Method for producing food or beverage

Abstract

To provide a technique for producing a functional substance, which uses a substance that can be efficiently fermented by anaerobic microorganisms and can also be used in the production of fermented foods.SOLUTION: A method for producing a functional substance comprises a step of, in a solution containing a material of a functional substance, allowing anaerobic microorganisms capable of producing the functional substance from the material to produce the functional substance from the material. The solution containing the material of the functional substance contains L-cystine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a functional substance.

Background Art

[0002] L-cysteine is known to be added to a medium for anaerobic microorganisms as a reducing active compound (reducing agent) so that anaerobic microorganisms can grow (Patent Document 1). Conventionally, L-cysteine, thioglycolic acid, ascorbic acid, mercaptoacetic acid, thioacetic acid, glutathione, sodium sulfide, etc. have been used as reducing agents used in the production of functional substances such as equol, urolithin, colloid, and sugar by fermentation (Patent Documents 2 to 4). However, for example, L-cysteine hydrochloride is a pharmaceutical product and has restrictions on its use as a food additive. Therefore, when it is assumed to be used in the production of fermented foods using anaerobic microorganisms, it has been difficult to use L-cysteine hydrochloride.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a manufacturing technique for a functional substance using a substance that can efficiently perform fermentation by anaerobic microorganisms and can also be used in the production of fermented foods.

Means for Solving the Problems

[0005] As a result of intensive studies, the inventors have conceived of L-cysteine as the substance and completed the present invention. The present invention is as follows.

[0006] [1] A method for producing a functional substance, comprising a step of causing an anaerobic microorganism having the ability to produce a functional substance from a material to produce the functional substance from the material in a solution containing the material of the functional substance, A production method, wherein the solution containing the material of the functional substance contains L-cysteine. [2] The production method according to [1], wherein the material of the functional substance is daidzein and the functional substance is equol. [3] The production method according to [2], wherein the anaerobic microorganism is a microorganism belonging to Adlercreutzia equolifaciens subsp. celatus and / or Adlercreutzia equolifaciens subsp. equolifaciens. [4] A step of causing an anaerobic microorganism having the ability to produce equol from daidzein to produce equol from daidzein in a solution containing daidzein, wherein the solution containing daidzein contains L-cysteine, and A method for producing a food or drink containing equol, comprising a step of blending the produced equol with a raw material of the food or drink. [5] The production method according to [1], wherein the material of the functional substance is ellagic acid and the functional substance is urolithin A. [6] The production method according to claim 5, wherein the anaerobic microorganism is a microorganism belonging to Gordonibacter pamelaeae and / or Clostridium bolteae. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a technique for producing a functional substance by using L-cystine as a substance that can also be used in the production of fermented foods and efficiently performing fermentation by anaerobic microorganisms. Further, since L-cystine is safe for food, a safe fermented food can be produced using the obtained functional substance.

Mode for Carrying Out the Invention

[0008] In this specification, the accession number of a strain starting with the phrase "JCM" is a number assigned to a microorganism stored in the Japan Collection of Microorganisms (Microbial Material Development Office, National Institute of Advanced Industrial Science and Technology, Bioresource Center, Postal Code: 305-0074, Address: 3-1-1 Takano-dai, Tsukuba City, Ibaraki Prefecture), and it can be obtained from the said institution. Further, in this specification, the accession number of a strain starting with the phrase "FERM" is a number assigned to a microorganism stored in the Patent Microorganism Depositary, National Institute of Advanced Industrial Science and Technology (currently, the Patent Microorganism Depositary, National Institute of Technology and Evaluation, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture), and it can be obtained from the said institution. Further, in this specification, the accession number of a strain starting with the phrase "DSM" is a number assigned to a microorganism stored in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), and it can be obtained from the said institution. Further, in this specification, the accession number of a strain starting with the phrase "KCCM" is a number assigned to a microorganism stored in the Korean Culture Center of Microorganisms (KCCM), and it can be obtained from the said institution.

[0009] The present invention relates to a method for producing a functional substance, which includes a step of causing an anaerobic microorganism having the ability to produce a functional substance from a material to produce the functional substance from the material in a solution containing the material of the functional substance, and the solution containing the material of the functional substance contains L-cysteine.

[0010] (Functional substance) The functional substance of the present invention is not particularly limited as long as it can cause an anaerobic microorganism having the ability to produce a functional substance from the material to produce the functional substance from the material. Examples of the functional substance of the present invention include equol, urolithin A, dihydroquercetin, naringenin, prenylnaringenin, dihydrogosipetin, dihydrokempferol, dihydromyricetin, 5-hydroxyequol, enterolactone, enterodiol, and tetrahydrocurcumin.

[0011] (Material of the functional substance) The material of the functional substance of the present invention is not particularly limited as long as the anaerobic microorganism can produce the functional substance of the present invention from the material, and it may be a raw material, a product between the raw material and the functional substance, or a precursor of the functional substance. Examples of the material of the functional substance when the functional substance of the present invention is equol include daidzein. Further, examples of the material of the functional substance when the functional substance of the present invention is urolithin A include ellagic acid.

[0012] (Anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance) The anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance in the present invention is not particularly limited.

[0013] When the functional substance of the present invention is equol, for example, microorganisms belonging to the genus Coriobacterium, microorganisms belonging to the genus Adlercreutzia, microorganisms belonging to the genus Atopobium, microorganisms belonging to the genus Collinsella, microorganisms belonging to the genus Cryptobacterium, microorganisms belonging to the genus Denitrobacterium, microorganisms belonging to the genus Eggerthella, microorganisms belonging to the genus Enterorhabdus, microorganisms belonging to the genus Gordonibacter, microorganisms belonging to the genus Olsenella, the genus Paraeggerthella, microorganisms belonging to the genus Slackia, microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Bacteroides, microorganisms belonging to the genus Eubacterium, microorganisms belonging to the genus Ruminococcus, and microorganisms belonging to the genus Streptococcus can be mentioned.

[0014] Preferably, it is a microorganism belonging to Adlercreutzia equolifaciens subsp. equolifaciens, a microorganism belonging to Adlercreutzia equolifaciens subsp. celatus, a microorganism belonging to Eggerthella sp., a microorganism belonging to Paraeggerthella sp., a microorganism belonging to Slackia isoflavoniconvertens, a microorganism belonging to Slackia equolifaciens, a microorganism belonging to Slackia sp., a microorganism belonging to Lactococcus garvieae, a microorganism belonging to Bacteroides ovatus, a microorganism belonging to Eubacterium sp., a microorganism belonging to Ruminococcus productus, or a microorganism belonging to Streptococcus intermedius.

[0015] More preferably, it includes Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain, Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain, Eggerthella sp. KCCM 10490 strain, Slackia isoflavoniconvertens DSM 22006 strain, Slackia equolifaciens DSM 24851 strain, Slackia sp. FERM AP-20729 strain, Lactococcus garvieae DSM 6783 strain. Regardless of the genus, species, and strain, the above microorganisms may be used alone or in combination of two or more.

[0016] As a preferred embodiment when the functional substance of the present invention is equol, for example, the material of the functional substance is daidzein, and the anaerobic microorganisms having the ability to generate the functional substance from the material of the functional substance are Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain and / or Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain.

[0017] In addition, when the functional substance of the present invention is urolithin A, examples include microorganisms belonging to the genus Coriobacterium, microorganisms belonging to the genus Eggerthella, microorganisms belonging to the genus Slackia, microorganisms belonging to the genus Gordonibacter, and microorganisms belonging to the genus Clostridium.

[0018] Preferably, they are microorganisms belonging to Eggerthella sp., microorganisms belonging to Gordonibacter pamelaeae, microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter faecihominis, microorganisms belonging to Clostridium bolteae, microorganisms belonging to Clostridium asparagiforme, microorganisms belonging to Clostridium citroniae, and microorganisms belonging to Clostridium sp.

[0019] More preferably, Eggerthella sp. DC3563 (NITE BP-02376) strain, Gordonibacter pamelaeae DSM 19378 strain, Gordonibacter urolithinfaciens DSM 27213 strain, Clostridium bolteae JCM 12243 strain, Clostridium bolteae DSM 15670 strain, Clostridium bolteae DSM 29485 strain, Clostridium asparagiforme DSM 15981 strain, Clostridium citroniae DSM 19261 strain, Clostridium sp. DC3656 strain can be mentioned. Regardless of the genus, species, and strain, the above microorganisms may be used alone or in combination of two or more.

[0020] As a preferred embodiment when the functional substance of the present invention is urolithin A, for example, the material of the functional substance is ellagic acid, and the anaerobic microorganism having the ability to produce the functional substance from the material of the functional substance is Gordonibacter pamelaeae DSM 19378 strain, and / or Clostridium bolteae JCM 12243 strain.

[0021] In addition, the anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance in the present invention is not limited to the same strain as the deposited strain, and may be a strain substantially equivalent to the deposited strain. A strain substantially equivalent means a microorganism whose nucleotide sequence of the 16S rRNA gene has a homology of 97.5% or more, preferably 98% or more, more preferably 99% with the nucleotide sequence of the 16S rRNA gene of the deposited strain. Furthermore, the anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc., as long as the effects of the present invention are not impaired.

[0022] (Anaerobic microorganism in a resting cell state having the ability to produce a functional substance from the material of the functional substance) The anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance in the present invention includes its resting cells. Resting cells refer to cells obtained by removing medium components from cultured microorganisms by operations such as centrifugation, washing with a salt solution such as water or physiological saline, or a buffer solution, and suspending them in the same solution as the washing solution, and cells in a non-growing state. In the present invention, it refers to cells having at least a metabolic system capable of producing a functional substance from the material of the functional substance. As the buffer solution, phosphate buffer, Tris-hydrochloric acid buffer, citrate-phosphate buffer, citrate buffer, MOPS buffer, acetate buffer, glycine buffer, etc. are preferable. The pH and concentration of the buffer solution can be appropriately prepared according to conventional methods.

[0023] (Solution containing the material of the functional substance) The solution containing the material of the functional substance in the present invention is not particularly limited as long as a functional substance can be produced from the material of the functional substance by an anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance in the solution. Preferably it is a medium, more preferably the medium described in the column of "Medium and Production of Functional Substances by Cultivation" described later. Also, when the anaerobic microorganism having the ability to produce a functional substance from the material of the functional substance is in a resting cell state, the above-mentioned water, salt solution, and buffer solution are preferable. As used herein, all "media" refer to solutions in which microorganisms can grow, including minimal media, and do not include solutions in which microorganisms cannot grow, such as the water, salt solutions, buffer solutions, etc. described above.

[0024] When adding the material of the functional substance to the solution, it may be added before the generation of the functional substance, during the process, and may be added all at once, sequentially, or continuously. The content of the functional substance in the solution is usually 0.01 g / L or more, preferably 0.1 g / L or more, more preferably 1 g / L or more. On the other hand, it is usually 100 g / L or less, preferably 20 g / L or less, more preferably 10 g / L or less.

[0025] The solution containing the material of the functional substance in the present invention contains L-cystine. The content of L-cystine in the solution is usually 0.001 g / L or more, preferably 0.01 g / L or more, more preferably 0.1 g / L or more. On the other hand, it is usually 5.0 g / L or less, preferably 3.0 g / L or less, more preferably 1.0 g / L or less.

[0026] (Medium and Generation of Functional Substance by Cultivation) In the step of causing anaerobic microorganisms having the ability to generate a functional substance from the material to generate the functional substance from the material in a solution containing the material of the functional substance, it is preferable that the solution is a medium. The medium is not particularly limited. For example, when the functional substance is equol, examples include BHI medium (manufactured by Difco) and the medium used in the examples. When the functional substance is urolithin A, examples include Wilkins-Chalgren Anaerobe Broth, Brain Heart Infusion Broth, and the medium used in the examples. Hereinafter, additives and conditions when the solution containing the material of the functional substance is a medium will be described, but they are also applicable when the solution containing the material of the functional substance is not a medium.

[0027] Water-soluble organic substances can be added to the medium as a carbon source. Examples of water-soluble organic substances include the following compounds. That is, saccharides such as glucose, arabinose, sorbitol, sorbose, fructose, mannose, sucrose, trehalose, xylose; alcohols such as methanol, glycerol; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, fumaric acid, or salts thereof, etc.

[0028] The concentration of the organic substance added to the medium as a carbon source can be appropriately adjusted for efficient growth. Generally, the addition amount can be selected from the range of 0.1 to 10 wt / vol%.

[0029] In addition to the above carbon source, a nitrogen source can be added to the medium. As the nitrogen source, various nitrogen compounds that can be used in ordinary fermentation can be used. Preferred inorganic nitrogen sources include ammonium salts, nitrates, etc., more preferably ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate, etc. Also, as the organic nitrogen source, amino acids, yeast extract, peptones (such as polypeptone N, soy peptone, etc.), meat extract (such as Ehrelich's bonito extract, Lab-Lemco powder, bouillon, etc.), seafood extract, liver extract, digested serum powder, fish oil, etc. can be mentioned. More preferably, arginine, cysteine, citrulline, lysine, yeast extract, peptones (such as polypeptone N, etc.).

[0030] Furthermore, in addition to the carbon source and nitrogen source, for example, by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium, growth and activity may be enhanced in some cases. For example, the following can be mentioned as microbial growth assisting factors derived from animals and plants, such as inorganic compounds, vitamins, fatty acids.

[0031] Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium sulfate Folic acid Manganese Sulfate Pyridoxine Sodium Chloride Thiamine Cobalt Chloride Riboflavin Calcium Chloride Nicotinic Acid Zinc Sulfate Pantothenic Acid Copper Sulfate Vitamin B12 Alum Thioctic Acid Sodium Molybdate p-Aminobenzoic Acid Potassium Chloride Vitamin K Boric Acid etc. Nickel Chloride Sodium Tungstate Sodium Selenate Ferrous Ammonium Sulfate Sodium Acetate Trihydrate Magnesium Sulfate Heptahydrate Manganese Sulfate Tetrahydrate

[0032] Methods for producing a medium by adding growth promoting factors derived from animals and plants, such as these inorganic compounds and vitamins, are known. The medium can be in the form of a liquid, semi-solid, or solid. A preferred form of the medium is a liquid medium.

[0033] In addition, dextrins can be included in the medium of the present invention. By culturing anaerobic microorganisms in a medium containing dextrins, even if dextrins are required in the culture solution after culturing, a solution containing a functional substance and dextrins can be obtained without adding dextrins. The addition of dextrins to the medium can be carried out before and during the culture of the microorganisms.

[0034] The anaerobic microorganisms of the present invention can be cultured according to known methods for culturing microorganisms. In industrial production, it is also possible to use a continuous fermentation system that can continuously supply the medium and substrate gas and is equipped with a mechanism for recovering the culture.

[0035] In the production of the functional substance of the present invention, it is necessary to prevent the mixing of oxygen into the continuous culture system. As the incubator, a commonly used culture tank can be used as it is. There are commercially available culture tanks that can also be used for culturing anaerobic microorganisms of the present invention. An anaerobic atmosphere can also be created by replacing the oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas.

[0036] Depending on the shape of the culture tank, in order to sufficiently stir the culture medium, a stirrer or the like can also be used. By stirring the culture in the culture tank, the opportunity for the culture medium components and the substrate gas to come into contact with the anaerobic microorganisms of the present invention can be increased, and the production efficiency of the functional substance can be optimized.

[0037] As the culture system, it may be carried out in a closed system such as a bottle or a test tube sealed with a rubber stopper without aeration, but it is preferably free of air or oxygen. For example, it may contain nitrogen and / or hydrogen in an arbitrary ratio, or nitrogen and / or carbon dioxide in an arbitrary ratio, and it is preferably a gas phase or an aqueous phase containing hydrogen. The method of making the gas phase and the aqueous phase during culture such an environment is not particularly limited. For example, a method of replacing the gas phase with the above gas before culture, and in addition, a method of supplying from the bottom of the incubator and / or supplying to the gas phase part of the incubator during culture, a method of bubbling the aqueous phase with the above gas before culture, etc. can be taken. The hydrogen may be used as hydrogen gas as it is. Also, a raw material of hydrogen such as formic acid and / or its salt may be added to the culture medium, and hydrogen may be generated during culture by the action of microorganisms.

[0038] For sufficient growth of the anaerobic microorganisms of the present invention, the pH of the culture is preferably 5.0 or more, more preferably 6.0 or more, still more preferably 6.5 or more, while on the other hand, it is preferably 8.0 or less, more preferably 7.5 or less. Also, the temperature of the culture tank is not particularly limited, but in order to increase the production efficiency of the functional substance, it is preferably 30°C or more, more preferably 33°C or more, while on the other hand, it is preferably 40°C or less, more preferably 38°C or less. The culture time can be appropriately set according to the production amount of the functional substance, the remaining amount of the material of the functional substance, etc. Usually, it is 8 hours or more, preferably 12 hours or more, more preferably 16 hours or more. On the other hand, usually it is 120 hours or less, preferably 72 hours or less, more preferably 60 hours or less. Also, in order to efficiently produce the functional substance, the medium can be continuously supplied. The aeration rate is preferably 0.01 - 2.0 vvm. Also, the mixed gas can be supplied as nanobubbles. The pressurization conditions of the incubator are not particularly limited as long as the anaerobic microorganisms of the present invention can grow, but are preferably 0.02 - 0.2 MPa.

[0039] (Production of Functional Substance by Stationary Cells) When the solution in the case where the anaerobic microorganism having the ability to produce the functional substance from the material of the functional substance is stationary cells, instead of the above-mentioned medium, water, salt solution, buffer solution described in the column of "Stationary Cells of Anaerobic Microorganisms Having the Ability to Produce Functional Substances from the Material of Functional Substances" is preferred. Regarding other conditions, the description in the column of "Medium and Production of Functional Substances by Culture" is incorporated by reference.

[0040] (Other Processes) The present invention may include, for example, a step of quantifying the obtained functional substance. The method can follow a conventional method. For example, a part of the culture solution is collected, appropriately diluted, stirred well, then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and the insoluble matter is removed and quantified by high performance liquid chromatography, etc. Further, the present invention may include a step of recovering the obtained functional substance. The recovery step may include a purification step, a concentration step, etc. As the purification treatment in the purification step, sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymer substances; extraction with organic solvents, ionic liquids, etc.; adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, etc. can be performed. Also, as the concentration treatment in the concentration step, concentration by an evaporator, a reverse osmosis membrane, etc. can be mentioned. Furthermore, the solution containing the obtained functional substance can be powdered by freeze-drying, spray-drying, etc. In powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

[0041] (Another aspect of the present invention) Another aspect of the present invention is a step of causing an anaerobic microorganism having the ability to generate a functional substance from a material to generate the functional substance from the material in a solution containing the material of the functional substance, wherein the solution containing the material of the functional substance contains L-cystine, and a step of blending the generated functional substance and a raw material of a food or drink, and is a method for producing a food or drink containing a functional substance. In addition, in this specification, even when described as "food", it represents "food and drink" including "beverage".

[0042] Regarding the details of the step of causing an anaerobic microorganism having the ability to generate a functional substance from a material to generate the functional substance from the material in a solution containing the material of the functional substance, wherein the solution containing the material of the functional substance contains L-cystine, the description of the method for producing the functional substance is incorporated by reference.

[0043] In addition, the method for manufacturing a food or beverage of the present aspect includes a step of blending the generated functional substance and the raw material of the food or beverage. The food or beverage is manufactured by blending the raw material of the food or beverage commonly used according to a conventional method and the functional substance manufactured in the above step, and the blending timing is not particularly limited. In addition, the raw material of the food or beverage includes food additives. Furthermore, if necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, box, pack, etc.

[0044] The food or beverage may be mainly composed of water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, etc. Examples of the protein include animal and plant proteins such as whole milk powder, skim milk powder, partially skimmed milk powder, casein, soy protein, egg protein, and meat protein, and their hydrolysates, butter, etc. Examples of the carbohydrates include saccharides, processed starches (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, etc. Examples of the lipids include vegetable oils and fats such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, their fractionated oils, hydrogenated oils, interesterified oils, etc. Examples of the vitamins include vitamin A, carotenoids, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc. Examples of the minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, whey minerals, etc. Examples of the organic acids include malic acid, citric acid, lactic acid, tartaric acid, etc. These components may be used in combination of two or more, or may be synthetic products.

[0045] The content of the functional substance produced in the above process relative to the total amount of the food or drink is not particularly limited, but it is preferably a content that can achieve the desired effect by the functional substance when the food or drink is ingested. The content of the functional substance relative to the total amount of the food or drink depends on the type of the functional substance and the desired effect. For example, it may be 10 -6 mass% to 50 mass% or less, etc.

[0046] When the food or drink is a supplement, its form may be any of solid, gel-like, or liquid forms. For example, it can be in the form of various processed foods or drinks, powders, tablets, pills, capsules, jelly, granules, etc. Furthermore, if necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, box, pack, etc. Supplements may contain excipients such as dextrin, preservatives such as vitamin C, flavoring agents such as vanilla, pigments such as safflower pigment, monosaccharides, oligosaccharides, and polysaccharides (e.g., glucose, fructose, sucrose, saccharose, and carbohydrates containing these), acidulants, fragrances, oils and fats, emulsifiers, whole milk powder, or additives such as agar. These components may be used in combination of two or more kinds, and may be synthetic products.

Examples

[0047] The present invention will be specifically described below using examples, but the present invention is not limited to these examples.

[0048] 〔Example 1〕 A medium adjusted to pH 6.9 with the composition shown in Table 1 was dispensed into 18 mm test tubes (manufactured by Sanshin Kogyo) for anaerobic microorganism culture, 10 mL each. While replacing the gas phase with nitrogen, a butyl rubber stopper and a plastic cap were put on, and it was sterilized at 115°C for 15 minutes. An Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain that had been cryopreserved at -80°C was inoculated into this medium. After replacing the gas phase with hydrogen gas passed through a sterile filter for 2 minutes or more, shaking culture was performed at 37°C and 200 spm for 18 hours to prepare a preculture solution.

[0049]

Table 1

[0050] 〔Example 2〕 0.5 g / L of daidzein was added to the composition shown in Table 1, and 5 mL of the medium adjusted to pH 6.9 was dispensed into 18 mm test tubes (manufactured by Sanshin Kogyo Co., Ltd.) for anaerobic microorganism culture. While replacing the gas phase with nitrogen, a butyl rubber stopper and a plastic cap were put on, and sterilization was carried out at 115 °C for 15 minutes. 0.1 mL of the preculture solution prepared in Example 1 was inoculated into this medium, and after replacing the gas phase with hydrogen gas passed through a sterile filter for 2 minutes or more, shaking culture was carried out at 37 °C and 200 spm for 28 hours. After 28 hours, 0.45 g / L of equol was produced in the culture solution.

[0051] 〔Example 3〕 Cultivation and reaction were carried out in the same manner as in Examples 1 to 2 except that the concentration of L-cystine added in Example 2 was changed to 0.34 g / L. As a result, 0.45 g / L of equol was produced in the culture solution.

[0052] 〔Example 4〕 Cultivation and reaction were carried out in the same manner as in Examples 1 to 2 except that the concentration of L-cystine added in Example 2 was changed to 0.7 g / L. As a result, 0.44 g / L of equol was produced in the culture solution.

[0053] 〔Example 5〕 Preculture was carried out in the same manner as in Example 1 except that the strain inoculated in Example 1 was changed to Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain. This preculture solution was inoculated into the medium prepared in the same manner as in Example 2. As a result, after 29 hours, 0.43 g / L of equol was produced in the culture solution.

[0054] 〔Comparative Example 1〕 The culture and reaction were carried out in the same manner as in Examples 1 to 2, except that L-cystine added in Example 2 was changed to L-cysteine hydrochloride. As a result, 0.46 g / L of equol was produced in the culture broth. That is, even when L-cystine was used, a product equivalent to that obtained when L-cysteine hydrochloride was used was obtained.

[0055] 〔Example 6〕 The medium adjusted to pH 7.3 with the composition shown in Table 2 was dispensed in 10 mL aliquots into 18 mm test tubes (manufactured by Sanshin Kogyo) for anaerobic microorganism culture, and a butyl rubber stopper and a plastic cap were fitted while replacing the gas phase with nitrogen, and sterilized at 115 °C for 15 minutes. To this medium, the Gordonibacter pamelaeae DSM 19378 strain and the Clostridium bolteae JCM 12243 strain, which had been cryopreserved at -80 °C, were inoculated, and after replacing the gas phase with nitrogen gas passed through a sterile filter for 2 minutes or more, shaking culture was performed at 37 °C and 200 spm for 2 days to prepare a preculture broth.

[0056]

Table 2

[0057] 〔Example 7〕 The medium with the ellagic acid concentration adjusted to 1.0 g / L and pH 7.3 with the composition shown in Table 2 was dispensed in 5 mL aliquots into 18 mm test tubes (manufactured by Sanshin Kogyo) for anaerobic microorganism culture, and a butyl rubber stopper and a plastic cap were fitted while replacing the gas phase with nitrogen, and sterilized at 115 °C for 15 minutes. To this medium, 0.5 mL of the preculture broth prepared in Example 6 was inoculated, and after replacing the gas phase with nitrogen gas passed through a sterile filter for 2 minutes or more, shaking culture was performed at 37 °C and 200 spm for 5 days. After 5 days, 0.25 g / L of urolithin A was produced in the culture broth.

[0058] 〔Comparative Example 2〕 The cultivation and reaction were carried out in the same manner as in Examples 6 to 7, except that the L-cystine added in Examples 6 and 7 was changed to 0.3 g / L of L-cysteine hydrochloride. As a result, 0.37 g / L of urolithin A was produced in the culture solution. That is, even when L-cystine was used, a product equivalent to that obtained when L-cysteine hydrochloride was used was obtained.

[0059] [Comparative Example 3] The cultivation and reaction were carried out in the same manner as in Examples 6 to 7, except that the L-cystine added in Examples 6 and 7 was changed to 0.3 g / L of sodium thioglycolate. As a result, 0.33 g / L of urolithin A was produced in the culture solution. That is, even when L-cystine was used, a product equivalent to that obtained when sodium thioglycolate was used was obtained.

Industrial Applicability

[0060] According to the present invention, it is possible to provide a method for producing a functional substance using a reducing agent that can also be used in the production of fermented foods. The functional substance obtained by the method of the present invention can be used according to the use of each functional substance.

Claims

1. A step of producing equol from daidzein in a solution containing daidzein using an anaerobic microorganism having an ability to produce equol from daidzein, the step including a step of adding 0.001 g / L or more and 5.0 g / L or less of L-cystine to the solution containing daidzein; and A method for producing a food or beverage containing equol, comprising the step of blending the produced equol with a raw material for the food or beverage (excluding an embodiment including the step of adding L-cysteine ​​hydrochloride to the solution containing daidzein).

2. The method according to claim 1, wherein the anaerobic microorganism is a microorganism belonging to Adlercreutzia equolifaciens subsp. celatus and / or a microorganism belonging to Adlercreutzia equolifaciens subsp. equolifaciens.

3. A step of causing an anaerobic microorganism capable of producing urolithin A from ellagic acid in a solution containing ellagic acid to produce urolithin A from ellagic acid, the step comprising the step of adding 0.001 g / L or more and 5.0 g / L or less of L-cystine to the solution containing ellagic acid; and A method for producing a food or beverage containing urolithin A, comprising a step of blending the produced urolithin A with raw materials for the food or beverage (excluding embodiments which include a step of adding L-cysteine ​​hydrochloride to a solution containing ellagic acid).

4. 4. The method according to claim 3, wherein the anaerobic microorganism is a microorganism belonging to the genus Gordonibacter pamelaeae and / or a microorganism belonging to the genus Clostridium bolteae.

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