Title:
Process for producing aglycon and flavor-improved food containing the aglycon by diglycosidase, and converting agent to be used in the process
Kind Code:
A1


Abstract:
A physiologically active substance of aglycon type, in particular, aglycon isoflavone, can be efficiently produced, without resort to any acid/alkali treatment or fermentation and substantially without changing the physical properties of a material, by treating the material with a sufficient amount of diglycosidase for a sufficient period of time at an appropriate temperature and pH so that a physiologically active substance of glycoside type contained in the material can be converted into the physiologically active substance of aglycon type. Moreover, by using diglycosidase and/or a specific enzyme preparation, the aglycon content in a protein or protein-containing food can be increased and the flavor thereof can be improved.



Inventors:
Tsuruhami, Kazutaka (Kakamigahara-shi, JP)
Toumoto, Atsuki (Kakamigahara-shi, JP)
Goto, Masataka (Kakamigahara-shi, JP)
Koikeda, Satoshi (Kakamigahara-shi, JP)
Application Number:
11/444358
Publication Date:
09/28/2006
Filing Date:
06/01/2006
Assignee:
AMANO ENZYME INC.
Primary Class:
Other Classes:
435/69.1, 426/29
International Classes:
A61K38/47; A23L1/30; A23L5/20; A23L7/10; C12N9/24; C12P17/06; C12P21/06
View Patent Images:
Related US Applications:



Primary Examiner:
PROUTY, REBECCA E
Attorney, Agent or Firm:
Locke Lord LLP (P.O. BOX 55874, BOSTON, MA, 02205, US)
Claims:
1. A process for producing a protein having an increased aglycon content or a food containing the protein, which comprises a step of treating a protein or protein-containing food with diglycosidase, wherein said diglycosidase has activity to act upon a disaccharide glycoside to release saccharides in a disaccharide unit.

2. The process for producing a protein having an increased aglycon content or a food containing the protein according to claim 1, wherein the protein or protein-containing food contains a glycoside containing an isoflavone as the aglycon.

3. The process for producing a protein having an increased aglycon content or a food containing the protein according to claim 1 or 2, wherein the protein or protein-containing food to be produced is a further flavor-improved one.

4. The process for producing a protein having an increased aglycon content or a food containing the protein according to claim 1 or 2, wherein the glycoside containing an isoflavone as the aglycon is one or more selected from the group consisting of daidzin, genistin, or glycitin and acetyl derivatives, succinyl derivatives, or malonyl derivatives thereof.

5. The process for producing a protein having an increased aglycon content or a food containing the protein according to claim 1 or 2, which further comprises a step of treating with an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, proteases, lipases, β-glucosidases, and yeast-dissolving enzymes.

6. The process for producing a protein having an increased aglycon content or a food containing the protein according to claim 1 or 2, wherein the improvement of flavor is reduction of bitterness and/or astringency.

7. A process for producing a flavor-improved protein or a food containing the protein, which comprises a step of treating a protein or protein containing food with a diglycosidase and an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, cellulases, pectinases, proteases, lipases, β-glucosidases, β-galactosidases, and yeast-dissolving enzymes, wherein said diglycosidase has activity to act upon a disaccharide glycoside to release an aglycon and saccharides in a disaccharide unit.

8. The process for producing a flavor-improved protein or a food containing the protein according to claim 7, wherein the protein or protein-containing food contains a glycoside containing a flavonoid as the aglycon.

9. The process for producing a flavor-improved protein or a food containing the protein according to claim 7 or 8, wherein the protein or protein-containing food contains a glycoside containing an isoflavone as the aglycon.

10. A method of administering diglycosidase orally to form an aglycon from a glycoside in a living body, wherein said diglycosidase has activity to act upon a disaccharide glycoside to release saccharides in a disaccharide unit.

11. The method according to claim 10, wherein diglycosidase is orally administered to form an aglycon in a living body from a glycoside containing an isoflavone as the aglycon.

12. A method of accelerating a bioabsorption of a physiologically active substance, which comprises administering diglycosidase orally before, during, or after the ingestion of a food containing a physiologically active substance of glycoside type, wherein said diglycosidase has activity to act upon a disaccharide glycoside to release saccharides in a disaccharide unit.

13. An agent for converting a physiologically active substance of glycoside type into the physiologically active substance of aglycon type, which contains at least a diglycosidase, wherein said diglycosidase has activity to act upon a disaccharide glycoside to release saccharides in a disaccharide unit.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 10/204,679 filed Aug. 23, 2002, which is a 371 of PCT international application No. PCT/JP01/02656 filed Mar. 29, 2001; the above-noted applications incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a process for producing an aglycon, a process for producing a protein having an increased aglycon content or a food containing said protein, a process for producing a flavor-improved protein or a food containing said protein, and a process for forming an isoflavone in a living body. The invention may be utilized, for example, for producing processed foods, health foods, dietary supplements, and medicaments.

BACKGROUND ART

Glycosides are chemical substances wherein a saccharide is bonded to a non-saccharide part called aglycon, and are widely present in nature.

Some glycosides are known to exhibit physiological activity through decomposition into aglycons by a glycosidase such as glucosidase produced by enteric bacteria though the glycosides themselves do not exhibit the physiological activity because of their poor enteric absorption, as phytochemicals (phytogenic functional ingredients) such as polyphenols having antioxidation action and phytoestrogens having a weak estrogenic action. Thus, in view of preventing and symptom-alleviating effects of life-style related diseases such as cancer, arteriosclerosis, osteoporosis, and climacteric disorder, and diseases owing to aging, attention has been focused on soybean protein concentrates, soybean-processed materials, and food containing the soybean protein concentrates.

Among the phytochemicals, glucosides that contain isoflavones as aglycons as represented by the following general formula (hereinafter, sometimes referred to as isoflavone glucosides) contain aglycons such as daidzein, genistein, glycitein, and it is revealed from cellular level investigations and epidemiological surveys that they inhibit growth of breast cancer and prostatic cancer cells, alleviate arteriosclerosis and osteoporosis, and also alleviate climacteric disorder owing to the female hormone-like estrogenic action. embedded image
(wherein R1 and R2 each is independently selected from the group consisting of H, OH, and OCH3, and R3 is selected from the group of H, COCH3, COCH2COOH, and COCH2CH2COOH)

However, there is a possibility that a sufficient amount of glycosidase cannot be produced by enteric bacteria in elderly persons, sick persons, and antibiotics-administered patients, and also a glycosidase is difficult to decompose glycosides modified with acetyl, or malonyl group and disaccharide or trisaccharide glycosides, so that it cannot be expected to absorb a sufficient amount of phytochemicals contained in soybean protein concentrates and the like.

Moreover, it is avoided to take foods containing soybean-processed materials such as soybean protein concentrates especially in Western countries owing to the distinctive smell and bitterness, and thus some limitation exists as sources of aglycon isoflavones.

Accordingly, a process for forming phytochemicals efficiently in a living body, and an improvement of flavor of a soybean-processed material rich in aglycon isoflavones, which are highly efficiently absorbed and capable of ingesting a sufficient amount of isoflavones by taking small amount of them, and having less smell and bitterness distinctive of soybean or of a food containing the soybean-processed material have been desired.

On the other hand, the method for converting an isoflavone glucoside into an aglycon isoflavone is known as described in JP-A-10-117792. In this method, a phytogenic protein extract is treated with an alkali to convert a modified glucoside isoflavone into a glucoside isoflavone, which is then subjected to a treatment with glycosidase. Such a treatment is carried out because conventional glycosidase cannot act directly on the modified glucoside isoflavone. Thus, the method is accompanied by the problems of requirement of two steps, enhancement of bitterness by the alkali-treatment, change in physical properties and ingredients, formation of by-products, waste liquid after the alkali-treatment, and the like. Also, glucosidase which takes charge of a main part of the action tends to be influenced by free glucose, so that the kind and concentration of the material used for the production may be limited.

Moreover, JP-A-8-214787 describes a process for converting a glucoside isoflavone into an aglycon isoflavone by fermentation using a microorganism. However, there are possibilities of decomposing the resulting aglycon isoflavone by the microorganism and of forming unexpected by-products, and therefore many problems may arise at the actual production.

Furthermore, a method of hydrolysis with an acid such as hydrochloric acid may be a candidate, but the decomposition of proteins, phospholipids, neutral lipids, and other ingredients may occur along with formation of by-products because of the severe conditions. Especially, the formation of chlorinated compounds such as MCP (monochloropropanol) and DCP (dichloropropanol) whose carcinogenicity has been reported cannot be avoided.

Therefore, an object of the invention is to provide a process for producing a phytogenic physiologically active substance of aglycon type efficiently without resort of any acid/alkali treatment or fermentation and substantially without changing the physical properties of a material. Moreover, other objects of the invention are to enhance the aglycon content in a protein or a protein-containing food by using diglycosidase and/or a specific enzyme preparation and to improve the flavor. These objects and other objects will be further clarified by the following detailed explanations.

DISCLOSURE OF THE INVENTION

As a result of extensive studies, we have found that diglycosidase discovered from origins of various microorganisms efficiently decompose glycosides which are difficult to decompose by conventional glycosidase and also acts even in a living body, and thus accomplished the invention. Furthermore, we have found that the invention can be conducted using any diglycosidase from any source.

Namely, the invention relates to the following.

(1) A process for producing an aglycon which comprises forming an aglycon by treating, with diglycosidase, a glycoside containing a compound selected from the group consisting of phytoestrogens, polyphenols, isoflavones, biochanin A, formononetin, cumestrol, and lignans as the aglycon.

(2) The process for producing an aglycon according to claim 1, wherein the aglycon is an isoflavone.

(3) The process for producing an aglycon as described above, wherein the glycoside containing an isoflavone as the aglycon is one or more selected from the group consisting of daidzin, genistin, or glycitin and acetyl derivatives, succinyl derivatives, or malonyl derivatives thereof.

(4) The process for producing an aglycon as described above, wherein the diglycosidase is a glucose-tolerant one.

(5) The process for producing an aglycon as described above, wherein the diglycosidase is diglycosidase produced by Penicillium multicolor IAM7153.

(6) A process for producing a protein having an increased aglycon content or a food containing the protein, which comprises a step of treating a protein or protein-containing food with diglycosidase.

(7) The process for producing a protein having an increased aglycon content or a food containing the protein as described above, wherein the protein or protein-containing food contains a glycoside containing an isoflavone as the aglycon.

(8) The process for producing a protein having an increased aglycon content or a food containing the protein as described above, wherein the protein or protein-containing food to be produced is a further flavor-improved one.

(9) The process for producing a protein having an increased aglycon content or a food containing the protein as described above, wherein the glycoside containing an isoflavone as the aglycon is one or more selected from the group consisting of daidzin, genistin, or glycitin and acetyl derivatives, succinyl derivatives, or malonyl derivatives thereof.

(10) The process for producing a protein having an increased aglycon content or a food containing the protein as described above, which further comprises a step of treating with an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, proteases, lipases, α-glucosidases, and yeast-dissolving enzymes.

(11) The process for producing a protein having an increased aglycon content or a food containing the protein as described above, wherein the improvement of flavor is reduction of bitterness and/or astringency.

(12) A process for producing a flavor-improved protein or a food containing the protein, which comprises a step of treating with an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, cellulases, pectinases, proteases, lipases, α-glucosidases, α-galactosidase, and yeast-dissolving enzymes.

(13) The process for producing a flavor-improved protein or a food containing the protein as described above, wherein the protein or protein-containing food contains a glycoside containing a flavonoid as the aglycon.

(14) The process for producing a flavor-improved protein or a food containing the protein as described above, wherein the protein or protein-containing food contains a glycoside containing an isoflavone as the aglycon.

(15) A method of administering diglycosidase orally to form an aglycon from a glycoside in a living body.

(16) The method as described above, wherein diglycosidase is orally administered to form an aglycon in a living body from a glycoside containing an isoflavone as the aglycon.

(17) A method of converting a physiologically active substance of glycoside type into a physiologically active substance of aglycon type, which comprises treating the physiologically active substance of glycoside type with diglycosidase.

(18) A process for producing a composition rich in a phytogenic physiologically active substance of aglycon type, which comprises treating a phytogenic material containing a phytogenic physiologically active substance of glycoside type with diglycosidase.

(19) A method of accelerating a bioabsorption of a physiologically active substance, which comprises administering diglycosidase orally before, during, or after the ingestion of a food containing a physiologically active substance of glycoside type.

(20) An agent converting a physiologically active substance of glycoside type into the physiologically active substance of aglycon type, which contains at least diglycosidase.

These embodiments and other embodiments of the invention will be further clarified by the following detailed explanations.

Diglycosidase described in the invention efficiently acts on the glycosides that contain a compound selected from the group consisting of phytoestrogens, polyphenols, isoflavones, biochanin A, formononetin, cumestrol, and lignans as the aglycon (hereinafter, also referred to as aglycon glycoside), can very efficiently act especially on the glycosides containing an isoflavone as the aglycon (hereinafter, also referred to as isoflavone glycosides), and is hardly influenced by free glucose. Therefore, the process can be advantageously carried out in the case that the isoflavone glycoside is daidzin, genistin, or glycitin, or an acetyl derivative, succinyl derivative, or malonyl derivative thereof. By the way, the isoflavone formed from the isoflavone glycoside is also referred to as aglycon isoflavone.

Moreover, by administering the diglycosidase, the preventive effect on various diseases can be enhanced through the formation of an isoflavone from an aglycon glycoside or a food containing the same which is orally ingested. Diglycosidase produced by Penicillium multicolor IAM7153 is preferably used as diglycosidase. β-Galactosidase derived from Penicillium multicolor is an enzyme which is described in Food Additives List and whose safety is recognized, and thus diglycosidase produced by such highly safety bacterium is estimated to be highly safe.

Furthermore, in the case of using soybean as a starting material, although the benefit of soybean protein concentrates and soybean-processed materials to health is reported, ingestion of them are avoided especially in Western countries owing to the distinctive smell and bitterness. The above-mentioned acetylglycoside isoflavones and malonylglycoside isoflavones have a strong bitterness but the aglycon isoflavones have less bitterness. Therefore, a food wherein bitterness of soybean protein concentrate or soybean-processed material is efficiently reduced can be provided by the conversion into aglycon isoflavones by diglycosidase.

When such soybean protein concentrate or soybean-processed material wherein isoflavones are concentrated as aglycons having higher absorption efficiency is used, a sufficient amount of isoflavones can be ingested through a little intake, and also the form can be changed to a form easily ingested by people who avoid the smell and strange taste distinctive of soybean.

Moreover, owing to the smell and strange taste of soybean protein concentrates or soybean-processed materials, the amount used is limited in the case of using them as food materials, and therefore soybean protein concentrates or soybean-processed materials have a limitation as isoflavone sources for foods. According to the invention, use of the soybean protein concentrates or soybean-processed materials, wherein isoflavone glycosides are digested with diglycosidase and concentrated as isoflavone aglycons which have higher absorption efficiency and less bitterness, enables supply of isoflavones to foods through a little use of the concentrates or materials, so that they can be utilized for many kinds of foods as isoflavone sources.

Diglycosidase for use in the invention is characterized in that it has an activity of acting on a disaccharide glycoside, which is difficult to utilize as a substrate by conventional glucosidase, to isolate a saccharide as a two-saccharide unit from the disaccharide glycoside and also to form an aglycon. Herein, an enzyme having the above activity is referred to as “diglycosidase”.

The diglycosidase in the invention is an enzyme which is classified into a saccharide-chain hydrolase but has a property different from the properties of conventional α- and β-glycosidases. Diglycosidase can utilize, as a substrate, so-called a glycoside wherein a linear or branched saccharide chain composed of single or two or more kinds of saccharides is bonded to a compound other than a saccharide through hydroxyl group in the saccharide chain, and recognizes the substrate at the two-saccharide unit to cleavage it, whereby corresponding disaccharide and an aglycon having a saccharide chain with two saccharide-smaller chain length are formed successively and finally, an aglycon is formed. Additionally, it also decomposes modified glucosides such as acetyl derivatives, succinyl derivatives, and malonyl derivatives, which are difficult to decompose by conventional glucosidase, into saccharides and aglycons. As representative examples of saccharides present in nature, starch, cellulose, polysaccharides constituting cell walls, and the like may be mentioned. Many kinds of saccharide chains may be suitable for the saccharide chains of glycosides, and examples thereof include 6-O-β-D-xylopyranosyl-β-D-glucopyranoside (β-primeveroside), 6-O-α-L-arabinopyranosyl-β-D-glucopyranoside (vicianoside), 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside, 6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside (rutinoside), 6-O-β-D-apiofuranosyl-β-D-glucopyranoside, 6-O-β-D-glucopyranosyl-β-D-glucopyranoside (gentiobioside), 4-O-α-glucopyranosyl-β-D-glucopyranoside (maltose), 2-O-α-L-rhamnopyranosyl-β-D-galactopyranoside (rhamninose), 6-O-α-L-rhamnopyranosyl-p-D-galactopyranoside (robinobioside), 2-O-β-D-xylopyranosyl-β-D-glucopyranoside (xylosylglucose), 4-O-β-D-glucopyranosyl-β-D-glucopyranoside (cellobioside), xylobioside, and the like. Other than the above-mentioned compounds, any combination of saccharides can be recognized as a substrate for the reaction as far as the combination has a disaccharide structure. Aglycon means a compound to be obtained from a glycoside by eliminating a saccharide of the glycoside. Aglycons of glycosides are widely present in nature, and examples thereof include volatile compounds in plants such as linalool, geraniol, citronellal, phenethyl alcohol, citronellol, jasmones, limonene, terpinene, citral, nerol, pinene, borneol, terpineol, methyl jasmonate, hexanol, hexenol, hexanal, hexenal, vanillin, benzaldehyde, eugenol, methyl salicylate, linalool oxide, benzyl alcohol, and vomifomitol; pigments in plants such as alizarin, purpurin, anthocyanidin including pellagonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin; and flavonoids such as nariltin, naringenin, hesperetin, neohesperetin, diosmetin, quercetin, campherol, myricetin, isorhamnetin, and syringenin; and the like. Other than the compounds mentioned herein, various compounds may be present as aglycons of glycosides or may become aglycons of glycosides.

Furthermore, diglycosidase can utilize so-called monosaccharide glycosides, wherein one molecule of saccharide is bonded to an aglycon, as substrates to form corresponding monosaccharides and aglycons, other than above-mentioned disaccharide-isolating activity. In particular, it is a characteristic that diglycosidase can act on monosaccharide glycosides which is resistant to hydrolysis by conventional β-glucosidase.

Diglycosidase for use in the invention can be obtained from microorganisms having ability of producing diglycosidase without requiring undue experimental burden from those skilled in the art (For example, cf. WO00/18931).

The microorganisms producing diglycosidase of the invention can be obtained by the following screening, for example. That is, an enrichment culture is carried out by inoculating a soil suspension to a liquid medium for separation containing eugenyl primeveroside or the like as sole carbon source, applying the culture liquid onto a similar plating agar medium for separation and selecting colonies grown. These strains are cultured in a suitable liquid medium and strains having pNP-isolating activity can be selected through cleavage of disaccharide from pNP-primeveroside or the like.

On these strains thus selected, microorganisms producing diglycosidase can be screened using pNP-primeveroside or the like as a substrate and disaccharide isolation as a measure.

The producing ability has been already confirmed on Aspergillus niger IFO4407 (available from Institute of Fermentation, 2-17-85, Juso-honmachi, Yodogawa-ku, Osaka), Aspergillus niger IAM 2020, Aspergillus fumigatus IAM2046, Penicillium multicolor IAM7153 (available from Institute of Molecular Cell Biology, the University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo), and the like.

Additionally, in other various microorganisms, the diglycosidase activity has been confirmed on various microorganisms such as the genus Aspergillus, the genus Penicillium, the genus Rhizopus, the genus Rhizomucor, the genus Talaromyces, the genus Mortierella, the genus Cryptococcus, the genus Microbacterium, the genus Corynebacterium, the genus Actinoplanes, and the like.

Any strain can be used in the invention as far as it has an ability of producing diglycosidase, and the strain is not limited to the above-mentioned strains. Furthermore, the process for producing diglycosidase usable in the invention includes mutant strains of the strains having a diglycosidase-producing ability, or various microorganisms or various cells (e.g., yeast cells, bacterial cells, higher plant cells, and animal cells) modified so as to be capable of producing diglycosidase by recombinant DNA method, and particularly preferred are those modified so as to be capable of producing diglycosidase with high productivity. In the case that a diglycosidase-producing ability is imparted by introducing a diglycosidase gene, the microorganism used as a host may not have a diglycosidase-producing ability.

For producing diglycosidase using the above various microorganisms, a method and conditions suitable for the culture of the microorganism can be set, and the method and conditions are not particularly limited. For example, any of liquid culture and solid culture may be used for culturing the above various strains, but liquid culture is preferably used. The liquid culture may be carried out as follows, for example.

The medium to be employed may be any medium as far as the microorganism producing diglycosidase is capable of growing in the medium. For example, there may be used media to which carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerol, dextrin, molasses, and organic acids; further nitrogen sources such as ammonium sulfate, ammonium carbonate, ammonium phosphonate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, and meat extract; and further inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphonates, manganese salts, iron salts, and zinc salts are added. Furthermore, for accumulating diglycosidase, various inducing substances may be added to the medium. As the inducing substances, saccharides may be used, for example, and there may be preferably used gentose (e.g., gentose #80, Nihon Shokuhin Kako Co., Ltd.), gentiobiose, genti-oligosaccharide (e.g., gentiologo etc., Wako Pure Chemical Industries, Ltd.), galactomannan, and the like. The adding amount of these inducing substances is not particularly limited as far as the productivity of aimed diglycosidase is enhanced, but the substance is preferably added in an amount of 0.01 to 10%.

The pH of the medium is adjusted to from about 3 to 8, preferably from about 5 to 6, and culture is carried out at a temperature of about 10 to 50° C., preferably about 25 to 30° C. for 1 to 15 days, preferably 4 to 7 days under aerobic conditions. As the culturing method, a shaking culture or an aerobic submerged culture by means of a jar fermenter may be utilized. However, the above various culturing conditions may be optionally changed, of course, depending on the microorganism or cell to be cultured, and the conditions are not particularly limited as far as diglycosidase of the invention is produced.

For isolation and purification of diglycosidase from the culture liquid obtained, using a diglycosidase activity as a measure, purified diglycosidase can be obtained by combining centrifugal separation, UF concentration, salting out, and various chromatography such as ion exchange resins, and treating in a usual manner (Referential document: Tanpakusitsu•Kouso no Kisojikkenhou (Basic experimental methods for proteins and enzymes), written by Takekazu Horio, Nankodo).

A culture liquid obtained by culturing the above microorganism may be utilized as such as the enzyme composition of the invention. Of course, the culture liquid may be optionally changed in the degree of purification according to the purpose used in the invention.

The invention provides a process for producing an aglycon which comprises forming an aglycon by treating, with diglycosidase, a glycoside containing a compound selected from the group consisting of phytoestrogens, polyphenols, isoflavones, biochanin A, formononetin, cumestrol, and lignans as the aglycon. The producing process includes the reaction of a phytogenic material containing the above compound as the aglycon with a sufficient amount of diglycosidase under weakly acidic conditions at an appropriate temperature and pH for a sufficient period of time so as to convert at least most of the glycoside in the starting material into an aglycon, whereby an aglycon is produced. The invention provides a producing process wherein diglycosidase is added to a plant extract in order to produce a plant extract rich in an aglycon.

The novel process is a one-step process of converting most of an aglycon glycoside into free aglycon by an enzyme preparation containing a hydrolase of disaccharide glycosides, i.e., diglycosidase. The process is effective for the aglycon glycosides present in phytogenic materials, preferably proteins or protein foods. Since the process is found to be capable of substantially complete conversion of modified glucoside isoflavones and glucoside isoflavones into aglycon isoflavones, it includes the conversion of modified glucoside isoflavones and glucoside isoflavones into aglycon isoflavones. In some phytogenic protein materials, particularly soybean protein materials, substantial part of total isoflavone contents in the phytogenic protein materials is present in the form of isoflavone glycosides. Therefore, not only the conversion of glycoside isoflavones into aglycon isoflavones but also the conversion of modified glycoside isoflavones into aglycon isoflavones are necessary for maximum increase of the amount of aglycon isoflavones obtainable from the phytogenic protein materials.

The starting material in a preferred embodiment is any protein or protein-containing food (more preferably a phytogenic material, phytogenic protein, or phytogenic protein-containing food) containing a physiologically active substance of glycoside type. Some processes in the following explanations are described using soybean products as examples, but the process of the invention can be generally applied to a wide range of proteins or protein-containing foods other than soybean and soybean products.

In the invention, the “protein or protein-containing food” preferably contains a physiologically active substance of glycoside type but is not particularly limited.

The “phytogenic material” in the invention means a whole plant body which is edible or used as a medicine, or a part thereof such as leaf, flower, fruit, stem, or root, or a processed product thereof. Examples thereof include whole plant bodies harvested, or parts thereof such as leaf, flower, fruit, stem, and root, and plant extracts and processed products thereof. Specific examples of the phytogenic material include the following materials: phytogenic proteins such as soybean protein, soymilk, juices (orange juice, grape juice, apple juice, pomegranate juice), herb tea, plant extracts such as herb extract, and processed products of the above materials such as juice drinks, wine, tea, black tea, and cocoa.

The “phytogenic protein” means a protein obtainable from the above “phytogenic material”, and may be a mixture with other ingredients derived from the phytogenic material.

In the invention, the “compound selected from the group consisting of phytochemicals, phytoestrogens, polyphenols, isoflavone, biochanin A, formononetin, cumestrol, and lignans” is not particularly limited as far as the compound falls within these conceptual range, but it is preferably a compound which exhibits a physiological activity or enhances a physiological activity in a living body (preferably a warm-blooded animal, more preferably human). The compound is preferably a flavonoid, more preferably an isoflavone, most preferably an isoflavone represented by the above structural formula.

The “physiologically active substance” in the invention means a substance, most of which is preferably present as a glycoside in a plant body and which exhibits a physiological activity or enhances a physiological activity in a living body upon the conversion into the aglycon type. Specifically, the physiologically active substance includes phytochemicals, phytoestrogens, polyphenols, isoflavone, biochanin A, formononetin, cumestrol, and lignans as mentioned above, and preferred are isoflavones.

The “physiologically active substance of glycoside type” means that the aglycon of the above glycoside is a physiologically active substance, and the saccharide chain is composed of one or more saccharide, preferably two or more saccharides. The two-saccharide chains include those mentioned above and the like.

The “enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, proteases, lipases, α-glucosidases, and yeast-dissolving enzymes” is not particularly limited as far as it mainly contains these enzymes, and commercially available enzymes may be employed. The following will illustrates those manufactured by Amano Enzyme Inc. Examples of amylase include Amylase AD “Amano” 1 (optimum pH: 6.0, optimum temperature: 70° C.), Gluczyme NL 4.2 (optimum pH: 4.5, optimum temperature: 65° C.), Transglucosidase L “Amano” (optimum pH: 5.0, optimum temperature: 60° C.), and the like. Examples of cellulase include Cellulase A “Amano” 3 (optimum pH: 4.5, optimum temperature: 55° C.), Cellulase T “Amano” 4, Hemicellulase “Amano” 90G (optimum pH: 4.5, optimum temperature: 50° C.), Hemicellulase GM “Amano”, and the like. Examples of pectinase include Pectinase PL “Amano” (optimum pH: 4.55-0, optimum temperature: 60-55° C.) and the like. Examples of protease include Umamizyme, Newlase F3G, Papain W-40, Pancreatin F, Protease B, Protease A “Amano” G, and the like, and examples of lipase include Lipase A “Amano” 6 (optimum pH: 6.5, optimum temperature: 45° C.), and the like. A yeast-dissolving enzyme preparation YL-15 (optimum pH: 7.0, optimum temperature: 50-55° C.) is mentioned as a yeast-dissolving enzyme, and ADG-S-DS (optimum pH: 4.5-5, optimum temperature: 50-60° C.) and the like are mentioned as an α-galactosidase.

The above enzymes and enzyme preparations can be produced by known methods. For example, an enzyme can be obtained by screening a microorganism producing a specific enzyme mentioned above in a similar manner to the production of diglycosidase and culturing the resulting enzyme-producing strain in a suitable medium. Examples of the above enzyme-producing strain include Bacillus subtillis, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, Rhizopus nivenus, Pseudomonas sp., and the like.

The following will explain the invention in further detail with regard to the process for producing a protein having an increased aglycon content or a food containing the protein, which comprises a step of treating a protein or protein-containing food with diglycosidase, by way of illustration of a phytogenic physiologically active ingredient (especially an isoflavone glycoside) derived from a phytogenic material, but the invention can be conducted using any above compound other than the phytogenic physiologically active ingredient derived from a phytogenic material. By the way, the term of soybean material used herein means any type of soybean or variants of soybeans.

Some different embodiments are possible as specific processes for carrying out the invention.

In the first embodiment, a phytogenic physiologically active substance of glycoside type is converted into a phytogenic physiologically active substance of aglycon type while the phytogenic physiologically active substance is left in the phytogenic material. Therefore, the formed phytogenic physiologically active substance of aglycon type may be left in the phytogenic material or may be suitably removed. The aglycon form of the phytogenic physiologically active substance may be generally removed by a solvent, hydrophobic effluence or extraction. The solvent suitable for the operation includes acetone, ethanol, and other similar organic solvents, but is not limited thereto.

In the second embodiment, a phytogenic physiologically active substance of glycoside type (e.g., isoflavone modified glycoside or isoflavone glucoside) in a phytogenic material is removed from the phytogenic material by aqueous effluence or extraction. The aqueous effluence is carried out through the effluence of relatively soluble phytogenic physiologically active substance of glycoside type by immersing the phytogenic material or by exposing the phytogenic material to or dipping it in water or a mixture of hydrophilic solvents such as ethanol or other alcohols. The pH of the resulting aqueous solution is from about pH 2 to about pH 5, preferably about pH 4. After removal, the phytogenic physiologically active substance of glycoside type is converted into the phytogenic physiologically active substance of aglycon type.

In the third embodiment, prior to all the operations for conversion, a phytogenic physiologically active substance of glycoside type is removed from a phytogenic material.

Depending on the type of phytogenic material containing a phytogenic physiologically active substance of glycoside type, in some cases, the phytogenic material is preferably processed to a finely crushed form. This operation is desirable for bringing a phytogenic physiologically active substance in the phytogenic material into contact with a reagent (diglycosidase) employed in the step which will be described in detail in the following. The material may be subjected to grinding, crushing, or other processing. When the phytogenic material is in condition that isoflavone compounds in the phytogenic material easily come into contact with an external reagent or reactant, e.g., a small leaf part in a plant, it is not necessary to subject the phytogenic material to the above processing.

The conversion of a phytogenic physiologically active substance of glycoside type into the phytogenic physiologically active substance of aglycon type is sometimes partially carried out by enzymes present in the mixture depending on the phytogenic material used. These enzymes may be present naturally in phytogenic protein materials or may be derived from microorganisms grown in the materials. Such enzymes are called as residual enzymes. However, there is a possibility that the conversion of the phytogenic physiologically active substance of glycoside type into the phytogenic physiologically active substance of aglycon type cannot be carried out sufficiently depending on the nature and concentration of the residual enzyme in the phytogenic protein materials. By adding an enzyme preparation containing an external enzyme, i.e., diglycosidase, maximum converting efficiency of the phytogenic physiologically active substance of aglycon type can be achieved.

In the invention, the amount of the enzyme to be added depends on various factors including the type of enzyme present, the distribution of enzyme concentration, the pH of reaction system, the activity of enzyme present, and temperature. In the case of adding an enzyme, typically preferred enzyme amount is preferably from 28 to 2800 AU, usually from 10 to 10000 AU relative to 100 g of a phytogenic material as total concentration of enzyme present based on dry weight thereof. When a sufficient concentration of enzymes including a residual enzyme, an additional enzyme or both enzymes are present in the system, a phytogenic physiologically active substance of glycoside type is brought into contact with the enzymes at an appropriate temperature and pH for a sufficient period of time so as to convert substantially all the phytogenic physiologically active substance of glycoside type in the mixture into the phytogenic physiologically active substance of aglycon type.

The conversion-production step is preferably carried out at a pH of about 2 to about 6. More preferred pH range for the conversion-production step is from about 3 to about 5. Depending on the phytogenic material used, the pH may be adjusted with an acidic reagent such as hydrochloric acid, phosphoric acid, acetic acid, or sulfuric acid, or an alkaline reagent such as sodium hydroxide. In many cases, it is assumed to use an acidic or alkaline reagent of food grade. The temperature to be used in the conversion-production step is preferably from about 25° C. to about 65° C. More preferred temperature is from about 30° C. to about 55° C. Throughout the reaction, the temperature is usually constant, but the temperature may be elevated or lowered according to the successive step and final intended use. Namely, it may be relatively freely-changed according to the various circumstances of the situation.

The period of time necessary for the conversion and production may be determined depending on complicated relationship between various factors of the kind, concentration, and physical properties of the material to be reacted, the concentration of the enzyme added, and further the temperature and pH of the reaction system. In most cases, the conversion-production can be substantially completely achieved within 6 to 12 hours. The period of time for the conversion-production can be shortened depending on the concentration of the diglycosidase preparation added. At the conversion-production step, most of the isoflavone glycoside in the mixture can be converted into the aglycon isoflavone. The efficiency of the conversion is usually at least about 50% or more, preferably about 70% or more. By adopting the above preferable reaction conditions, nearly complete conversion can be achieved.

By adopting conditions similar to the above, it is possible to carry out a process of the invention for producing an aglycon which comprises forming an aglycon by treating, with diglycosidase, a glycoside containing a compound selected from the group consisting of phytoestrogens, polyphenols, isoflavones, biochanin A, formononetin, cumestrol, and lignans as the aglycon.

In addition to the above step, in the invention, the process may further comprise a step of treating with an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, proteases, lipases, α-glucosidase, and yeast-dissolving enzymes. This step may be conducted before or after the step of treating with diglycosidase, or the treatment with diglycosidase and the enzyme preparation may be carried out at the same time. In this case, the treatment with diglycosidase and the enzyme preparation at the same time is carried out under the conditions similar to those in the case of using diglycosidase solely. Moreover, when the treatment with an enzyme preparation is carried out before or after the treatment with diglycosidase, the pH, temperature, period of time, and the like may be selected in consideration of optimum pH and optimum temperature of the above each enzyme preparation. This process is also accompanied by the effects of increasing aglycon content in a protein or protein-containing food and of improving flavor through the reduction of bitterness and/or astringency.

Additionally, in the invention, during the process of finding the above effects of the combined use with diglycosidase, it was found that flavor is improved by treating a protein or protein-containing food with a specific enzyme preparation alone, i.e., an enzyme preparation containing mainly at least one enzyme selected from the group consisting of amylases, cellulases, pectinases, proteases, lipases, α-glucosidase, α-galactosidases, and yeast-dissolving enzymes. With regard to the treating conditions in this case, the pH is preferably from 3 to 8, more preferably from 5 to 7.5, and the treating temperature and treating time are similar to the case of the combined use with diglycosidase. By the way, the treated product may be optionally adjusted to a desired pH.

By treating a protein or protein-containing food as mentioned above, the flavor of the protein or protein-containing food can be improved and particularly, bitterness and/or astringency can be reduced.

In addition, by administering the phytogenic physiologically active substance of aglycon type produced as above or a composition rich in the phytogenic physiologically active substance of aglycon type as such or as a mixture with a food or drink, the effect derived from the phytogenic physiologically active substance can be attained. The effects of the phytogenic physiologically active substance include effects of preventing various diseases (cancer, life-style related diseases, osteoporosis, a burning sensation in climacteric disorder, and the like), and of regulation of intestinal function, immunostimulation, and biophylactic action. Moreover, other than the administration of the phytogenic physiologically active substance which is converted into aglycon type beforehand, by administering orally a phytogenic physiologically active substance of glycoside type and/or a phytogenic material containing a phytogenic physiologically active substance of glycoside type together with diglycosidase, the phytogenic physiologically active substance of glycoside type is converted into the phytogenic physiologically active substance of aglycon type in a living body, for example in stomach or intestines and the absorption of the phytogenic physiologically active substance of aglycon type and the migration into blood are accelerated, whereby preventive effect of the phytogenic physiologically active substance to various diseases can be enhanced.

The method of accelerating a bioabsorption of a physiologically active substance according to the invention, which comprises administering diglycosidase orally before, during, and/or after the ingestion of a food containing physiologically active substances of glycoside type, (preferably, a method of forming an isoflavone from a glycoside containing an isoflavone as the aglycon in a living body), is conducted as follows.

The target is a warm-blooded animal, preferably human or livestock.

As far as the phytogenic physiologically active substance of glycoside type comes into contact with diglycosidase in stomach and intestines, diglycosidase may be administered at any time before, during, or after the ingestion of a food containing physiologically active substances of glycoside type. Preferred is between just after a meal and one hour after the meal.

The dose of diglycosidase is not particularly limited as far as the conversion of the phytogenic physiologically active substance of glycoside type into the phytogenic physiologically active substance of aglycon type occurs in a living body, but diglycosidase is orally administered in an amount of usually from 10 mg/day to 500 mg/day, preferably from 30 mg/day to 300 mg/day, more preferably 100 mg/day to 200 mg/day. The number of dose is not particularly limited but is preferably from once per several days to several times per day, particularly preferably three times per day (i.e., after every meal).

Moreover, the ingesting amount of the physiologically active substance is not particularly limited as far as its effect is attained, but the substance is ingested in an amount of preferably 10 mg/day or more, more preferably 50 mg/day or more, further preferably 50 mg/day to 100 mg/day.

Furthermore, diglycosidase may be administered solely, as an enzyme preparation, and/or as a mixture with conventional glycosidase (e.g., glucosidase, galactosidase, etc.).

Diglycosidase may be used as an enzyme preparation. In this case, the enzyme preparation contains diglycosidase as the essential ingredient, and may further contain various enzymes, stabilizers, and the like.

Additionally, in the case of the administration as an enzyme preparation mixed with conventional glycosidase, examples of the conventional glycosidase include glucosidase, galactosidase, xylosidase, and rhamnosidase, and the dose of diglycosidase is preferably from 10 mg/day to 50 mg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of Example 7.

FIG. 2 is a graph showing the results of Example 8.

FIG. 3 is a graph showing the results of Example 14.

FIG. 4 is a graph showing the results of Example 15.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will be explained in detail with illustrating Examples using soybean materials as phytogenic materials. Examples are illustrated for the purpose of explanation only and they by no means restrict the scope of the invention.

The phytogenic materials, for example defatted soybean, soymilk, concentrated soybean protein, and various soybean products contain 12 kinds of isoflavone compounds. Specifically, they contain aglycons of glycitein, daidzein, and genistein; glucoside glycosides of glycitin, daidzin, and genistin; acetylglycitin, acetyldaidzin, and acetylgenistin having O-acetyl group at 6-position of the glucose residue; and malonylglycitin, malonyldaidzin, and malonylgenistin having O-malonyl group at 6-position of the glucose residue. The existing ratio of these compounds is characteristic to each of the difference of varieties of soybean and the difference of treatment in the production steps.

Unless otherwise stated, ratio, part(s), percent, and the like are herein based on weight.

By the way, the measured activities of various enzymes herein are shown as values obtainable by the method described below unless otherwise stated.

Diglycosidase Activity

The activity was measured on an automatic chemical analyzing apparatus (TBA-30R manufactured by Toshiba Corporation). Thirty μL of an enzyme sample was mixed with 200 μL of 2 mM solution of p-nitrophenyl (pNP) primeveroside used as a substrate of disaccharide glycoside, which is obtained by dissolving the compound in an acetate buffer (pH 5.5), followed by reaction at 40° C. for 9.75 minutes at the cycle time of 22.5 seconds. Then, 250 μL of sodium carbonate was added thereto and then absorbance at 412 nm was measured. A blank derived from the sample was measured similarly using 20 mM acetate buffer (pH 5.5) instead of the substrate solution.

The enzyme amount increasing the absorbance by 1 under the conditions is defined as 1 AU.

The pNP-primeveroside used herein can be synthesized, for example, by reacting pNP-glucoside (manufactured by Merck) with xylo-origosaccharide (manufactured by Wako Pure Chemical Industries, Ltd.) using an enzyme, xylosidase (manufactured by Sigma) to bond xylose to pNP-glucoside in β-1,6-manner via one residue transfer.

Example 1

Production of Diglycosidase by Penicillium Multicolor IAM7153

Culture of Diglycosidase

A medium for growth (pH 5.6) containing 2.0% of defatted soybean, 3.0% of glucose, 0.5% of potassium dihydrogen phosphate, 0.4% of ammonium sulfate, 0.3% of dry yeast was sterilized at 121° C. for 20 minutes. To 100 mL of the sterilized medium was inoculated 1 oese of Penicillium multicolor IAM7153, followed by pre-culture at 27° C. at the shaking rate of 140 min−1. After 5 days, 20 L of a main medium of pH 4.9 containing 1.0% of Sunfiber R, 2.0% of potassium dihydrogen phosphate, 1.0% of ammonium sulfate, and 3.13% of meast P1G was sterilized in a 30 L jar fermenter at 121° C. for 20 minutes while stirring at 150 min−1. The pre-medium was inoculated at a rate of 1.5% and the whole was cultured at a stirring number of 250 min−1, an aeration of 0.75 vvm (15 L/min), an inner pressure of 0.5 kg/cm2 (48 kPa), and a temperature of 27±1° C. for 8 days.

Purification of Diglycosidase

To the culture broth were added 2% by weight each, based on total liquid amount, of Zemlite Super 56M and Fineflow A as filtration aids and filtration through diatomaceous earth was carried out. The filtrate was concentrated by a factor of 20 using an ultrafiltration membrane UF AIP-2020 (MW 6,000) and also the substitution by 20 mM acetate buffer of pH 4.7 was conducted. Ammonium sulfate was added to the above ultrafiltration concentrate to conduct 50% ammonium sulfate-salting out. The resulting precipitate was removed and ammonium sulfate was further added to the supernatant to conduct 80% ammonium sulfate-salting out. The precipitate was recovered and dissolved in 20 mM acetate buffer of pH 4.7. The solution was passed through a 10-DG column (BioRad Co.) to exchange the buffer for 20 mM acetate buffer of pH 4.7 containing 30% saturated ammonium sulfate (this solution is also referred to as “crude diglycosidase”). This solution was applied to a hydrophobic chromatography (HiLoad 16/10 Phenyl Sepharose High Performance (Pharmacia)) to separate a fraction showing diglycosidase activity from fractions showing β-glucosidase and β-xylosidase activities. Elution was started at room temperature with 20 mM acetate buffer containing 30% saturated ammonium sulfate at a flow rate of 2 mL/min and elution was carried out by linear gradient of 30 to 0%. A fraction showing diglycosidase activity was eluted at 10 to 12.5% saturated ammonium sulfate concentration. The diglycosidase fraction recovered was concentrated and a centrifuged supernatant was charged onto 10-DG column to exchange the solution for 25 mM tris-hydrochloride buffer of pH 7.1. This liquid was applied to an isoelectric chromatography (Mono-P HR5/20 (Pharmacia)) and elution was started at room temperature with polybuffer 74 of pH 5.0 at 1 mL/min. The aimed diglycosidase activity was eluted from pH 6.2 to pH 6.3. Since a single band was obtained on an SDS electrophoresis of the fraction, it was proved that diglycosidase (hereinafter, also referred to as “purified diglycosidase”) could be purified.

Example 2

Reactivity of Diglycosidase Toward Various Isoflavone Glycosides

Diglycosidase was diluted with an acetate buffer of pH 4.0 to prepare a 0.75 AU/mL enzyme solution. As references, a similar operation was conducted using β-glucosidase (manufactured by Fluka) derived from Aspergillus niger, β-glucosidase (manufactured by Sigma) derived from almond, β-xylosidase derived from pectinase G (manufactured by Amano Enzyme Inc.). Each of purified products of isoflavone glycosides (glycitin, acetylglycitin, malonylglycitin, daidzin, acetyldaidzin, malonyldaidzin, genistin, acetylgenistin, malonylgenistin, all manufactured by Nacalai Tesque, Inc.) was dissolved in methanol to prepare each 2 mM substrate solution. The reaction was carried out by mixing 10 μL of a substrate solution, 200 μL of 20 mM acetate buffer (pH 4.0), and 40 μL of each purified enzyme solution at a total liquid volume of 250 μL. The reaction was carried out at 55° C. and isolation of an aglycon isoflavone from an isoflavone glycoside in the reaction mixture was detected by HLPC at 0, 1, 3, and 6 hours of the reaction.

HLPC Analysis

To the reaction mixture was added 700 μL of ethanol, followed by stirring and ultrasonication. After centrifugation at 15,000 rpm and 4° C. for 10 minutes, the supernatant was filtered through a filter and then the filtrate was applied to HPLC.

The isoflavone glycoside and aglycon isoflavone contained in the filtrate was separated and detected by a high performance liquid chromatography (HPLC, Shimadzu CLASS LC-10 system) using TOSOH TSK gel ODS-80TM column (manufactured by Tosoh Corporation). The filtrate containing an isoflavone glycoside and an aglycon isoflavone was injected into the column by means of an auto-injector (Shimadzu, SIL-10AXL) and elution was started with a solution containing 2% of eluting solution A (acetonitrile) and 98% of eluting solution B (10% acetic acid solution), and after 5 minutes, continued by a linear concentration gradient finishing with a solution of 50% of eluting solution A and 50% of eluting solution B. Total flow rate was 0.8 mL/min and 12 kinds of isoflavone glycosides and aglycon isoflavones, i.e., glycitin, daidzin, genistin, 6″-O-acetylglycitin, 6″-O-acetyldaidzin, 6″-O-acetylgenistin, 6″-O-malonylglycitin, 6″-O-malonyldaidzin, 6″-O-malonylgenistin, glycitein, daidzein, and genistein can be separated. The absorbance at 260 nm was detected by a UV detector (Shimadzu, SPD-10AV). Using purified products (manufactured by Nacalai Tesque, Inc.) of the above isoflavone glycosides and aglycon isoflavones, the isoflavone glycoside and aglycon isoflavone were quantitatively determined according to a calibration curve method. By the way, unless otherwise stated, the measuring conditions of HPLC herein mean those described in the example.

As a result, β-glucosidase derived from Aspergillus niger could act well on 3 types of glucoside isoflavones of glycitin, daidzin, and genistin, but the reactivity on modified glycosides was very low. β-Glucosidase derived from a plant exhibits a low efficiency of decomposing glycoside isoflavones and no action was observed on modified isoflavones. On the other hand, diglycosidase could cleave all the isoflavone glycosides at a high efficiency. Of these, a high efficiency was observed toward acetylglucoside isoflavones. From the above results, diglycosidase was found to isolate aglycon isoflavones from isoflavone glycosides through cleavage very efficiently (Table 1).

In addition, it was found that the efficiency of decomposing isoflavone glucosides, especially genistin could be further enhanced by the combined use of β-glucosidase in addition to the enzyme.

TABLE 1
Reaction time (h)
0136
Purified enzymeSubstrateglucosideaglyconglucosideaglyconglucosideaglyconglucosideaglycon
Diglycosidase derived fromglycitin10002.197.90.299.80.399.7
P. multicolordaidzin99.10.929.970.118.281.811.588.5
genistin1000100010001000
malonylglycitin100021.778.315.584.51486
malonyldaidzin100049.250.839.960.138.561.5
malonylgenistin100025.874.217.982.117.782.3
acetylglycitin10002982.197.92.597.5
acetyldaidzin100010.489.63.896.23.896.2
acetylgenistin10000.299.80.199.90.299.8
Diglycosidase derived fromglycitin100099.50.510001000
A. fumigatusdaidzin99.10.992.97.185.314.775.224.8
genistin1000100010001000
malonylglycitin100099.60.499.20.898.61.4
malonyldaidzin1000901079.520.567.732.3
malonylgenistin100097.62.494.85.291.88.2
acetylglycitin100094686.513.577.322.7
acetyldaidzin100073.926.152.847.233.566.5
acetylgenistin100087.412.677.122.964.835.2
β-Xylosidase derived fromglycitin100099.20.897.62.495.54.5
pectinase Gdaidzin99.10.990.99.1762457.942.1
genistin100098.51.598.21.898.21.8
malonylglycitin10001000100099.60.4
malonyldaidzin100099.60.498.41.696.73.3
malonylgenistin1000100099.40.6982
acetylglycitin1000100010001000
acetyldaidzin100090.39.798.71.397.72.3
acetylgenistin1000100099.80.299.50.5
β-Glucosidase derived fromglycitin100050.549.511.788.34.595.5
A. nigerdaidzin1000010001000100
genistin1000010001000100
malonylglycitin100098.81.295.64.490.99.1
malonyldaidzin100095.54.590.79.38416
malonylgenistin100096.93.193787.912.1
acetylglycitin1000100099198.11.9
acetyldaidzin100095.64.493.66.4919
acetylgenistin100098.61.496.73.393.86.2
β-Glucosidase derived fromglycitin100098.41.698.11.998.11.9
almonddaidzin99.10.990.99.189.210.888.711.3
genistin100090.69.488.611.488.211.8
malonylglycitin1000100010001000
malonyldaidzin1000100010001000
malonylgenistin1000100010001000
acetylglycitin1000100010001000
acetyldaidzin99.80.299.50.599.40.699.40.6
acetylgenistin1000100010001000

Example 3

Influence of Free Glucose on Diglycosidase Activity

Purified diglycosidase was diluted with 20 mM acetate buffer of pH 4.0 to prepare a 0.75 AU/mL enzyme solution.

With a glucose solution was mixed 10 μL of each 2 mM substrate solution described in Example 2, and 20 mM acetate buffer of pH 4.0 was added thereto to be a liquid volume of 210 μL. Further, 40 μL of the enzyme solution was added and reaction was carried out at a final liquid volume of 250 μL. Adjustment of the glucose solution to be added allows glucose to exist in the reaction mixture in the range of 0 to 20%. The reaction was carried out at 55° C. and the existence of isoflavone glycosides and aglycon isoflavones was detected by HLPC at 0, 0.5, 1, and 3 hours of the reaction.

When the results were compared assuming that isolated aglycon amount at the glucose concentration of 0% and the reaction time of 0.5 hour is 100% using each isoflavone glycoside as the substrate, the converting efficiency of diglycosidase into aglycon isoflavone is hardly inhibited by the increase of free glucose concentration. To the contrary, increase of the converting efficiency was observed until 8% glucose concentration. Therefore, in the conversion into aglycon isoflavone by diglycosidase, no inhibition by glucose was observed up to 20% concentration (Table 2).

Moreover, β-glucosidase used in the conventional converting method into aglycon isoflavone is inhibited by glucose and large decrease of the reaction efficiency was observed, but diglycosidase of the invention was found to be hardly inhibited by glucose. Therefore, the amount, kind, and usage of phytogenic materials, which are starting materials for converting into aglycon isoflavone, are restricted in the case of the conventional glycosidase, but there is no such restriction in the case of diglycosidase of the invention and the efficiency of the conversion into isoflavone aglycon is remarkably enhanced.

TABLE 2
Isolation of isoflavone aglycons by diglycosidase in presence of glucose
Glucose
Concen-
trationIsoflavone glycoside
(%)glycitindaidzinacetylglycitinacetylgenistinacetyldaidzinmalonylglycitinmalonylgenistinmalonyldaidzin
 0100100100100100100100100
 296128.1100.9104.1108.1121.4152125.3
 499.7126100.9104.1112.2127.6164.7132.1
 8100.1129.1100.9104.1115.8128.6163.5125.4
2092.8111.9100.9104.1114.5116.4139.598.1

Example 4

Examination of Temperature in the Conversion into Isoflavone Aglycons by Diglycosidase Using Soybean Materials

Into 400 μL of 20 mM acetate buffer of pH 4.0 was suspended 50 mg of each of various soybean materials (roasted soy flour (manufactured by Fuji Shokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.), defatted soybean (manufactured by Fuji Seiyu K.K.), concentrated soybean protein (manufactured by Fuji Seiyu K.K.)), whereby a substrate solution was prepared. Crude diglycosidase was diluted with 20 mM acetate buffer of pH 4.0 to be the glycosidase activity of 1.88 AU/mL. Fifty μL of the enzyme solution was mixed with the substrate solution and the whole was reacted at 80, 65, 55, 45, 37, or 30° C. at the total volume of 500 μL. To the reaction mixture was added 700 μL of ethanol after 0, 1, 3, and 6 hours of the reaction. After stirring and ultrasonication, the mixture was subjected to centrifugal separation at 15,000 rpm and 4° C. for 10 minutes. The supernatant was filtered through a filter and the existence of isoflavone glycosides and aglycon isoflavones contained in the reaction mixture was detected by HLPC.

The decomposition efficiency from isoflavone glycosides into aglycon isoflavones by an enzyme preparation containing diglycosidase activity was investigated during the treatment of 4 kinds of soybean materials (roasted soy flour, soymilk, concentrated soybean protein, defatted soybean) with the enzyme, the isoflavone compounds being separated into three groups. Namely, the conversion efficiency was analyzed upon three groups of glycitin family (glycitin, malonylglycitin, acetylglycitin, glycitein), genistin family (genistin, malonylgenistin, acetylgenistin, genistein), and daidzin family (daidzin, malonyldaidzin, malonyldaidzin, acetyldaidzin, daidzein (Tables 3 to 14).

TABLE 3
Decomposition efficiency of glycitin family in roasted soy flour
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
30no050.9not detected38.710.4
134.8not detected33.831.5
3not detectednot detected33.266.8
6not detectednot detected19.180.9
yes651.4not detected37.511.1
45no051.3not detected38.310.4
112.4not detected27.460.2
312.4not detected20.367.4
65.9not detected9.185.0
yes649.8not detected36.813.4
55no050.5not detected39.110.4
1not detectednot detected30.169.9
3not detectednot detected12.187.9
6not detectednot detected6.094.0
yes651.7not detected37.311.0
65no050.9not detected38.710.4
1not detectednot detected34.565.5
3not detectednot detected31.069.0
6not detectednot detected29.071.0
yes650.3not detected39.310.4
80no051.3not detected38.310.4
119.2not detected40.140.8
320.5not detected38.441.1
633.6not detected35.730.7
yes650.6not detected39.49.9

TABLE 4
Decomposition efficiency of genistin family in roasted soy flour
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
30no049.7not detected45.54.9
125.7not detected45.329.0
312.0not detected43.844.2
65.5not detected40.653.8
yes650.3not detected43.85.8
45no050.2not detected45.34.4
13.7not detected43.652.7
35.6not detected38.555.9
61.2not detected31.767.1
yes651.7not detected39.58.8
55no049.7not detected45.74.6
11.2not detected42.556.2
3not detectednot detected34.066.0
6not detectednot detected27.372.7
yes654.0not detected39.96.2
65no049.7not detected45.54.9
1not detectednot detected43.656.4
3not detectednot detected40.559.5
6not detectednot detected38.861.2
yes650.7not detected44.35.0
80no050.2not detected45.34.4
17.8not detected48.543.6
39.1not detected48.242.7
618.4not detected47.733.9
yes652.2not detected43.44.3

TABLE 5
Decomposition efficiency of daidzin family in roasted soy flour
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
35no048.6not detected47.83.6
111.7not detected46.941.4
3not detectednot detected45.354.7
6not detectednot detected39.660.4
yes650.3not detected43.85.8
45no048.6not detected47.73.7
13.6not detected42.953.5
32.7not detected37.959.4
65.4not detected28.566.2
yes649.4not detected42.58.1
55no048.8not detected47.53.7
1not detectednot detected42.857.2
3not detectednot detected32.567.5
6not detectednot detected26.773.3
yes651.1not detected43.65.3
65no048.6not detected47.83.6
1not detectednot detected43.756.3
3not detectednot detected40.259.8
6not detectednot detected37.862.2
yes649.7not detected46.14.2
80no048.6not detected47.73.7
13.2not detected49.147.7
34.4not detected47.947.7
612.5not detected46.740.8
yes651.3not detected45.03.8

TABLE 6
Decomposition efficiency of glycitin family in soymilk
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
30no043.948.0not detected8.1
1not detected51.0not detected49.0
3not detected39.6not detected60.4
6not detected28.4not detected71.6
yes644.547.7not detected7.9
45no042.649.1not detected8.2
1not detected46.2not detected53.8
3not detected29.9not detected70.1
6not detected15.3not detected84.7
yes645.447.2not detected7.4
55no047.844.6not detected7.6
1not detected39.1not detected60.9
3not detected27.7not detected72.3
6not detected16.5not detected83.5
yes653.539.9not detected6.7
65no043.948.0not detected8.1
1not detected42.4not detected57.6
3not detected34.3not detected65.7
6not detected25.5not detected74.5
yes656.836.0not detected7.2
80no042.649.1not detected8.2
110.743.6not detected45.7
322.532.6not detected44.9
644.221.3not detected34.4
yes671.419.3not detected9.3

TABLE 7
Decomposition efficiency of genistin family in soymilk
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
30no030.758.50.910.0
1not detected56.5not detected43.5
3not detected47.9not detected52.1
6not detected36.1not detected63.9
yes631.257.70.910.1
45no031.057.80.910.3
1not detected49.4not detected50.6
3not detected34.7not detected65.3
6not detected20.7not detected79.3
yes634.457.20.67.9
55no031.659.10.68.7
1not detected46.9not detected53.1
3not detected32.5not detected67.5
6not detected20.4not detected79.6
yes637.453.30.78.6
65no030.758.50.910.0
1not detected48.9not detected51.1
3not detected40.6not detected59.4
6not detected31.8not detected68.2
yes644.444.61.19.9
80no031.057.80.910.3
110.650.40.938.0
324.337.71.037.0
643.924.12.329.6
yes664.522.92.510.1

TABLE 8
Decomposition efficiency of daidzin family in soymilk
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
30no034.156.8not detected9.1
1not detected58.2not detected41.8
3not detected52.8not detected47.2
6not detected46.2not detected53.8
yes634.856.1not detected9.1
45no033.957.2not detected8.9
16.850.2not detected43.0
36.941.1not detected52.0
66.930.2not detected62.8
yes636.055.8not detected8.2
55no034.257.3not detected8.6
1not detected50.9not detected49.1
3not detected41.1not detected58.9
6not detected31.1not detected68.9
yes640.750.8not detected8.6
65no034.156.8not detected9.1
1not detected51.7not detected48.3
3not detected43.9not detected56.1
6not detected35.3not detected64.7
yes648.742.4not detected8.8
80no033.957.2not detected8.9
18.950.8not detected40.3
323.936.4not detected39.7
642.923.0not detected34.1
yes669.521.8not detected8.8

TABLE 9
Decomposition efficiency of glycitin family in concentrated soybean protein
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
30no052.20.436.011.4
147.10.535.017.5
339.10.533.426.9
628.40.530.840.3
yes652.80.435.411.3
45no052.30.435.911.4
130.20.634.135.2
318.80.631.049.6
67.50.627.164.9
yes653.20.535.011.3
55no052.30.436.011.2
114.20.533.951.4
3not detected0.529.869.7
6not detected0.526.872.7
yes652.90.435.311.3
65no052.20.436.011.4
15.20.535.758.6
3not detected0.535.564.0
6not detected0.435.863.8
yes652.50.435.711.4
80no052.30.435.911.4
139.70.436.023.8
340.50.435.723.4
641.70.335.222.8
yes653.60.334.811.4

TABLE 10
Decomposition efficiency of genistin family in concentrated soybean protein
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
30no049.8not detected43.17.1
141.1not detected43.215.7
330.3not detected43.026.7
619.7not detected42.537.8
yes651.8not detected41.56.7
45no049.7not detected43.07.2
122.2not detected42.835.0
315.6not detected41.043.4
65.3not detected39.655.1
yes654.5not detected38.96.5
55no050.1not detected42.97.0
17.0not detected43.149.8
30.8not detected41.158.1
6not detectednot detected39.061.0
yes652.3not detected41.16.6
65no049.8not detected43.17.1
1not detectednot detected45.254.8
3not detectednot detected44.056.0
6not detectednot detected43.456.6
yes650.8not detected42.36.9
80no049.7not detected43.07.2
129.6not detected44.026.4
330.3not detected43.626.1
632.3not detected43.424.3
yes651.9not detected41.46.7

TABLE 11
Decomposition efficiency of daidzin family
in concentrated soybean protein
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
30no052.3not detected44.63.0
129.5not detected44.426.2
313.4not detected43.942.7
64.0not detected43.552.5
yes654.5not detected42.33.2
45no052.4not detected44.53.1
110.6not detected44.145.3
39.6not detected41.548.8
61.7not detected42.955.5
yes694.3not detected0.05.7
55no052.5not detected44.53.0
11.0not detected44.055.0
3not detectednot detected40.659.4
6not detectednot detected37.762.3
yes654.6not detected42.33.2
65no052.3not detected44.63.0
11.8not detected44.353.9
31.7not detected42.955.4
61.7not detected41.656.7
yes653.2not detected43.63.2
80no052.4not detected44.53.1
122.6not detected45.531.9
323.3not detected44.732.0
625.5not detected43.830.7
yes654.5not detected42.33.2

TABLE 12
Decomposition efficiency of glycitin family in
defatted soybean
Heat
ReactiontreatmentReaction
temperatureoftimeGlycosideAglycon
(° C.)enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
30no0not detected61.3not detected38.7
1not detected48.3not detected51.7
3not detected44.6not detected55.4
6not detected45.6not detected54.4
yes6not detected45.1not detected54.9
45no0not detected65.2not detected34.8
1not detected44.3not detected55.7
3not detected44.8not detected55.2
6not detected41.0not detected59.0
yes6not detected44.2not detected55.8
55no0not detected61.3not detected38.7
1not detected44.8not detected55.2
3not detected42.6not detected57.4
6not detected42.7not detected57.3
yes6not detected45.5not detected54.5
65no0not detected65.2not detected34.8
1not detected45.3not detected54.7
3not detected42.1not detected57.9
6not detected40.0not detected60.0
yes6not detected48.8not detected51.2
80no0not detected65.2not detected34.8
1not detected46.1not detected53.9
3not detected45.0not detected55.0
6not detected40.3not detected59.7
yes6not detected48.1not detected51.9

TABLE 13
Decomposition efficiency of genistin family in
defatted soybean
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
30no038.047.01.713.3
1not detected51.31.347.4
3not detected49.10.750.2
67.645.60.146.6
yes6not detected49.50.450.2
45no037.047.01.514.5
10.949.00.849.2
31.046.80.351.8
61.044.20.254.6
yes61.348.10.250.4
55no038.047.01.713.3
10.048.80.750.5
30.046.10.253.7
60.043.40.156.5
yes60.047.80.451.9
65no037.047.01.514.5
10.048.91.249.9
31.845.21.251.8
63.341.11.154.5
yes628.239.71.330.7
80no037.047.01.514.5
19.643.81.844.9
322.132.52.143.3
635.320.92.541.3
yes657.820.12.419.6

TABLE 14
Decomposition efficiency of daidzin family in
defatted soybean
ReactionHeatReaction
temperaturetreatmenttimeGlycosideAglycon
(° C.)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
30no042.243.3not detected14.5
1not detected46.6not detected53.4
3not detected45.2not detected54.8
6not detected45.1not detected54.9
yes6not detected45.4not detected54.6
45no040.543.3not detected16.2
1not detected45.5not detected54.5
3not detected43.7not detected56.3
6not detected41.7not detected58.3
yes6not detected44.3not detected55.7
55no042.243.3not detected14.5
1not detected45.0not detected55.0
3not detected42.7not detected57.3
6not detected40.3not detected59.7
yes6not detected44.1not detected55.9
65no040.543.3not detected16.2
1not detected44.7not detected55.3
3not detected41.6not detected58.4
6not detected37.3not detected62.7
yes632.835.2not detected32.0
80no040.543.3not detected16.2
17.839.8not detected52.4
319.228.8not detected52.0
630.417.8not detected51.7
yes660.917.4not detected21.7

All three groups of isoflavone glucosides were decomposed at all the temperature ranges tested, and promptly at 37 to 65° C., particularly 55° C. Furthermore, in defatted soybean, endogenous β-glucosidase also participates in the decomposition. It is revealed that the decomposition of modified glucoside glycosides, most of which is considered to occur by the action of diglycosidase, easily occurs at a temperature of 37 to 55° C. Among three groups of aglycon isoflavones, maximum isolation of aglycons was observed in the reaction at 55° C. for 6 hours. In the case of soy flour, glycitein was about 94%, genistein about 74%, and daidzein about 73%. In the case of soymilk, glycitein was about 84%, genistein about 80%, and daidzein about 70%. In the case of concentrated soybean protein, glycitein was about 73%, genistein about 61%, and daidzein about 62%. In the case of defatted soybean, glycitein was about 57%, genistein about 57%, and daidzein about 60%.

From these results, it was revealed that the decomposition of modified glucoside glycosides by diglycosidase efficiently occurred at a temperature of 37 to 65° C., particularly around 55° C.

Example 5

Examination of pH in the Conversion into Isoflavone Aglycons by Diglycosidase Using Soybean Materials

A substrate solution of 450 μL was prepared by suspending 50 mg of each soybean material (roasted soy flour (manufactured by Fuji Shokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.), defatted soybean (manufactured by Fuji Seiyu K.K.), concentrated soybean protein (manufactured by Fuji Seiyu K.K.)), and adjusting the pH to 2 to 11 with hydrochloric acid or sodium hydroxide. Each enzyme solution of pH 2-11 wherein diglycosidase activity of crude diglycosidase was adjusted to 1.88 AU/mL was added thereto in an amount of 50 μL and the whole was reacted at 55° C. at the total volume of 500 μL. To the reaction mixture was added 700 μL of ethanol after 0, 1, 3, and 6 hours of the reaction. After stirring and ultrasonication, the mixture was subjected to centrifugal separation at 15,000 rpm and 4° C. for 10 minutes. The supernatant was filtered through a filter and the existence of isoflavone glycosides and aglycon isoflavones contained in the reaction mixture was detected by HLPC (Tables 15 to 26).

TABLE 15
Decomposition efficiency of glycitin family in
roasted soy flour
Reaction
ReactionHeat treatmenttimeGlycosideAglycon
pHof enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
pH2no056.8not detected35.37.9
156.6not detected34.09.4
356.3not detected34.29.5
654.4not detected35.510.1
yes654.4not detected35.210.4
pH3no056.8not detected35.37.9
1not detectednot detected38.761.3
3not detectednot detected30.869.2
6not detectednot detected31.768.3
yes655.1not detected35.69.3
pH4no056.6not detected34.88.6
1not detectednot detected18.881.2
3not detectednot detected8.291.8
6not detectednot detectednot detected100.0
yes655.1not detected35.99.0
pH5no056.6not detected34.88.6
1not detectednot detected22.977.1
3not detectednot detected6.593.5
6not detectednot detectednot detected100.0
yes655.4not detected35.29.4
pH6.5no055.9not detected35.48.8
1not detectednot detected35.065.0
3not detectednot detected19.780.3
6not detectednot detected11.988.1
yes654.8not detected35.49.8
pH8.5no059.4not detected31.09.6
139.4not detected32.428.2
329.3not detected30.640.1
622.4not detected30.247.4
yes659.2not detected31.79.1

TABLE 16
Decomposition efficiency of genistin family
in roasted soy flour
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)genistinmalonylgenistinacetylgenistingenistein
pH2no050.0not detected45.24.8
149.7not detected45.05.3
350.7not detected43.16.2
652.7not detected40.76.5
yes652.8not detected41.26.0
pH3no050.0not detected45.24.8
13.4not detected45.251.4
3not detectednot detected41.958.1
6not detectednot detected43.556.5
yes649.9not detected45.15.0
pH4no049.9not detected45.44.7
1not detectednot detected36.363.7
3not detectednot detected23.476.6
6not detectednot detected13.486.6
yes650.2not detected45.44.4
pH5no049.9not detected45.44.7
1not detectednot detected39.960.1
3not detectednot detected26.973.1
6not detectednot detected17.682.4
yes652.6not detected42.05.3
pH6.5no049.4not detected45.84.8
1not detectednot detected45.354.7
3not detectednot detected38.861.2
6not detectednot detected34.066.0
yes654.8not detected39.26.0
pH8.5no055.8not detected39.54.6
128.7not detected39.531.8
319.2not detected39.241.6
614.5not detected37.348.2
yes659.5not detected35.25.3

TABLE 17
Decomposition efficiency of daidzin family
in roasted soy flour
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
pH2no049.1not detected47.43.5
147.6not detected46.95.4
350.3not detected45.04.8
653.1not detected42.44.5
yes653.8not detected42.43.8
pH3no049.1not detected47.43.5
1not detectednot detected46.054.0
3not detectednot detected39.660.4
6not detectednot detected40.759.3
yes649.0not detected47.43.7
pH4no048.7not detected47.73.5
1not detectednot detected34.465.6
3not detectednot detected20.579.5
6not detectednot detected11.188.9
yes648.6not detected47.93.5
pH5no048.7not detected47.73.5
1not detectednot detected38.561.5
3not detectednot detected24.775.3
6not detectednot detected15.284.8
yes650.6not detected45.04.4
pH6.5no048.4not detected48.23.4
1not detectednot detected45.454.6
3not detectednot detected39.260.8
6not detectednot detected34.465.6
yes651.9not detected43.44.7
pH8.5no054.1not detected42.13.8
120.4not detected42.736.9
313.3not detected40.346.4
610.2not detected38.051.8
yes658.8not detected37.33.9

TABLE 18
Decomposition efficiency of glycitin
family in soymilk
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
pH2.3no050.242.6not detected7.2
1not detected51.3not detected48.7
3not detected47.7not detected52.3
6not detected46.5not detected53.5
yes651.741.3not detected6.9
pH3.5no050.242.6not detected7.2
1not detected21.9not detected78.1
3not detectednot detectednot detected100.0
6not detectednot detectednot detected100.0
yes654.238.2not detected7.6
pH4.8no050.743.2not detected6.2
1not detected37.0not detected63.0
3not detected21.1not detected78.9
6not detected8.3not detected91.7
yes654.339.2not detected6.5
pH6.2no050.743.2not detected6.2
1not detected46.4not detected53.6
3not detected34.5not detected65.5
6not detected29.6not detected70.4
yes654.237.6not detected8.2
pH7.2no050.043.9not detected6.1
1not detected48.8not detected51.2
3not detected46.2not detected53.8
6not detected41.6not detected58.4
yes656.937.5not detected5.6
pH11.6no054.339.4not detected6.2
1not detected50.4not detected49.6
3not detected46.5not detected53.5
6not detected44.0not detected56.0
yes659.734.4not detected5.8

TABLE 19
Decomposition efficiency of genistin family
in soymilk
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)genistinmalonylgenistinacetylgenistingenistein
pH2.3no031.558.20.69.8
1not detected57.50.741.8
3not detected54.10.845.1
6not detected52.10.647.3
yes635.554.30.69.6
pH3.5no031.558.20.69.8
1not detected27.9not detected72.1
3not detected7.5not detected92.5
6not detected2.7not detected97.3
yes637.052.70.59.8
pH4.8no031.758.60.59.1
1not detected41.0not detected59.0
3not detected23.9not detected76.1
6not detected11.3not detected88.7
yes637.851.90.69.7
pH6.2no031.758.60.59.1
1not detected52.6not detected47.4
3not detected41.2not detected58.8
6not detected35.8not detected64.2
yes638.351.50.69.5
pH7.2no031.257.00.711.1
1not detected56.9not detected43.1
3not detected51.5not detected48.5
6not detected46.4not detected53.6
yes638.951.10.79.3
pH11.6no034.854.20.510.5
1not detected57.7not detected42.3
3not detected52.6not detected47.4
6not detected49.5not detected50.5
yes641.347.70.910.1

TABLE 20
Decomposition efficiency of daidzin family
in soymilk
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
pH2.3no034.455.1not detected10.5
1not detected54.7not detected45.3
3not detected51.1not detected48.9
6not detected48.5not detected51.5
yes638.851.0not detected10.2
pH3.5no034.455.1not detected10.5
1not detected35.6not detected64.4
3not detected15.1not detected84.9
6not detected7.4not detected92.6
yes640.449.1not detected10.6
pH4.8no034.956.2not detected9.0
1not detected47.4not detected52.6
3not detected33.9not detected66.1
6not detected20.3not detected79.7
yes641.249.7not detected9.1
pH6.2no034.956.2not detected9.0
1not detected54.1not detected45.9
3not detected46.4not detected53.6
6not detected41.9not detected58.1
yes640.649.7not detected9.7
pH7.2no034.956.1not detected9.0
1not detected56.4not detected43.6
3not detected52.4not detected47.6
6not detected48.2not detected51.8
yes641.548.3not detected10.2
pH11.6no038.852.5not detected8.7
1not detected56.4not detected43.6
3not detected50.4not detected49.6
6not detected47.7not detected52.3
yes644.646.1not detected9.3

TABLE 21
Decomposition efficiency of glycitin
family in concentrated soybean protein
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
pH1.6no052.30.435.911.3
152.90.435.111.6
354.30.433.511.7
656.10.431.811.7
yes657.90.430.910.8
pH2.7no052.30.435.911.3
138.00.536.625.0
337.20.536.525.7
633.70.536.629.2
yes652.70.435.411.5
pH3.7no052.40.436.111.1
1not detected0.532.966.7
3not detected0.426.173.5
6not detected0.422.177.5
yes652.40.435.911.3
pH5.1no052.40.436.111.1
14.10.633.262.1
3not detected0.626.073.4
6not detected0.621.078.5
yes652.30.435.811.5
pH6.6no052.20.436.111.3
122.60.434.842.2
36.90.432.460.3
62.00.429.568.1
yes653.30.435.211.1
pH8.6no054.90.433.011.7
151.60.432.615.4
345.90.432.621.0
636.70.432.030.9
yes656.80.331.611.3

TABLE 22
Decomposition efficiency of genistin family in
concentrated soybean protein
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)genistinmalonylgenistinacetylgenistingenistein
pH1.6no049.7not detected42.57.8
150.2not detected42.27.6
351.6not detected40.67.8
653.3not detected38.97.9
yes654.4not detected37.77.9
pH2.7no049.7not detected42.57.8
123.1not detected44.332.6
322.1not detected43.734.2
617.5not detected44.038.4
yes650.5not detected42.57.0
pH3.7no050.7not detected43.16.2
1not detectednot detected43.556.5
3not detectednot detected36.963.1
6not detectednot detected30.769.3
yes650.6not detected43.06.4
pH5.1no050.7not detected43.16.2
1not detectednot detected45.254.8
3not detectednot detected40.259.8
6not detectednot detected35.664.4
yes651.4not detected42.46.2
pH6.6no050.4not detected43.06.5
113.9not detected43.642.4
33.8not detected42.853.3
61.7not detected41.057.2
yes653.2not detected40.66.2
pH8.6no053.8not detected39.76.5
150.4not detected38.810.8
345.1not detected39.015.8
636.3not detected38.525.2
yes656.2not detected37.66.2

TABLE 23
Decomposition efficiency of daidzin family
in concentrated soybean protein
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
pH1.6no052.5not detected44.53.0
153.0not detected43.73.3
355.0not detected41.73.3
657.2not detected39.43.4
yes659.0not detected38.12.9
pH2.7no052.5not detected44.53.0
112.9not detected46.440.7
312.5not detected46.041.6
69.3not detected45.845.0
yes653.0not detected43.93.2
pH3.7no052.5not detected44.63.0
1not detectednot detected41.658.4
3not detectednot detected33.067.0
6not detectednot detected25.274.8
yes652.5not detected44.43.1
pH5.1no052.5not detected44.63.0
1not detectednot detected43.456.6
3not detectednot detected37.063.0
6not detectednot detected31.268.8
yes653.4not detected43.43.2
pH6.6no052.1not detected44.83.0
15.0not detected43.951.0
3not detectednot detected42.957.1
6not detectednot detected41.258.8
yes655.1not detected41.83.2
pH8.6no056.1not detected40.73.2
150.8not detected39.29.9
345.0not detected39.015.9
634.5not detected38.127.4
yes659.1not detected37.73.2

TABLE 24
Decomposition efficiency of glycitin
family in defatted soybean
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
pH2.6no0not detected100.0not detectednot detected
1not detected100.0not detectednot detected
3not detected100.0not detectednot detected
6not detected100.0not detectednot detected
yes6not detected100.0not detectednot detected
pH3.4no0not detected100.0not detectednot detected
1not detected100.0not detectednot detected
3not detected100.0not detectednot detected
6not detected100.0not detectednot detected
yes6not detected100.0not detectednot detected
pH4.8no0not detected64.0not detected36.0
1not detected40.1not detected59.9
3not detected37.5not detected62.5
6not detected31.9not detected68.1
yes6not detected56.2not detected43.8
pH5.4no0not detected64.0not detected36.0
1not detected45.1not detected54.9
3not detected100.0not detectednot detected
6not detected32.3not detected67.7
yes6not detected39.3not detected60.7
pH6.6no0not detected39.6not detected60.4
1not detected52.9not detected47.1
3not detected53.6not detected46.4
6not detected58.1not detected41.9
yes6not detected50.3not detected49.7
pH7.8no0not detected66.8not detected33.2
1not detected57.5not detected42.5
3not detected53.2not detected46.8
6not detected49.1not detected50.9
yes6not detected55.8not detected44.2

TABLE 25
Decomposition efficiency of genistin
family in defatted soybean
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)genistinmalonylgenistinacetylgenistingenistein
pH2.6no042.043.81.812.4
136.344.41.717.7
320.345.41.732.7
620.143.51.934.6
yes644.342.01.612.1
pH3.4no042.043.81.812.4
1not detected41.61.556.9
3not detected36.41.162.5
6not detected30.40.868.8
yes644.339.81.814.1
pH4.8no040.543.21.814.5
1not detected42.81.056.1
3not detected37.50.661.9
6not detected31.10.468.5
yes631.841.71.325.2
pH5.4no040.543.21.814.5
1not detected48.00.551.5
3not detected44.6not detected55.4
6not detected39.7not detected60.3
yes66.245.8not detected48.0
pH6.6no039.247.11.712.0
1not detected50.10.649.3
3not detected48.30.351.4
6not detected46.10.253.7
yes67.745.70.246.4
pH7.8no040.445.81.612.2
115.746.61.036.7
312.344.40.842.5
611.442.20.745.7
yes637.539.90.622.0

TABLE 26
Decomposition efficiency of daidzin family
in defatted soybean
Heat
treatmentReaction
ReactionoftimeGlycosideAglycon
pHenzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
pH2.6no045.841.0not detected13.2
137.541.2not detected21.3
317.341.8not detected40.9
616.940.2not detected42.9
yes648.038.9not detected13.0
pH3.4no045.841.0not detected13.2
1not detected39.4not detected60.6
3not detected34.5not detected65.5
6not detected29.0not detected71.0
yes647.837.1not detected15.1
pH4.8no043.540.7not detected15.7
1not detected40.5not detected59.5
3not detected36.7not detected63.3
6not detected30.5not detected69.5
yes637.138.4not detected24.5
pH5.4no043.540.7not detected15.7
1not detected44.2not detected55.8
3not detected41.5not detected58.5
6not detected36.9not detected63.1
yes6not detected44.9not detected55.1
pH6.6no043.643.1not detected13.2
1not detected45.9not detected54.1
3not detected44.0not detected56.0
6not detected41.8not detected58.2
yes69.341.2not detected49.5
pH7.8no044.742.0not detected13.3
113.343.6not detected43.1
310.141.4not detected48.4
69.438.7not detected51.9
yes641.636.7not detected21.7

From these results, maximum pH was found to be in the range of 3.5 to 5. Specifically, in the case of roasted soy flour, after the reaction at pH 4 for 6 hours, the existing ratio of aglycon of each isoflavone family was as follows: glycitein 100%, genistein 87%, and daidzein 89%. In the case of soymilk, almost complete decomposition of isoflavone glycosides occurred after the reaction at pH 3.5 for 6 hours, and glycitein was 100%, genistein 97%, and daidzein 93%. In the case of concentrated soybean protein, after the reaction at pH 3.7 for 6 hours, glycitein was 78%, genistein 69%, and daidzein 75%. In the case of defatted soybean, maximum isolation of aglycons was observed after the reaction at pH 3.4 for 6 hours, and glycitein was 68%, genistein 69%, and daidzein 71%.

Example 6

Examination of Substrate Concentration in the Conversion into Isoflavone Aglycons by Diglycosidase Using Soybean Materials

Into 20 mM acetate buffer of pH 4.0 was suspended 0.1 g, 0.25 g, 0.5 g, 1.0 g, or 1.5 g of each of various soybean materials (roasted soy flour (manufactured by Fuji Shokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.), defatted soybean (manufactured by Fuji Seiyu K.K.), concentrated soybean protein (manufactured by Fuji Seiyu K.K.)). The pH of the suspension was measured and the liquid volume was adjusted to 4.5 mL while the pH was adjusted to 4.0 with 1N hydrochloric acid. An enzyme solution wherein diglycosidase activity of crude diglycosidase was adjusted to 1.88 AU/mL was added thereto in an amount of 0.5 mL, whereby the final liquid volume was 5.0 mL. Namely, the ratio of the soybean material in the reaction mixture was 2%, 5%, 10%, 20%, or 30% (w/v). The whole was reacted at 55° C. under shaking. To 5 mL of the reaction mixture was added 7 mL of ethanol after 0, 1, 3, and 6 hours of the reaction. After ultrasonication, the mixture was thoroughly mixed. It was subjected to centrifugal separation at 2,000 rpm and room temperature for 5 minutes. Then, 1 μL of the supernatant was placed in a 1.5 mL microtube and was subjected to centrifugal separation at 15,000 rpm and 4° C. for 10 minutes. The supernatant was filtered through a filter and each sample was suitably diluted by a factor of 1 to 6 depending on the substrate concentration. Fifty μL of the diluted solution was analyzed by HLPC (Tables 27 to 38).

TABLE 27
Decomposition efficiency of glycitin family in
roasted soy flour
SubstrateHeatReactionGlycoside
concentrationtreatmenttimeacetyl-Aglycon
(%)of enzyme(h)glycitinmalonylglycitinglycitinglycitein
2no050.2not detected33.616.2
1not detectednot detectednot detected100.0
3not detectednot detectednot detected100.0
6not detectednot detectednot detected100.0
yes653.4not detected34.412.2
5no054.8not detected33.811.4
1not detectednot detected11.888.2
3not detectednot detected7.292.8
6not detectednot detectednot detected100.0
yes654.8not detected33.811.4
10no070.3not detected24.25.5
1not detectednot detected16.783.3
3not detectednot detected9.790.3
6not detectednot detectednot detected100.0
yes655.0not detected35.19.9
20no059.6not detected31.88.6
1not detectednot detected22.977.1
3not detectednot detected13.886.2
6not detectednot detected10.090.0
yes656.0not detected35.09.1
30no055.9not detected35.38.8
1not detectednot detected28.471.6
3not detectednot detected24.475.6
6not detectednot detected13.786.3
yes656.9not detected34.48.7

TABLE 28
Decomposition efficiency of genistin family in
roasted soy flour
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
2no033.2not detected44.722.1
1not detectednot detected10.689.4
3not detectednot detected5.095.0
6not detectednot detected1.598.5
yes648.4not detected45.06.7
5no047.8not detected44.77.5
1not detectednot detected22.177.9
3not detectednot detected15.284.8
6not detectednot detected9.290.8
yes647.8not detected44.77.5
10no059.5not detected38.22.3
1not detectednot detected33.366.7
3not detectednot detected25.374.7
6not detectednot detected18.781.3
yes649.5not detected45.94.6
20no050.5not detected46.13.4
1not detectednot detected44.155.9
3not detectednot detected35.864.2
6not detectednot detected28.671.4
yes649.5not detected46.24.3
30no049.4not detected46.73.8
15.7not detected46.647.7
39.6not detected41.448.9
62.0not detected37.860.2
yes650.1not detected46.13.8

TABLE 29
Decomposition efficiency of daidzin family in
roasted soy flour
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
2no030.2not detected47.622.3
1not detectednot detected9.290.8
3not detectednot detectednot detected100.0
6not detectednot detectednot detected100.0
yes646.6not detected47.26.2
5no046.1not detected46.17.7
1not detectednot detected20.979.1
3not detectednot detected12.587.5
6not detectednot detected6.893.2
yes646.1not detected46.17.7
10no060.1not detected38.51.4
1not detectednot detected30.569.5
3not detectednot detected21.578.5
6not detectednot detected14.985.1
yes647.5not detected47.74.8
20no050.3not detected46.13.7
1not detectednot detected40.759.3
3not detectednot detected32.667.4
6not detectednot detected25.174.9
yes648.6not detected47.24.2
30no047.7not detected47.64.7
1not detectednot detected46.453.6
3not detectednot detected42.657.4
6not detectednot detected34.365.7
yes649.0not detected47.43.6

TABLE 30
Decomposition efficiency of glycitin family in
soymilk
Heat
SubstratetreatmentReaction
concentrationoftimeGlycosideAglycon
(%)enzyme(h)glycitinmalonylglycitinacetyl-glycitinglycitein
2no071.828.2not detectednot detected
1not detectednot detectednot detected100.0
3not detectednot detectednot detected100.0
6not detectednot detectednot detected100.0
yes651.848.2not detectednot detected
5no049.450.6not detectednot detected
1not detected24.6not detected75.4
3not detected7.5not detected92.5
6not detectednot detectednot detected100.0
yes656.743.3not detectednot detected
10no052.541.1not detected6.4
1not detected26.4not detected73.6
3not detected10.5not detected89.5
6not detected4.2not detected95.8
yes656.837.0not detected6.2
20no052.441.3not detected6.3
1not detected27.8not detected72.2
3not detected9.4not detected90.6
6not detected5.9not detected94.1
yes655.538.8not detected5.7
30no050.943.0not detected6.1
1not detected33.1not detected66.9
3not detected15.9not detected84.1
6not detected7.7not detected92.3
yes655.837.5not detected6.7

TABLE 31
Decomposition efficiency of genistin family in soymilk
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)genistinmalonylgenistinacetyl-genistingenistein
 2no054.535.0not detected10.5
1not detected15.3not detected84.7
3not detected3.5not detected96.5
6not detected0.0not detected100.0
yes638.049.5not detected12.5
 5no031.558.2not detected10.3
1not detected24.9not detected75.1
3not detected6.9not detected93.1
6not detected2.1not detected97.9
yes637.952.5not detected9.7
10no032.956.50.510.1
1not detected31.5not detected68.5
3not detected11.9not detected88.1
6not detected4.8not detected95.2
yes637.651.20.510.6
20no033.356.30.69.9
1not detected37.6not detected62.4
3not detected15.7not detected84.3
6not detected10.7not detected89.3
yes638.651.00.69.8
30no033.655.90.69.9
1not detected42.5not detected57.5
3not detected25.4not detected74.6
6not detected17.1not detected82.9
yes638.650.00.610.9

TABLE 32
Decomposition efficiency of daidzin family in soymilk
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)daidzinmalonyldaidzinacetyl-daidzindaidzein
 2no053.532.2not detected14.4
1not detected27.5not detected72.5
3not detected9.9not detected90.1
6not detected2.9not detected97.1
yes639.847.0not detected13.1
 5no034.253.6not detected12.2
1not detected34.2not detected65.8
3not detected15.9not detected84.1
6not detected6.1not detected93.9
yes640.547.6not detected12.0
10no034.653.4not detected12.0
1not detected37.4not detected62.6
3not detected20.5not detected79.5
6not detected10.7not detected89.3
yes639.447.8not detected12.8
20no034.153.6not detected12.3
1not detected41.2not detected58.8
3not detected21.5not detected78.5
6not detected16.4not detected83.6
yes639.848.1not detected12.1
30no034.953.5not detected11.6
1not detected43.8not detected56.2
3not detected29.7not detected70.3
6not detected21.9not detected78.1
yes640.046.9not detected13.1

TABLE 33
Decomposition efficiency of glycitin family in concentrated soybean protein
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
 2no052.20.435.811.6
1not detected0.414.385.3
3not detected0.25.394.4
6not detectednot detected0.0100.0
yes653.40.435.011.2
 5no052.90.435.611.2
1not detected0.527.172.4
3not detected0.417.881.9
6not detected0.212.387.4
yes654.00.434.411.3
10no052.90.335.711.0
11.60.431.266.8
3not detected0.326.273.5
6not detected0.323.975.8
yes653.40.335.211.1
20no053.10.235.910.8
113.20.335.451.2
3not detected0.233.666.2
6not detected0.330.569.2
yes652.80.435.811.1
30no057.50.433.98.2
12.00.635.961.5
3not detected0.530.768.9
6not detected0.328.371.4
yes658.00.433.48.2

TABLE 34
Decomposition efficiency of genistin family in concentrated soybean protein
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
 2no050.5not detected42.96.6
11.2not detected20.078.7
3not detectednot detected8.391.7
6not detectednot detected3.396.7
yes651.6not detected41.37.0
 5no051.2not detected43.15.8
1not detectednot detected36.463.6
3not detectednot detected26.373.7
6not detectednot detected17.882.2
yes652.2not detected41.76.2
10no050.6not detected43.45.9
11.3not detected42.855.8
3not detectednot detected37.362.7
6not detectednot detected33.266.8
yes650.9not detected42.86.3
20no050.4not detected43.75.9
16.0not detected47.046.9
3not detectednot detected44.655.4
6not detectednot detected42.357.7
yes650.8not detected43.26.1
30no054.6not detected43.51.9
12.8not detected65.331.9
3not detectednot detected61.039.0
6not detectednot detected55.944.1
yes655.6not detected42.42.0

TABLE 35
Decomposition efficiency of daidzin family in concentrated soybean protein
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)daidzinmalonyldaidzinacetyl-daidzindaidzein
 2no052.8not detected44.03.2
1not detectednot detected17.882.2
3not detectednot detected5.394.7
6not detectednot detectednot detected100.0
yes652.6not detected43.24.2
 5no053.0not detected44.03.1
1not detectednot detected32.667.4
3not detectednot detected20.579.5
6not detectednot detected12.088.0
yes653.5not detected43.03.5
10no052.8not detected44.23.0
12.2not detected39.758.1
3not detectednot detected32.967.1
6not detectednot detected26.973.1
yes653.3not detected43.43.3
20no052.9not detected44.13.0
10.7not detected46.452.9
3not detectednot detected41.558.5
6not detectednot detected37.962.1
yes652.8not detected44.03.2
30no049.2not detected49.21.7
12.7not detected56.640.7
3not detectednot detected49.950.1
6not detectednot detected42.857.2
yes649.8not detected48.41.8

TABLE 36
Decomposition efficiency of glycitin family in defatted soybean
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)glycitinmalonylglycitinacetylglycitinglycitein
 2no0not detectednot detectednot detectednot detected
1not detectednot detectednot detectednot detected
3not detectednot detectednot detectednot detected
6not detectednot detectednot detectednot detected
yes6not detectednot detectednot detectednot detected
 5no0not detected58.4not detected41.6
1not detected41.6not detected58.4
3not detected31.8not detected68.2
6not detected19.5not detected80.5
yes6not detected58.5not detected41.5
10no0not detected53.0not detected47.0
1not detected54.7not detected45.3
3not detected36.6not detected63.4
6not detected27.9not detected72.1
yes6not detected57.6not detected42.4

TABLE 37
Decomposition efficiency of genistin family in defatted soybean
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)genistinmalonylgenistinacetylgenistingenistein
 2no037.745.41.815.1
1not detected31.4not detected68.6
3not detected17.0not detected83.0
6not detected11.4not detected88.6
yes638.741.81.518.0
 5no040.043.91.914.2
1not detected38.00.861.2
3not detected29.60.569.9
6not detected19.00.380.7
yes641.841.11.815.4
10no042.441.61.914.1
1not detected41.51.357.2
3not detected35.10.964.0
6not detected29.10.670.3
yes644.339.41.714.5

TABLE 38
Decomposition efficiency of daidzin family in defatted soybean
SubstrateHeatReaction
concentrationtreatmenttimeGlycosideAglycon
(%)of enzyme(h)daidzinmalonyldaidzinacetyldaidzindaidzein
 2no042.342.2not detected15.5
1not detected35.2not detected64.8
3not detected21.2not detected78.8
6not detected16.5not detected83.5
yes643.738.1not detected18.2
 5no044.041.1not detected14.9
1not detected37.7not detected62.3
3not detected30.6not detected69.4
6not detected21.0not detected79.0
yes645.838.1not detected16.1
10no045.639.7not detected14.7
1not detected39.5not detected60.5
3not detected34.2not detected65.8
6not detected29.0not detected71.0
yes647.537.0not detected15.5

By combining maximum reaction temperature and pH, in all the materials examined, isolation of each isoflavone glucoside was found to be 70% or more when the material concentration is 10% or less. In particular, when the material concentration ranges from 2% to 5%, it was revealed that almost 100% conversion of isoflavone glycosides into aglycons occurred.

Example 7

Influence of Commercially Available Enzyme Preparations Accelerating the Conversion into Aglycon Isoflavones by Diglycosidase

Soyaflavone (manufactured by Fuji Seiyu K.K.) was suspended into 0.1 M sodium acetate of pH 3.0 and the substrate concentration was adjusted to 30% (w/v) and pH of the solution to 5.0. The suspension was pre-incubated at 50° C. for 1 hour, whereby the temperature of the suspension was elevated to 50° C. To the suspension was added each commercially available enzyme preparation (Amylase AD “Amano” 1, YL-15, Gluczyme NL4.2, Transglucosidase L “Amano”, all manufactured by Amano Enzyme Inc.) solely or in combination with diglycosidase (0.3 AU) so as to be 0.1% (w/v). The whole was reacted at 50° C. for 6 hours and the change of the composition of isoflavone glycosides and aglycon isoflavones was analyzed by HPLC. Isoflavone glycosides and aglycon isoflavones were quantitatively determined. Among them, relative values of the aglycon isoflavones were shown in FIG. 1, ideal values of the aglycon isoflavones being 100%.

The ideal value of each aglycon isoflavone was calculated as follows based on the content of each isoflavone glycoside and aglycon isoflavone in the case that no enzyme was added.
Ideal value of aglycon isoflavone=AG+G1×MAG/MG1+G2×MAG/MG2+ . . .
AG; amount of aglycon isoflavone, G1; amount of isoflavone glycoside, MAG; molecular weight of aglycon isoflavone, MG; molecular weight of isoflavone glycoside

Each commercially available enzyme preparation it self could hardly hydrolyze glycosides but the combination with diglycosidase obviously increased the amount of isolated aglycon isoflavones. For example, the combination of diglycosidase with Amylase AD “Amano” 1 resulted in 2.1-fold increase of glycitein and 1.3-fold increase of genistein as compared with the action of diglycosidase alone. However, about 1.1-fold increase was observed for daidzein. It was revealed that the combination of diglycosidase with YL-15 resulted in 1.8-fold increase of glycitein, 1.2-fold increase of genistein, and 1.1-fold increase of daidzein, the combination of diglycosidase with Gluczyme NL-4.2 resulted in 0.8-fold increase of glycitein, 1.1-fold increase of genistein, and 1.0-fold increase of daidzein, and the combination of diglycosidase with Transglucosidase L “Amano” resulted in 1.6-fold increase of glycitein, 1.0-fold increase of genistein, and 1.0-fold increase of daidzein.

Example 8

Examination of Effective Amount of Amylase AD “Amano” 1 Accelerating the Conversion into Aglycon Isoflavones by Diglycosidase

Roasted soy flour (manufactured by Fuji Shokuhin K.K.) was suspended into 0.1 M sodium acetate adjusted to pH 3.15, the substrate concentration was adjusted to 30% (w/v), and the pH of the solution to 5.0. The suspension was pre-incubated at 50° C. for 1 hour, whereby the temperature of the suspension was elevated to 50° C. To the suspension were added diglycosidase so as to be a final concentration of 0.1% (w/v) (0.3 AU) and Amylase AD “Amano” 1 (manufactured by Amano Enzyme Innc.) so as to be 0.1, 0.05, 0.01, 0.005, and 0.0001% (w/v). The whole was reacted at 50° C. for 3 hours and the change of isoflavone composition was analyzed by HPLC.

As shown in FIG. 2, it is found that the isolated amount of aglycon rather increased at a concentration lower than 0.1% (w/v). An effect was observed even at a mall amount of 0.0001% (w/v). At 0.005% (w/v), glycitein and genistein increased by a factor of 1.23 and 1.20, respectively. In the case of daidzein, 1.2-fold increase was observed. By the way, 0% means the results in the case of diglycosidase alone.

Example 9

Flavor Improvement of Soybean Protein by Diglycosidase or each Commercially Available Enzyme Preparation

To 10 g of Fujipro (separated soybean protein, manufactured by Fuji Seiyu K.K.) was added 90 mL of water, and the whole was thoroughly mixed to obtain a soybean protein solution. Thereto was added diglycosidase or each of commercially available enzyme preparation (Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS, Lactase F, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, Protease B, YL-15, Pectinase PL “Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2, all manufactured by Amano Enzyme Inc.) so that diglycosidase (0.5 AU) or each enzyme preparation was contained in the soybean protein solution in a concentration of 0.25% (w/v). The treatment was carried out at 50° C. for 5 hours. By the way, the reaction pH was 7.1. For a sensory test, pH was not adjusted for avoiding the influence of a buffer (the same shall apply to Examples 10 to 12).

Sensory Test (Flavor Improvement of Separated Soybean Protein Treated with Diglycosidase or Each Commercially Available Enzyme Preparation)

For carrying out a sensory test, each soybean protein treated with diglycosidase or each commercially available enzyme preparation was subjected to centrifugal separation at 1500×g and 4° C. for 20 minutes. The precipitate was removed and pH of the supernatant was adjusted to 6.0 with hydrochloric acid. Using a solution obtained by two-fold dilution of the solution, the test was carried out. The evaluation was conducted by five expert panelists, and the deliciousness, bitterness•astringency, and aftertaste were compared with those of Control untreated with the enzyme (Table 39).

TABLE 39
Flavor-improving effect of enzyme preparation on separated soybean protein
Panelist APanelist BPanelist C
bitter-bitterbitter-
deli-ness.deli-ness.deli-ness.
cious-astrin-after-cious-astrin-after-cious-astrin-after-
Enzyme namenessgencytastenessgencytastenessgencytaste
Untreated (Control)±±±±±±±±±
Diglycosidase±−−+±+±−−±
Amylase AD “Amano” 1±−−+±−−±±±
ADG-D-DS±−−+±±±++
Lipase A “Amano” 6±±+±+±±+
Lactase F-DS±±±±±±±±
Lactase F “Amano”±±±±±±±±±
Cellulase A “Amano” 3±−−+±−−±±±
Hemicellulase “Amano” 90G±−−+±−−±±±
Protease B±+±±±±±±±
YL-15±++++±+++±±+++±
Pectinase PL±±+±±±±±±
Trans-glucosidase L±±±±±±±±±
Gluczyme±±±±±±±±±
Panelist DPanelist E
bitterness.bitterness.after-
Enzyme namedeliciousnessastringencyaftertastedeliciousnessastringencytaste
Untreated (Control)±±±±±±
Diglycosidase+±++±
Amylase AD “Amano” 1±±±±±
ADG-D-DS+±+±±
Lipase A “Amano” 6±±+±±±
Lactase F-DS±±±±±
Lactase F “Amano”±±±±±±
Cellulase A “Amano” 3+±+−−±
Hemicellulase “Amano” 90G±±±±+
Protease B±±±±±±
YL-15±+++±±+±
Pectinase PL±±±±±±
Transglucosidase L±±±±±±
Gluczyme±±±±±±

±; No change is observed as compared with Control.

+; This means that “deliciousness” and “aftertaste” are improved and “bitterness.astringency” is strengthened.

−; This means that “deliciousness” and “aftertaste” become worse and “bitterness.astringency” is reduced.

The increase in number of + and − means that the tendency becomes strong.

As a result, the bitterness•astringency was reduced or disappeared by diglycosidase, Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Cellulase A “Amano” 3, or Hemicellulase “Amano” 90G.

Also, it was revealed that the treatment with diglycosidase, Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, YL-15, or Pectinase PL “Amano” was effective for improving aftertaste. It was revealed that these enzyme preparations improve overall flavor, for example, appearance of sweetness and reduction of smelling of grass, other than the examined articles.

Example 10

Flavor Improvement of Soybean Protein by Diglycosidase and each Commercially Available Enzyme Preparation

To 10 g of Fujipro (separated soybean protein, manufactured by Fuji Seiyu K.K.) was added 90 mL of water, and the whole was thoroughly mixed to obtain a soybean protein solution. Thereto was added diglycosidase and each of commercially available enzyme preparation (Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS, Lactase F, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, Protease B, YL-15, Pectinase PL “Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2, all manufactured by Amano Enzyme Inc.) so that diglycosidase was contained 6.5 AU in the soybean protein solution and each enzyme preparation in a concentration of 0.25% (w/v). The treatment was carried out at 50° C. for 5 hours to obtain an enzyme-treated soybean protein. By the way, the reaction pH was 7.1.

Sensory Test (Flavor Improvement of Separated Soybean Protein Treated with Diglycosidase and Each Commercially Available Enzyme Preparation)

For carrying out a sensory test, each soybean protein treated with enzymes was subjected to centrifugal separation at 1500×g and 4° C. for 20 minutes. The precipitate was removed and pH of the supernatant was adjusted to 6.0 with hydrochloric acid. Using a solution obtained by two-fold dilution of the solution, the test was carried out. The evaluation was conducted by five expert panelists, and the deliciousness, bitterness•astringency, and aftertaste were compared with those of Control untreated with the enzyme (Table 40).

TABLE 40
Flavor improvement of separated soybean protein by combination of enzymes
Panelist APanelist BPanelist C
bitter-bitter-bitter-
deli-ness.deli-ness.deli-ness.
cious-astrin-after-cious-astrin-after-cious-astri-after-
Enzyme namenessgencytastenessgencytastenessgencytaste
Untreated (Control)±±±±±±±±±
Diglycosidase + Amylase AD±−−−++−−−+±−−±
“Amano”1
Diglycosidase + ADG-S-DS+−−−±+±++−−++
Diglycosidase + Lipase A+−−−±±±+±+
“Amano”6
Diglycosidase + Lactase F-DS±±±±+±±
Diglycosidase + Lactase F±±++±+±+
“Amano”
Diglycosidase + Cellulase A±−−−+++−−−++±
“Amano”3
Diglycosidase +±−−−+++−−±+±
Hemicellulase “Amano”90G
Diglycosidase + Protease B±−−−±+±±−−±
Diglycosidase + YL-15±++++++±+
Diglycosidase +±+++±+−−±
Pectinase PL “Amano”
Diglycosidase +±±±±±±
Transglucosidase L “Amano”
Diglycosidase + Gluczyme±±±+± :±±±
NL4.2
Panelist DPanelist E
bitterness.bitterness.after-
Enzyme namedeliciousnessastringencyaftertastedeliciousnessastringencytaste
Untreated (Control)±±±±±±
Diglycosidase + Amylase AD++±±+−−++
“Amano”1
Diglycosidase + ADG-S-DS++++±−−+
Diglycosidase + Lipase A++±+±+
“Amano”6
Diglycosidase + Lactase F-DS±±±+±
Diglycosidase + Lactase F+±+±−−++
“Amano”
Diglycosidase +++±+−−−+
Cellulase A “Amano”3
Diglycosidase ++++++±
Hemicellulase “Amano” 90G
Diglycosidase + Protease B++±±±+
Diglycosidase + YL-15++++±±+
Diglycosidase + Pectinase PL++±+±+
“Amano”
Diglycosidase ++±±+±
Transglucosidase L “Amano”
Diglycosidase + Gluczyme+±± :±±
NL4.2

As a result, it was revealed that effects, for example, appearance of sweetness, reduction of bitterness-astringency, or improvement of aftertaste were observed in all the combinations of diglycosidase and each commercially available enzyme preparation (diglycosidase and Amylase AD “Amano” 1, diglycosidase and ADG-S-DS, diglycosidase and Lipase A “Amano” 6, diglycosidase and Lactase F-DS, diglycosidase and Lactase F “Amano”, diglycosidase and Cellulase A “Amano” 3, diglycosidase and Hemicellulase “Amano” 90G, diglycosidase and Protease B, diglycosidase and YL-15, diglycosidase and Pectinase PL “Amano”, diglycosidase and Transglucosidase L “Amano”, diglycosidase and Gluczyme NL4.2) shown in the table. From these facts, it was evident that flavor-improving effect was stronger in the combination of diglycosidase and each commercially available enzyme preparation than in the case of diglycosidase alone.

Example 11

Flavor Improvement of Soymilk by Diglycosidase or Each Commercially Available Enzyme Preparation

To 20 mL of an ingredient-unadjusted soymilk (Gitoh Shokuhin K.K.) was added diglycosidase or each of commercially available enzyme preparation (ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, all manufactured by Amano Enzyme Inc.) so that diglycosidase was contained 6.5 AU in the soymilk or each enzyme preparation in a concentration of 0.25% (w/v). The treatment was carried out at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated by heat treatment at 70° C. for 1 hour. By the way, the reaction pH was 6.6.

Sensory Test (Flavor Improvement of Soymilk Treated with Diglycosidase or Each Commercially Available Enzyme Preparation)

The enzyme-treated liquid thus obtained was diluted with water by a factor of 3, and then subjected to a sensory test. The evaluation was conducted by five expert panelists, and the deliciousness, bitterness-astringency, and aftertaste were compared with those of Control untreated with the enzyme (Table 41).

TABLE 41
Flavor-improving effect of enzyme preparation on soymilk
Panelist APanelist BPanelist C
bitter-bitter-bitter-
sweet-ness.astrin-after-sweet-ness.astrin-after-sweet-ness.astrin-after-
Enzyme namenessgencytastenessgencytastenessgencytaste
Untreated±±±±±±±±±
(Control)
Diglycosidase±±±±±+++
ADG-D-DS+±++±±
Amylase AD+−−++++±
“Amano” 1
Cellulase A±±±++±±
“Amano” 3
Hemicellulase±±±++±±±
“Amano” 90G
Panelist E
Panelist Dafter-
Enzyme namesweetnessbitterness.astringencyaftertastesweetnessbitterness.astringencytaste
Untreated±±±±±±
(Control)
Diglycosidase+±±+±±
ADG-D-DS+±±+±±
Amylase AD±±±±±±
“Amano” 1
Cellulase A±±±++
“Amano” 3
Hemicellulase±±±++
“Amano” 90G

It was revealed that appearance of sweetness, reduction of bitterness•astringency, and improvement of aftertaste were effected by diglycosidase or a commercially available enzyme preparation.

Example 12

Flavor Improvement of Soymilk by Diglycosidase and Each Commercially Available Enzyme Preparation

To 20 mL of an ingredient-unadjusted soymilk (Gitoh Shokuhin K.K.) was added diglycosidase and each of commercially available enzyme preparation (ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3, or Hemicellulase “Amano” 90G, all manufactured by Amano Enzyme Inc.) so that diglycosidase was contained 6.5 AU in the soymilk and each enzyme preparation in a concentration of 0.25% (w/v). The treatment was carried out at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated by heat treatment at 70° C. for 1 hour. By the way, the reaction pH was 6.6.

Sensory Test (Flavor Improvement of Soymilk Treated with Diglycosidase and Each Commercially Available Enzyme Preparation)

The enzyme-treated liquid thus obtained was diluted with water by a factor of 3, and then subjected to a sensory test. The evaluation was conducted by five expert panelists, and the deliciousness, bitterness-astringency, and aftertaste were compared with those of Control untreated with the enzyme (Table 42).

TABLE 42
Flavor improvement of soymilk by combination of enzymes
Panelist APanelist BPanelist C
bitter-bitter-bitter-
sweet-ness.astrin-after-sweet-ness.astrin-after-sweet-ness.astrin-after-
Enzyme namenessgencytastenessgencytastenessgencytaste
Untreated (Control)±±±±±±±±±
Diglycosidase + ADG-S-DS+−−±++++−−+
Diglycosidase + Amylase+−−−++−−±+−−±
AD “Amano” 1
Diglycosidase + Cellulase A±±+++++−−+
“Amano” 3
Diglycosidase + Hemicellulase+±+++±
“Amano” 90G
Panelist E
Panelist Dafter-
Enzyme namesweetnessbitterness.astringencyaftertastesweetnessbitterness.astringencytaste
Untreated (Control)±±±±±±
Diglycosidase + ADG-S-DS+±++±±
Diglycosidase + Amylase AD+++±±
“Amano” 1
Diglycosidase + Cellulase A+++++
“Amano” 3
Diglycosidase + Hemicellulase+±±+++
“Amano” 90G

As a result of the sensory test, it was revealed that effects, for example, appearance of sweetness, reduction of bitterness-astringency, or improvement of aftertaste were observed in all the combinations of diglycosidase and each commercially available enzyme preparation (diglycosidase and ADG-S-DS, diglycosidase and Amylase AD “Amano” 1, diglycosidase and Cellulase A “Amano” 3, diglycosidase and Hemicellulase “Amano” 90G) shown in the table. Furthermore, it was revealed that such a treatment with the enzymes improves overall flavor, for example, reduction of smelling of grass. From these facts, it was evident that flavor-improving effect was stronger in the combination of diglycosidase and each commercially available enzyme preparation than in the case of diglycosidase alone or a commercially available enzyme preparation alone.

Example 13

Formation of Aglycon Isoflavones from Defatted Soybean Protein by Diglycosidase at the pH in a Stomach

Into 20 mM acetate buffer of pH 4.0 was suspended 0.05 g of defatted soybean protein (Fuji Seiyu K.K.). The pH of the suspension was measured and the liquid volume was adjusted to 4.5 mL while the pH was adjusted to 4.0 with 1N hydrochloric acid. An enzyme solution wherein the diglycosidase activity of crude diglycosidase was adjusted to 1.88 AU/mL was added thereto in an amount of 0.5 mL, whereby the final liquid volume was 5.0 mL (concentration of defatted soybean protein: 1% (w/v)), followed by treatment at 37° C. for 3 hours. After the treatment, 75 μL of methanol and 500 μL of water were added to 25 μL of the reaction mixture. The whole was filtered through a 0.2 μm filter and then the filtrate was further diluted with water by a factor of 2.5, followed by HPLC analysis.

As a result of treating defatted soybean protein at pH 4 which was the pH range in a stomach during a meal as described above, no isolation of aglycon isoflavones was observed in the treatment at pH 4 without adding the enzyme, but isolation of aglycon isoflavones was observed in the product treated with diglycosidase. Therefore, it was proved that diglycosidase could convert isoflavone glycosides into aglycon isoflavones under the pH condition in a stomach.

Example 14

Formation of Aglycon Isoflavones from Roasterd Soy Flour by Diglycosidase at the pH in a Stomach

Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 2.5 g of roasted soy flour (manufactured by Kakudai Sangyo).

To 3 mL of the suspension was added 0.001, 0.002, 0.005, 0.01, 0.025, 0.05, 0.075, 0.15, 0.374, 0.75, or 1.5 mg of diglycosidase (290 AU/g), followed by incubation under shaking at 37° C. for 30 minutes. After the reaction, 10 mL of methanol was added thereto, and aglycon isoflavones were extracted and analyzed by HPLC.

As described above, the formation of aglycon isoflavones from isoflavone glycosides contained in soy flour was investigated after the reaction at pH 5 and 37° C. for 30 minutes on the assumption of the environment in a stomach. As a result, as shown in Table 43 and FIG. 3, 80% or more of the isoflavone glycosides could be converted into aglycon isoflavones. This result suggests that aglycon isoflavones may be formed from isoflavone glycosides in a stomach when diglycosidase is orally administered.

TABLE 43
Relationship between added amount of diglycosidase and
formation (%) of aglycon isoflavones
aglycon
isoflavones
diglycosidase(%)
1.50086.1%
0.75081.3%
0.37474.7%
0.15065.4%
0.07556.3%
0.05051.1%
0.02544.3%
0.01031.0%
0.00520.3%
0.00212.8%
0.0019.2%
05.0%

Example 15

Formation of Aglycon Isoflavones from an Isoflavone Preparation by Diglycosidase at the pH in a Stomach

Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 150 mg of an isoflavone preparation (manufactured by Nature's Bountry, USA). To 3 mL of the suspension was added 0, 0.00045, 0.00113, 0.00225, 0.0045, 0.009, 0.0225, 0.045, 0.09, 0.18, 0.45, or 0.9 mg of diglycosidase (290 AU/g), followed by incubation under shaking at 37° C. for 30 minutes. After the reaction, 10 mL of methanol was added thereto, and aglycon isoflavones were extracted and analyzed by HPLC.

As described above, the formation of aglycon isoflavones from isoflavone glycosides contained in the isoflavone preparation was investigated after the reaction at pH 5 and 37° C. for 30 minutes on the assumption of the environment in a stomach. As a result, as shown in Table 44 and FIG. 4, 80% or more of the isoflavone glycosides could be converted into aglycon isoflavones. This result suggests that aglycon isoflavones may be formed from isoflavone glycosides in a stomach when diglycosidase is orally administered.

TABLE 44
Relationship between added amount of diglycosidase and
formation (%) of aglycon isoflavones
diglycosidaseaglycon isoflavones
(mg)(%)
0.9000079.4%
0.4500078.7%
0.1800075.3%
0.0900070.7%
0.0450064.6%
0.0225059.2%
0.0090047.5%
0.0045038.9%
0.0022530.3%
0.0011325.6%
0.0004521.5%
0.0000020.0%

INDUSTRIAL APPLICABILITY

A physiologically active substance of aglycon type can be efficiently produced, without resort to any acid/alkali treatment or fermentation and substantially without changing the physical properties of a material.

Since diglycosidase has a nature of well acting on 6″-O-acetyl and 6″-o-malonylglucosides which are resistant to decomposition by conventional glucosidase, the process can be conducted at one-step without requiring the process of converting decomposition-resistant isoflavone glycosides into isoflavone glycosides decomposable by glucosidase, the process being described in JP-A-10-117792. Moreover, the present process hardly causes change of physical properties of a starting material derived from decomposition of proteins or phospholipids, the decomposition being caused during the process of hydrolysis with a strong acid. Furthermore, by using diglycosidase and/or a specific enzyme preparation, the aglycon content in a protein or protein-containing food can be increased and also the flavor thereof can be improved.