect of soy isoflavones in improving bone density and lipid metabolism was examined by dividing postmenopausal women into two groups: women who produce equol and women who do not produce equol. A significant improvement was observed in women producing equol.

Equol is produced by the metabolism of daidzein by enteric bacteria. The ability to produce equol varies among individuals, and the percentage of japanese people who produce equol is reported to be about 50%. That is, about 50% of japanese people cannot produce equol (non-equol-producing individuals). Such individuals would not benefit from any beneficial physiological effects based on the equol effect even when eating soy and processed soy foods. Thus, in order to obtain beneficial physiological effects based on the action of equol in non-equol producing individuals, it is believed that the ingestion of equol itself is also effective.

Non-patent document 1: morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, Ogawa S, Inoue S, Muramatsu M, Masamune Y.interaction of phytoestogens with oligonucleotide receptors α and β. Biol Pharm Bull 24 (4): 351-356, 2001

Non-patent document 2: maubach J, Bracke ME, Heyerick A, Depyperse HT, Serreyn RF, Mareel MM, Keukeleire DD. quantity of soy-derivative biotechnologies in human break tissue and biological fluids by high-performance Chromatography, J Chromatography B784: 137-144, 2003

Non-patent document 3: morton MS, Chan PSF, Cheng C, Blacklock N, Matos-Ferreira A, Abrances-Monteiro L, Correia R, Lloyd S, Griffiths K.Lignans and isoflavone in plasma and prestatic fluid in men: samplefrom Portugal, Hong Kong, and the United kingdom. state 32: 122-128, 1997

Non-patent document 4: tammy EH, Paul DM, Paul GF, Robert D, Stephen B, Kenneth J, Ray M, Lorraine GO, krithiina W, Holly MS, Karen jg, long-term specificity happy extract of soybean metabolism and metabolism in calcium medium J Nutr 135: 1400-1406, 2005.

Disclosure of Invention

Problems to be solved by the invention

The present inventors have now found that a fermented soybean hypocotyl obtained by fermenting a soybean hypocotyl with an equol-producing microorganism can be used as an equol-containing food material. The soybean hypocotyl ferment contains not only equol but also isoflavone, saponin and similar beneficial ingredients derived from soybean. The fermented soybean hypocotyls can obtain such effective physiological effects due to these beneficial ingredients, and thus can be used as functional materials. The present inventors have also found that a fermented soybean hypocotyl can be used as an allergen-reducing material because of the reduction of allergens attributable to the soybean hypocotyl therein. The present inventors found that the fermented soybean hypocotyl exhibits physiological effects due to the beneficial ingredients and is hypoallergenic, so that it can be used as a functional food material.

In contrast, the content of equol in the fermented soybean hypocotyl varies depending on the type of soybean hypocotyl used for preparing the fermented soybean hypocotyl, the type of equol-producing microorganism, and the like, but is generally about 1% by weight. If a material with an increased equol content could be provided, a variety of different types of equol-containing food products could be readily provided for the variety of food products. However, the fermented soybean hypocotyl is not a known product per se, and an effective method for efficiently extracting useful components from the fermented soybean hypocotyl has not yet been found.

The fermented soybean hypocotyl, which is the equol-containing substance obtained by the fermentation method as described above, is safer than that obtained by a chemical synthesis method and is suitable for industrial production. However, the equol-containing material obtained by the fermentation method described above contains metabolites other than equol and various components derived from the raw material remaining therein. Depending on the type of raw material used for the fermentation, the equol-containing substance obtained by the fermentation process may comprise a material that may become an allergen. Therefore, in order to use equol as an additive for foods or medicines, a technique of not only producing equol but also purifying an equol-containing substance to obtain high-purity equol is required. However, many techniques for purifying equol have not been reported, and there is a need to establish a technique which is industrially applicable and which can purify equol easily and efficiently.

The fermented soybean hypocotyl has a bitter taste due to soybean hypocotyl or equol. Therefore, when the fermented soybean hypocotyl is used as a food material, sufficient attention must be paid to the flavor of the resulting food. The soybean hypocotyl fermentation product is a novel food material which has not been reported previously, and it is not known which method is effective for improving its flavor. In particular, when the fermented soybean hypocotyl is used as a material for baked confectionery, it is prone to a problem of flavor deterioration after undergoing a baking step. Therefore, it is a very difficult technical object to exhibit good flavor.

The object of the present invention is to provide an extract derived from equol-containing fermented soybean hypocotyl containing useful equol components, and a method for producing the extract. The present invention provides a method for purifying an equol-containing material to efficiently obtain high-purity equol. It is another object of the present invention to provide a food material obtained by fermenting equol-containing soybean hypocotyl fermented product with an equol-producing microorganism, in which the flavor is improved. It is still another object of the present invention to provide a food product (in particular, a baked confectionery) comprising the equol-containing fermented soybean hypocotyl and exhibiting an excellent flavor. It is still another object of the present invention to provide various forms of foods containing the equol-containing fermented soybean hypocotyl or its extract.

Means for solving the problems of the present invention

The present inventors have conducted intensive studies to achieve the above object and found that an equol-containing component can be efficiently extracted from a fermented soybean hypocotyl by extracting the fermented soybean hypocotyl containing equol using an aqueous ethanol solution as an extractant. The present inventors have further found that by sequentially extracting an equol-containing fermented soybean hypocotyl using an aqueous ethanol solution and ethanol, the content of saponins causing astringency, bitterness, harshness and the like unpleasant taste can be reduced while efficiently extracting equol and glycitein.

The present inventors have also found that high-purity equol can be obtained by suspending an equol-containing fermented soybean hypocotyl in an aqueous solution having a pH of 2 to 6, collecting insoluble matter, and then extracting the resulting insoluble residue with an aqueous ethanol solution.

The present inventors have further found that high-purity equol can be efficiently and conveniently obtained by performing the following steps (1) to (5):

step (1): extracting equol-containing material with ethanol aqueous solution;

step (2): further extracting the obtained extract with ethanol;

And (3): extracting the obtained extract with hexane;

and (4): extracting the resulting insoluble residue with a mixture of hexane and ether; and

and (5): subjecting the obtained liquid extract to silica gel column chromatography to obtain equol-containing fraction.

The present inventors have further found that high purity equol can be efficiently and conveniently obtained by performing the following steps (i) to (iii):

(i) extracting equol-containing material with a specific organic solvent or an aqueous organic solvent solution, and concentrating the obtained liquid extract;

(ii) subjecting the obtained equol concentrate to silica gel column chromatography to obtain equol-containing fraction; and

(iii) the solvent was removed from the obtained equol-containing fraction and the obtained residue was recrystallized.

The present inventors have also found that, in a food material obtained by dispersing an equol-containing fermented soybean hypocotyl or an extract thereof in a cocoa mass, bitterness due to the equol-containing fermented soybean hypocotyl can be reduced and excellent flavor can be exhibited. The present inventors have also found that the food material can be used in various food products, including baked confectionery, without negatively affecting flavour.

The present inventors have succeeded in developing various foods to which equol-containing fermented soybean hypocotyl or an extract thereof is added.

The present invention has been accomplished based on these findings and has made some improvements thereto.

The present invention provides a method for obtaining an equol-containing extract.

1-1. A method for producing an equol-containing extract, comprising the step of I-1:

extracting the equol-containing fermented soybean hypocotyl with ethanol aqueous solution as extractant, and collecting the obtained liquid extract.

Item 1-2. the method according to item 1-1, further comprising step I-2:

removing the extractant from the liquid extract obtained in step I-1;

extracting with ethanol as extractant; and collecting the liquid extract.

Item 1 to 3 the process according to item 1 to 1, wherein the ethanol concentration of the aqueous ethanol solution used in step I-1 is 20 to 98 vol%.

The method according to any one of items 1-1 to 1-3, wherein the equol-containing fermented soybean hypocotyl is obtained by fermenting a soybean hypocotyl with an equol-producing microorganism.

1-5. A method for producing an equol-containing extract from an equol-containing soybean hypocotyl fermented product, comprising the steps of II-1 and II-2:

Step II-1: suspending the equol-containing fermented soybean hypocotyl in water, adding acid to adjust pH to 2-6, and collecting insoluble residue; and

step II-2: extracting the insoluble residue obtained in step II-1 with ethanol aqueous solution as extractant, and collecting the obtained liquid extract.

1-6 the process of item 1-5, wherein the acid used in step II-1 is acetic acid.

The present invention provides the following equol-containing extracts.

The equol-containing extract obtained by extracting an equol-containing fermented soybean hypocotyl using an aqueous ethanol solution as an extractant.

Item 2-2. the equol-containing extract obtained by sequentially extracting the equol-containing fermented soybean hypocotyl using an aqueous ethanol solution and ethanol.

Item 2-3. the equol-containing extract obtained by the method of any one of items 1-1 to 1-6.

The present invention provides the following method for purifying equol.

3-1. A method for purifying an equol-containing material to obtain equol, comprising the following steps 1-1 to 1-5:

step 1-1: extracting equol-containing material with ethanol aqueous solution as extractant, and collecting the obtained liquid extract;

Step 1-2: removing the solvent from the liquid extract obtained in step 1-1, extracting the residue with ethanol as an extractant, and collecting the liquid extract;

step 1-3: removing the solvent from the liquid extract obtained in step 1-2, suspending the resulting residue in hexane, and then collecting the insoluble residue;

step 1-4: extracting the insoluble residue obtained in step 1-3 with a mixture of hexane and ether as an extractant, and collecting the liquid extract; and

step 1-5: subjecting the liquid extract obtained in steps 1-4 to silica gel column chromatography to obtain equol-containing fraction.

The method according to item 3-2, wherein the equol-containing material is a fermented product comprising equol.

3-3 the method according to item 3-2, wherein the equol-containing fermented product is an equol-containing fermented soybean hypocotyl product obtained by fermenting a soybean hypocotyl with an equol-producing microorganism.

Item 3-4 the method according to any one of items 3-1 to 3-3, wherein the extractant used in step 1-4 is a mixture of hexane and diethyl ether.

Item 3 to 5 the process according to any one of items 3 to 1 to 3 to 4, wherein the extractant used in the step 1 to 4 is a mixture of hexane and ether in a volume ratio of 10: 90 to 20: 80.

Item 3-6 the method according to any one of items 3-1 to 3-5, wherein the aqueous ethanol solution used in step 1-1 has an ethanol concentration of 20 to 98 vol%.

3-7. A method for purifying an equol-containing material, comprising the following steps 2-1 to 2-3:

step 2-1: extracting the equol-containing material with an organic solvent or an aqueous organic solvent solution of at least one member selected from the group consisting of ethyl acetate, alcohol, acetone, dioxane, acetonitrile, diethyl ether and toluene as an extractant, and collecting the resulting liquid extract;

step 2-2: performing silica gel column chromatography treatment on the liquid extract obtained in the step 2-1 to obtain a fraction containing equol; and

step 2-3: removing the solvent from the equol-containing fraction obtained in the step 2-2, recrystallizing the obtained residue, and collecting the precipitated crystals.

3-8 the method according to the item 3-7, wherein the step 2-3 comprises performing recrystallization using an aqueous alcohol solution after recrystallization using a mixed solution of ethyl acetate and hexane.

The present invention provides the following food materials and food products using such food materials.

4-1. A food material obtained by dispersing a fermented product of soybean hypocotyl containing equol or an extract thereof in cocoa mass.

The food material according to item 4-1, which comprises 10 to 2000 parts by weight of the cocoa mass per 100 parts by weight of the equol-containing fermented soybean hypocotyl or the extract thereof.

The food material according to item 4-1 or 4-2, wherein the equol-containing fermented soybean hypocotyl is obtained by fermenting soybean hypocotyls with an equol-producing microorganism.

The food material according to item 4-3, wherein the equol-producing microorganism is a microorganism that can metabolize at least one daidzein compound selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein to produce equol.

Item 4-5 the food material according to any one of items 4-1 to 4-4, which is in granular form, sliced form or disk form.

An equol-containing food comprising the food material of any one of items 4-1 to 4-5.

4-7 the equol-containing food according to any one of items 4 to 6, which is a baked dessert.

The present invention provides the following food products:

5-1A food product comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

A beverage comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

A dietary supplement comprising equol-containing fermented soybean hypocotyl or extract thereof.

A cream food comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

5-5A dessert comprising equol-containing fermented soybean hypocotyl or an extract thereof.

A snack comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

A seasoning comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

5-8A retort-packed food (retort-packed food) comprising equol-containing fermented soybean hypocotyl or an extract thereof.

5-9A processed meat product comprising equol-containing fermented soybean hypocotyl or an extract thereof.

5-10A fish paste (fish paste) comprising equol-containing fermented soybean hypocotyl or an extract thereof.

5-11A processed egg product comprising equol-containing fermented soybean hypocotyl or an extract thereof.

A canned food comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

A bread food comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

5-14A frozen dessert comprising equol-containing fermented soybean hypocotyl or extract thereof.

A processed soybean food comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

A cooked rice product comprising equol-containing fermented soybean hypocotyl or an extract thereof.

A soup comprising a fermented product of soybean hypocotyl containing equol or an extract thereof.

The food according to any one of claims 5-1 to 5-17, wherein the equol-containing fermented soybean hypocotyl is obtained by fermenting soybean hypocotyls with an equol-producing microorganism.

Item 5-19 the food product according to item 5-18, wherein the microorganism has an ability to metabolize at least one daidzein compound selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein to produce equol.

Effects of the invention

The method for preparing the equol-containing extract of the present invention can prepare the equol-containing extract useful as a functional food material by efficiently extracting useful components containing equol from the equol-containing soybean hypocotyl fermented product. By sequentially extracting the equol-containing fermented soybean hypocotyl using an aqueous ethanol solution and ethanol, an equol-containing extract containing high concentrations of equol and glycitein, which has a reduced amount of saponin causing an unpleasant taste, can be obtained. Thus, the equol-containing extract can be added to food products without negatively affecting the taste.

The purification method of the present invention enables to obtain equol of high purity from equol-containing material conveniently and efficiently. In particular, the purification method of the present invention enables to obtain high-purity equol even when the equol-containing substance contains isoflavones having a structure similar to that of equol. Therefore, the purification method of the present invention can be suitably used for obtaining equol from an equol-containing fermented product containing a large portion of isoflavones.

Further, the food material obtained by dispersing the equol-containing soybean hypocotyl fermented product or extract thereof in the cocoa mass has reduced bitterness and exhibits excellent flavor, although it contains the equol-containing soybean hypocotyl fermented product or extract thereof. The food material of the present invention can be used in various foods without adversely affecting the flavor thereof. When conventional food materials are added to the baked snack in granular or sliced form, these ingredients tend to diffuse throughout the baked snack during the baking step, and thus the desired flavor cannot be obtained. In the food material of the present invention, the equol-containing fermented soybean hypocotyl is masked with cocoa mass. Therefore, even when the food material of the present invention is added to the baked snack in a granular form or a sliced form, the diffusion of the effective ingredient within the food material can be suppressed, and thus a desired flavor can be obtained.

The beneficial physiological effects of the food product of the present invention can be obtained based on the beneficial physiological activities of the equol-containing fermented soybean hypocotyl or extract thereof.

The equol-containing fermented soybean hypocotyl or its extract has reduced allergen derived from soybean hypocotyl, and therefore the food material and various foods of the present invention can be safely consumed by or administered to a person suffering from soybean allergy.

Detailed Description

The present invention will be described in detail below.

1. Preparation method of extract containing equol

The present invention provides a method for producing an equol-containing extract using an equol-containing fermented soybean hypocotyl as a starting material. The preparation method of the equol-containing extract of the present invention is divided into the following preparation methods I and II. The equol-containing soybean hypocotyl fermentation product used as a raw material for the method of the present invention is explained in detail below and preparation methods I and II are described in detail.

Equol-containing fermented soybean hypocotyl

In the method for producing an equol-containing extract according to the invention, an equol-containing fermented soybean hypocotyl is used as a starting material. The equol-containing fermented soybean hypocotyl is described below.

The equol-containing fermented soybean hypocotyl is a fermented soybean hypocotyl produced by fermenting soybean hypocotyls using an equol-producing microorganism.

The equol-producing microorganism used for preparing the equol-containing soybean hypocotyl fermented product is selected from microorganisms having an ability to produce equol (metabolic activity) by metabolizing at least one daidzein compound selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein. Here, examples of the daidzein glycoside include daidzin (daidzin), malonyl daidzin, and acetyl daidzin.

The equol-producing microorganism is not limited, and may be any microorganism having the aforementioned ability. For example, any publicly known microorganism or any microorganism obtained by a general screening method may be used. Such equol-producing microorganisms are known to be present in microorganisms belonging to the genus Lactococcus (Lactococcus), such as Lactococcus garvieae (Lactococcus garvieae), microorganisms belonging to the genus Streptococcus (Streptococcus), such as Streptococcus intermedius (Streptococcus intermedius) or Streptococcus constellations (Streptococcus constellatus), or microorganisms belonging to the genus Bacteroides (Bacteroides), such as Bacteroides ovatus (Bacteroides ovatus). Among various different kinds of equol-producing microorganisms, the present invention preferably selects lactic acid bacteria belonging to the genus lactococcus or streptococcus, more preferably lactic acid bacteria belonging to the genus lactococcus, and particularly lactic acid bacteria belonging to the genus lactococcus garvieae. Equol-producing microorganisms can be isolated from, for example, human feces based on the presence index of equol-producing characteristics. The inventors of the present invention deposited some identified bacteria isolated from human feces, 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), which can be provided as equol-producing microorganisms. Of these, lactococcus 20 to 92 is particularly preferred in the present invention.

The equol-containing fermented soybean hypocotyl is prepared using the soybean hypocotyl as a fermentation material. The soybean hypocotyl refers to a portion corresponding to the germ or radicle at the time of soybean germination, and is known to contain a large amount of daidzein compounds such as daidzein glycoside or daidzein. The soybean hypocotyls used in the present invention do not limit the producing area of soybeans or are processed as long as the daidzein compound in the interior thereof is not significantly lost. For example, the equol-containing soybean hypocotyl fermentation product may be an untreated hypocotyl, or may be an hypocotyl isolated from heated, dried or cooked soybeans, or an hypocotyl obtained by heating, drying or cooking an hypocotyl isolated from untreated soybeans. In addition, the soybean hypocotyls may be treated by degreasing or deproteinization. The form of the soybean hypocotyl is not particularly limited, and may be in the form of powder, chunks, or pulverized or broken particles. The powdery soybean hypocotyl is particularly preferable because it enables efficient production of equol. The fermentation of soybean hypocotyls is accomplished by first adjusting the water content of the soybean hypocotyls by adding an appropriate amount of water and then inoculating the hypocotyls with equol-producing microorganisms.

The amount of water added to the soybean hypocotyl is adjusted according to the type of equol-producing microorganism or the type of fermenter. The ratio of water to the soybean hypocotyl at the start of fermentation is 400-4000 parts by weight, preferably 500-2000 parts by weight, more preferably 600-1000 parts by weight, per 100 parts by weight (dry weight) of the soybean hypocotyl.

In addition, if necessary, certain additives may be added to the soybean hypocotyl as a raw material in fermentation to increase the fermentation efficiency or improve the product flavor. Examples of the additives include nitrogen sources (such as yeast extract, polypeptone, or meat extract), carbon sources (such as glucose or sucrose), inorganic salts (such as phosphate, carbonate, or bisulfate), vitamins, and nutrients (such as amino acids). In particular, when an equol-producing microorganism which converts arginine to ornithine (hereinafter referred to as "ornithine/equol-producing microorganism") is used, arginine is added to the soybean hypocotyl before fermentation so that the fermented substance contains ornithine. In this case, the amount of arginine is, for example, about 0.5 to 3 parts by weight based on 100 parts by weight (dry weight) of the soybean hypocotyl. The ornithine/equol-producing microorganism can be obtained by publicly known screening methods using the equol-producing ability and the index of arginine-ornithine converting ability. The ornithine/equol-producing microorganism may be selected from the group of lactococcus garvieae, which is represented by lactococcus 20-92 (FERMBP-10036).

In addition, the pH of the fermented material (soybean hypocotyl-containing substance) is not particularly limited in the range in which the equol-producing microorganism grows, but to ensure good proliferation of the equol-producing microorganism, the pH of the fermented material is preferably within about 6 to 7, more preferably about 6.3 to 6.8.

In addition, isoflavone containing the above daidzein compound can be added to fermented material (containing soybean hypocotyl). Thus, inclusion of isoflavones in the fermentation material increases the equol content of the fermented soybean hypocotyl, thereby improving the usability of the fermented soybean hypocotyl.

The fermentation of soybean hypocotyls is carried out under appropriate conditions according to the growth characteristics of the equol-producing microorganism. For example, when the equol-producing microorganisms listed above are used, the fermentation of soybean hypocotyls is carried out under anaerobic conditions.

The fermentation temperature is not limited within a suitable range for growth of the equol-producing microorganism. The temperature is generally 20 to 40 ℃, preferably 35 to 40 ℃, more preferably 36 to 38 ℃.

The fermentation time is determined according to the desired amount of equol produced, the residual amount of daidzein compound, the type of equol-producing microorganism, and the like. The fermentation time is generally 1 to 10 days, preferably 2 to 7 days, more preferably 3 to 5 days.

In the fermented soybean hypocotyl obtained by fermentation under the aforementioned conditions, the produced equol is accumulated, and thus a beneficial physiological activity of equol can be exhibited. The equol content in the fermented soybean hypocotyl varies depending on the type of equol-producing microorganism or the fermentation conditions. The equol content in the fermented soybean hypocotyl is generally 1 to 20mg, preferably 2 to 12mg, more preferably 5 to 8mg per gram (dry weight) of the fermented soybean hypocotyl.

In addition to equol, the equol-containing fermented soybean hypocotyl contains various isoflavones, such as daidzein compounds including daidzin, malonyl daidzin, acetyl daidzin, daidzein, or dihydrodaidzein (hereinafter, these compounds are referred to as "daidzein compounds"), genistein compounds including malonyl genistin, acetyl genistin, genistein, and dihydrogenistein (hereinafter, these compounds are referred to as "genistein compounds"), or glycitein compounds including glycitin, malonyl glycitin, acetyl glycitin, glycitein, and dihydroglycitein (hereinafter, these compounds are referred to as "glycitein compounds"). Isoflavones also exhibit beneficial physiological activity. The content of each isoflavone (including equol) in the fermented soybean hypocotyl is generally 5 to 20mg, preferably 5 to 15mg, more preferably 8 to 15mg per gram (dry weight) of the fermented soybean hypocotyl containing equol.

The equol-containing fermented soybean hypocotyl is also different from a general soybean hypocotyl in the composition of isoflavones other than equol. In particular, the total content of genistein compounds in the equol-containing fermented soybean hypocotyl is preferably not more than 15% by weight, more preferably not more than 12% by weight. Due to this low content of genistein compounds, the equol-containing fermented soybean hypocotyl is more advantageous in the composition of isoflavones than the unfermented soybean hypocotyl. It should be noted that genistein is a kind of isoflavone and is a chemical substance suspected of destroying endocrine, and thus it is required to reduce the concentration of genistein compounds in food (Young HJ, Jodi F, Kimberly FA, Daniel RD, William GH. effects of both dietary daidzein and Itsometabolite, equiol, at physical concentrations on the growth of the food-dependent human breath sampler (MCF-7) moved expressed induced fermented product microorganism 127 (4): 856-863, 2006).

The following is an example of the isoflavone composition of equol-containing fermented soybean hypocotyl (the unit "mg" represents the total amount of each isoflavone per gram (dry weight) of fermented soybean hypocotyl).

Equol: 1-20mg, preferably 2-12mg

Daidzein compound: 0.1-30mg, preferably 0.1-1.5mg

Genistein compounds: 0.05-2.5mg, preferably 0.05-2mg

Glycitein compound: 0.1-4mg, preferably 2-3.5 mg.

The following is an example of the composition ratio of isoflavones in the equol-containing soybean hypocotyl fermented product (the unit "wt%" represents the ratio relative to the total amount of isoflavones in the equol-containing soybean hypocotyl fermented product).

Equol: 30 to 75% by weight, preferably 40 to 70% by weight, more preferably 45 to 70% by weight

Daidzein compound: 1 to 20% by weight, preferably 2 to 15% by weight, more preferably 4 to 12% by weight

Genistein compounds: 0.1 to 20% by weight, preferably 1 to 15% by weight, more preferably 1 to 10% by weight

Glycitein compound: 10-50 wt.%, preferably 15-35 wt.%, more preferably 25-35 wt.%.

Since this equol-containing soybean hypocotyl fermented product has a specific isoflavone composition not found in any prior art, it can be defined as an isoflavone-containing substance determined by the aforementioned isoflavone composition.

Lactococcus 20-92(FERM BP-10036) is particularly preferable as an equol-producing microorganism in the preparation of an equol-containing soybean hypocotyl fermented product having the aforementioned isoflavone composition.

The equol-containing soybean hypocotyl fermented product also contains saponin derived from soybean hypocotyl. The equol-containing soybean hypocotyl fermented product generally contains the saponin in an amount of 10 to 80mg, preferably 20 to 50mg, more preferably 30 to 40mg per gram (dry weight) of the equol-containing soybean hypocotyl fermented product.

Further, as described above, ornithine is contained in a fermented soybean hypocotyl product prepared by adding arginine to soybean hypocotyl and then fermenting with an ornithine/equol-producing microorganism. The ornithine content of the equol-containing soybean hypocotyl fermented product is 5 to 20mg, preferably 8 to 15mg, more preferably 9 to 12mg per gram (dry weight) of the equol-containing soybean hypocotyl fermented product.

The equol-containing fermented soybean hypocotyl obtained by fermentation under the aforementioned conditions may be used as it is as a material for the production method of the present invention after fermentation, or may be dried to a dry solid after fermentation before being used as a material for the production method of the present invention.

In addition, the equol-containing fermented soybean hypocotyl obtained by fermentation under the aforementioned conditions may be processed, for example, defatted, before the following step I-1. Defatting can be performed by a method using hexane as an extractant.

1-1 preparation method I

In preparation method I, the equol-containing extract is prepared by performing step I-1 described below.

Step I-1

The preparation method I for preparing the equol-containing extract comprises the step I-1 of extracting the equol-containing fermented soybean hypocotyl using an aqueous ethanol solution as an extractant and collecting the liquid extract.

The ethanol concentration of the aqueous ethanol solution (mixed solution of ethanol and water) used in step I-1 is generally 20 to 98% by volume, preferably 45 to 95% by volume, and more preferably 65 to 85% by volume. In accordance with this concentration range, it becomes possible to extract useful components (including equol) from the equol-containing fermented soybean hypocotyl.

The extraction in step I-1 uses, for example, an aqueous ethanol solution in an amount of 1 to 1000 parts by weight, preferably 2 to 500 parts by weight, more preferably 5 to 20 parts by weight, per part by weight (dry weight) of the equol-containing soybean hypocotyl fermented product.

The extraction conditions of step I-1 are not particularly limited, and a warm or cold extraction method, a diafiltration method, or the like may be used. More specifically, an aqueous ethanol solution is added to the equol-containing fermented soybean hypocotyl, and the mixture is allowed to stand or stir at room temperature for at least one minute, preferably 5 minutes to 48 hours at room temperature.

After this extraction, the liquid extract is subjected to separation to obtain an equol-containing extract. The collection of the liquid extract is carried out by a publicly known method such as filtration or centrifugation.

The liquid extract thus separated in step I-1 contains useful components including equol and glycitein, and the amount of oil is lower than that in the equol-containing fermented soybean hypocotyl. The liquid extract thus obtained in step I-1 may be added directly to a food or a pharmaceutical product as an equol-containing liquid extract, or may be solidified by removing the extractant before adding to the food or pharmaceutical product. In addition, the liquid extract thus obtained in step I-1 may be subjected to equol purification (in the form of a liquid or solid equol-containing extract).

The liquid extract isolated in step I-1 comprises equol, glycitein and saponins (which may cause undesirable astringency, bitterness or harshness). It is therefore preferable to subject the liquid extract separated in step I-1 to the following step I-2 to remove saponins and increase the concentration of useful components including equol.

Step I-2

Step I-2 comprises removing the extractant from the liquid extract obtained in step I-1, performing another extraction using ethanol as the extractant, and collecting the extract.

In step I-2, the removal of the extractant from the extract obtained in step I-1 can be carried out by publicly known methods such as drying.

The solid obtained by removing the extractant from the extract obtained in step I-1 (this solid may also be referred to as "solid material" hereinafter) is extracted using ethanol as the extractant. The ethanol used as the extractant in step I-2 may be anhydrous ethanol, but the anhydrous ethanol may inevitably contain moisture.

The extraction in step I-2 uses, for example, 1 to 1000 parts by weight, preferably 5 to 500 parts by weight, and more preferably 5 to 20 parts by weight of ethanol per part by weight (dry weight) of the solid material.

The conditions of the extraction in step I-2 are not particularly limited, and may be carried out by a warm or cold extraction method, a diafiltration method, or the like. More specifically, ethanol is added to the solid material and the mixture is allowed to stand or stir at room temperature for at least one minute, preferably 5 minutes to 48 hours at room temperature. The extraction of step I-2 may be combined with sonication to improve equol recovery.

After this extraction, the extract is subjected to separation to obtain an equol-containing extract. The collection of the extract is carried out by a publicly known method such as filtration or centrifugation.

The extract thus isolated in step I-2 contains a greater amount of useful components (including equol and glycitein) and a lesser amount of saponins. The effectiveness of this extract is therefore higher than the extract obtained in step I-1. The extract thus obtained in step I-2 may be added directly to a food or a pharmaceutical product as a liquid equol-containing extract, or may be solidified by removing the extractant before adding to the food or pharmaceutical product. The extract thus obtained in step I-2 can be subjected to equol purification (in the form of a liquid or solid equol-containing extract). Note that, in order to improve the storage stability of the equol-containing extract, it is more preferable to solidify the liquid extract by removing the extractant.

By preparing the formulaComposition and use of equol-containing extract obtained by Process I

The equol content of the liquid extract obtained in step I-1 or step I-2 is 0.2 to 40% by weight, preferably 0.4 to 24% by weight, and more preferably 1 to 16% by weight, based on the total amount of the dry solid product (100% by weight). The glycitein content of the liquid extract obtained in step I-1 or step I-2 is 0.01-8 wt%, preferably 0.08-5 wt%, and more preferably 0.2-3 wt% based on the total amount of the dry solid product.

The equol-containing extract obtained in step I-1 or step I-2 contains equol at a high concentration and exhibits a beneficial activity based on equol. More specifically, the equol-containing extract obtained in step I-1 or step I-2 has the effect of preventing or treating various diseases or symptoms, including climacteric disorder, osteoporosis, prostatic hypertrophy, and metabolic syndrome; or for blood cholesterol level reduction, skin whitening, pimples, intestinal control (intestinalcontrol), obesity and polyuria. Since the beneficial activity is exhibited as such, the equol-containing extract obtained in step I-1 or step I-2 can be added to a food or a pharmaceutical product.

When used as a food material, the equol-containing extract obtained in step I-1 or step I-2 is usually added to beverages, granules, fine granules, capsules, sheets, powders, dairy products, gums, puddings, bars (bars), and other various dry foods. The food containing the equol-containing extract can be used as a general food, and also as a specific health food, a nutritional supplement, a functional food, a weak food, and the like. In particular, any food product comprising the fermented soybean hypocotyl of the present invention can be used as a nutritional supplement. Note that details of the equol-containing extract-containing food material obtained in step I-1 or step I-2 are described later.

When the equol-containing extract obtained in step I-1 or step I-2 is added to a food material, the proportion of the equol-containing extract in the food product varies depending on the type of the food, the equol content, the age and sex of the consumer, the desired effect, and the like. Generally, the total amount (dry weight) of the equol-containing extract is 0.1-90g per 100g of the food.

When used in medical products, the equol-containing extract obtained in step I-1 or step I-2 is often incorporated into tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, or suppositories. Such medical products comprising the equol-containing extract have the effect of preventing or treating various diseases or symptoms, including climacteric disorder (involuntary climacteric complaint), osteoporosis, hyperlipidemia, osteoporosis, prostatic hypertrophy, and metabolic syndrome; or for blood cholesterol level reduction, skin whitening, pimples, bowel control, obesity and diuresis. In particular, medical products comprising an equol-containing extract are suitable for preventing or treating an indeterminate disease (indefinite complex) or a postmenopausal syndrome (e.g. osteoporosis, climacteric complaints) in middle-aged women.

The dosage of the medical product comprising the equol-containing extract obtained in step I-1 or step I-2 cannot be uniformly defined, because it varies depending on the content of equol in the equol-containing extract, the age, body weight, symptoms, or the number of administrations of the subject. The daily adult dose generally corresponds to an amount (dry weight) of the equol-containing extract of 1-2000mg, preferably 5-1000mg, and more preferably 40-50 mg. It is particularly preferred that the dosage of the equol-containing extract is determined such that 2-30mg of equol is administered daily.

1-2 preparation method II

Preparation method II an equol-containing extract was prepared by performing the following steps II-1 and II-2. Preparation process II is described in detail below.

Step II-1

Preparation method II the equol-containing fermented soybean hypocotyl is first suspended in water, an acid is added to the suspension to adjust the pH to a range of 2-6, and the insoluble residue is separated (step II-1). In other words, in step II-1, the equol-containing fermented soybean hypocotyl is suspended in an acid-containing aqueous solution (pH 2-6), followed by collecting the insoluble residue.

In step II-1, the equol-containing fermented soybean hypocotyl is suspended in water, and an acid is added to the suspension to adjust the pH to a range of 2 to 6.

The acid used in step II-1 may be an organic acid or an inorganic acid. The organic acid is not limited as long as it can be adjusted to the aforementioned pH range. Examples of the organic acids include citric acid, L-tartaric acid, fumaric acid, L-ascorbic acid, gluconic acid, acetic acid, lactic acid, DL-malic acid, oxalic acid, formic acid, propionic acid, butyric acid and methanesulfonic acid (methanesulfole acid). Similarly, there is no limitation on the inorganic acid as long as it can be adjusted to the aforementioned pH range. Examples of the inorganic acid include hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid. Among them, acetic acid, hydrochloric acid and sulfuric acid are preferable.

In step II-1, the amount of water in which the equol-containing soybean hypocotyl fermented product is suspended is, for example, 3 to 30 parts by weight, more preferably 5 to 20 parts by weight, per part by weight (dry weight) of the equol-containing soybean hypocotyl fermented product.

In step II-1, the equol-containing fermented soybean hypocotyl is mixed in an aqueous solution of pH 2 to 6, and the solution is stirred or allowed to stand at room temperature for at least 30 minutes, more preferably 2 to 24 hours.

This treatment makes equol a part of the insoluble residue, and equol can be obtained by collecting the insoluble residue using publicly known methods such as filtration or centrifugation.

The insoluble residue thus obtained in step II-1 is then subjected to the following step II-2.

Step II-2

The insoluble residue thus obtained in step II-1 was subjected to another extraction using an aqueous ethanol solution as an extractant. The resulting liquid extract is subjected to separation (step II-2).

The insoluble residue obtained in step II-1 may be subjected to step II-2, whether it is a dry product or a wet product. However, when the insoluble residue is a wet product, it is preferable to adjust the water content of the aqueous ethanol solution (described later) in consideration of the measured water content in the insoluble matter.

The ethanol concentration of the ethanol aqueous solution (mixed solution of ethanol and water) used in step II-1 is, for example, 50 to 95% by volume, more preferably 70 to 90% by volume. In this concentration range, the extraction of equol from the insoluble residue in step II-2 can be performed efficiently.

The extraction in step II-2 uses an aqueous ethanol solution in an amount of, for example, 2 to 30 parts by weight, more preferably 4 to 10 parts by weight, per part by weight (dry weight) of the insoluble residue obtained in step II-1.

The conditions of the extraction in step II-2 are not particularly limited, and a warm or cold extraction method, a percolation method, or the like may be used. More specifically, an aqueous ethanol solution is added to the insoluble residue obtained in step II-1, and the mixture is allowed to stand or stir at room temperature for at least 30 minutes, preferably 1 to 50 hours at room temperature.

After this extraction, the liquid extract is subjected to separation to obtain an equol-containing fraction. The collection of the extract is carried out by a publicly known method such as filtration or centrifugation.

Since the liquid extract thus separated in step II-2 contains equol in a high concentration and useful components such as glycitein compounds, which is contained in concentrated equol, the extract is more useful than equol-containing fermented soybean hypocotyl. The liquid extract obtained in step II-2 may be added directly to a food or a pharmaceutical as a liquid equol-containing extract, or may be processed into a powder by removing the extractant before adding to the food or pharmaceutical. The liquid extract thus obtained in step II-2 may be subjected to equol purification (in the form of a liquid or solid equol-containing extract). Note that, in order to improve the storage stability of the equol-containing extract, it is more preferable to solidify the liquid extract by removing the extractant.

The removal of the extractant from the liquid extract obtained in step II-2 may be carried out under the same conditions as in step I-2 of preparation Process I; however, the removal step is preferably carried out by spray drying.

Composition and use of equol-containing extract obtained by preparation method II

By drying the liquid extract obtained in the process II (the liquid extract obtained in the aforementioned step II-2), it is possible to isolate equol with a recovery rate of 90% or more. Thus obtained is an equol-containing powder containing ten times more equol than in the equol-containing soybean hypocotyl fermented product. By this high equol content, the equol-containing powder more effectively exhibits the beneficial activity derived from equol. The purpose or method of using the equol-containing powder obtained in method II is the same as that of the equol-containing powder obtained in method I.

2. Equol purification method

The invention also provides a method for purifying an equol-containing material to obtain equol. The equol purification method of the present invention is divided into the following purification methods 1 and 2. The equol-containing fermented soybean hypocotyl to be purified by the purification method of the present invention is described in detail below and the purification methods 1 and 2 are described in detail.

Equol-containing substance

In the purification method of the present invention, the equol-containing material to be subjected to the purification treatment is not limited as long as it contains equol. For example, the equol-containing material used in the method may be a reaction product containing equol produced by chemical synthesis or an equol-containing fermented material produced by fermentation. The equol-containing fermented material produced by fermentation contains many kinds of isoflavones having a structure similar to that of equol. However, since the method of the present invention can advantageously separate equol from isoflavones in an efficient manner, an equol-containing fermented material is suitable as the equol-containing material for the purification method of the present invention.

The equol-containing fermented material is explained below.

The equol-containing fermented material is produced by a publicly known fermentation method using an equol-producing microorganism. More specifically, a microorganism having an ability (metabolic activity) to produce equol by metabolizing at least one daidzein compound selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein is inoculated in a fermentation material (material to be fermented) containing soy flavonoids. The sample is then fermented (incubated) under conditions suitable for the growth of the microorganism. The obtained fermented material contains equol.

The equol-producing microorganism is selected from the list of "equol-containing soybean hypocotyl fermentation product" in section "1. preparation method of equol-containing extract".

The fermentation material comprising the daidzein compound is not limited by other factors; however, it is preferable that the material be approved for safety as a food material. Examples of the fermented material containing the soybean flavone compound include soybeans, soybean hypocotyls, soybean hypocotyl extracts, tofu, fried tofu, soybean milk, fermented soybeans, soy sauce, soybean paste, fermented soybean and red clover (red clover) or extracts thereof, alfalfa or extracts thereof. A suitable fermentation material comprising soy flavonoid compounds is soy hypocotyls because of its high soy flavonoid content.

In addition, isoflavones containing any of the above-described daidzein compounds may be added in advance to a fermentation material containing daidzein compounds. By adding isoflavones to the fermented material, the equol content of the obtained fermented material is increased.

In addition, if necessary, certain additives may be used in the fermented material containing the daidzein compound to improve the fermentation efficiency and the like. Examples of the additives include nitrogen sources (such as yeast extract, polypeptone or meat extract), carbon sources (such as glucose or sucrose), inorganic salts (such as phosphate, carbonate or hydrogen sulfate), vitamins and nutrients (such as amino acids).

Various fermentation conditions (including water content, time, temperature, atmosphere) for preparing the equol-containing fermented material are determined depending on the type of equol-producing microorganism, the type of fermented material, the amount of equol produced, the amount of residual soy flavone compounds, and the like.

As the equol-containing substance used in the purification method of the present invention, the equol-containing fermented soybean hypocotyl described in the section "1. method for producing equol-containing extract" is particularly preferable.

2-1 purification method 1

Purification method 1 equol was purified by performing the following steps 1-1 to 1-5.

Step 1-1

The method of the present invention extracts the equol-containing material using an aqueous ethanol solution as an extractant, and then collects the resulting liquid extract (step 1-1).

The typical ethanol concentration of the ethanol aqueous solution used in step 1-1 is 20 to 98 vol%, preferably 45 to 95 vol%, and more preferably 45 to 60 vol%. By ensuring this ethanol concentration, it is possible to efficiently extract equol from an equol-containing material.

The extraction in the step 1-1 uses, for example, an aqueous ethanol solution in an amount of 1 to 1000 parts by weight, preferably 2 to 500 parts by weight, more preferably 5 to 20 parts by weight, per part by weight (dry weight) of the equol-containing substance.

The conditions of the extraction in step 1-1 are not particularly limited, and a warm or cold extraction method, a percolation method, or the like may be used. More specifically, an aqueous ethanol solution is added to the equol-containing substance, and the mixture is allowed to stand or stir at room temperature for at least one minute, more preferably 5 minutes to 48 hours at room temperature.

After this extraction, the liquid extract is subjected to separation to obtain an equol-containing fraction. The collection of the liquid extract is carried out by a publicly known method such as filtration or centrifugation.

The liquid extract thus separated in step 1-1 comprises a reduced amount of oil in the equol-containing material and contains isoflavones (including glycitein and saponins), as well as equol.

The liquid extract thus obtained in step 1-1 is subjected to the following step 1-2.

Step 1-2

After removing the extractant from the liquid extract obtained in step 1-1, the residue is extracted using ethanol as the extractant, and the liquid extract is separated (step 1-2).

In step 1-2, the extractant is removed from the liquid extract obtained in step 1-1, and the residue thus obtained is subjected to another extraction using ethanol, and the liquid extract is collected under the same conditions as in step I-2 of preparation Process I in section "1. preparation Process of equol-containing extract".

The liquid extract collected in step 1-2 contains reduced amounts of saponins and contains isoflavones (including glycitein compounds) and equol. The liquid extract thus obtained in step 1-2 is subjected to the following steps 1-3.

Steps 1 to 3

After removing the extractant from the liquid extract obtained in step 1-2, the residue was suspended in hexane to separate insoluble substances (step 1-3).

In step 1-3, the removal of the extractant from the liquid extract obtained in step 1-2 can be carried out by publicly known methods such as distillation under reduced pressure, drying, etc.

The extractant is removed from the liquid extract obtained in step 1-2, and hexane is added to the residue (hereinafter sometimes referred to as "second material residue") to suspend the residue in hexane.

The extraction in steps 1-3 uses hexane in an amount of 1-50 parts by weight, and more preferably 5-10 parts by weight, per part by weight (dry weight) of the second material residue.

The conditions for the extraction in steps 1 to 3 are not particularly limited. For example, warm or cold extraction methods may be employed. More specifically, hexane is added to the second material residue, and the mixture is allowed to stand or stir at room temperature for at least 5 minutes, preferably 3 minutes to 1 hour at room temperature.

After this suspension with hexane, equol was extracted as an insoluble material. Thus, the equol-containing extract is obtained by collecting the insoluble material. The collection of the insoluble residue is carried out by publicly known methods such as filtration or centrifugation.

Fat-soluble impurities are removed from the liquid extract obtained in step 1-2 through step 1-3. The insoluble residue obtained in step 1-3 is subjected to the following steps 1-4.

Steps 1 to 4

Step 1-4 comprises extracting the insoluble residue obtained in step 1-3 with a mixture of hexane and ether as an extractant, and collecting the resulting liquid extract (step 1-4).

The ether as one of the components of the extractant used in steps 1 to 4 is not particularly limited. Examples of the ether include dimethyl ether, methyl ethyl ether and ethyl ether, and they may be used alone or in combination. Among these, diethyl ether is particularly preferable because of its equol purification efficiency.

The mixing ratio of the mixed solution of hexane and ether used in steps 1 to 4 is not particularly limited; however, to obtain equol with the desired purity, the volume ratio of hexane to ether is 10: 90 to 20: 80, and preferably 30: 70 to 25: 75.

The extraction in the steps 1-4 uses a mixed solution of hexane and ether in an amount of 50-5000 parts by weight, more preferably 200-1000 parts by weight, per part by weight (dry weight) of the insoluble residue obtained in the steps 1-3.

The conditions for the extraction in steps 1 to 4 are not particularly limited. For example, a warm or cold leaching extraction process or a percolation process may be employed. More specifically, a mixed solution of hexane and ether is added to the insoluble matter obtained in steps 1 to 3, and the mixture is allowed to stand or stir at room temperature for at least 10 minutes, preferably 15 minutes to 30 hours at room temperature.

After this extraction, the liquid extract is subjected to separation to obtain equol. The collection of the liquid extract is carried out by a publicly known method such as filtration or centrifugation.

Steps 1 to 5

The liquid extract obtained in steps 1-4 is subjected to silica gel chromatography to obtain an equol-containing fraction (steps 1-5).

Steps 1 to 5 may employ any kind of silica gel chromatography using separation columns each containing silica gel as a packing agent. Examples of silica gel column chromatography include open column chromatography in which a solvent is made to flow by gravity drop, flash column chromatography in which a solvent is made to flow by a pump, and high performance liquid chromatography. Open column chromatography and flash column chromatography are particularly preferred as they can be conveniently accomplished in an industrial manner. When silica gel column chromatography is performed, it is preferable to use a mixed solution of hexane and ether as a mobile phase (developing solvent) to further increase the purification degree of equol. There is no limitation on the ether used for the mobile phase. Examples of the ether include dimethyl ether, methyl ether (methyl ether), and diethyl ether, and they may be used alone or in combination. To increase the purification degree of equol, the ether is preferably the same as that of the mixed solution of hexane and ether used in steps 1 to 4. In the elution of equol by silica gel column chromatography in steps 1 to 5, it is preferable to use, as the initial mobile phase, a mixed solution of hexane and ether having the same composition as the mixed solution of hexane and ether in steps 1 to 4, and the ratio of ether is gradually increased according to a gradient method. This increase in the ether ratio in the mobile phase allows equol to be separated from other impurities along with the mobile phase (in which hexane is mixed with ether in a ratio of 40: 60 to 20: 80, more preferably 30: 70 to 25: 50). Thereafter, the fraction containing equol that had been eluted by silica gel column chromatography was separated to obtain high-purity equol. Then, the extractant is removed from the equol-containing fraction by publicly known methods such as distillation under reduced pressure, drying, and the like. This removed most of the impurities from the stream, leaving a high purity equol solid.

2-2 purification method 2

Step 2-1

The purification method 2 of the present invention extracts the equol-containing material using an organic solvent or an aqueous organic solvent solution of at least one member selected from the group consisting of ethyl acetate, alcohol, acetone, dioxane, acetonitrile, diethyl ether and toluene as an extractant, and then concentrates the liquid extract organic solvent (step 2-1).

In step 2-1, the extractant may be an organic solvent of at least one member selected from the group consisting of ethyl acetate, alcohol, acetone, dioxane, acetonitrile, diethyl ether, and toluene. However, in order to ensure efficient extraction of equol, it is more preferable to use an aqueous organic solvent solution obtained by adding water to the aforementioned organic solvent.

Examples of alcohols useful as extractants include methanol, ethanol, isopropanol, and n-propanol.

The typical concentration of the aqueous organic solvent solution is 80 to 99% by volume, preferably 90 to 97% by volume. Ensuring this concentration makes it possible to obtain a liquid extract with a high equol content.

One preferred extract for use in step 2-1 is an aqueous ethyl acetate solution.

The extraction conditions are not particularly limited, and a warm or cold extraction method or the like may be used. More specifically, the extractant is added to the equol-containing substance, and the mixture is allowed to stand or stir at room temperature for at least 30 minutes, more preferably 1 hour to 50 hours at room temperature.

The conditions of the extraction in the step 2-1 are also not particularly limited, and a warm or cold extraction method, a percolation method, or the like may be used.

After such extraction, equol precipitated in the liquid extract is separated by a publicly known solid-liquid separation method such as filtration or centrifugation. Then, the obtained liquid extract is concentrated, for example, under reduced pressure to obtain an equol concentrate. The equol concentrate thus obtained was subjected to the following step 2-2.

Step 2-2

The equol concentrate obtained in step 2-1 was subjected to silica gel column chromatography to obtain an equol-containing fraction (step 2-2).

In step 2-2, the equol concentrate obtained in step 2-1 may be directly subjected to silica gel column chromatography.

In addition, if the extraction in step 2-1 is performed using an aqueous solvent containing alcohol, ethyl acetate and water in equal amounts of 5-10 times the volume of the concentrated liquid extract are added to the concentrate, and the ethyl acetate fraction is separated and concentrated to perform column chromatography. This column chromatography in step 2-2 may be any kind of chromatography using separation columns each containing silica gel as a filler. Examples of silica gel column chromatography include open column chromatography in which a solvent is made to flow by gravity drop, flash column chromatography in which a solvent is made to flow by a pump, and high performance liquid chromatography. Open column chromatography and flash column chromatography are particularly preferred as they can be conveniently accomplished in an industrial manner.

In the silica gel column chromatography of step 2-2, a mixed solution of an ether or ethyl acetate solution (hereinafter referred to as solution A) and an n-hexane, petroleum ether or n-heptane solution (hereinafter referred to as solution B) is preferably used as a mobile phase. The ratio of the solution B in the mixed solution is 2 to 50 parts by volume, more preferably 3 to 10 parts by volume per part by volume of the solution A.

Thus, the liquid extract obtained in step 2-1 was subjected to silica gel column chromatography to separate the equol-containing fraction.

The equol-containing fraction obtained in step 2-2 was subjected to step 2-3.

Step 2 to 3

The solvent was removed from the equol-containing fraction obtained in step 2-2, and the resulting residue was recrystallized. The precipitated crystals are then separated (step 2-3).

The solvent removal from the equol-containing fraction obtained in step 2-2 can be carried out by a publicly known solvent removal method such as concentration under reduced pressure.

The solvent used for the recrystallization in step 2-3 is not particularly limited as long as it can be used for the recrystallization of equol. Typical solvents are a mixed solution of ethanol and water (ethanol aqueous solution), ethyl acetate, a mixed solution of ethyl acetate and hexane, or a mixed solution of ethanol and hexane. A mixed solution of ethyl acetate and hexane and an aqueous ethanol solution are particularly preferred. More specifically, the mixed solution of ethyl acetate and hexane generally contains ethyl acetate and hexane in a volume ratio of 10: 1 to 1: 10. The ethanol concentration of the ethanol aqueous solution used for recrystallization is usually 30 to 90% by volume, preferably 40 to 70% by volume. The purity of the obtained equol was further improved by recrystallization using a mixed solution of ethyl acetate and hexane or an aqueous ethanol solution adjusted to the aforementioned concentration.

The recrystallization in step 2-3 can be carried out using a conventional method. For example, the solvent for recrystallization is added to the residue resulting from the removal of the solvent from the equol-containing fraction obtained in step 2-2, and the residue is dissolved in the solvent by heating. The solution was then cooled to precipitate equol crystals. To further promote or accelerate equol crystallization, equol seeds can be seeded into the sample.

The temperature conditions for crystallization are determined according to the boiling point and freezing point of the solvent. Typically, the residue is dissolved at about the boiling point of the solvent and the crystals precipitate at about 0-25 ℃.

3. Food material

The present invention further provides a food material in which the equol-containing fermented soybean hypocotyl or its extract is dispersed in the cocoa mass. Details of the food material of the present invention, such as ingredients, are described below.

Equol-containing fermented product of soybean hypocotyl and its extraction

The equol-containing soybean hypocotyl fermented product used for the food material of the present invention is the same as the equol-containing soybean hypocotyl fermented product described in the section "1. preparation method of equol-containing extract" above. The equol-containing fermented soybean hypocotyl can be used for the food material of the present invention immediately after fermentation or after drying to a dry solid as required. In order to improve the storage stability of the equol-containing fermented soybean hypocotyl, it is more preferable to solidify the equol-containing fermented soybean hypocotyl by hot air drying or the like. The air-dried equol-containing fermented soybean hypocotyl can be further processed into powder as required.

The extract of equol-containing fermented soybean hypocotyl is an equol-containing extract obtained by solvent extraction of equol-containing fermented soybean hypocotyl.

The extract of equol-containing fermented soybean hypocotyl is not particularly limited; however, the extract is preferably an equol-containing extract obtained by the method described in the section "1. method for producing an equol-containing extract" above, and more preferably an equol-containing extract is obtained by the above-mentioned production method I. The extract of the equol-containing fermented soybean hypocotyl product of the invention can be used in a liquid state; however, it is more preferred to solidify the liquid extract by removing the extractant.

The food material of the present invention may comprise equol-containing fermented soybean hypocotyl or an extract thereof; however, equol-containing soybean hypocotyl fermentations are nutritionally preferred.

Cocoa mass

Cocoa mass is a food material used primarily as a chocolate ingredient. Cocoa mass is obtained by processing cocoa beans by fermentation, roasting and grinding. The cocoa mass used as the food material of the present invention is not particularly limited as long as its safety as a food material is recognized.

Equol-containing fermented soybean hypocotyl or its extract and cacao mass at a ratio

In the food material of the present invention, the ratio of the equol-containing fermented soybean hypocotyl or its extract to the cacao mass is not limited; however, in order to improve the flavor of food, the proportion of the cocoa mass is more preferably 10 to 2000 parts by weight (dry weight), preferably 20 to 900 parts by weight, more preferably 20 to 400 parts by weight, based on 100 parts by weight of the equol-containing fermented soybean hypocotyl or the extract thereof. In particular, when the equol-containing soybean hypocotyl fermented product is used, the proportion of the cocoa mass is generally 100-2000 parts by weight (dry weight), preferably 200-900 parts by weight, and more preferably 200-400 parts by weight, based on 100 parts by weight of the equol-containing soybean hypocotyl fermented product or extract thereof.

The food material of the present invention may consist of only the equol-containing fermented soybean hypocotyl or its extract and cocoa mass, but may contain other ingredients as described in detail below. The total proportion of the equol-containing fermented soybean hypocotyl or its extract and cocoa mass is 40 to 100% by weight, preferably 60 to 95% by weight, and more preferably 70 to 95% by weight based on the total amount of the food material of the present invention. By thus adjusting the ratio of the ingredients, the beneficial biological activity derived from the equol-containing soybean hypocotyl fermented product can be effectively expressed, and the food product will have good flavor.

Optional ingredients

The food material of the present invention may contain various ingredients in addition to (a) the equol-containing fermented soybean hypocotyl or its extract and (b) the cocoa mass, as long as the effects of the present invention are not affected.

Suitable ingredients for the food material of the present invention include sweeteners such as sugar, trehalose, acesulfame K, xylitol, sucralose, erythritol, saccharin and starch syrup; oils and fats such as cocoa butter or vegetable oil; emulsifiers such as soybean lecithin; a fragrance; a fragrance formulation; milk and water.

Forms of food materials

The shape of the food material of the present invention is not particularly limited, and may be in the form of powder, granules, slices or disks, depending on the form of the target product of the food. In order to make good use of the textural characteristics of the food material of the invention in the product, the food material is preferably processed in the form of granules or slices, particularly preferably in the form of granules or slices of 30-1000mg per slice.

The method for producing the food material of the present invention

The food material of the present invention is prepared by mixing the equol-containing fermented soybean hypocotyl or its extract, the cocoa mass, and other essential ingredients at a temperature at which the cocoa butter in the cocoa mass is dissolved (preferably 30 to 50 ℃), and then cooling and solidifying the mixture. As a result, the food material of the present invention in which the equol-containing fermented soybean hypocotyl is dispersed in the cocoa mass is prepared. If desired, the food material of the present invention may be subjected to crushing, grinding, sieving, etc. to form a desired shape. Alternatively, the food material of the present invention may be molded using extrusion granulation (extrusion granulation) during the preparation process.

Use of food material

The food material of the present invention can be added to various kinds of foodstuffs as a raw material for equol-containing foods or as a food additive. The invention also provides the food containing the equol, which is prepared from the food material.

The equol-containing food product of the invention is not particularly limited, and includes baked snacks and frozen desserts.

Examples of baked snacks include various glutinous rice biscuits, wheat biscuits (such as wafers or wafers), biscuits, cookies, soda biscuits, pies, cakes, donuts and bread. Examples of frozen confections include ice cream, water ice and ice lollies (ice pops).

Among them, baked snacks are one of the suitable forms of equol-containing food products. In particular, baked snacks made from soy flour contain not only equol but also other useful ingredients derived from soy; therefore, baked snacks are very advantageous, providing excellent health preserving effects.

One preferred example of a baked snack made from soy flour comprises soy flour, egg and butter, but no wheat. The soy flour used for this type of biscuit is preferably treated by heating or the like to inactivate or reduce the lipoxygenase and to remove or reduce the beany flavor. Such baked snack can be prepared, for example, by using 0-35 parts by weight of egg, 10-65 parts by weight of butter, 0.1-50 parts by weight of the food material of the present invention and other ingredients per 100 parts by weight of soybean flour. These ingredients are mixed with an appropriate amount of water to make a dough. The dough is then formed into a predetermined shape and baked at about 60-250 c for 2-60 minutes.

The proportion of the food material of the present invention in the equol-containing food is not particularly limited, and is adjusted according to the equol content in the food material of the present invention, the form of the equol-containing food, and the like. The proportion of the food material of the present invention is generally 3 to 30% by weight, preferably 5 to 20% by weight, and more preferably 5 to 8% by weight, based on the total amount of the material of the equol-containing food.

In addition, the proportion of equol in the equol-containing food product is generally 0.002 to 0.1% by weight, preferably 0.004 to 0.05% by weight, and more preferably 0.005 to 0.03% by weight, based on the total amount of the material of the equol-containing food product. By ensuring this ratio of the food material of the present invention, the beneficial biological activity derived from the equol-containing soybean hypocotyl fermented product is exhibited in the food, while maintaining the good flavor of the equol-containing food.

The equol-containing food is prepared by mixing the food material of the present invention and predetermined amounts of other ingredients, and shaping, baking, cooling, etc., the mixture according to the target form of the food product. The equol-containing food is prepared from equol-containing fermented soybean hypocotyl, and contains various useful bioactive materials (including equol) to exhibit various biological activities or pharmacological activities. Therefore, the equol-containing food can be used not only as a general food but also as a food for specified health uses, a nutritional supplement, a functional food, a weak food, and the like. The food comprising the fermented soybean hypocotyl of the present invention is particularly useful as a nutritional health product.

For example, equol-containing foods have an effect of preventing or treating various diseases or symptoms, including climacteric disorder, osteoporosis, prostatic hypertrophy and metabolic syndrome; or for blood cholesterol level reduction, skin whitening, pimples, bowel control, obesity and diuresis. In particular, the equol-containing food is suitable for the prevention and treatment of indefinite diseases or postmenopausal syndromes (e.g., osteoporosis, menopausal disorders) in middle-aged women.

The daily intake of the food containing equol is determined by the equol content of the food, the age, weight, symptoms or frequency of administration of the consumer.

4. Food products in various forms

The present invention further provides various forms of food products comprising the equol-containing fermented soybean hypocotyl or extract thereof. Examples of the food of the present invention include beverages, dietary supplements, cream foods, desserts, snacks, seasonings, retort pouch foods, processed meat products, fish pastes, processed egg products, canned foods, bread, frozen desserts, processed soybean foods, cooked rice products and soups.

The equol-containing fermented soybean hypocotyl and its extract and the various forms of the food provided by the present invention are explained in detail below.

Equol-containing fermented soybean hypocotyl and extract thereof

The equol-containing fermented soybean hypocotyl or its extract used in the present invention is the same as that used in the section "3. food material".

The food product of the present invention may comprise any one of the aforementioned lists of equol-containing fermented soybean hypocotyl or an extract thereof. The food product of the present invention preferably comprises a fermented soybean hypocotyl containing equol.

Food product form

The equol-containing fermented soybean hypocotyl or its extract is effective in preventing or treating various diseases or symptoms including climacteric disorder, osteoporosis, prostatic hyperplasia and metabolic syndrome; or for blood cholesterol level reduction, skin whitening, pimples, bowel control, obesity and diuresis. In particular, the equol-containing food is suitable for preventing or treating an indefinite disease or postmenopausal syndrome (e.g., osteoporosis, menopausal disorder) in middle-aged women. Therefore, the food of the present invention in various forms can be used as a functional food effective for those diseases or symptoms. More specifically, the food of the present invention comprising the fermented product of soybean hypocotyl can be used not only as general foods but also as foods for specified health uses, nutritional supplements, functional foods, dietary foods for the weak, and the like. The food product of the invention comprising a fermented soybean hypocotyl is particularly useful as a nutritional supplement.

Specific examples of the food product of the present invention are explained below.

(1) Beverage and its preparing process

The present invention provides a beverage comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The beverage of the present invention may be any kind of beverage; however, it is applicable to a fruit juice-containing beverage, a vegetable juice-containing beverage, a carbonated beverage (such as carbonated drinks, colas, fruit juice-containing carbonated beverages, ginger carbonated drinks (ginger ale), soda), a soft drink (such as sports drinks, coffee drinks such as coffee or cappuccino), tea (such as english tea, green tea or oolong tea), a cocoa drink, a lactic acid bacteria drink, a milk drink (such as milk, fruity milk or yogurt drink), an alcoholic beverage (such as beer, Japanese sake (distilled liquor), whisky, cocktail or Japanese sake mixed with soda.

The content of the equol-containing fermented soybean hypocotyl product or extract thereof in each beverage of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl product or extract thereof, the type of beverage, the intended effect, and the like. For example, the content of the soybean hypocotyl fermented product or extract thereof in the beverage is generally 0.02 to 15% by weight, preferably 0.1 to 1% by weight, and more preferably 0.2 to 0.5% by weight (dry weight).

(2) Dietary supplement

The present invention provides a dietary supplement comprising equol-containing soybean hypocotyl ferment or extract thereof.

The form of the dietary supplement of the present invention is not limited and includes soft gels (softgels), tablets, powders, and the like, with soft gels being particularly preferred. The soft gel or powder is preferably contained in a predetermined amount in the capsule.

One suitable embodiment of the soft gel of the present invention is a soft gel comprising gelatin, water and glycerin and a fermented product or extract of soybean hypocotyl containing equol therein. The soft gel may further comprise other additives such as soybean oil, beeswax, lecithin or beta-carotene.

The content of the equol-containing soybean hypocotyl fermented product or extract thereof in each dietary supplement of the present invention is not limited, and is individually determined according to the equol content of the soybean hypocotyl fermented product or extract thereof, the type of supplement, the expected effect, and the like. For example, the content of the soybean hypocotyl ferment or extract thereof in the dietary supplement is 2-100 wt% (dry weight).

(3) Cream food

The present invention provides a cream food comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The cream food of the present invention is not particularly limited and may include whipped cream (whipped cream), custard cream (custard cream), butter cream (butter cream), fresh cream (fresh cream), and margarine (butter cream).

The content of the equol-containing fermented soybean hypocotyl product or extract thereof in each cream food of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl product or extract thereof, the type of cream food, the expected effect, and the like. For example, the content of the soybean hypocotyl fermentation product or the extract thereof in the cream food is generally 0.03 to 5% by weight, preferably 0.15 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(4) Dessert

The present invention provides a dessert comprising equol-containing fermented soybean hypocotyl or an extract thereof.

The dessert is not particularly limited and may include yogurt, jelly, pudding, mousse, and the like.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each dessert of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or extract thereof, the type of dessert, the expected effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the dessert is generally 0.03 to 5% by weight, preferably 0.15 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(5) Snack food

The present invention provides a snack containing an equol-containing fermented soybean hypocotyl or an extract thereof.

The snack is not particularly limited and can include any western or japanese candy, including baked or steamed snacks. Examples of the snacks of the present invention include candy, lollipop, toffee, pretzel (pretzel), snack chips (tablet), chewing gum, chocolate, cookies, bean snacks, wafers, cheese crackers, gummy candy, common rice crackers, and Suhama (japanese traditional snack), dry snacks, and Yokan (red bean jelly), bean-jam snacks (bean-jam bun), fern starch dumplings, pound cakes, apple pie fillings, vanilla (savory), and snack glutinous rice crackers. Among the suitable embodiments of the snack are baked snacks comprising soy flour.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each snack of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or extract thereof, the type of snack, the intended effect, and the like. For example, the content of the soybean hypocotyl fermented product or extract thereof in the snack is generally 0.01 to 99% by weight, preferably 0.04 to 36% by weight, and more preferably 0.08 to 18% by weight (dry weight).

(6) Seasoning

The present invention provides a seasoning comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The seasoning is not particularly limited and may include barbecue sauce, sauce of roasted eel (Kabayaki) (roasted seafood), squid-flavor (squid-chinmi) sauce, tomato sauce, tsuyu (soup), seasoning, sesame dipping sauce (sea spreading sauce), snack seasoning, and soy sauce.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each seasoning of the present invention is not limited, and is individually determined depending on the equol content in the fermented soybean hypocotyl or extract thereof, the type of the seasoning, the intended effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the seasoning is generally 0.04 to 40% by weight, preferably 0.2 to 8% by weight, and more preferably 0.4 to 4% by weight (dry weight).

(7) Sterilization bagged food

The present invention provides a retort pouch food (retort pouch food) containing equol-containing fermented soybean hypocotyl or an extract thereof.

The retort pouch is not particularly limited and may include various seasoning materials including premixes for curry sauce, shredded rice sauce (shredded rice sauce), stew, bouillon (eat sauce) or white sauce; premixes for chinese rice covered (donburi), beef rice covered, and chicken and egg rice covered; a premix for a beancurd with a large number of peppers and bean paste and a premix for a rice in a pot (Kamameshi) (a japanese rice dish cooked with various kinds of meat, seafood, and vegetables).

The content of the equol-containing fermented soybean hypocotyl product or the extract thereof in each sterilized pouched food of the present invention is not limited, and is determined individually according to the equol content in the fermented soybean hypocotyl product or the extract thereof, the type of the sterilized pouched food, the expected effect, and the like. For example, the content of the soybean hypocotyl fermentation product or the extract thereof in the retort pouch food is generally 0.003to 4% by weight, preferably 0.01 to 0.8% by weight, and more preferably 0.03 to 0.4% by weight (dry weight).

(8) Processed meat product

The present invention provides a processed meat product comprising equol-containing fermented soybean hypocotyl or an extract thereof.

The processed meat product of the present invention is not particularly limited and may include sausages (dry, semi-dry, bolonia, vienna, smoked sausages), liver paste, salted pork, shouldered pork (shoulderbacon), ham, hamburger steak and meatballs.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each processed meat product of the present invention is not limited and is individually determined depending on the equol content in the fermented soybean hypocotyl or extract thereof, the type of meat product, the expected effect, and the like. For example, the content of the soybean hypocotyl fermentation product or extract thereof in the processed meat product is generally 0.02 to 0.4% by weight, preferably 0.1 to 0.8% by weight, and more preferably 0.2 to 0.4% by weight (dry weight).

(9) Fish paste product

The present invention provides a fish paste product comprising equol-containing fermented soybean hypocotyl or an extract thereof.

The surimi product of the present invention is not particularly limited and may include boiled surimi and fish cake rolls (fish cake tubes).

The content of the equol-containing fermented soybean hypocotyl or its extract in the fish-paste product of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or its extract, the type of fish-paste product, the intended effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the fish-paste product is generally 0.03 to 5% by weight, preferably 0.1 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(10) Processed egg product

The present invention provides a processed egg product comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The processed egg product of the present invention is not particularly limited and may include japanese omelette, thick japanese omelette, western omelette and omelet.

The content of the equol-containing fermented soybean hypocotyl or its extract in each processed egg product of the present invention is not limited, and is individually determined depending on the equol content in the fermented soybean hypocotyl or its extract, the type of processed egg product, the expected effect, and the like. For example, the content of the soybean hypocotyl fermentation product or the extract thereof in the processed egg product is generally 0.03 to 5% by weight, preferably 0.1 to 5% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(11) Canned food

The present invention provides a canned food containing a fermented product of soybean hypocotyl containing equol or an extract thereof.

The can food of the present invention is not particularly limited and may include fruit cans such as orange cans or peach cans, seafood cans such as tuna cans or sardine cans, jam cans, soup cans, and sauce cans.

The content of the equol-containing fermented soybean hypocotyl or its extract in each of the canned foods of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or its extract, the type of the canned food, the expected effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the canned food is generally 0.03 to 5% by weight, preferably 0.1 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(12) Bread

The present invention provides bread containing equol-containing fermented soybean hypocotyl or an extract thereof.

The bread of the present invention is not particularly limited and may include bar bread, french bread, stuffed bread, melon bread (melon break), bean paste bread, cream bread, jam bread, rolls, and croissants.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each bread of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or extract thereof, the type of bread, the expected effect, and the like. For example, the content of the soybean hypocotyl ferment or the extract thereof in bread is generally 0.01 to 50% by weight, preferably 0.05 to 0.6% by weight, and more preferably 0.1 to 0.3% by weight (dry weight).

(13) Frozen dessert

The present invention provides a frozen dessert comprising equol-containing fermented soybean hypocotyl or an extract thereof.

The frozen dessert of the present invention is not particularly limited and may include ice cream and sorbet (sherbet).

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each frozen dessert of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or extract thereof, the type of frozen dessert, the expected effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the frozen dessert is generally 0.03 to 5% by weight, preferably 0.1 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(14) Processed soybean food

The present invention provides a processed soybean food comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The processed soybean food of the present invention is not particularly limited and may include bean curd, thick fried bean curd, fried bean curd mixed with vegetables, bean milk skin, fermented soybeans and soymilk.

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each processed soybean food of the present invention is not limited, and is individually determined depending on the equol content in the fermented soybean hypocotyl or extract thereof, the type of the processed soybean food, the intended effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the processed soybean food is generally 0.03 to 0.5% by weight, preferably 0.1 to 1% by weight, and more preferably 0.3 to 0.5% by weight (dry weight).

(15) Cooked rice product

The present invention provides a cooked rice product comprising equol-containing fermented soybean hypocotyl or an extract thereof. The cooked rice product of the present invention is not particularly limited and may include fried rice, meated rice (pilaf), rice porridge, steamed glutinous rice with chestnuts, boiled rice, red bean rice, and rice steamed with meat and vegetables.

The content of the equol-containing fermented soybean hypocotyl or its extract in each cooked rice product of the present invention is not limited, and is individually determined depending on the equol content in the fermented soybean hypocotyl or its extract, the type of cooked rice product, the expected effect, and the like. For example, the content of the fermented soybean hypocotyl or its extract in the cooked rice product is generally 0.01 to 2.5% by weight, preferably 0.02 to 0.5% by weight, and more preferably 0.04 to 0.25% by weight (dry weight).

(16) Soup

The present invention provides a soup comprising an equol-containing fermented soybean hypocotyl or an extract thereof.

The soup of the present invention is not particularly limited and may include corn butter soup (corn creamshou), bouillon stew (consomm é), vegetable puree (minestron), onion soup and gravy puree (potage).

The content of the equol-containing fermented soybean hypocotyl or extract thereof in each soup of the present invention is not limited, and is individually determined according to the equol content in the fermented soybean hypocotyl or extract thereof, the type of soup, the intended effect, and the like. For example, the content of the soybean hypocotyl fermentation product or extract thereof in the soup is generally 0.02 to 15% by weight, preferably 0.1 to 1% by weight, and more preferably 0.2 to 0.5% by weight (dry weight).

Examples

The present invention is explained more specifically below with reference to reference examples, and the like. The present invention is not limited to these examples.

Reference examples 1-1 to 1-3

Preparation of Equol-containing fermented product of soybean hypocotyl

Soybean hypocotyl powder, arginine and water were mixed to prepare a soybean hypocotyl solution (material) having a composition shown in table 1. Lactococcus 20-92 strain (FERM BP-10036) was inoculated into 5ml of this soybean hypocotyl solution, and the sample was subjected to static culture (static culture) at a temperature of 37 ℃ under anaerobic conditions for 96 hours. Thereafter, the resulting fermentation solution (culture solution) was sterilized by heating at 100 ℃ for 1 minute, followed by drying at 80 ℃. The dried product was processed into powder using a homogenizer to obtain soybean hypocotyl ferment powder.

Table 1 shows the viable cell count and culture pH after 96 hours of culture, the amount of soybean hypocotyl ferment powder collected, and the equol content of the soybean hypocotyl ferment powder. These data show that fermentation of soybean hypocotyl powder with equol-producing bacteria produces equol with high efficiency.

TABLE 1

Each example was performed using three batches of soybean hypocotyl powder (N ═ 3). In the analysis of isoflavone composition in the table, the left-hand value represents the content (mg) of each isoflavone per gram of soybean hypocotyl fermentation product, and the right-hand parenthesized value represents the ratio (wt%) of each isoflavone to the total isoflavone amount (100 wt%) in the soybean hypocotyl fermentation product. "n.d." means no measurement was made and "-" in parentheses means no calculation was made.

Reference examples 1 to 4

Preparation of Equol-containing fermented product of soybean hypocotyl

Lactococcus 20-92 strain (FERM BP-10036) was inoculated to 5ml of a soybean hypocotyl solution containing 10% by weight of soybean hypocotyl powder and 0.1% by weight of arginine, and the sample was fermented by static culture at 37 ℃ for 96 hours under anaerobic conditions. Thereafter, the resulting fermentation solution (culture solution) was sterilized by heating at 100 ℃ for 1 minute, followed by drying at 80 ℃. The dried product was processed into powder using a homogenizer to obtain a powder of soybean hypocotyl ferment.

The respective ingredients of the soybean hypocotyl powder ("before fermentation" in tables 2 and 3) and the soybean hypocotyl fermented product powder ("after fermentation" in tables 2 and 3) were analyzed. Table 2 shows the analysis on soy isoflavones and table 3 shows the analysis on nutritional ingredients. These tables show that the soybean hypocotyl fermentate obtained by fermentation of soybean hypocotyls using lactococcus 20-92 strain has a high equol content. In addition, the oligosaccharide content of oligosaccharides like raffinose or stachyose remained essentially the same after fermentation, indicating that the fermentation did not affect the oligosaccharide content. Also, these tables indicate that arginine is converted to ornithine by fermentation. Therefore, by adding arginine to the soybean hypocotyl before fermentation using lactococcus 20-92 strain, the resulting fermented substance contains not only equol but also ornithine.

TABLE 2

N.D. means "not detected"

TABLE 3

N.D. means "not detected"

Reference examples 1-5 to 1-11

Preparation of Equol-containing fermented product of soybean hypocotyl

In these examples, soybean hypocotyl fermented product powders (reference examples 1-5 to 1-11) were prepared in the same manner as in reference example 1-3 except that seven batches of soybean hypocotyl powders different from the three batches of soybean hypocotyl powders in reference example 1-3 were used. The composition of isoflavones in the obtained fermented soybean hypocotyl was analyzed as shown in Table 4. This result also indicates that each of the fermented soybean hypocotyls of reference examples 1-5 to 1-11 had a high equol content and an isoflavone ratio not achieved by any known technique.

TABLE 4

The values in the upper row represent the content of each isoflavone (mg) per gram of soybean hypocotyl fermentation product, and the values in the lower row represent the ratio (wt%) of each isoflavone to the total isoflavone content (100 wt%) in the soybean hypocotyl fermentation product.

Reference Experimental example 1

Allergen confirmation test

Soybean hypocotyls are known to contain allergens including Gym4, Gm30K, Gm28K, 7S globulin mixture (β -conglycinin), oleosin (oleosin), and trypsin inhibitors. The presence of the allergen in the equol-containing soybean hypocotyl fermented product prepared in reference example 1-1 was examined using the following test.

First, an appropriate amount of equol-containing soybean hypocotyl fermented product obtained in reference example 1-1 was added to an extraction buffer (Tris HCl, pH 7.5, containing 1M EDTA and an appropriate amount of protease inhibitor). The sample was stirred well to extract the water soluble components. The resulting solution was filtered to remove the solid content to obtain a liquid extract. All proteins in this liquid extract were detected using a protein analysis system (Bio-Rad). In addition, the liquid extract contained major allergens (Gym4, Gm30K, Gm28K, 7S globulin mixture, oleosins and trypsin inhibitors) that were detected using western blotting. In addition, for comparison, total protein and allergen were detected by using soybean cotyledon powder and soybean hypocotyl powder instead of soybean hypocotyl fermentation product in the same manner.

Fig. 1 to 3 show the results. FIG. 1 shows the results of total protein detection; FIG. 2 shows the results of Gym4, Gm30K, and Gm28K assays; and figure 3 shows the results of 7S globulin mixture, oleosin and trypsin inhibitor detection.

These results indicate that the major allergen content of soybean or soybean hypocotyl is reduced in equol-containing soybean hypocotyl fermentations.

Example 1: preparation of equol-containing extract

Example 1-1: preparation of equol-containing extract by preparation method I

1 g of equol-containing soybean hypocotyl fermented powder prepared under the same conditions as in reference examples 1 to 3 was added to 20ml of an extractant according to Table 5; and shaken at room temperature for 120 minutes. Subsequently, the residue (hereinafter referred to as "residue 1") was removed by centrifugation to separate a liquid extract (hereinafter referred to as "liquid extract 1-1"). Subsequently, the liquid extract 1-1 was distilled to dryness using an evaporator, 5ml of ethanol was added thereto, and shaking was performed at room temperature for 10 minutes. Subsequently, the residue (hereinafter referred to as "residue 2") was removed by centrifugation to separate a liquid extract (hereinafter referred to as "liquid extract 1-2"). The liquid extract 1-2 was then distilled to dryness using an evaporator to obtain a solid extract.

In addition, another 20ml of the same composition of the extractant was added to residue 1 and shaken at room temperature for 120 minutes. Subsequently, the residue was removed by centrifugation to separate a liquid extract (hereinafter referred to as liquid extract 2-1). The liquid extract 2-1 was then distilled to dryness using an evaporator. 5ml of ethanol was added thereto, and shaking was performed at room temperature for 10 minutes. Subsequently, the residue was removed by centrifugation to separate an extract (hereinafter referred to as "liquid extract 2-2"). The liquid extract 2-2 was distilled to dryness using an evaporator to obtain a solid extract.

TABLE 5

Name of the extracting agent	Composition of extracting agent
		25Et	Ethanol solution containing 25 vol% ethanol and 75 vol% purified water

50Et	Ethanol solution containing 50 vol% ethanol and 50 vol% purified water
		75Et	Ethanol solution containing 75% by volume ethanol and 25% by volume purified water
100Et	100% by volume of ethanol

The liquid extracts 1-1 and 1-2, the liquid extracts 2-1 and 2-2, and the residue 2 were subjected to Thin Layer Chromatography (TLC) analysis (as a mobile phase, hexane: chloroform: ethyl acetate 1: 1 (volume ratio) for equol detection, and ethyl acetate: propanol: chloroform: methanol: 0.9% KCl 25: 10: 9 (volume ratio) for saponin detection) to measure the saponin content and the equol content. The weight of the solid extract obtained from the liquid extract 1-2 and the equol concentration thereof were measured. The results are shown in fig. 4 to 7 and table 6. FIG. 4 shows the results of TLC analysis of the equol content in the liquid extracts 1-2 and 2-2; FIG. 5 shows the results of TLC analysis of the saponin contents in liquid extracts 1-2 and 2-2; FIG. 6 shows the results of TLC analysis of the equol content in the liquid extracts 1-2 and the residue 2; and fig. 7 shows the results of TLC analysis of the saponin content in the liquid extracts 1-2 and residue 2.

As shown in the figure, it was found that when the equol-containing fermented soybean hypocotyl was extracted with ethanol, the extract contained a large amount of oil, and the recovery rate of equol was low.

In contrast, when the equol-containing fermented soybean hypocotyl is extracted with an aqueous ethanol solution, the oil content decreases and the recovery rate of equol increases. In particular, it is clear that the use of an aqueous ethanol solution having an ethanol concentration of 75 vol% significantly improves the equol recovery. In addition, it was also found that extraction using ethanol with 75 vol.% minimized the extraction of fat components. In addition, based on the fact that a concentrated amount of saponin was detected in the residue 2, saponin could be removed by extraction of the residue (which was obtained by removing the solvent from the liquid extract 1-1) with ethanol without reducing the amount of equol.

TABLE 6

EQL stands for equol. The "concentration rate" represents the EQL concentration rate of the solid extract when the EQL concentration of the EQL-containing soybean hypocotyl ferment was assumed to be 1.

Examples 1 to 2

Preparation of equol-containing extract by preparation method I

In this example, equol-containing soybean hypocotyl fermented product powders were prepared under the same conditions as in reference examples 1 to 3, except that different batches of soybean hypocotyl powders were used.

30g of equol-containing soybean hypocotyl fermented product powder was added to 150ml of 70 vol% ethanol aqueous solution (100 ml consisting of ethanol and 30ml of water) or 150ml of 90 vol% ethanol aqueous solution (100 ml consisting of ethanol and 10ml of water); and stirred at room temperature for eight hours. After removing the residue by filtration, the residue was washed with 20ml of an aqueous ethanol solution having the same composition as the extractant. The filtrate and the washed liquid were collected to obtain a liquid extract. The liquid extract was concentrated under reduced pressure to obtain a solid extract.

The weight of the obtained solid extract and the weight of equol contained therein and the concentrations of daidzein, dihydrodaidzein, genistein, dihydrogenistein, daidzein flavone, dihydrodaidzein flavone were measured, as shown in tables 7 and 8.

TABLE 7

Extraction results with 70 vol.% ethanol solution

EQL for equol; DZN represents daidzein; DHD represents dihydrodaidzein; GNT represents genistein; DHG represents dihydrogenistein; GLY represents daidzein (glystein); and DHG represents dihydrodaidzein (dihydroglystein).

Isoflavone content (mg) represents the total weight of each isoflavone contained in the EQL-containing fermented soybean hypocotyl (material: 30g) or in the obtained isolated extract (8000 mg); and the isoflavone concentration (% by weight) represents the proportion of each isoflavone contained in the EQL-containing soybean hypocotyl fermented product (material) or the obtained isolated extract. The "concentration ratio" represents the concentration ratio of each component in the solid extract when the concentration of the component in the EQL-containing soybean hypocotyl fermented product is assumed to be 1.

TABLE 8

Extraction results with 90 vol.% ethanol solution

EQL for equol; DZN represents daidzein; DHD represents dihydrodaidzein; GNT represents genistein; DHG represents dihydrogenistein; GLY represents daidzein; and DHG represents dihydrodaidzein flavone.

Isoflavone content (mg) represents the total weight of each isoflavone contained in the EQL-containing fermented soybean hypocotyl (material: 30g) or in the obtained isolated extract (4100 mg); and the isoflavone concentration (% by weight) represents the proportion of each isoflavone contained in the EQL-containing soybean hypocotyl fermented product (material) or in the obtained isolated extract. The "concentration ratio" represents the concentration ratio of each component in the solid extract when the concentration of the component in the EQL-containing soybean hypocotyl fermented product is assumed to be 1.

It is shown that by extracting the equol-containing fermented soybean hypocotyl with an aqueous ethanol solution, the equol recovery rate was improved and the daidzein was also collected. At the same time, the concentration of other isoflavones is very low in this extract.

Examples 1 to 3

Preparation of equol-containing extract by preparation method I

In this example, equol-containing soybean hypocotyl fermented product powders were prepared under the same conditions as in reference examples 1 to 3, except that different batches of soybean hypocotyl powders were used.

Mixing 50g of equol-containing fermented soybean hypocotyl powder with 7ml of purified water, and further mixing with 250ml of n-hexane; and stirred at room temperature for seven hours. The insoluble residue was separated by filtration and dried at 60 ℃ for seven hours to obtain defatted equol-containing soybean hypocotyl fermented product powder.

20g of defatted equol-containing soybean hypocotyl fermented product powder to 100ml of an 80 vol% ethanol aqueous solution (100ml composed of ethanol and 20ml of water) or 100ml of a 90 vol% ethanol aqueous solution (100ml composed of ethanol and 10ml of water) and stirred at room temperature for fourteen hours. After removing the residue by filtration, the residue was washed with 10ml of an aqueous ethanol solution having the same composition as the extractant. Separating the filtrate and the washed liquid to obtain a liquid extract.

The liquid extract was dried at 60 ℃ for six hours to obtain a solid extract.

The weight of the obtained solid extract and the weight of equol contained therein and the concentrations of daidzein, dihydrodaidzein, genistein, dihydrogenistein, daidzein flavone, dihydrodaidzein flavone were measured, as shown in tables 9 and 10

TABLE 9

Extraction results with 80 vol.% ethanol solution

EQL for equol; DZN represents daidzein; DHD represents dihydrodaidzein; GNT represents genistein; DHG represents dihydrogenistein; GLY represents daidzein; and DHG represents dihydrodaidzein flavone.

Isoflavone content (mg) represents the weight of each isoflavone contained in the defatted EQL-containing soybean hypocotyl fermented product (material: 20g) or in the collected extract (4000 mg); and the isoflavone concentration (% by weight) represents the proportion of each isoflavone contained in the defatted EQL-containing soybean hypocotyl fermented product (material) or the collected extract. The "concentration ratio" represents the concentration ratio of each component in the solid extract when the concentration of the component of the defatted EQL-containing soybean hypocotyl fermented product is assumed to be 1.

Watch 10

Extraction results with 90 vol.% ethanol solution

EQL for equol; DZN represents daidzein; DHD represents dihydrodaidzein; GNT represents genistein; DHG represents dihydrogenistein; GLY represents daidzein; and DHG represents dihydrodaidzein flavone.

Isoflavone content (mg) represents the total weight of each isoflavone contained in the defatted EQL-containing soybean hypocotyl ferment (material: 20g) or in the collected extract (2200 mg); and the isoflavone concentration (% by weight) represents the proportion of each isoflavone contained in the defatted EQL-containing soybean hypocotyl fermented product (material) or the collected extract. The "concentration rate" represents the concentration rate of each component in the solid extract when the concentration of the component in the defatted EQL-containing soybean hypocotyl fermented product is assumed to be 1.

It is shown in the table that the high recovery rate of equol obtained in example 1-2 can be ensured even when the equol-containing fermented soybean hypocotyl is defatted beforehand.

Examples 1 to 4

Preparation of equol-containing extract by preparation method II

In this example, equol-containing soybean hypocotyl fermented product powder (equol concentration of 6.2mg/g) was prepared under the same conditions as in reference examples 1 to 3, except that different batches of soybean hypocotyl powder were used.

80g of equol-containing fermented soybean hypocotyl powder was suspended in 900ml of water. While the suspension was stirred, 5ml of acetic acid was added until the pH became about 4.0. After standing at room temperature for 24 hours, the insoluble residue was collected by filtration and dried. Thus 50.33g of an insoluble residue were obtained.

Then, 250ml of an aqueous ethanol solution (ethanol concentration 75 vol%) was added to 50.33g of the insoluble residue, and stirring was performed at room temperature for two days. After removing the residue by filtration under reduced pressure, the filtrate was collected. The filtrate was distilled to dryness to a solid with an evaporator to obtain 7.53g of equol-containing powder.

The equol concentration of the obtained equol-containing powder was 60.0mg/g, which was about ten times the equol concentration (6.2mg/g) of the equol-containing soybean hypocotyl fermented product used as a starting material. The equol recovery was 91%.

Example 2: equol purification

Example 2-1: equol purification method 1

Equol-containing soybean hypocotyl fermented product powder (equol concentration of 6.2mg/g) was prepared under the same conditions as in reference examples 1 to 3, except that different batches of soybean hypocotyl powder were used.

10g of equol-containing fermented soybean hypocotyl powder was added to 50ml of 50 vol% ethanol aqueous solution, and stirred at room temperature for two hours. Subsequently, the residue (hereinafter referred to as "residue 1") was removed by centrifugation (2500rpm, 25 minutes) to separate a liquid extract (hereinafter referred to as "liquid extract A-1"). Subsequently, 50ml of 50 vol% aqueous ethanol solution was added to residue 1, and stirring was performed at room temperature for one hour. Subsequently, the residue was removed by centrifugation (2500rpm, 25 minutes) to separate a liquid extract (hereinafter referred to as "liquid extract A-2"). The extracts A-1 and A-2 were mixed, and then distilled to dryness using an evaporator to obtain a residual solid (hereinafter referred to as "residual solid 1").

Then, 10ml of ethanol was added to the residual solid 1, and stirring was performed at room temperature for 30 minutes under sonication conditions. Subsequently, the residue was removed by centrifugation (1000rpm, 10 minutes) to separate a liquid extract (hereinafter referred to as "liquid extract B"). The liquid extract B was distilled to dryness using an evaporator to obtain about 1g of a residual solid (hereinafter referred to as "residual solid 2").

Subsequently, 10ml of hexane was added to the 1g of the residual solid 2, and stirring was performed at room temperature for 30 minutes. Then, the liquid extract was removed by centrifugation (1000rpm, 10 minutes) to collect 0.3g of insoluble residue.

To 0.3mg of the obtained insoluble residue was added 2ml of a mixed solution of hexane and dimethyl ether (hexane: dimethyl ether: 2: 3 (volume ratio)), and stirring was performed at room temperature for 30 minutes. Then, the residue was removed by centrifugation (1000rpm, 10 minutes) to collect a liquid extract. The obtained liquid extract was subjected to silica gel chromatography under the following conditions.

Column: a 2cm internal diameter column, packed with silica gel.

Mobile phase:

sequential provision of

200ml of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 40: 60 (volume ratio));

200ml of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 30: 70 (volume ratio));

200ml of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 25: 75 (volume ratio));

200ml of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 20: 80 (volume ratio));

and 200ml of hexane.

Collect each 50ml eluate from the column. The fractions eluted from the mixed solution of hexane and ether (hexane: ether: 40: 60 (volume ratio)) and the mixed solution of hexane and ether (hexane: ether: 25: 75 (volume ratio)) contained 40mg of equol (recovery: 80%), and the purity of equol was 99.9% on a dry solid basis. FIG. 8 shows TLC analysis of fractions eluted using silica gel chromatography. As shown in the figure, fractions eluted from a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 30: 70 (volume ratio)) and a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 25: 75 (volume ratio)) contained high-purity equol. Table 11 details the samples provided in the lanes of fig. 8.

TABLE 11

Traces in FIG. 8	Details of the samples
		Way 1	Equol standard substance
Way 2	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 40: 60 (volume ratio)) was in the range of 0 to 50ml
		Way 3	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 40: 60 (volume ratio)) was in the range of 50 to 100ml
Way 4	Elution flow obtained when the elution amount of the mixed solution of hexane and diethyl ether (hexane: diethyl ether ═ 30: 70 (volume ratio)) was in the range of 0 to 50mlPortions are
		Way 5	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 30: 70 (volume ratio)) was in the range of 50 to 100ml
Way 6	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 25: 75 (volume ratio)) was in the range of 0 to 50ml
		Way 7	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether: 25: 75 (volume ratio)) was in the range of 50 to 100ml
Way 8	Elution fractions obtained when the elution amount of a mixed solution of hexane and diethyl ether (hexane: diethyl ether ═ 20: 80 (volume ratio)) was in the range of 0 to 50ml

Example 2-2: equol purification method 2

Equol-containing soybean hypocotyl fermented product powder (equol concentration of 6.2mg/g) was prepared under the same conditions as in reference examples 1 to 3, except that different batches of soybean hypocotyl powder were used.

100g of equol-containing soybean hypocotyl fermented product powder was added to 500ml of a 95 vol% ethyl acetate aqueous solution, and stirring was performed at room temperature for four hours. Subsequently, the insoluble residue was removed by filtration to collect the extract. The separated insoluble residue was washed with 80ml of ethyl acetate, and the washed liquid was mixed with the filtrate, and the mixture was concentrated under reduced pressure to collect 12g of an oily substance. The oily substance was subjected to silica gel chromatography.

Column: a2 cm internal diameter column was packed with 70g of silica gel using hexane.

Mobile phase: 500ml of hexane was supplied, followed by a mixed solution of ethyl acetate and hexane (ethyl acetate: hexane: 1: 5 (volume ratio)).

The solvent was distilled off from the equol-containing elution solvent thus obtained, whereby 630mg of a yellow crystalline solid was obtained.

The crystalline solid was added to 2ml of ethyl acetate and dissolved by heating at 80 ℃. 8ml of hexane was added thereto and stirred for 30 minutes while cooling the mixture with ice to precipitate equol crystals. The obtained equol crystals were collected by filtration and washed with hexane. The crystals were then dried to obtain 530mg of an equol-containing solid (pale yellow crystals).

In addition, 530mg of the equol-containing solid was added to 4ml of an aqueous ethanol solution (ethanol concentration ═ 62.5 vol%), and heated to 80 ℃ to be dissolved in the solution. The solution was allowed to stand at 10 ℃ for 12 hours to recrystallize equol, which was then filtered and dried. As a result, 390mg of white needle-like equol crystals were obtained.

Rotation angle [ alpha ] of equol crystals obtained in 25 ℃ ethanol]_D ²⁵Is-21.6 deg. The melting point was 188-. The equol obtained was confirmed to be nearly 100% pure.

Example 3: food material comprising equol-containing fermented soybean hypocotyl or extract thereof, and food product comprising the food material

Example 3-1 preparation of food containing Equol-containing fermented product of soybean hypocotyl and cacao mass Material

The equol-containing soybean hypocotyl fermented product prepared under the same conditions as in reference examples 1 to 3 was mixed with cocoa mass dissolved by heating in a ratio of 1: 3 (weight ratio). The mixture is cooled to obtain a food material in which the equol-containing fermented soybean hypocotyl is dispersed in the cacao mass.

The taste of the food material thus prepared was evaluated by 10 panelists, and from the results of all panelists, it was concluded that the bitterness produced by the equol-containing soybean hypocotyl fermented product was suppressed and the food material had a satisfactory flavor.

Example 3-2 food Material comprising extract of Equol-containing fermented product of soybean hypocotyl

A solid extract obtained by evaporating the liquid extract 2-1 prepared with 75Et in example 1-1 (hereinafter referred to as "75 Et extract") and cocoa mass dissolved by heating were mixed in a ratio of 1: 24 (weight ratio). The mixture was cooled to obtain a food material in which the 75Et extract was dispersed in cocoa mass.

The taste of the food material thus prepared was evaluated by 10 panelists, and from the results of all panelists, it was concluded that the bitter taste produced by the 75Et extract was suppressed and the food material had a satisfactory flavor.

Examples 3-3 baked snack

The ingredients shown in table 12 were mixed, and the mixture was shaped into a rectangle (about 1.5cm × 1.5cm × 10cm) and baked in an oven at 180 ℃ to produce a baked snack.

The taste of the baked snack thus prepared was evaluated by 10 panelists, and from the results of all panelists, it was concluded that the bitterness produced by the equol-containing soybean hypocotyl fermentation was suppressed, and that the baked snack had a satisfactory chocolate flavor and an originally satisfactory flavor.

TABLE 12

	Proportion (wt%)
		Sliced food Material obtained in example 3-1 (about 0.1g per slice)	5
Soybean flour	25
		Raisin	q.s.
Butter oil	q.s.
		Candy	q.s.
Egg	q.s.
		Orange peel	q.s.
Pineapple	q.s.
		Indigestible dextrins	q.s.
Cocoa mass	q.s.
		Sliced apricot kernel	q.s.
Cocoa powder	q.s.
		Salt (salt)	q.s.
Fermentation powder	q.s.
		Perfume	q.s.
Total of	100

Comparative example 3-1: baked snack

A baked snack was prepared under the same conditions as in example 3-3, except that 1g of the equol-containing soybean hypocotyl fermented product prepared under the same conditions as in example 3-1 was mixed with 5g of chocolate chips instead of the chip-shaped food material obtained in example 3-1.

The taste of the baked snack thus prepared was evaluated by 10 panelists, and from the results of all panelists, it was concluded that the satisfactory taste disappeared while the bitter taste produced from the equol-containing soybean hypocotyl fermentation remained throughout the center.

Examples 3 to 4: baked snack

A baked snack was prepared under the same conditions as in example 3-3, except that the same amount of the chip-shaped food material obtained in example 3-2 was contained instead of the chip-shaped food material obtained in example 3-1.

The taste of the baked snack thus prepared was evaluated by 10 panelists, and from the results of all panelists, it was concluded that the bitterness produced by the 75Et extract was suppressed and the baked snack had a satisfactory chocolate flavor and an original satisfactory flavor.

Example 4 food containing Equol-containing fermented product of soybean hypocotyl or extract thereof

Example 4ABeverage and its preparing process

In the following, specific preparation examples of the beverage according to the invention are given.

Example 4-A-1Soda water

Carbonated water was obtained by preparing a mint-flavored syrup having the following composition, and mixing the resulting mint-flavored syrup with carbonated water (carbonated water) in a 3: 1 volume ratio.

Composition of mint flavored syrup

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.27(kg)

Sugar 0.60

Citric acid (Crystal) 0.01

Salt 0.012

Potassium chloride 0.005

Calcium lactate 0.008

Mint extract 0.03

Mint flavor 0.20

Balance of water

Total 100.00L

Example 4A-2Soda water

Soda water was prepared under the same conditions as in example 4-A-1, except that mint-flavored syrup was prepared by adding 0.034kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness, instead of the equol-containing soybean hypocotyl fermented product.

Example 4-A-3Orange flavored carbonated beverage without orange juice

An orange-flavored carbonated beverage without orange juice was obtained by preparing an orange-flavored syrup having the following composition and mixing the resulting orange-flavored syrup with carbonated water in a 9: 11 volume ratio.

Orange flavored syrup composition

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.20(kg)

Sugar 10.00

Citric acid (Crystal) 0.03

DL-malic acid 0.07

Edible pigment 0.05

Perfume 0.10

Balance of water

Total 45.00L

Example 4-A-4Orange flavored carbonated beverage without orange juice

An orange-flavored carbonated beverage containing no orange juice was prepared under the same conditions as in example 4-A-3, except that the orange-flavored syrup was prepared by adding 0.025kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness, instead of the equol-containing soybean hypocotyl fermented product.

Example 4-A-5Low calorie cola

Sugarless cola was obtained by preparing a cola syrup having the following composition and mixing the resulting cola syrup with carbonated water in a volume ratio of 8: 12.

Cola syrup composition

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.20(kg)

Sucralose 0.02

Phosphoric acid 0.05

Citric acid (Crystal) 0.07

Caffeine 0.10

Caramel color 0.20

Spice (Cola flavor) 0.10

Balance of water

40.00L in total

Example 4-A-6Low calorie cola

Diet cola was prepared under the same conditions as in example 4-A-5, except that cola syrup was prepared by adding 0.03kg of solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness in place of the equol-containing soybean hypocotyl ferment.

Example 4-A-7Carbonated apple beverage

The carbonated apple beverage was obtained by preparing an apple-flavored syrup having the following composition, and mixing the resulting apple-flavored syrup with carbonated water in a volume ratio of 13: 7.

Composition of apple-flavored syrup

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.20(kg)

5 times concentrated apple juice 11.00

Fructose/glucose syrup 5.00

Citric acid (Crystal) 0.10

Edible pigment 0.05

Perfume 0.10

Balance of water

65.00L in total

Example 4-A-8Carbonated apple beverage

A carbonated apple beverage was prepared under the same conditions as in example 4-A-7, except that an apple-flavored syrup was prepared by adding 0.03kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness, instead of the equol-containing soybean hypocotyl fermented product.

Example 4-A-9Carbonated grape beverage

Carbonated grape beverage was obtained by preparing a grape-flavored syrup having the following composition, and mixing the resulting grape-flavored syrup with carbonated water in a 9: 11 volume ratio.

Composition of grape flavored syrup

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.20(kg)

4.40 times concentrated clarified grape juice

Citric acid (Crystal) 0.25

Edible pigment 0.05

Perfume 0.20

Balance of water

Total 45.00L

Example 4-A-10Carbonated grape beverage

A carbonated grape beverage was prepared under the same conditions as in example 4-A-9, except that a grape-flavored syrup was prepared by adding 0.03kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-11Beverage containing apple juice

An apple juice-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

5 times concentrated apple juice 22.00

Perfume 0.05

Balance of water

Total 100.00L

Example 4-A-12Beverage containing apple juice

A beverage containing apple juice was prepared under the same conditions as in example 4-A-11, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-13Beverage containing orange juice

An orange juice-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Concentrated Valencia orange juice (Brix: 55 degree) 4.40

Sugar 5.00

Citric acid (Crystal) 0.16

0.025% original gellan gum

Vitamin C0.03

Pectin 0.0025

Orange essence 0.25

Balance of water

Total 100.00L

Example 4-A-14Beverage containing orange juice

An orange juice-containing beverage was prepared under the same conditions as in example 4-A-13, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-15Low calorie beverage containing fruit juice

A low-calorie beverage containing fruit juice having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

4.40 times of concentrated succus Citri Tangerinae (citrus juice)

Sucralose 0.009

Citric acid (Crystal) 0.18

L-ascorbic acid 0.03

Gellan gum 0.024

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-16Low calorie beverage containing fruit juice

A diet drink containing fruit juice was prepared under the same conditions as in example 4-A-15, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-17Sugar-free Near Water (Sugarless Near Water) (Water-like soft drink)

A sugarless near water (soft drink water similar to water) having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Sucralose 0.008

Polydextrose 2.20

0.44 times of concentrated succus Citri Grandis

Citric acid (Crystal) 0.53

Calcium lactate 0.06

Potassium chloride 0.01

L-ascorbic acid 0.03

Dibenzoyl thiamine hydrochloride 0.0002

Pyridoxine hydrochloride 0.00015

Nicotinamide 0.0015

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-18Sugar-free near water (Water-like soft drink)

A sugar-free near-water (water-like soft drink) was prepared under the same conditions as in example 4-A-17, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-A-19Sports beverage

A sports beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Clear lemon juice 0.50

Sucralose 0.02

Milk minerals (latex minerals) 1.00

Vitamin mixture 0.25

L-ascorbic acid sodium salt 0.05

Citric acid 0.125

Balance of water

Total 100.00L

Example 4-A-20Sports beverage

A sports beverage was prepared under the same conditions as in example 4-A-19, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4A-21Powdered green tea-milk beverage

A powdered green tea-milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Milk 10.00

Skimmed milk 3.50

Powdered Green tea (RS-20, produced by ITO EN, LTD.) 0.50

Sugar 7.00

Emulsifier 0.70

Edible pigment 0.03

Perfume 0.20

Sodium bicarbonate for adjusting pH to 6.8

Balance of water

Total 100.00L

Example 4A-22Powdered green tea-milk beverage

A powdered green tea-milk beverage was prepared under the same conditions as in example 4-A-21, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-23Sugar-free cheese beverage

A sugar-free cheese beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Fermented milk (SNF 20%) 40.00

Water soluble soybean polysaccharide 0.20

Pectin 0.20

Perfume 0.20

Lactic acid for adjusting pH to 4.2

Balance of water

Total 100.00L

Example 4-A-24Sugar-free cheese beverage

A sugar-free cheese beverage was prepared under the same conditions as in example 4-A-23, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-25Pasteurized lactic acid bacteria beverage

A pasteurized lactic acid bacteria beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Fermented milk (SNF 20%) 15.00

Water soluble soybean polysaccharide 0.40

Sugar 8.00

Perfume 0.10

Lactic acid for adjusting pH to 3.8

Balance of water

Total 100.00L

Example 4-A-26Pasteurized lactic acid bacteria beverage

A pasteurized lactic acid bacteria beverage was prepared under the same conditions as in example 4-A-25, except that 0.05kg of a solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-27Low-calorie yogurt drink

A low-calorie yogurt drink having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Skimmed milk 3.50

Water soluble soybean polysaccharide 0.40

Sugar 5.00

Sucralose 0.0006

Fructose/glucose syrup (75%) 5.00

Citric acid (Crystal) 0.45

Perfume 0.10

Lactic acid for adjusting pH to 3.8

Balance of water

Total 100.00L

Example 4-A-28Low-calorie yogurt drink

A diet yogurt drink was prepared under the same conditions as in example 4-A-27, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-A-29Strawberry-flavored milk beverage

A strawberry flavored milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Skimmed milk 2.80

Whole milk powder 3.00

Sugar 12.00

Emulsifier 0.06

Edible pigment 0.05

Spice (strawberry essence) 0.10

Balance of water

Total 100.00L

Example 4-A-30Strawberry-flavored milk beverage

A strawberry-flavored milk beverage was prepared under the same conditions as in example 4-A-29, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-31Fat-free milk

A fat-free milk having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

Skimmed milk 12.00

Egg calcium (Egg calcium) 1.00

Emulsifier 0.06

Perfume 0.10

Balance of water

Total 100.00L

Example 4A-32Fat-free milk

Fat-free milk was prepared under the same conditions as in example 4-A-31, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4A-33Perfumed grass beverage (Cream Soda)

An aromatised grass beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.20(kg)

Defatted sweetened condensed milk 3.00

Citric acid (Crystal) 0.04

Sugar 15.00

Edible pigment 0.60

Perfume 0.10

Balance of water

40.00L in total

Example 4-A-34Adding vanilla beverage

A flavored grass beverage was prepared under the same conditions as in example 4-A-33, except that 0.025kg of a solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness as in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-35Powdered green tea-milk beverage

A powdered green tea-milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Skimmed milk 3.50

Milk 10.00

Emulsifier 0.70

Sugar 6.00

Balance of water

Total 100.00L

Example 4A-36Powdered green tea-milk beverage

A powdered green tea-milk beverage was prepared under the same conditions as in example 4-A-29, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-37Black tea

Black tea having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

20.00 times of Dajiling tea extract (40 times)

Sugar 5.00

Perfume 0.05

Balance of water

Total 100.00L

Example 4A-38Black tea

Black tea was prepared under the same conditions as in example 4-A-37, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-39Beverage containing apple juice

An apple juice-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

2.20 times concentrated clear apple juice

Oligosaccharide 1.00

Reducing maltose syrup 1.50

Vitamin C0.05

Citric acid 0.10

Water-soluble dietary fiber 8.00

Balance of water

Total 100.00L

Example 4-A-40Beverage containing apple juice

A beverage containing apple juice was prepared under the same conditions as in example 4-A-39, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4A-41Soft drink

A soft drink having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Fructose 3.70

Fructose/glucose syrup 8.00

Sugar 0.30

DL-malic acid 0.07

Sodium citrate 0.03

Emulsifier 0.052

Perfume 0.22

Balance of water

Total 100.00L

Example 4-A-42Soft drink

A soft drink was prepared under the same conditions as in example 4-A-41, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-43Beverage containing orange juice

An orange juice-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Fructose/glucose syrup 2.50

Beet oligosaccharide 0.026

Sugar 4.00

Concentrated orange juice 4.40

Citric acid 0.026

Sodium citrate 0.003

L-ascorbic acid 0.01

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-44Beverage containing orange juice

An orange juice-containing beverage was prepared under the same conditions as in example 4-A-43, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-45Beverage containing apple juice

An apple juice-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Concentrated apple juice 4.40

Sugar 8.00

Citric acid 0.15

Fragrance 0.12

Balance of water

Total 100.00L

Example 4-A-46Beverage containing apple juice

A beverage containing apple juice was prepared under the same conditions as in example 4-A-.45, except that 0.05kg of a solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-A-47-1Vegetable juice

A vegetable juice having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Vegetable juice 20.00

Balance of water

Total 100.00L

Example 4-A-47-2Vegetable juice

A vegetable juice was prepared under the same conditions as in example 4-A-47-1, except that 0.05kg of a solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-48-1Sports beverage

A sports beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Milk minerals (1 active minerals) 1.00

Vitamin mixture 0.25

Citric acid 0.125

Ascorbic acid sodium salt 0.05

Lemon juice 0.50

Sugar 12.00

Balance of water

Total 100.00L

Example 4-A-48-2Sports beverage

The beverage was sports-served under the same conditions as in example 4-A-48-1 except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-49Oolong tea

Oolong tea having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Oolong tea extract 3.00

Ascorbic acid sodium salt 0.01

Perfume 0.20

Balance of water

Total 100.00L

Example 4-A-50Oolong Tea

Oolong tea was prepared under the same conditions as in example 4-A-49, except that 0.05kg of solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-A-51Lemon added tea

Lemon-added tea having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Black tea extract 6.00

Reducing maltose syrup powder 3.00

Sugar 3.00

Lemon juice 0.50

L-ascorbic acid 0.02

Perfume 0.10

Balance of water

Total 100.00L

Example 4A-52Lemon added tea

Lemon-added tea was prepared under the same conditions as in example 4-A-51, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-53Milk tea is added

Milk tea with the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Milk 7.00

Whole milk powder 0.25

Sugar 4.00

Tea extract 35.00

Emulsifier 0.05

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-54Milk tea is added

Milk tea was prepared under the same conditions as in example 4-A-53, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-55Coffee beverage

A coffee beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

Coffee extract 30.00

Milk 25.00

Emulsifier 0.10

Sodium bicarbonate 0.12

Stevia (Stevia) 0.01

Sugar 3.00

L-rhamnose 0.01

Perfume 0.05

Balance of water

Total 100.00L

Example 4A-56Coffee beverage

A coffee beverage was prepared under the same conditions as in example 4-A-55, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-57Cocoa milk beverage

A cocoa milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

Milk 10.00

Skimmed milk 3.50

Fructose 5.50

Sugar 0.40

Cocoa powder 1.00

Emulsifier 0.45

Fragrance 0.08

Balance of water

Total 100.00L

Example 4-A-58Cocoa milk beverage

A cocoa milk beverage was prepared under the same conditions as in example 4-A-57, except that the liquid extract 1-2 obtained by evaporation with 75Et in example 1-1 to 0.05kg of a solid extract prepared dry were used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-59Powdered green tea-milk beverage

A powdered green tea-milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

Milk 10.00

Skimmed milk 3.50

Fructose 5.80

Sugar 0.40

Powdered green tea 0.90

Emulsifier 0.48

Fragrance 0.27

Edible pigment 0.03

Balance of water

Total 100.00L

Example 4-A-60Powdered green tea-milk beverage

A powdered green tea-milk beverage, excepting for using, instead of the equol-containing soybean hypocotyl fermented product, was prepared under the same conditions as in example 4-A-59, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-61Yoghourt beverage

A yogurt drink having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Skimmed milk 1.50

Fructose 2.50

Sugar 0.50

Concentrated strawberry juice 0.22

Sodium citrate 0.32

Emulsifier 0.40

Fragrance 0.12

Balance of water

Total 100.00L

Example 4-A-62Yoghourt beverage

A yogurt drink was prepared under the same conditions as in example 4-A-61, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-63Beverage containing vitamins

A vitamin-containing beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Sugar 24.00

Citric acid 0.20

L-ascorbic acid 0.30

Vitamin mixture 0.06

Perfume 0.10

Balance of carbonated water

Total 100.00L

Example 4-A-64Beverage containing vitamins

A vitamin-containing beverage was prepared under the same conditions as in example 4-A-63, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-65Red bean soup

A red bean soup having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Red bean paste 10.00

Sugar alcohol 6.50

Sugar 24.00

Salt 0.10

Emulsifier 0.60

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-66Red bean soup

A red bean soup was prepared under the same conditions as in example 4-A-65, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-67Green tea

Green tea having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40(kg)

Green tea extract 3.00

Ascorbic acid sodium salt 0.01

Perfume 0.20

Balance of water

Total 100.00L

Example 4-A-68Green tea

Green tea was prepared under the same conditions as in example 4-A-67, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-A-69Powdered orange juice beverage

A powdered orange juice beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Fructose/glucose syrup 2.50

Beet oligosaccharide 0.026

Sugar 4.00

Powdery orange juice 2.00

Citric acid 0.026

Sodium citrate 0.003

L-ascorbic acid 0.01

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-70Powdered orange juice beverage

A powdered orange juice beverage was prepared under the same conditions as in example 4-A-69, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-71Amino acid beverage

An amino acid beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Sugar 15.00

Fructose 15.00

Citric acid 4.00

Sodium citrate 1.00

L-valine 2.00

L-leucine 4.00

L-isoleucine 2.00

L-arginine 2.00

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-72Amino acid beverage

An amino acid beverage was prepared under the same conditions as in example 4-A-71, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-73Soy milk beverage

A soy milk beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Soymilk 100.00

Example 4-A-74Soy milk beverage

A soybean milk beverage was prepared under the same conditions as in example 4-A-73, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-A-75Energy beverage

An energy beverage having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Taurine 1.00

Nicotinamide 0.02

Vitamin B10.005

Vitamin B20.005

Vitamin B60.005

Caffeine 0.05

Sugar 15.00

Fructose 15.00

Citric acid 0.10

Perfume 0.10

Balance of water

Total 100.00L

Example 4-A-76Amino acid beverage

An amino acid beverage was prepared under the same conditions as in example 4-A-75, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-BDietary supplement

In the following, specific preparation examples of the dietary supplement according to the present invention are given.

Example 4-B-1Soft gels

The equol-containing fermented soybean hypocotyl (reference examples 1 to 3), gelatin, glycerin, citric acid, soybean oil, beeswax, lecithin, beta-carotene and water were used to prepare a soft gel according to a known method.

Example 4-B-2Soft gels

A soft gel was prepared under the same conditions as in example 4-B-1, except that the solid extract prepared by evaporating the liquid extract 1-2 obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-CCream food

In the following, specific preparation examples of the cream food according to the present invention are given.

Example 4-C-1Whipped cream

A whipped cream having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(g)

Coconut oil (mp: 35 deg.C) 27.00

Glycerin fatty acid ester 0.10

Skimmed milk 3.30

Sucralose 0.012

Reducing starch sugar (powder sugar) 25.00

Lecithin 0.15

Emulsifier 0.70

Sodium metaphosphate 0.10

Balance of water

A total of 100.00g

Example 4-C-2Whipped cream

Whipped cream was prepared under the same conditions as in example 4-C-1 except that 0.05g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4C-3Artificial cream

A margarine having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(g)

Purified coconut oil 36.00

Sodium caseinate 4.50

Skimmed milk 5.00

Lecithin 0.05

Trisodium citrate 0.20

Sucrose fatty acid ester 0.40

Milk essence 0.10

Balance of water

A total of 100.00g

Example 4C-4Artificial cream

A margarine was prepared under the same conditions as in example 4-C-3, except that 0.05g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4C-5Milk cake

A custard having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Margarine 20.00

Starch syrup 5.00

Whole egg 3.00

Corn starch 4.00

Modified starch 6.00

Milk protein (Lactic protein) 2.00

Sugar 30.00

Balance of water

A total of 100.00kg

Example 4C-6Milk cake

A custard was prepared under the same conditions as in example 4-C-5, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4C-7Milk cake

A custard having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Carrageenan 0.40

Gelatin 0.50

Corn starch 4.00

Starch syrup dry powder 3.00

Granulated sugar 10.00

Glucose 7.00

Skimmed milk 4.00

Whole egg 3.50

Isomerized sugar (isometrized sugar) 7.00

Defatted sweetened condensed milk 3.00

Fresh butter (unaltered butter) 6.50

50.00 g of water

Example 4C-8Milk cake

A custard was prepared under the same conditions as in example 4-C-7, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-DDessert

In the following, specific preparation examples of the dessert according to the present invention are given.

Example 4D-1Hard cheese (Hard Yogurt)

Hard cheese was prepared by homogenizing the ingredients of the following composition and adding cheese starter (starteroygort) thereto.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Skimmed milk 9.00

Fresh butter 5.00

Sugar 6.50

Gelling agent 0.60

Balance of water

A total of 100.00kg

Example 4D-2Hard cheese

Hard cheese was prepared under the same conditions as in example 4-D-1, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl ferment.

Example 4D-3Soft cheese (Soft Yogurt)

Soft cheese was prepared by homogenizing the ingredients of the following composition and adding a cheese starter thereto.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Skimmed milk 9.00

Butter 5.25

Sugar 6.50

Locust Bean Gum 0.30

Balance of water

A total of 100.00kg

Example 4-D-4Soft cheese

Soft cheese was prepared under the same conditions as in example 4-D-3, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-D-5White peach jelly (white peach jelly)

The following ingredients were used to prepare a white peach jelly according to the general preparation method of the jelly.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Sugar 14.40

K-carrageenan 0.60

White peach jam 20.00

Balance of water

Total of 100.00 parts by weight

Example 4-D-6White peach jelly

A white peach jelly was prepared under the same conditions as in example 4-D-5, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4D-7Shaddock jelly

The grapefruit jelly was prepared according to the general preparation method of jelly using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Sugar 18.00

Gellan gum 0.25

Grapefruit juice 20.00

Calcium lactate 0.10

Citric acid 0.20

Perfume 0.10

Balance of water

Total of 100.00 parts by weight

Example 4D-8Shaddock jelly

A grapefruit jelly was prepared under the same conditions as in example 4-D-7, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-D-9Coffee jelly

The coffee jelly was prepared according to the preparation method of the jelly using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Reducing starch syrup 10.00

Xylitol 5.00

K-carrageenan 0.50

Agar 0.20

Locust Bean Gum 0.20

Sugar 3.00

Fragrance 0.15

Balance of water

Total of 100.00 parts by weight

Example 4D-10Coffee jelly

A coffee jelly was prepared under the same conditions as in example 4-D-9, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4D-11Strawberry milk jelly

The strawberry milk jelly was prepared according to the general preparation method of jelly using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Purified coconut oil 30.00

Emulsifier 0.25

Skimmed milk 3.30

Sugar 3.50

Reducing starch syrup 20.00

Lactitol 8.00

Stabilizer 0.70

Xylitol 5.00

Gelatin 0.90

Strawberry jam 5.00

Concentrated strawberry juice 5.00

Citric acid 0.10

Balance of water

Total of 100.00 parts by weight

Example 4D-12Strawberry milk jelly

A strawberry milk jelly was prepared under the same conditions as in example 4-D-11, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Examples 4 to ESnack food

In the following, specific preparation examples of snacks according to the present invention are given.

Example 4E-1Hard candy

Hard candy was prepared according to the general preparation method of hard candy using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(g)

Sugar 70.00

Starch syrup 40.00

Water 20.00

Example 4E-2Hard candy

A hard candy was prepared under the same conditions as in example 4-E-1, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-3Ginger-flavored sugar

Ginger-flavored candy was prepared according to the general preparation method of candy using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Ginger juice 1.50

Reducing lactose 60.00

Reducing maltose syrup 56.00

Sucralose 0.03

Perfume 0.075

Water 30.00

Example 4-E-4Ginger-flavored sugar

A ginger-flavored sugar was prepared under the same conditions as in example 4-E-3, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness as in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-5Throat-moistening herbal medicine sugar (Throat Soothing Herb Candy)

Throat-moistening herbal candy was prepared using the ingredients of the following composition according to the general preparation method of candy.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 4.00(g)

Mixed herb extract 0.60

Sugar 40.00

Reducing maltose syrup 56.00

Mint flavor 0.20

Caramel color 0.20

Water 20.00

Example 4E-6Throat-moistening herbal medicine sugar

Throat-soothing herbal candy was prepared under the same conditions as in example 4-E-5, except that 0.5g of solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of equol-containing soybean hypocotyl ferment.

Examples 4 to E-7Lemon chewing gum

Lemon gummy candies were prepared according to the general preparation method of gummy candies using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 4.00(g)

Acidic gelatin (250blooms) 7.00

Reducing starch syrup 100.00

4 times concentrated lemon juice 1.25

Citric acid (Crystal) 1.00

Sucralose 0.02

Edible pigment 0.02

Perfume 0.02

12.00 g of water

Example 4E-8Lemon chewing gum

Lemon gummy candies were prepared under the same conditions as in example 4-E-7, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-9Maltose (Caramel Candy)

Maltose was prepared using the following ingredients according to a general method for preparing maltose.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Sweetened condensed milk 45.00

Starch syrup 45.00

Vegetable fats and oils 3.50

Lecithin 0.20

Soft candy material 5.00

Spice (caramel flavor) 0.10

Example 4E-10Maltose

Maltose was prepared under the same conditions as in example 4-E-9, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-11Spiced salt crisp cake

The following ingredients were mixed to make a dough, which was baked in an oven to make a pretzel.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Strong flour 100.00

Baking powder 1.35

Fresh butter 25.00

Salt 2.00

Sugar 17.50

Soybean dietary fiber 0.80

Sucrose fatty acid ester 0.30

Perfume 0.20

Water 40.00

Example 4E-12Spiced salt crisp cake

Pretzels were prepared under the same conditions as in example 4-E-11, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4E-13Mint Tablet (Mint Tablet)

The following ingredients were used to prepare mint tablets according to the general preparation method of tablets.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(g)

Sorbitol 90.00

Sucralose 0.15

Mint flavor 5.00

Sucrose fatty acid ester 1.00

Example 4E-14Mint tablet

Mint sheets were prepared under the same conditions as in example 4-E-13, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-15Cheese slice

Cheese tablets were prepared according to the general preparation method of tablets using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 4.00(g)

Trehalose (granular) 88.60

Sucralose 0.08

Powdered fermented milk 5.00

Citric acid anhydrous 1.00

Cheese flavor (Yogurt flavor) 0.20

Sucrose fatty acid ester 1.50

Example 4E-16Cheese slice

Cheese pieces were prepared under the same conditions as in example 4-E-15, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-17Lemon slice

Lemon tablets were prepared using the ingredients of the following composition according to the general preparation method of tablets.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Sugar (granular) 89.50

Powdery lemon juice 2.00

L-ascorbic acid 3.00

Citric acid anhydrous 1.00

Sucrose fatty acid ester 1.00

Example 4E-18Lemon slice

Lemon slices were prepared under the same conditions as in example 4-E-17, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-19Mint chewing gum

Mint chewing gum was prepared according to the general preparation method of chewing gum using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Gum matrix 25.00

Lactitol 63.20

Sugar alcohol 5.00

Sucralose 0.13

0.50% of Glycerol

Peppermint oil 1.50

Mint essence 0.50

Edible pigment 0.20

Example 4E-20Mint chewing gum

A mint chewing gum was prepared under the same conditions as in example 4-E-19, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-21Apple bubble gum

Apple bubble gum was prepared according to the general preparation method of chewing gum using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 4.00(g)

Gum matrix 30.00

Palatinit 70.00

Sucralose 0.084

0.50% of Glycerol

Citric acid (Crystal) 1.00

Apple essence 0.80

Example 4E-22Apple bubble gum

Apple bubble gum was prepared under the same conditions as in example 4-E-21, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl ferment.

Example 4E-23Strawberry chewing gum

Strawberry chewing gum was prepared according to the general preparation method of chewing gum using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(g)

Gum matrix 23.00

Powder candy 62.00

Starch syrup 5.00

Sucralose 0.001

Citric acid (Crystal) 1.20

Red cabbage pigment 0.20

Strawberry essence 0.20

Example 4E-24Strawberry chewing gum

A strawberry chewing gum was prepared under the same conditions as in example 4-E-23, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-25Chocolate

Chocolate was prepared according to the general preparation method of chocolate using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(g)

Cocoa mass 40.00

Cocoa butter 25.00

Reducing sugar powder 50.00

Sucralose 0.03

Lecithin 0.40

Vanillin 0.05

Example 4E-26Chocolate

Chocolate was prepared under the same conditions as in example 4-E-25, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-27Strawberry flavored chocolate

A strawberry flavored chocolate was prepared according to the general preparation method of chocolate using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(g)

Cocoa butter 25.00

Hard butter (Hard button) 7.00

Whole milk powder 27.00

Sugar 41.00

Strawberry essence 0.20

Beet Red 0.50

Sucralose 0.001

Lecithin 0.30

Vanillin 0.02

Example 4E-28Strawberry flavored chocolate

A strawberry-flavored chocolate was prepared under the same conditions as in example 4-E-27, except that 0.5g of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-29Small cookie

The following ingredients were mixed to make a dough, which was baked in an oven to make a cookie.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Soft flour 48.50

0.30 of baking powder

Fresh margarine 24.00

Salt 0.20

Sugar 12.00

Yolk 4.70

Trehalose 22.00

Perfume 0.20

Water 40.00

Example 4E-30Small cookie

A cookie was prepared under the same conditions as in example 4-E-29, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-31Steamed bread (Manju) crust (bun stuffed with steamed red beans)

Steamed bread scalp (bun filled with steamed red bean filling) was prepared according to the general preparation method of steamed bread scalp using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference example 1-2) 4.00 (parts by weight)

Soft flour 500.00

2.50 of baking powder

Xanthan gum 1.00

Salt 6.00

Sugar 30.00

Trehalose 30.00

Dry yeast 7.50

Lard 15.00

Water 200.00

Example 4E-32Steamed bread scalp (bun stuffed with steamed red beans)

Steamed bread scalp (bun stuffed with steamed red beans) was prepared under the same conditions as in example 4-E-31 except that 0.5 part by weight of the solid extract prepared by steaming 1-2 parts of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4E-33Apple pie filling

Apple pie fillings were prepared using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40 parts by weight

Apple 200.00

Maltose 150.00

Sucralose 0.10

Lemon juice 10.00

Walnut 60.00

Raisin 60.00

Apple essence 0.20

Example 4E-34Apple pie filling

An apple pie filling was prepared under the same conditions as in example 4-E-33, except that 0.5 parts by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-35Pound cake

The following ingredients were mixed to make a dough, which was baked in an oven to make a pound cake.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40 parts by weight

Butter 100.00

Reducing maltose syrup 100.00

Sucralose 0.03

Stevia rebaudiana Bertoni 0.03

Whole egg 100.00

Perfume 0.22

Soft flour 120.00

1.50 of baking powder

Example 4E-36Pound cake

Pound cakes were prepared under the same conditions as in example 4-E-35, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4E-37Fern starch dumpling

Fern starch dumplings were prepared using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 0.40 parts by weight

Rice starch 20.00

Sweet potato starch 40.00

Sugar 6.00

Balance of water

Total of 100.00 parts by weight

Example 4E-38Fern starch dumpling

Fern starch dumplings were prepared under the same conditions as in example 4-E-37, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl ferment.

Example 4E-39Bean dessert

The bean snack was prepared using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 6.00 (parts by weight)

Kanbaiko (ground baked Rice cake) 7.00

Sugar 53.00

Kinako (roasted soybean meal) 4.00

Fermented flour q.s.

A flavorant q.s.

A sweetener q.s.

Total of 100.00 parts by weight

Example 4E-40Bean dessert

A bean snack was prepared under the same conditions as in example 4-E-39, except that 0.75 parts by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-41 Bean dessert

The bean snack was prepared using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 12.00 (parts by weight)

Rice flour 13.00

Sugar 45.00

Kinako (roasted soybean meal) 15.00

Powdered green tea 15.00

Fermented flour q.s.

A flavorant q.s.

A sweetener q.s.

Total of 100.00 parts by weight

Example 4E-42Bean dessert

A bean snack was prepared under the same conditions as in example 4-E-41, except that 1.5 parts by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-43Bean dessert

The bean snack was prepared using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference example 1-1) 18.00 (parts by weight)

Kanbaiko (ground baked Rice cake) 15.00

Sugar 52.00

Kinako (roasted soybean meal) 15.00

Powdered green tea 12.00

Fermented flour q.s.

A flavorant q.s.

A sweetener q.s.

Total of 100.00 parts by weight

Example 4E-44Bean dessert

A bean snack was prepared under the same conditions as in example 4-E-43, except that 2.25 parts by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-45Bean dessert

The bean snack was prepared using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 6.00 (parts by weight)

Starch 20.00

Sugar 32.00

Kinako (roasted soybean flour) 10.00

Black sesame 20.00

Fermented flour q.s.

A flavorant q.s.

A sweetener q.s.

Total of 100.00 parts by weight

Example 4E-46Bean dessert

A bean snack was prepared under the same conditions as in example 4-E-45, except that 0.75 parts by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-47Nama-fu dessert (fresh gluten cake)

Nama-fu snacks (fresh gluten cakes) were prepared using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 10.00 (parts by weight)

Gluten 20.00

Rice flour 15.00

Kinako (roasted soybean meal) q.s.

An emulsifier q.s.

And (4) honey q.s.

Sorbitol 20.00

Chocolate 30.00

Example 4E-48Nama-fu dessert (fresh gluten cake)

A nama-fu snack (fresh gluten cake) was prepared under the same conditions as in example 4-E-47, except that 1.25 parts by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-49Thin pancake (dense type)

The following ingredients were used to make wafers (thick) according to the general preparation method of wafers.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Wheat flour 100.00

Sugar 2.00

Fats and oils 3.00

Whole egg 3.00

Skimmed milk 2.00

Sodium bicarbonate 0.30

Milk 50.00

Water 100.00

Example 4E-50Thin pancake (dense type)

A pancake (concentrated type) was prepared under the same conditions as in example 4-E-49, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-51Thin pancake (common)

The following ingredients were used to prepare wafers (normal) according to the general preparation method of wafers.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Wheat flour 100.00

Fats and oils 2.50

0.30 whole egg

Ammonium carbonate 0.40

Sodium bicarbonate 0.30

Lecithin 0.05

150.00 g of water

Example 4E-52Thin pancake (common)

A wafer (ordinary) was prepared under the same conditions as in example 4-E-51, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing fermented soybean hypocotyl material.

Example 4E-53Cheese biscuit

Cheese crackers were prepared according to the general preparation method using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Wheat flour 100.00

Fats and oils 9.00

Malt extract 1.30

Sodium bicarbonate 0.60

Spice 0.10

Cheese powder 13.00

Sugar 80.00

Salt 0.90

Ammonium carbonate 0.60

Lecithin 0.05

Water 33.00

Example 4E-54Cheese biscuit

Cheese crackers were prepared under the same conditions as in example 4-E-53, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl ferment.

Example 4E-55Yokanan (Red bean jelly)

Yokanan (red bean jelly) was prepared according to the general preparation method using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

Agar 7.50

Granulated sugar 320.00

Red bean paste 620.00

Starch syrup 50.00

Water 300.00

Example 4E-56Yokanan (Red bean jelly)

Yokan (red bean jelly) was prepared under the same conditions as in example 4-E-55, except that 0.5 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-57Muffin (Soft Cookie)

Muffins were prepared according to the general preparation method using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

Wheat flour 200.00

Butter 150.00

Sugar 80.00

Whole egg 150.00

Example 4E-58Muffin

Muffins were prepared under the same conditions as in example 4-E-57, except that 0.5 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-59Rice cake

The following ingredients were used to prepare rice cakes according to the general preparation method.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Normal rice 100.00

Hot water q.s.

Example 4E-60Rice cake

A rice cake was prepared under the same conditions as in example 4-E-59, except that 0.5 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-61Suhama (Japanese traditional dessert)

Suhama (japanese traditional snack) was prepared according to the general preparation method using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

White high-quality soft white sugar 600.00

Kinako (roasted soybean meal) 400.00

Starch syrup 130.00

180.00 g of water

Example 4E-62Suhama (Japanese traditional dessert)

Suhama (japanese traditional snack) was prepared under the same conditions as in example 4-E-61, except that 0.5 parts by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4E-63Dry dessert

The following ingredients were used to prepare dry desserts according to the general preparation method.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

And three basins (Wasanbon) of sugar 500.00

Shitori (for wetting ingredients during processing) 25.00

Kanbaiko (ground baked Rice cake) 75.00

Example 4E-64Dry dessert

A dry dessert was prepared under the same conditions as in example 4-E-63, except that 0.5 parts by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-65Fruit chewing gum

The following ingredients were used to prepare fruit gummies according to the general preparation method.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

Sugar 30.00

Starch syrup 50.00

Gelatin 7.00

Fruit juice 5.00

Citric acid 1.50

Perfume 0.20

Example 4E-66Fruit chewing gum

A fruit gummy candy was prepared under the same conditions as in example 4-E-65, except that 0.5 parts by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by Et 75 in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4E-67Baked snack containing soybean flour

1.25% by weight of equol-containing fermented soybean hypocotyl (reference example 1 to 3) and 25% by weight of soybean flour, and raisin, butter, sugar, egg, orange peel, pineapple, indigestible dextrin, cacao mass, almond pieces, cocoa powder, salt, baking powder and spices were mixed in appropriate amounts. The resulting mixture (100 wt%) was then molded into a rectangle (about 1.5cm × 1.5cm × 10cm) using a mold to be baked in an oven at 180 ℃, thereby preparing a baked snack containing soybean flour.

Example 4E-68Containing soybean flourBaked snack

A baked snack containing soybean flour was prepared under the same conditions as in example 4-E-67, except that 0.25 parts by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-FSeasoning

In the following, specific preparation examples of the seasoning according to the present invention are given.

Example 4-F-1Barbecue Sauce (Barbecue Sauce)

A barbecue sauce having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(kg)

Reducing starch syrup 20.00

Trehalose 0.50

Mirin wine (Mirin) 4.00

Soy sauce (dark) 24.00

Apple pulp 19.00

Garlic syrup 4.50

Fresh ginger pulp 4.50

Sesame oil 0.10

Xanthan gum 0.50

Capsicum powder 0.10

Black pepper powder 0.05

Citric acid (Crystal) 0.20

Roasted sesame 0.40

Balance of water

A total of 100.00kg

Example 4-F-2Barbecue sauce

Barbecue sauce was prepared under the same conditions as in example 4-F-1, except that 0.5kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-F-3Tomato sauce

A tomato paste having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 4.00(kg)

Tomato sauce 40.00

Wine vinegar 18.00

Fructose/glucose syrup 5.00

Soy sauce (dark) 24.00

2.50 portions of common salt

Garlic powder 0.07

Onion powder 0.10

Cortex Cinnamomi Japonici powder 0.02

Clove powder 0.01

Balance of water

A total of 100.00kg

Example 4-F-4Tomato sauce

Tomato paste was prepared under the same conditions as in example 4-F-3, except that 0.5kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by Et 75 in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-F-5Tsuyu (dipping sauce)

Tsuyuu (dip for tempura, i.e., japanese dry fried food) having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 4.00(kg)

Sugar 7.00

Soy sauce (dark) 26.00

Soy sauce (light) 10.00

Fish sauce (Fish sauce) 5.00

Milin wine 10.00

Sodium L-glutamate monohydrate 3.00

Salt 0.60

Balance of water

A total of 100.00kg

Example 4-F-6Tsuyu (soup)

Tsuyu (soup) was prepared under the same conditions as in example 4-F-5, except that 0.5kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-F-7Seasoning

Having the following composition and mixing the aqueous phase and the oil phase in a weight ratio of 7: 3 to prepare a seasoning (separation type).

Composition of the aqueous phase

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(kg)

Sugar 6.00

Soy sauce (dark) 20.00

Brewed Vinegar (acidity: 10%) 8.50

Fish sauce 3.00

Lemon juice 2.00

Salt 2.00

Sodium L-glutamate monohydrate 0.20

Capsicum powder 0.05

Roasted sesame 0.30

Xanthan gum 0.10

Balance of water

A total of 100.00kg

Composition of oil phase

Sesame oil 20.00(kg)

Corn salad oil 79.00

Condiment 1.00

A total of 100.00kg

Practice ofExample 4-F-8Seasoning

A seasoning (isolated type) was prepared under the same conditions as in example 4-F-7, except that 0.5kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-F-9Sesame dipping sauce for Japanese chafing dish (Shabu-Shabu) (boiling sliced meat and vegetables in hot water rapidly)

Sesame dipping sauce for japanese chafing dish having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40(kg)

Soy sauce (light) 12.00

Brewed Vinegar 11.00

0.10% of garlic powder

Ginger powder 0.10

White pepper 0.10

Chicken meat extract 3.00

Yeast extract 3.00

Sesame oil 1.00

Fructose/glucose syrup 8.00

15.00% mirin wine

White miso 10.00

Thickener 3.50

Sugar 8.00

Salt 10.00

Fragrance 0.15

Balance of water

A total of 100.00kg

Example 4-F-10Sesame dipping sauce for Japanese chafing dish (boiling sliced meat and vegetables in hot water rapidly)

A sesame dip for Japanese chafing dish was prepared under the same conditions as in example 4-F-9, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4F-11Barbecue sauce (miso flavor)

A barbecue paste (miso flavor) having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 4.00(kg)

Soy sauce (dark) 25.00

Miso 14.00

D-sorbitol 12.00

Common salt 3.20

Garlic powder 3.30

Garlic syrup 2.00

Beef extract 1.00

Roasted sesame 0.50

Sodium L-glutamate monohydrate 0.50

Ginger juice 0.40

Amino acid flavoring agent 0.01

Thickener 3.50

Sugar 10.00

Balance of water

A total of 100.00kg

Example 4-F-12Barbecue sauce (miso wind)Flavor)

Barbecue paste (miso flavor) was prepared under the same conditions as in example 4-F-11, except that 0.05kg of the solid extract prepared by evaporating 1-2 to dryness of the liquid extract obtained with 75Et in example 1-1 was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-F-13Snack seasoning

A snack condiment having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Common salt 14.00

Tricalcium phosphate 3.00

Disodium succinate 0.20

Sodium L-glutamate monohydrate 7.00

Powdery soy sauce 3.00

Yeast extract 1.50

Onion powder 1.50

Capsicum powder 1.50

Garlic powder 6.00

Capsicum powder 0.50

Chicken soup 21.00

Beef soup 4.00

Stevia rebaudiana 0.60

Glucose 13.00

Spice 0.01

Corn starch 23.00

Sugar 2.00

Example 4-F-14Snack seasoning

A snack seasoning was prepared under the same conditions as in example 4-F-13, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-F-15Emulsified soy sauce

An emulsified soy sauce having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Salad oil 34.00

Soy sauce (dark) 53.00

Arabic gum 1.00

Sugar 12.00

Example 4-F-16Emulsified soy sauce

An emulsified soy sauce was prepared under the same conditions as in example 4-F-15, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained by using 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-F-17Seasoning

A seasoning (emulsified type) having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Salad oil 35.00

Vinegar 10.00

Sugar 11.00

Salt 4.00

Xanthan gum 0.30

Balance of water

Total of 100.00 parts by weight

Example 4-F-18Seasoning

A seasoning (emulsion type) was prepared under the same conditions as in example 4-F-17, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-F-19Sauce for roasted eel (roasted seafood)

A sauce for roasted eel (roasted seafood) having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

D-sorbitol 10.00

Thickener 0.30

Soy sauce (dark) 38.00

20.00% mirin wine

Umami extract 2.00

Common salt 1.80

Sodium L-glutamate monohydrate 1.45

Inosinic acid sodium 0.025

Guanylate 0.025

Trehalose 15.00

Sugar 6.00

Caramel color 0.50

Onion pigment 0.10

Perfume 0.50

Balance of water

Total of 100.00 parts by weight

Example 4-F-20Sauce for roasted eel (roasted seafood)

A dressing for roasted eel (roasted seafood) was prepared under the same conditions as in example 4-F-19 except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-F-21Seasoning for squid delicacy

A seasoning for squid taste having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference example 1-2) 0.40 parts by weight

Soy sauce (dark) 20.00

Soy sauce (light) 10.00

Fish sauce 5.00

Milin wine 10.00

Brewing vinegar 2.00

Sugar 38.00

Common salt 1.00

Sodium L-glutamate monohydrate 2.00

Umami extract 1.40

Disodium succinate 0.20

0.05% of garlic powder

Balance of water

Total of 100.00 parts by weight

Example 4-F-22Seasoning for squid delicacy

A seasoning for squid flavor was prepared under the same conditions as in example 4-F-21, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-GSterilization bagged food

In the following, specific preparation examples of the retort pouch food according to the present invention are given.

Example 4-G-1Sterilization bagged curry

The following composition was used to prepare retort curry in accordance with the general preparation method.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Curry powder 1.80

Onion 7.00

Fresh butter 4.50

Bulbus Allii pulp 0.60

Ginger pulp 0.50

Wheat flour 4.50

Fruit hot and sour sauce 3.00

Tomato sauce 0.50

Salt 0.55

Sodium L-glutamate monohydrate 0.48

Nucleic acid seasoning 0.015

Semi-iced sauce flavor 1.00

Pork extract flavoring agent 0.50

Bouillon flavor 1.00

Potato 7.00

Carrot 5.00

Beef 10.00

Balance of water

A total of 100.00kg

Example 4-G-2Sterilization bagged curry

Sterilized bagged curry was prepared under the same conditions as in example 4-G-1, except that 0.05kg of the solid extract prepared by evaporating 1-2 to dryness of the liquid extract obtained with 75Et in example 1-1 was used instead of the equol-containing fermented soybean hypocotyl.

Example 4-G-3Chinese rice-covered premix in sterilization bag

The following composition was used to prepare a retort pouch of chinese rice meal premix according to the general preparation method.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Soy sauce (light) 72.00

Sake (sake) 46.00

Salt 4.00

Sodium L-glutamate monohydrate 1.30

Nucleic acid seasoning 0.07

Chicken extract powder 1.00

Sugar 7.00

Water 209.00

Pork 200.00

Loligo chinensis 120.00

Shrimp 80.00

Shiitake mushroom (Lentinus edodes) 70.00

Boiled bamboo shoot 160.00

Carrot 60.00

Onion 240.00

Example 4-G-4Chinese rice-covered premix in sterilization bag

A retort pouch Chinese rice covered premix was prepared under the same conditions as in example 4-G-3, except that 0.05kg of a solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-HProcessed meat product

In the following, specific examples of processed meat products according to the invention are given.

Example 4-H-1Sausage

The sausage was prepared according to the general preparation method of sausage using the ingredients of the following composition.

Meat part

5 mm-chopped pork (front leg) 70.00(kg)

5 mm-chopped pork (lard) 10.00

Ice Water 20.00

A total of 100.00kg

Auxiliary ingredients

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Common salt 1.70

Sodium nitrite 0.012

Polyphosphate salt 0.30

Ascorbic acid sodium 1.00

Sodium caseinate (Sodium casein) 0.50

White pepper powder 0.10

Potassium sorbate 0.20

Amino acid flavoring agent 0.30

Spice mixture 0.50

Example 4-H-2Sausage

Sausages were prepared under the same conditions as in example 4-H-1, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-H-3Smoked sausage

The meat portion and the seasoning, each having the following ingredients, were mixed in a ratio of 16: 1. Using the resulting mixture, smoked sausages were prepared according to the general preparation method of smoked sausages.

Meat part

Chopped pork (front leg) 85.00(kg)

Whole egg 4.00

Milk 3.00

Ice Water 7.00

Seasoning

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Salt 24.00

Amino acid seasoning 3.20

Spice 8.65

Chopped onion 16.00

Sugar 48.00

Example 4-H-4Smoked sausage

Smoked sausages were prepared under the same conditions as in example 4-H-3, except that 0.05kg of the solid extract prepared by evaporating 1-2 to dryness of the liquid extract obtained with 75Et in example 1-1 was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-H-5Hamburger steak

Hamburger steak was prepared according to the general preparation method using the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Ground beef and pork mixture 45.00

Pork 9.00

Onion 12.00

Whole egg 5.00

Granular soy protein 9.00

Casein sodium salt 5.00

Carrageenan 1.50

Locust Bean Gum 0.50

Spice 0.30

Yeast extract 0.20

Common salt 1.00

Sugar 1.20

Water soluble dietary fiber 1.00

Balance of water

Total of 100.00 parts by weight

Example 4-H-6Hamburger steak

Hamburger steak was prepared under the same conditions as in example 4-H-5 except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-IFish paste

In the following, specific preparation examples of the fish paste according to the present invention are given.

Example 4-I-1Fried fish cake (Kamaboko) (fish paste cake)

The fried fish (fish paste cake) was prepared according to a general preparation method using the following ingredients.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Minced fish (Surimi) 50.00

Common salt 1.50

Amino acid flavoring agent 0.30

Glycine 0.50

Anhydrous sodium acetate 0.30

Potato starch 8.00

1.00% Milin wine

Water balance

A total of 100.00kg

Example 4-I-2Fried fish cake (Fish sauce cake)

A fried fish cake (fish-paste cake) was prepared under the same conditions as in example 4-I-1, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-JProcessed egg product

In the following, specific preparation examples of processed egg products according to the invention are given.

Example 4-J-1Thick Japanese omelet

The thick japanese omelet was prepared according to the general preparation method of the thick japanese omelet using the ingredients of the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Whole egg 70.00

Salt 0.40

Starch 2.00

Soup blend 2.00

Sugar 1.20

Sodium acetate 1.00

Balance of water

Total of 100.00 parts by weight

Example 4-J-2Thick Japanese omelet

Prepared under the same conditions as in example 4-J-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-KCanned food

In the following, specific preparation examples of the canned food according to the present invention are given.

Example 4-K-1Orange can

Canned citrus was prepared by filling citrus pulp and a syrup having the following composition.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Sugar 10.00

Fructose/glucose syrup 10.00

Citric acid 0.25

Trisodium citrate 0.10

Balance of water

Total of 100.00 parts by weight

Example 4-K-2Orange can

A citrus jar was prepared under the same conditions as in example 4-K-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-LBread product

In the following, specific preparation examples of the bread according to the invention are given.

Example 4L-1Bar-shaped bread

Loaves were prepared using a dough having the following composition.

Hair-like dough

Strong flour 70.00(kg)

Yeast food 0.10

Live yeast 2.00

Water 40.00

Major dough

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40(kg)

Strong flour 30.00

Sugar 3.00

Salt 2.00

Skimmed milk 2.00

Shortening 5.00

Water 25.00

Example 4L-2Bar-shaped bread

Loaves were prepared under the same conditions as in example 4-L-1, except that 0.05kg of the solid extract prepared by evaporating 1-2 of the liquid extract obtained by 75Et in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl ferment.

Example 4-MFrozen dessert

In the following, specific preparation examples of frozen desserts according to the invention are given.

Example 4M-1Ice cream

An ice cream having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Coarse butter 8.80

Butter 5.00

Starch syrup 16.00

Skimmed milk 6.50

Trehalose 7.00

Sugar 5.00

Stabilizer 0.20

Fragrance 0.15

Balance of water

Total of 100.00 parts by weight

Example 4M-2Ice cream

An ice cream was prepared under the same conditions as in example 4-M-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained by Et 75 in example 1-1 to dryness was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-NProcessed soybean food

Next, specific preparation examples of the processed soybean food according to the present invention are given.

Example 4N-1Fermented soybean

99.6 parts by weight of steamed soybeans and 0.4 part by weight of equol-containing fermented soybean hypocotyls were mixed (refer to examples 1 to 3). Then inoculating the obtained mixture with Bacillus natto (Bacillus natto) for fermentation; thereby preparing fermented soybeans.

Example 4N-2Fermented soybean

Fermented soybeans were prepared under the same conditions as in example 4-N-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl ferment.

Example 4-N-3Bean curd

A bean curd having the following composition was prepared.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Soymilk 99.30

Magnesium chloride 0.20

Potassium sorbate 0.10

Total of 100.00 parts by weight

Example 4-N-4Bean curd

Bean curd was prepared under the same conditions as in example 4-N-3, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained by 75Et to dryness in example 1-1 was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-OCooked rice product

In the following, specific preparation examples of cooked rice products according to the invention are given.

Example 4-O-1Rice gruel

The rice gruel was prepared using the ingredients of the following composition according to the general preparation method of rice gruel.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Rice 160.00

Common salt 1.00

Water 900.00

Example 4-O-2Rice gruel

Rice porridge was prepared under the same conditions as in example 4-O-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Example 4-O-3Steamed glutinous rice with chestnut

The steamed glutinous rice with chestnut was prepared according to the general preparation method using the following ingredients.

Equol-containing fermented product of soybean hypocotyl (reference examples 1 to 3) 4.00 (parts by weight)

Glutinous rice 1000.00

Peeled chestnut 300.00

Soup stock 230.00

Semen Sesami Niger 30.00

Salt solution 6.00

Example 4-O-4Steamed glutinous rice with chestnut

Steamed glutinous rice with chestnuts was prepared under the same conditions as in example 4-O-3, except that 0.5 parts by weight of the solid extract prepared by evaporating 1-2 parts by weight of the liquid extract obtained with 75Et to dryness in example 1-1 was used instead of the equol-containing soybean hypocotyl fermented product.

Example 4-PSoup

In the following, specific preparation examples of the soup according to the invention are given.

Example 4P-1 Corn butter soup

The following ingredients were used to prepare corn cream soup according to the general preparation method.

Equol-containing fermented soybean hypocotyl (reference examples 1 to 3) 0.40 parts by weight

Milk 180.00

Corn 190.00

Stewed broth 20.00

Salad oil 15.00

Common salt q.s.

Pepper q.s.

Water 360.00

Example 4P-2Corn butter soup

Corn cream soup was prepared under the same conditions as in example 4-P-1, except that 0.05 part by weight of the solid extract prepared by evaporating 1-2 parts of the liquid extract obtained with 75Et in example 1-1 to dryness was used in place of the equol-containing soybean hypocotyl fermented product.

Brief description of the drawings

FIG. 1 is an electrophoretogram showing total proteins contained in the equol-containing soybean hypocotyl fermented product, soybean cotyledon and soybean hypocotyl obtained in reference example 1-1.

Fig. 2 is an electrophoretogram showing the results of detection of major allergens (Gym4, Gm30K, and Gm28K) contained in equol-containing soybean hypocotyl ferment, soybean cotyledon, and soybean hypocotyl obtained in reference example 1-1.

FIG. 3 is an electrophoresis chart showing the results of detection of major allergens (7S globulin mixture, oleosin and trypsin inhibitor) contained in the equol-containing soybean hypocotyl fermented product, soybean cotyledon and soybean hypocotyl obtained in reference example 1-1.

FIG. 4 shows the results of TLC analysis showing the equol content of the liquid extracts 1-2 and 2-2 in example 1-1.

FIG. 5 shows the results of TLC analysis showing the saponin contents of the liquid extracts 1-2 and 2-2 in example 1-1.

FIG. 6 shows the results of TLC analysis showing the equol contents of the liquid extracts 1-2 and the residue 2 in example 1-1.

FIG. 7 shows the results of TLC analysis showing the saponin contents of the liquid extracts 1-2 and the residue 2 in example 1-1.

FIG. 8 shows the result of TLC analysis of the solution eluted by silica gel column chromatography in example 2-1.

Claims (10)

1. A method for preparing an equol-containing and saponin-reduced extract comprising:

step I-1: extracting the equol-containing fermented soybean hypocotyl with an aqueous ethanol solution as an extractant, and collecting the resultant liquid extract, and

step I-2: removing the extractant from the liquid extract obtained in step I-1, performing extraction using ethanol as the extractant, and collecting the resulting liquid extract.

2. The process for producing an equol-containing and saponin-reduced extract according to claim 1, wherein the ethanol concentration of the ethanol aqueous solution used in step I-1 is 45 to 95 vol%.

3. The method for preparing an equol-containing and saponin-reduced extract according to claim 1, further comprising:

step I-3: removing the solvent from the liquid extract obtained in step I-2, suspending the resulting residue in hexane, and then collecting the insoluble residue;

step I-4: extracting the insoluble residue obtained in step I-3 using a mixture of hexane and ether as an extractant, and then collecting a liquid extract; and

step I-5: subjecting the liquid extract obtained in step I-4 to silica gel column chromatography, and obtaining an equol-containing fraction.

4. The method for producing an equol-containing and saponin-reduced extract according to any one of claims 1 to 3, wherein the equol-containing soybean hypocotyl fermented product is obtained by fermenting a soybean hypocotyl with an equol-producing microorganism.

5. The method of claim 3, wherein the extractant used in step I-4 is a mixture of hexane and diethyl ether.

6. The method according to claim 3, wherein the extractant used in step I-4 is a mixture of hexane and ether in a volume ratio of 10: 90 to 20: 80.

7. The method according to claim 5, wherein the extractant used in step I-4 is a mixture of hexane and diethyl ether in a volume ratio of 10: 90 to 20: 80.

8. An equol-containing extract obtained by the method of any one of claims 1-3.

9. An equol-containing food material obtained by the method of any one of claims 1 to 3.

10. A material comprising equol and glycitein compounds obtained by the method of any one of claims 1 to 3.