Method for producing a composition, method for producing a food or drink, and a fermentation composition

Abstract

The present invention is to provide at least a production method of a composition containing equol and / or equol derivative, which does not undergo browning even when heated as it is or in the presence of an additional amino acid or the like. The problem is solved by a production method of a composition including the following (a) process, (b1) process and / or (b2) process, and (c) process. (a) Process of generating glucose and isoflavone aglycone from isoflavone glycoside; (b1) Process of causing a microorganism having equol production ability to produce equol from the isoflavone aglycone; (b2) Process of causing a microorganism having equol derivative production ability to produce equol derivative from the isoflavone aglycone; and (c) Process of decomposing the glucose so that the content of the glucose in the composition is 0 g / L or more and less than 2.0 g / L, or 30 g / kg or less relative to the amount of solid components of the composition.

Description

Technical Field

[0001] This disclosure relates to a method for manufacturing a composition, a method for manufacturing a food or beverage, and a fermentation composition. Background Technology

[0002] Isoflavone glycosides contained in soybean hypocotyl and soybean hypocotyl extract are converted into aglycones by β-glucosidase, and then further metabolized into equol and equol derivatives. Due to the strong physiological effects of equol, its use in preventing and improving menopausal symptoms, osteoporosis (Patent Document 1), preventing and treating skin aging and wrinkles (Patent Document 2), and relieving allergy symptoms (Patent Document 3) has been proposed.

[0003] Estrol derivatives, especially 5-hydroxyestrol, are known to have antioxidant effects (Non-Patent Literature 1) and life-extending effects (Non-Patent Literature 2).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Publication No. 2001-523258

[0007] Patent Document 2: Japanese Patent Publication No. 2002-511860

[0008] Patent Document 3: Japanese Patent No. 4479505

[0009] Non-patent literature

[0010] Non-patent literature 1: Archives Biochem. Biophys. Vol. 356, pp. 133-141 (1998)

[0011] Non-patent literature 2: J. Chin. Pharm. Sci. Vol. 23, pp. 378-384 (2014) Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] When the inventors manufactured compositions containing equol and / or equol derivatives from isoflavone glycosides, they found that the compositions browned upon heating. Furthermore, when these compositions were mixed with raw materials containing proteins or the like for food production and heated, browning also occurred upon heating. This is believed to be caused by the reaction between reducing sugars and amino acids, known as the Maillard reaction. Moreover, the Maillard reaction may produce byproducts considered carcinogenic, such as acrylamide.

[0014] The problem with this disclosure is to provide at least one method for manufacturing a composition comprising equol and / or equol derivatives, wherein the composition does not brown even when heated in its original state or in the presence of added amino acids, etc.

[0015] Solution for solving the problem

[0016] The inventors conducted in-depth research and discovered that when isoflavone glycosides are converted into aglycones, glucose, a reducing sugar, is produced. This glucose remains in the manufactured composition, causing the Maillard reaction to occur upon heating, resulting in browning of the composition. Furthermore, it was found that the above problem can be solved by implementing a step that decomposes the glucose contained in the composition.

[0017] That is, this disclosure includes at least the following.

[0018] [1] A method of manufacturing a composition, comprising: step (a); step (b1) and / or step (b2); and step (c).

[0019] (a) a step of generating glucose and isoflavone aglycone from isoflavone glycosides; (b1) a step of generating equol from the isoflavone aglycone using microorganisms capable of equol production; (b2) a step of generating equol derivatives from the isoflavone aglycone using microorganisms capable of equol derivative production; and (c) a step of decomposing the glucose so that the glucose content in the composition is 0 g / L or more and less than 2.0 g / L, or 30 g / kg or less relative to the solid content of the composition.

[0020] [2] According to the manufacturing method of [1], wherein step (c) is a step of decomposing the glucose by using fermentation of microorganisms with sugar assimilation ability.

[0021] [3] According to the manufacturing method described in [2], the microorganism having sugar assimilation ability is selected from one or more microorganisms in the group consisting of butyric acid bacteria, lactic acid bacteria and yeast.

[0022] [4] According to the manufacturing method described in [3], wherein the microorganism having sugar assimilation ability is selected from one or more microorganisms belonging to the genus Clostridium, the genus Lactobacillus, the genus Lactococcus, the genus Leuconostoc, and the genus Saccharomyces.

[0023] [5] According to the manufacturing method described in [1], wherein step (c) is a step of using an enzyme that breaks down glucose to break down the glucose.

[0024] [6] According to the manufacturing method described in [5], wherein the enzyme that breaks down glucose is selected from one or more of the group consisting of glucose oxidase and glucose dehydrogenase.

[0025] [7] The manufacturing method according to any one of [1] to [6], wherein the (b1) step and / or the (b2) step and the (c) step are performed simultaneously.

[0026] [8] The manufacturing method according to any one of [1] to [7], wherein the isoflavone glycoside is daidzein and the isoflavone aglycone is daidzein.

[0027] [9] The manufacturing method according to any one of [1] to [8], wherein step (a) is a step of causing β-glucosidase to act on isoflavone glycosides to generate glucose and isoflavone aglycones.

[0028]

[10] A method of manufacturing a food product, comprising: the following steps (a); (b1) and / or (b2); (c); and (d).

[0029] (a) a step of generating glucose and isoflavone aglycone from isoflavone glycosides; (b1) a step of generating equol from the isoflavone aglycone using microorganisms capable of equol production; (b2) a step of generating equol derivatives from the isoflavone aglycone using microorganisms capable of equol derivative production; (c) a step of decomposing the glucose such that the glucose content is 0 g / L or more and less than 2.0 g / L in a composition manufactured by the method comprising steps (a), (b1) and / or (b2) and (c), or less than 30 g / kg relative to the solid content of the composition; and (d) a step of manufacturing a food or beverage using a composition manufactured by the method comprising steps (a), (b1) and / or (b2) and (c).

[0030]

[11] A fermentation composition comprising equol and / or equol derivatives and substantially free of glucose.

[0031]

[12] The fermentation composition according to

[11] , wherein "substantially glucose-free" means containing more than 0 g / L and less than 2.0 g / L of glucose.

[0032]

[13] A fermentation composition comprising equol and / or equol derivatives and glucose, wherein the glucose content is less than 30 g / kg relative to the solid content of the fermentation composition.

[0033]

[14] The fermentation composition according to any one of

[11] to

[13] , wherein the fermentation composition is used in food and beverage products.

[0034] Invention Effects

[0035] This disclosure achieves at least the following effects: it provides a method for manufacturing a composition comprising equol and / or equol derivatives, said composition reducing the amount of glucose, and does not undergo Maillard reaction or browning even when heated in the presence of the original or added amino acids, etc. Detailed Implementation

[0036] The various components and combinations thereof in each embodiment are merely examples, and appropriate additions, omissions, substitutions, and other modifications may be made to the components without departing from the spirit of this disclosure. This disclosure is not limited to the embodiments but only to the claims. Furthermore, the various solutions disclosed in this specification can also be combined with any other features disclosed in this specification.

[0037] In addition, the numerical range indicated by “~” refers to the range of values ​​recorded before and after “~” as the lower and upper limits. “A~B” means above A and below B.

[0038] In this specification, the accession number of strains beginning with the letters DSM is the number assigned to microorganisms deposited in DSMZ (Germany Microbial Culture Collection: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH).

[0039] The accession number of strains beginning with the letters JCM is a number assigned to microorganisms preserved at the Japan Collection of Microorganisms (Microbial Materials Development Laboratory, Research Center for Biological Resources, RIKEN, 305-0074, Ibaraki Prefecture, Japan), and can be obtained from that institution.

[0040] The accession number of strains beginning with the letters FERM is a number assigned to microorganisms preserved at the National Institute of Advanced Industrial Science and Technology (IAIST) Patent Biology Collection Center (now the Patent Biology Collection Center of the Technical Base for Product Evaluation, IAIST, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture), and can be obtained from that institution.

[0041] The accession number of strains beginning with the letters KCCM is a number assigned to microorganisms preserved at the Korean Culture Center of Microorganisms (KCCM), and can be obtained from that institution.

[0042] In this specification, “microorganism” means true bacteria, archaea, or fungi, and does not include viruses, plants, or animals.

[0043] In this specification, glucose decomposition refers to the inactivation of the reducing end of glucose in a manner that does not involve Maillard reaction with amino acids, including glucose oxidation and its conversion into other compounds through metabolism.

[0044] Furthermore, there are no particular limitations on the definition of amino acids, as long as they are compounds that can undergo a Maillard reaction with glucose; compounds containing an amino group can be listed. Examples of compounds containing an amino group include amino acids, peptides, and proteins.

[0045] One embodiment of this disclosure is a method of manufacturing a composition, comprising: step (a), step (b1) and / or step (b2) described below; and step (c).

[0046] (a) a step of generating glucose and isoflavone aglycone from isoflavone glycosides; (b1) a step of generating equol from the isoflavone aglycone using microorganisms capable of equol production; (b2) a step of generating equol derivatives from the isoflavone aglycone using microorganisms capable of equol derivative production; and (c) a step of decomposing the glucose so that the glucose content in the composition is 0 g / L or more and less than 2.0 g / L, or 30 g / kg or less relative to the solid content of the composition.

[0047] (a) Process

[0048] The manufacturing method of this embodiment includes: (a) a step of generating glucose and isoflavone aglycone from isoflavone glycosides.

[0049] Isoflavone glycosides refer to glycosides of isoflavones. Isoflavones are a type of polyphenol, and are flavonoids with isoflavones as their basic backbone.

[0050] Isoflavones are mostly found in legumes such as soybeans, kudzu, red clover, and licorice. As isoflavones in this embodiment, examples include isoflavones derived from legumes. Specifically, examples include: isoflavones derived from soybeans (sometimes referred to as "soy isoflavones" or "soy isoflavones" in this technical field and market, and treated as having the same meaning in this disclosure), isoflavones derived from kudzu (sometimes referred to as "kudzu isoflavones" or "kudzu isoflavones" in this technical field and market, and treated as having the same meaning in this disclosure), isoflavones derived from red clover (sometimes referred to as "red clover isoflavones" or "red clover isoflavones" in this technical field and market, and treated as having the same meaning in this disclosure), and isoflavones derived from licorice (sometimes referred to as "licorice isoflavones" or "licorice isoflavones" in this technical field and market, and treated as having the same meaning in this disclosure).

[0051] Examples of isoflavone glycosides include genistin, gycitin, and daidzin. In this embodiment, a mixture of two or more may also be used. For example, it could be a mixture of daidzin, gycitin, and genistin. Since these are contained in soybean hypocotyl, soybean hypocotyl extract, etc., soybean hypocotyl extract can be used, for example, as a mixture of isoflavone glycosides.

[0052] Examples of isoflavone aglycones include: daidzein, 6-hydroxydaidzein, dihydroxydaidzein, genistein, stigmacin, chickpea sprout extract A, formononetin, orobol, and coumestrol. In this embodiment, a mixture of two or more isoflavone aglycones may also be used. It should be noted that the term "isoflavone aglycone" in this disclosure is sometimes referred to as "aglycone of isoflavones" or similar terms in the art and market, and is used interchangeably in this disclosure.

[0053] In this embodiment, the isoflavone glycoside is preferably daidzein, and the isoflavone aglycone is daidzein; or the isoflavone glycoside is genistein, and the isoflavone aglycone is genistein. More preferably, the isoflavone glycoside is daidzein, and the isoflavone aglycone is daidzein.

[0054] Methods for generating glucose and isoflavone aglycones from isoflavone glycosides include methods that involve β-glucosidase acting on isoflavone glycosides and methods that involve culturing microorganisms that produce β-glucosidase in a culture medium containing isoflavone glycosides.

[0055] β-glucosidase is an enzyme that catalyzes the hydrolysis of β-glycosidic bonds in sugars. There are no specific limitations on the type of β-glucosidase that possesses the aforementioned enzymatic activities; examples include β-glucosidases derived from microorganisms, higher plants, and animals, such as those belonging to the genera *Aspergillus*, *Penicillium*, *Rhizopus*, *Pseudomonas*, *Pichia*, and *Lactococcus*. Additionally, commercially available enzyme preparations containing β-glucosidase, such as pectinase G (manufactured by Amano Enzyme), can also be used.

[0056] To induce β-glucosidase to act on isoflavone glycosides, β-glucosidase is simply added to the solution containing the isoflavone glycosides. The amount added can be adjusted appropriately, typically 10 U or more and 3000 U or less relative to 1 g of isoflavone glycosides, preferably 20 U or more and 1000 U or less, and more preferably 50 U or more and 500 U or less.

[0057] The conditions for β-glucosidase to act on isoflavone glycosides are not particularly limited as long as β-glucosidase acts on isoflavone glycosides and catalyzes the release of glucose. The temperature is usually above 15°C and below 65°C, preferably above 20°C and below 55°C, and more preferably above 30°C and below 45°C.

[0058] In addition, the pH is generally 2 or higher and 9 or lower, preferably 3 or higher and 7 or lower, and more preferably 4 or higher and 6 or lower.

[0059] There are no particular restrictions on the type of microorganism that produces β-glucosidase; one or more microorganisms can be used, regardless of genus, species, or strain.

[0060] For example, microorganisms belonging to the genera Aspergillus, Lactococcus, Streptococcus, Lactobacillus, Bifidobacterium, Penicillium, Pichia, Pseudomonas, and Rhizopus can be listed.

[0061] Preferred species include bacteria belonging to the genera Aspergillus, Lactococcus, Streptococcus, Lactobacillus, and Bifidobacterium.

[0062] Further selection can include microorganisms belonging to Aspergillus niger, Aspergillus oryzae, Aspergillus aculeatus, Lactococcus lactis, Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium bifidum.

[0063] As for the culture conditions of the microorganisms that produce β-glucosidase, the culture conditions commonly used in the culture of said microorganisms, or culture conditions with appropriate modifications, can be used.

[0064] As a culture medium, for example, BHI medium manufactured by Difco, ANAEROBE BASAL BROTH (ABB medium) manufactured by Oxoid, Wilkins-Chalgren Anaerobe Broth (CM0643) manufactured by Oxoid, GAM medium manufactured by Nissui Pharmaceutical Co., Ltd., and modified GAM medium, etc., can be used. It should be noted that in this specification, culture medium refers to a solution containing a basic culture medium in which microorganisms can proliferate, excluding solutions in which microorganisms cannot proliferate, such as water, salt solutions, buffer solutions, etc.

[0065] Isoflavone glycosides are added to the culture medium. The addition of isoflavone glycosides to the culture medium can be carried out before or during the culture of the microorganisms.

[0066] The content of isoflavone glycosides is not particularly limited, but is usually above 0.5 g / L and below 100 g / L, preferably above 1 g / L and below 50 g / L, and more preferably above 2 g / L and below 20 g / L.

[0067] Water-soluble organic compounds can be added to the culture medium as a carbon source. Examples of water-soluble organic compounds include: sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, and xylose; alcohols such as glycerol; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, fumaric acid, and succinic acid; and polysaccharides such as dextrin.

[0068] The concentration of organic matter added to the culture medium as a carbon source can be appropriately adjusted to ensure effective growth. Typically, the addition amount can be selected from the range of 0.1 to 10 wt / vol%.

[0069] In addition to the carbon sources mentioned above, nitrogen sources can also be added to the culture medium. Various nitrogen compounds commonly used in fermentation can be used as nitrogen sources. For example, inorganic and organic nitrogen sources can be listed.

[0070] Examples of inorganic nitrogen sources include ammonium salts and nitrates. Preferred inorganic nitrogen sources include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, or sodium nitrate.

[0071] In addition, examples of organic nitrogen sources include: amino acids (glutamic acid, arginine, ornithine, etc.), oils such as oleic acid, yeast extracts, peptones (e.g., polypeptone N, soybean peptone, etc.), meat extracts (e.g., Ehrlich bonito extract, Lab-Lemco, broth culture medium, etc.), fish and shellfish extracts, liver extracts, digested serum, fish oil, etc. More preferably, arginine, cysteine, citrulline, lysine, yeast extracts, and peptones (e.g., polypeptone N, etc.).

[0072] Furthermore, in addition to carbon and nitrogen sources, sometimes cofactors such as vitamins and inorganic compounds such as various salts are added to the culture medium to enhance proliferation and activity. For example, the following can be listed as cofactors for the proliferation of microorganisms derived from plants and animals, such as inorganic compounds and vitamins.

[0073]

[0074] The method of adding these inorganic compounds, vitamins, and other plant and animal-derived growth promoters to prepare culture media is well known. The culture medium can be liquid, semi-solid, or solid. Liquid culture medium is preferred.

[0075] Furthermore, the culture medium disclosed herein can contain dextrins. By culturing anaerobic microorganisms in a dextrin-containing culture medium, functional substances and dextrin-containing solutions can be obtained without adding dextrins, even if dextrins are required in the post-culturing culture medium. The addition of dextrins to the culture medium can be carried out before or during the cultivation of the microorganisms.

[0076] In addition, under anaerobic conditions, it is possible to improve growth by adding reducing agents such as cysteine, cystine, sodium sulfide, sulfite, ascorbic acid, glutathione, thioglycolic acid, and rutin, as well as enzymes that decompose reactive oxygen species such as catalase and superoxide dismutase to the culture medium.

[0077] In anaerobic culture, the gaseous and aqueous phases used in the culture preferably do not contain air or oxygen. Examples include nitrogen and / or hydrogen in any ratio, and nitrogen and / or carbon dioxide in any ratio. They can be supplied in gaseous form.

[0078] The proportion of hydrogen in the gas phase is not particularly limited, but is usually 0.5% or more and 100% or less, preferably 1.0% or more and 20% or less, and more preferably 2.0% or more and 10% or less.

[0079] The pH of the culture medium is preferably 5.0 or higher, more preferably 6.0 or higher, even more preferably 6.5 or higher, and on the other hand, preferably 8.0 or lower, more preferably 7.5 or lower.

[0080] The preferred culture temperature is 20℃~45℃, more preferably 25℃~40℃, and even more preferably 30℃~37℃.

[0081] The pressure conditions of the incubator can be any conditions that allow growth, without any particular limitation. A range of 0.001 to 1 MPa can be listed, with 0.01 to 0.5 MPa being preferred.

[0082] Examples of cultivation time include typically 8 to 340 hours, preferably 12 to 170 hours, and more preferably 16 to 120 hours.

[0083] [(b1) Process]

[0084] The manufacturing method of this embodiment includes: (b1) a step of causing a microorganism capable of producing equol to produce equol from the isoflavone aglycone and / or the step described later (b2).

[0085] Examples of microorganisms capable of producing equol include: those belonging to the genus *Adlercreutzia*, the genus *Atopobium*, the genus *Bacteroides*, the genus *Collinsella*, the genus *Coriobacterium*, the genus *Cryptobacterium*, the genus *Denitrifying Bacteria*, and the genus *Eggerthella*. Microorganisms belonging to the genera *Enterobacter*, *Eubacterium*, *Gordonibacter*, *Lactococcus*, *Olsenella*, *Paraeggerthella*, *Ruminococcus*, *Slackia*, and *Streptococcus*.

[0086] Microorganisms belonging to the following genera are preferred: *Adlercreutzia equolifaciens* subsp. *equolifaciens*, *Adlercreutzia equolifaciens* subsp. *celatus*, *Bacteroides ovatus*, *Eggerthella* sp., *Eubacterium* sp., *Lactococcus garvieae*, *Paraeggerthella* sp., *Ruminococcus productus*, *Slackia isoflavoniconvertens*, *Slackia equolifaciens*, *Slackia* sp., and *Streptococcus intermedius*.

[0087] Further preferred strains include: Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450, Adlercreutzia equolifaciens subsp. celatus DSM 18785, Eggerthella sp. KCCM 10490, Lactococcus garvieae DSM 6783, Slackia isoflavoniconvertens DSM 22006, Slackia equolifaciens DSM 24851, and Slackia sp. FERM AP-20729.

[0088] The above microorganisms, regardless of genus, species, or strain, can be used alone or in combination.

[0089] Furthermore, in this embodiment, the *Adlercreutzia equolifaciens* subsp. *equolifaciens* DSM 19450 strain is not limited to the same strain as the aforementioned preserved strain, but can be a strain substantially equivalent to the aforementioned preserved strain. A substantially equivalent strain refers to a strain belonging to the same genus or species as the aforementioned preserved strain and possessing the ability to produce equol. Furthermore, a substantially equivalent strain refers to a microorganism whose 16S rRNA gene sequence has at least 97%, preferably at least 97.5%, more preferably at least 98%, further preferably at least 98.7%, and even more preferably at least 99% homology to the 16S rRNA gene sequence of the aforementioned preserved strain. Moreover, the microorganism capable of producing equol can be a strain obtained from the aforementioned preserved strain or a substantially equivalent strain through mutation treatment, gene recombination, selection of natural mutant strains, etc., provided that it does not impair the effects of this disclosure.

[0090] This also applies to other preserved strains in this specification.

[0091] In the case where a microorganism capable of producing equol produces β-glucosidase, the microorganism producing β-glucosidase can be the same as the microorganism capable of producing equol. In this case, steps (a) and (b1) are carried out continuously in the same system.

[0092] As culture conditions, the culture conditions commonly used in the culture of the microorganisms, or culture conditions with appropriate modifications, can be used. Examples of the same conditions as those for the culture of microorganisms that produce β-glucosidase can be listed.

[0093] (b2) Process

[0094] The manufacturing method of this embodiment includes: step (b1) and / or step (b2) in which a microorganism capable of producing equol derivatives produces equol derivatives from the isoflavone aglycone. 5-hydroxyequol is an example of an equol derivative.

[0095] In the case where the equol derivative is 5-hydroxyequol, microorganisms belonging to the genera Adlercreutzia, Eggerthella, and Slackia can be listed as having the ability to produce 5-hydroxyequol.

[0096] As a microorganism belonging to the genus Adlercreutzia, it is preferred to be a microorganism belonging to the equolifaciens species, among which, Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 or Adlercreutzia equolifaciens subsp. celatus DSM 18785 are preferred.

[0097] As microorganisms belonging to the genus Eggerthella, the following microorganisms belonging to the genus Eggerthella sp. can be listed. Among them, strain KCCM 10490 of Eggerthella sp. is preferred.

[0098] As microorganisms belonging to the genus Slackia, examples include Slackia isoflavoniconvertens, Slackiaequolifaciens, and Slackia sp.

[0099] In the case where a microorganism capable of producing equol derivatives produces β-glucosidase, the microorganism producing β-glucosidase can be the same as the microorganism capable of producing equol derivatives. In this case, steps (a) and (b2) are carried out continuously in the same system.

[0100] As culture conditions, the culture conditions commonly used in the culture of the microorganisms, or culture conditions with appropriate modifications, can be used. Examples of the same conditions as those for the culture of microorganisms that produce β-glucosidase can be listed.

[0101] (c) Process

[0102] The manufacturing method of this embodiment includes: (c) a step of decomposing the glucose to a glucose content in the composition of 0 g / L or more and less than 2.0 g / L, or a solid content relative to the composition of 30 g / kg or less. Decomposing glucose refers to converting glucose into a compound having fewer carbon atoms.

[0103] (c) The process may be a process of breaking down the glucose by fermentation using microorganisms with sugar assimilation capabilities, or a process of breaking down the glucose using enzymes that break down glucose.

[0104] When fermentation with microorganisms capable of sugar assimilation is used in step (c), the microorganisms capable of sugar assimilation are preferably selected from one or more microorganisms in the group consisting of butyric acid bacteria, lactic acid bacteria, and yeast. More preferably, they are selected from one or more microorganisms in the group consisting of microorganisms belonging to the genus Clostridium, microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Leuconostoc, and microorganisms belonging to the genus Saccharomyces.

[0105] Microorganisms belonging to the genus Clostridium include: Clostridium asparagiforme DSM strain 15981, Clostridium bolteae JCM strain 12243, etc.

[0106] As microorganisms belonging to the genus Lactobacillus, examples include Lactobacillus brevis DSM 20054.

[0107] As microorganisms belonging to the genus Lactococcus, examples include Lactococcus lactis strain DSM 20481.

[0108] As microorganisms belonging to the genus Leuconostoc, examples include Leuconostoc mesenteroides subsp. mesenteroides DSM 20343.

[0109] In the case where a microorganism with sugar assimilation capacity produces β-glucosidase, the microorganism producing β-glucosidase can be the same as the microorganism with sugar assimilation capacity. In this case, steps (a) and (c) can be carried out continuously in the same system.

[0110] Microorganisms capable of sugar assimilation can be the same species as microorganisms capable of equol production. That is, microorganisms capable of sugar assimilation can be microorganisms capable of both equol production and sugar assimilation. In this case, steps (b1) and (c) are carried out continuously in the same system.

[0111] Microorganisms capable of sugar assimilation can be the same species as those capable of producing equol derivatives. That is, microorganisms capable of sugar assimilation can be microorganisms capable of both producing equol derivatives and sugar assimilation. In this case, steps (b2) and (c) are carried out continuously in the same system.

[0112] As culture conditions, the culture conditions commonly used in the culture of the microorganisms, or culture conditions with appropriate modifications, can be used. Examples of the same conditions as those for the culture of microorganisms that produce β-glucosidase can be listed.

[0113] In step (c), when an enzyme that breaks down glucose is used, glucose oxidase and glucose dehydrogenase can be listed as enzymes that break down glucose. They can be used alone or in combination of two or more. Glucose oxidase and glucose dehydrogenase are enzymes that catalyze the reaction of oxidizing β-D-glucose to D-gluconic acid-1,5-lactone.

[0114] There are no restrictions on whether glucose oxidase and glucose dehydrogenase have the above-mentioned enzyme activities. Examples include glucose oxidase and glucose dehydrogenase derived from Aspergillus niger and Gluconobacter oxydans, respectively.

[0115] When using an enzyme that breaks down glucose to break it down, the enzyme is simply added to a solution (culture medium) containing glucose. The amount added can be adjusted appropriately, typically 1 U or more and 10,000 U or less relative to 1 g of glucose, preferably 10 U or more and 5,000 U or less, and more preferably 100 U or more and 2,000 U or less.

[0116] There is no particular limitation on the temperature during glucose decomposition, but it is usually above 15°C and below 60°C, preferably above 25°C and below 50°C, more preferably above 30°C and below 40°C, and particularly preferably 35°C.

[0117] The pH value for glucose decomposition is not particularly limited, but is generally 4 or higher and 9 or lower, preferably 4 or higher and 7 or lower, and more preferably 5 or higher and 6 or lower.

[0118] When performing steps (b1) and (b2), there is no restriction on the order of these steps. Step (b2) can be performed after step (b1), step (b1) can be performed after step (b2), or steps (b1) and (b2) can be performed simultaneously.

[0119] There is no restriction on the order of process (b1) and / or process (b2) and process (c). Process (c) can be performed after process (b1) and / or process (b2), after process (c), or process (b1) and / or process (b2) can be performed simultaneously.

[0120] As a scheme for simultaneously performing step (b1) and / or step (b2) and step (c), specifically, the following schemes can be listed: adding an enzyme that breaks down glucose to a culture medium containing glucose, isoflavone aglycones, and microorganisms capable of producing equol; co-culturing microorganisms capable of producing equol and microorganisms capable of assimilating sugar in a culture medium containing glucose and isoflavone aglycones; culturing microorganisms capable of producing equol and assimilating sugar in a culture medium containing glucose and isoflavone aglycones, etc.

[0121] In step (c), the glucose is decomposed such that the glucose content in the composition is 0 g / L or more and less than 2.0 g / L, or the amount of solids in the composition is 30 g / kg or less.

[0122] To keep the glucose content within the above range, the culture conditions and the treatment conditions of the enzymes that break down glucose can be adjusted appropriately. For example, extending the culture time or treatment time can be considered.

[0123] The glucose content in the composition is preferably 0 g / L or more and less than 2.0 g / L, more preferably 0 g / L or more and less than 1.0 g / L, even more preferably 0 g / L or more and less than 0.5 g / L, and particularly preferably 0 g / L or more and less than 0.2 g / L. Furthermore, the amount of solids relative to the composition is preferably 30 g / kg or less, more preferably 10 g / kg or less, and even more preferably 4 g / kg or less. The amount of solids is determined by measuring the residual weight after drying the composition. Examples of drying methods include vacuum drying, atmospheric pressure drying, and freeze-drying.

[0124] The glucose content can be determined by the electrode method.

[0125] When the composition is in solid form or does not contain water, water can be added to the composition to dissolve the glucose in the water before measurement, and the glucose content relative to the amount of solid component can be determined.

[0126] [Other processes]

[0127] The manufacturing method of this embodiment may include, for example, a step of quantifying the obtained equol and / or equol derivatives. This method can follow conventional procedures. For example, it may involve collecting a portion of the culture medium and diluting it appropriately, stirring thoroughly, filtering it using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and quantifying the removed insoluble substances using high-performance liquid chromatography (HPLC).

[0128] Furthermore, the manufacturing method of this embodiment may include a step of recovering the obtained equol and / or equol derivatives. The recovery step includes a purification step, a concentration step, etc. As a purification process, the following treatments may be performed: sterilization of microorganisms based on heat, etc.; sterilization based on microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymers; extraction based on organic solvents, ionic liquids, etc.; adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, etc. Furthermore, as a concentration process, concentration based on evaporators, reverse osmosis membranes, etc., can be cited as examples.

[0129] Furthermore, the resulting solution containing equol and / or equol derivatives can be pulverized by freeze-drying, spray drying, or other methods. Excipients such as lactose, dextrin, and corn starch can also be added during pulverization.

[0130] [Methods for manufacturing food and beverages]

[0131] The composition manufactured by the method of this embodiment does not undergo the Maillard reaction or browning even when heated in its original state or in the presence of added amino acids, etc., and therefore can be preferably included in food and beverage products. It should be noted that, in this specification, food and beverage products include supplements.

[0132] That is, another embodiment of this disclosure is a method for manufacturing a food product, which includes: the following steps (a); (b1) and / or (b2); (c); and (d).

[0133] (a) a step of generating glucose and isoflavone aglycone from isoflavone glycosides; (b1) a step of generating equol from the isoflavone aglycone using microorganisms capable of equol production; (b2) a step of generating equol derivatives from the isoflavone aglycone using microorganisms capable of equol derivative production; (c) a step of decomposing the glucose such that the glucose content is 0 g / L or more and less than 2.0 g / L in a composition manufactured by the method comprising steps (a), (b1) and / or (b2) and (c), or less than 30 g / kg relative to the solid content of the composition; and (d) a step of manufacturing a food or beverage using a composition manufactured by the method comprising steps (a), (b1) and / or (b2) and (c).

[0134] The food and beverage manufactured by the manufacturing method of this embodiment contains equol and / or equol derivatives. In addition to ordinary food and beverages, the aforementioned food and beverages can also be used as specific health foods, nutritional supplements, functional foods, patient foods, food additives, etc. (including beverages). As a form of food and beverage, for example, after adding appropriate additives, it can be shaped into an edible form using conventional methods, such as granules, pellets, tablets, capsules, pastes, etc., for consumption. Furthermore, it can be added to various foods, such as processed meat products like ham and sausages; processed seafood products like fish cakes and chikuwa; bread, pastries, butter, milk powder, and fermented dairy products; or added to beverages such as water, fruit juice, milk, and soft drinks.

[0135] The aforementioned food and beverage products can include water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavorings. Examples of proteins include: whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, meat protein, and other animal and plant proteins; their hydrolysates; and butter. Examples of carbohydrates include: sugars, processed starches (excluding dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fiber. Examples of lipids include: lard, safflower oil, corn oil, rapeseed oil, coconut oil; their fractionated oils, hydrogenated oils, transesterified oils, and other vegetable oils. Examples of vitamins include: vitamin A, carotene, B vitamins, vitamin C, vitamin D, vitamin E, vitamin K, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these components can be used in combination, or synthetic products and / or foods and beverages containing a significant amount of these components can be used.

[0136] With respect to step (a); step (b1) and / or step (b2); and step (c), the description is referenced in the method of manufacturing the composition according to one embodiment of this disclosure. Furthermore, the method of manufacturing the food and beverage according to this embodiment may include the other steps described above.

[0137] (d) The process of manufacturing a food and beverage using the composition produced by the method comprising steps (a), (b1), and / or (b2) and (c) can be carried out according to conventional methods. Furthermore, the amount of equol and / or equol derivatives, the method of formulation, and the timing of formulation can be appropriately selected. Moreover, the aforementioned food and beverage can be sealed in containers such as bottles, bags, cans, boxes, and packs as needed.

[0138] [Fermentation Composition]

[0139] By the method of manufacturing the composition according to one embodiment of the present disclosure, a composition containing equol and / or equol derivatives can be obtained that reduces the amount of glucose and does not undergo Maillard reaction even when heated in the presence of the original or added amino acids.

[0140] That is, another embodiment of this disclosure is a fermentation composition comprising equol and / or equol derivatives, and substantially free of glucose. Alternatively, another embodiment of this disclosure is a fermentation composition comprising equol and / or equol derivatives, wherein the glucose content is less than 30 g / kg relative to the solids content of the fermentation composition. 5-Hydroxyequol is an example of an equol derivative.

[0141] Furthermore, the fermentation composition of this embodiment does not undergo the Maillard reaction or browning even when heated in its original state or in the presence of added amino acids, etc., and therefore can be preferably included in food and beverage products. That is, the above-described fermentation composition can be used in food and beverage products.

[0142] "Substantially glucose-free" means that the glucose content is low enough not to cause browning of the composition due to the Maillard reaction. Specifically, this includes cases where the glucose content is 0 g / L or more but less than 2.0 g / L.

[0143] The glucose content in the fermentation composition is preferably 0 g / L or more and less than 2.0 g / L, more preferably 0 g / L or more and less than 1.0 g / L, even more preferably 0 g / L or more and less than 0.5 g / L, and particularly preferably 0 g / L or more and less than 0.2 g / L. The amount of solids relative to the fermentation composition is preferably 30 g / kg or less, more preferably 10 g / kg or less, and even more preferably 4 g / kg or less. The amount of solids is determined by measuring the residual weight after drying the fermentation composition. Examples of drying methods include vacuum drying, atmospheric pressure drying, and freeze-drying.

[0144] The glucose content can be determined by the electrode method.

[0145] When the fermentation composition is in solid form and does not contain water, water can be added to the fermentation composition to dissolve the glucose in the water before measurement, and the glucose content relative to the amount of solid component can be determined.

[0146] The content of equol and / or equol derivatives relative to the solid content of the fermentation composition is preferably 1 g / kg or more, more preferably 5 g / kg or more, and even more preferably 10 g / kg or more.

[0147] The content of equol and / or equol derivatives can be determined by the above HPLC.

[0148] The fermentation composition may include other ingredients that can be contained in the above-mentioned culture medium.

[0149] In addition to ordinary food and beverage products, food and beverage products containing fermented compositions can also be used as specific health foods, nutritional supplements, functional foods, patient foods, food additives, etc. (including beverages). As food and beverage forms, they can be shaped into edible forms, such as granules, tablets, capsules, pastes, etc., after adding appropriate additives, using conventional methods for consumption. They can also be added to various foods, such as processed meat products like ham and sausages; processed seafood products like fish cakes and chikuwa; bread, pastries, butter, milk powder, and fermented dairy products; or beverages like water, fruit juice, milk, and soft drinks.

[0150] The aforementioned food and beverage products can include water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavorings. Examples of proteins include: whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, meat protein, and other animal and plant proteins; their hydrolysates; and butter. Examples of carbohydrates include: sugars, processed starches (excluding dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fiber. Examples of lipids include: lard, safflower oil, corn oil, rapeseed oil, coconut oil; their fractionated oils, hydrogenated oils, transesterified oils, and other vegetable oils. Examples of vitamins include: vitamin A, carotene, B vitamins, vitamin C, vitamin D, vitamin E, vitamin K, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these components can be used in combination, or synthetic products and / or foods and beverages containing a significant amount of these components can be used.

[0151] The aforementioned food and beverage products can be manufactured using conventional methods. Furthermore, the amount, method, and timing of the formulation of equol and / or equol derivatives can be appropriately selected. Subsequently, the aforementioned food and beverage products can be sealed in containers such as bottles, bags, jars, boxes, and packs as needed.

[0152] Example

[0153] The present disclosure will be further described in detail below through specific embodiments, but the present disclosure is not limited to these embodiments.

[0154] (Methods for determining glucose concentration)

[0155] Take 1 mL of the liquid containing the test sample and centrifuge to allow the bacterial cells to settle. Filter the supernatant through a 0.45 μm filter and analyze the filtrate under the following conditions. If the concentration exceeds the detection range (10 mg / dL to 600 mg / dL), dilute the filtrate with Milli-Q water and analyze.

[0156] Inspection device: Glutest Neo alpha manufactured by Sanwa Chemical Research Institute Co., Ltd.

[0157] Chip: Glutest Neo sensor manufactured by Sanwa Chemical Research Institute Co., Ltd.

[0158] The concentration of solid components (kg / L) in the liquid of the test subject was measured using an electronic moisture meter (Shimadzu MOC-120H) relative to the content of solid components (g / kg), and the glucose concentration (g / L) was calculated by dividing the above glucose concentration by the concentration of solid components.

[0159] (Methods for determining equol concentration and equol derivatives)

[0160] Take 20 μL of the test liquid and dilute it 50 times with a diluent consisting of ethanol:Milli-Q water = 70:30 (v / v). Filter the diluted solution through a 0.45 μm filter and analyze the filtrate under the following HPLC conditions.

[0161] (S)-Estradiol manufactured by FUJIFILM Wako Pure Chemical Co., Ltd. was used as the estradiol standard, and 5-hydroxyestradiol manufactured by Toronto Research Chemicals Co., Ltd. was used as the 5-hydroxyestradiol standard.

[0162] {HPLC conditions}

[0163] Column: Phenomenex SYNERGI, 4μm, POLAR-R, 150mm×4.6mm.

[0164] Eluent: Distilled water / methanol = 55 / 45 (v / v).

[0165] Temperature: 40℃.

[0166] Detection wavelength: 280nm.

[0167] Flow rate: 1.0 mL / min.

[0168] Injection: 10 μL.

[0169] Time: 30 minutes.

[0170] [Experimental Example 1]

[0171] (Preparation of pre-culture medium)

[0172] Dissolve 35.4 g of Anaerobe Basal Broth (ABB) medium (Thermo Scientific) in 1 L of water to prepare a pre-culture medium. Dispense 10 mL of the pre-culture medium into test tubes and seal with butyl rubber stoppers. After purging with nitrogen, sterilize in an autoclave.

[0173] (Enzyme treatment)

[0174] Add 0.16 g / L of pectinase G Amano (manufactured by Amano Enzyme) to 16 g / L of soybean hypocotyl extract (containing 80% of daidzein, genistein, genistein and other isoflavone glycosides), and keep at 50°C overnight while stirring, thereby releasing the sugar from the isoflavone glycosides.

[0175] (Preparation of formal culture medium)

[0176] The enzyme treatment solution was added to a final concentration of 35.4 g / L ABB medium, 1 g / L arginine, and 16 g / L β-cyclodextrin, and 1 L was dispensed into each of the 2 L mini jars. After purging with nitrogen, the mixture was autoclaved.

[0177] (Pre-culture)

[0178] After inoculating the microorganisms in the pre-culture medium with the combinations shown in Table 1, the medium was replaced with anaerobic gas and cultured at 37°C and 200 sppm for 2 days.

[0179] As microorganisms capable of producing equol, any species of *Adlercreutzia equolifaciens* subsp. *celatus* DSM 18785 (referred to as AC in the table) and *Adlercreutziaequolifaciens* DSM 19450 (referred to as AE in the table) are used. They also have the ability to produce equol derivatives (especially 5-hydroxyequol).

[0180] In addition, the following microorganisms with sugar assimilation capabilities were used: Clostridium asparagiforme DSM 15981 (referred to as CA in the table), Clostridium bolteae JCM12243 (referred to as CB in the table), Lactobacillus brevis DSM 20054 (referred to as LB in the table), Lactococcus lactis DSM 20481 (referred to as LL in the table), and Leuconostoc mesenteroides subsp. mesenteroides DSM 20343 (referred to as LM in the table).

[0181] (Formal training)

[0182] After inoculating the pre-culture medium into the formal culture medium, the culture was carried out at 37°C and 500 rpm for 2 days while anaerobic gas was introduced through a 0.22 μm filter.

[0183] (result)

[0184] After cultivation, the concentrations of equol, 5-hydroxyequol, and glucose were measured. The results are shown in Table 1. It should be noted that the glucose concentration shown in "g / kg" represents the glucose content relative to the solid content.

[0185] [Table 1]

[0186]

[0187] As can be seen from Comparative Examples 1, 2, and Examples 1-6, equol and 5-hydroxyequol were produced by culturing microorganisms capable of producing equol. Furthermore, as can be seen from Examples 1-6, sugar was assimilated by co-culturing microorganisms capable of sugar assimilation, and no glucose remained in the culture medium.

[0188] [Experimental Example 2]

[0189] (Preparation of pre-culture medium)

[0190] The pre-culture medium was prepared in the same manner as in Experiment 1.

[0191] (Enzyme treatment)

[0192] Add 0.16 g / L of pectinase G Amano to 16 g / L of soybean hypocotyl extract (containing 80% of daidzein, genistein, genistein and other isoflavone glycosides), and keep at 50°C overnight while stirring, thereby releasing the sugar from the isoflavone glycosides.

[0193] (Preparation of formal culture medium)

[0194] The enzyme treatment solution was added to aliquots of 50 mL each into vials, with the final concentrations being 35.4 g / L ABB medium, 1 g / L arginine, and 16 g / L β-cyclodextrin. The vials were then sealed with butyl rubber stoppers. After purging with nitrogen, the solution was autoclaved.

[0195] (Pre-culture)

[0196] After inoculating the pre-culture medium with the estrol-producing microorganisms shown in Table 2, the medium was replaced with anaerobic gas and cultured at 37°C and 200 sppm for 2 days.

[0197] (Formal training)

[0198] After inoculating the pre-culture medium into the formal culture medium, the medium was replaced with anaerobic gas and cultured at 37°C and 200 sppm for 3 days.

[0199] The samples cultured as described above were used as Comparative Examples 3 and 4, and the concentrations of equol, 5-hydroxyequol, and glucose were measured.

[0200] (Yeast treatment)

[0201] In Comparative Examples 3 and 4, samples stored overnight at 30°C were used as Examples 7 and 8, respectively, with 1 g of baker's yeast (SUPER CAMELLIA Dry Yeast manufactured by Nisshin Seifun Welna Co., Ltd.) added per 100 mL of culture medium.

[0202] (result)

[0203] The results of the determination of equadol concentration, 5-hydroxyequadol concentration and glucose concentration of Comparative Example 3, Comparative Example 4, Example 7 and Example 8 are shown in Table 2.

[0204] [Table 2]

[0205]

[0206] Compared to Comparative Examples 3 and 4 after formal culture, the glucose concentrations in Examples 7 and 8 after yeast treatment were significantly reduced. This suggests that glucose was assimilated by the yeast. Furthermore, the yeast-treated samples exhibited a characteristic yeast odor and good flavor.

[0207] [Experimental Example 3]

[0208] The liquids from Comparative Example 3 and Example 7 were centrifuged to remove bacterial cells and insoluble components, and the supernatant was recovered. The absorbance of the supernatant at 420 nm was measured. Similarly, the absorbance was measured after heating at 80°C for 2 hours. The results are shown in Table 3.

[0209] [Table 3]

[0210]

[0211] In Comparative Example 3, the absorbance increased upon heating. This was attributed to the Maillard reaction proceeding due to residual glucose, resulting in browning. On the other hand, in Example 7, the glucose concentration decreased to below 0.2 g / L due to yeast action, therefore no increase in absorbance was observed even after heat treatment.

[0212] [Experimental Example 4]

[0213] After adding glycine to the liquids of Comparative Example 3 and Example 7 to a concentration of 2 g / L, centrifugation was performed to remove bacterial cells and insoluble components, and the supernatant was recovered. The absorbance of the supernatant was measured at 420 nm. Similarly, after adding glycine to the liquids of Comparative Example 3 and Example 7 to a concentration of 2 g / L, the mixture was heated at 80°C for 2 hours, and the absorbance was measured. The results are shown in Table 4.

[0214] [Table 4]

[0215]

[0216] In Comparative Example 3, the absorbance increased upon heating. This was attributed to browning caused by the Maillard reaction proceeding due to residual glucose and the added glycine. On the other hand, in Example 7, the glucose concentration decreased to below 0.2 g / L due to yeast action; therefore, no increase in absorbance was observed after heat treatment even with the addition of glycine.

[0217] [Experimental Example 5]

[0218] The same procedure as in Example 2 was followed until formal culture. After culture, the production of equol and 5-hydroxyequol was confirmed by HPLC analysis, and the glucose concentration was measured. After confirming glucose residue, commercially available glucose oxidase (AmanoEnzyme Co., Ltd.) and peroxidase (FUJIFILM Wako Pure Chemical Co., Ltd.) were added, and the mixture was stored at 30°C overnight. Then, the concentrations of equol, 5-hydroxyequol, and glucose were measured. The culture medium before enzyme addition treatment was used as Comparative Examples 5 and 6, and the culture medium after enzyme addition treatment was used as Examples 9 and 10, as shown in Table 5.

[0219] It can be seen that glucose is broken down by enzyme addition, and no glucose remains in the treated liquid.

[0220] [Table 5]

[0221]

[0222] [Experimental Example 6]

[0223] (Preparation of pre-culture medium)

[0224] The pre-culture medium was prepared in the same manner as in Experiment 1.

[0225] (Enzyme treatment)

[0226] Add 0.16 g / L of pectinase G Amano (manufactured by Amano Enzyme) to 16 g / L of soybean hypocotyl extract (containing 44% daidzein, genistein, genistein and other isoflavone glycosides), and keep at 50°C overnight while stirring, thereby releasing the sugar from the isoflavone glycosides.

[0227] (Preparation of formal culture medium)

[0228] The enzyme was added to the enzyme treatment solution at a final concentration of 35.4 g / L ABB medium, 1 g / L arginine, and 16 g / L β-cyclodextrin, and 1 L was dispensed into each of the 2 L flasks. After purging with nitrogen, the mixture was autoclaved.

[0229] (Pre-culture)

[0230] After inoculating the pre-culture medium with the microorganisms shown in Table 5, the medium was replaced with anaerobic gas and cultured at 37°C and 200 sppm for 2 days. The microorganisms used were the same as those in Experimental Example 1.

[0231] (Formal training)

[0232] After inoculating the pre-culture medium into the formal culture medium, the culture was carried out at 37°C and 500 rpm for 2 days while anaerobic gas was introduced through a 0.22 μm filter.

[0233] (result)

[0234] After culture, the concentrations of equol, 5-hydroxyequol, and glucose were measured. The results are shown in Table 6.

[0235] [Table 6]

[0236]

[0237] As can be seen from Comparative Examples 7, 8, and Examples 11-16, equol and 5-hydroxyequol were produced by culturing microorganisms capable of producing equol. Furthermore, as can be seen from Examples 11-16, sugar was assimilated by co-culturing microorganisms capable of sugar assimilation, and no glucose remained in the culture medium.

[0238] [Experimental Example 7]

[0239] Commercially available glucose oxidase (Amano Enzyme Co., Ltd.) and peroxidase (FUJIFILM Wako Pure Chemical Co., Ltd.) were added to Comparative Examples 7 and 8, and the mixtures were stored at 30°C overnight. Then, the concentrations of equol, 5-hydroxyequol, and glucose were determined.

[0240] (result)

[0241] The culture medium after enzyme addition treatment was used as Examples 17 and 18. The results of the determination of equol concentration, 5-hydroxyequol concentration and glucose concentration of Comparative Examples 7, 8 and Examples 17 and 18 are shown in Table 7.

[0242] [Table 7]

[0243]

[0244] It can be seen that glucose is broken down by enzyme addition, and no glucose remains in the treated liquid.

[0245] [Experimental Example 8]

[0246] The liquids from Comparative Example 7 and Example 17 were centrifuged to remove bacterial cells and insoluble components, and the supernatant was recovered. The absorbance of the supernatant at 420 nm was measured. Similarly, the absorbance was measured after heating at 80°C for 2 hours. The results are shown in Table 8.

[0247] [Table 8]

[0248]

[0249] In Comparative Example 7, the absorbance increased upon heating. This was attributed to the Maillard reaction proceeding due to residual glucose, resulting in browning. On the other hand, in Example 17, the glucose concentration decreased to below 0.1 g / L due to enzymatic action, therefore no increase in absorbance was observed even after heat treatment.

Claims

1. A method for manufacturing a composition, comprising: The following processes: (a) process; (b1) process and / or (b2) process; and process (c), (a) The process of generating glucose and isoflavone aglycones from isoflavone glycosides; (b1) A process of producing equadol from said isoflavone aglycone by microorganisms capable of producing equadol. (b2) A process of causing microorganisms capable of producing equol derivatives to produce equol derivatives from the isoflavone aglycone. as well as (c) A step of decomposing the glucose so that the glucose content in the composition is 0 g / L or more and less than 2.0 g / L, or 30 g / kg or less relative to the solid content of the composition.

2. The manufacturing method according to claim 1, wherein, Step (c) is a process of breaking down glucose by using fermentation of microorganisms with sugar assimilation capabilities.

3. The manufacturing method according to claim 2, wherein, The microorganisms with sugar assimilation ability are selected from one or more microorganisms in the group consisting of butyric acid bacteria, lactic acid bacteria and yeast.

4. The manufacturing method according to claim 3, wherein, The microorganisms with sugar assimilation ability are selected from one or more microorganisms belonging to the genus Clostridium, the genus Lactobacillus, the genus Lactococcus, the genus Leuconostoc, and the genus Saccharomyces.

5. The manufacturing method according to claim 1, wherein, Step (c) is a step of using an enzyme that breaks down glucose to break down the glucose.

6. The manufacturing method according to claim 5, wherein, The enzyme that breaks down glucose is selected from one or more of the group consisting of glucose oxidase and glucose dehydrogenase.

7. The manufacturing method according to any one of claims 1 to 6, wherein, The (b1) process and / or (b2) process are performed simultaneously with the (c) process.

8. The manufacturing method according to any one of claims 1 to 6, wherein, The isoflavone glycoside is daidzein, and the isoflavone aglycone is daidzein.

9. The manufacturing method according to any one of claims 1 to 6, wherein, Step (a) is a step in which β-glucosidase acts on isoflavone glycosides to generate glucose and isoflavone aglycones.

10. A method for manufacturing a food product, comprising: The following processes: (a); (b1) and / or (b2); (c); and (d), (a) The process of generating glucose and isoflavone aglycones from isoflavone glycosides; (b1) A process of producing equadol from said isoflavone aglycone by microorganisms capable of producing equadol. (b2) A process of causing microorganisms capable of producing equol derivatives to produce equol derivatives from the isoflavone aglycone. (c) A step of decomposing the glucose to such that the glucose content in a composition manufactured by the method comprising steps (a), (b1), and / or (b2), and (c) is 0 g / L or more and less than 2.0 g / L, or less than 30 g / kg relative to the solid content of the composition; and (d) A process of manufacturing a food or beverage using a composition manufactured by a method comprising steps (a), (b1), and / or (b2) and (c).

11. A fermentation composition comprising equol and / or equol derivatives and substantially free of glucose.

12. The fermentation composition according to claim 11, wherein, "Substantially glucose-free" means containing more than 0 g / L and less than 2.0 g / L of glucose.

13. A fermentation composition comprising equol and / or equol derivatives and glucose, The glucose content is less than 30 g / kg relative to the solid content of the fermentation composition.

14. The fermentation composition according to claim 11 or 13, wherein, The fermentation composition is used in food and beverage products.

Images