Manufacturing method of functional substances

Abstract

To provide a method 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 technology]

[0002] It is known that L-cysteine ​​is 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, thiolacetic acid, glutathione, sodium sulfide, and the like have been used as reducing agents when producing functional substances such as equols, urolithins, corrinoids, and sugars 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, so that it has been difficult to use L-cysteine ​​hydrochloride when it is intended to be used in the production of fermented foods using anaerobic microorganisms. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 5546037 [Patent Document 2] Patent No. 5996187 [Patent Document 3] Patent No. 5943326 [Patent Document 4] Special Publication No. 6-098015 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a technology for producing functional substances that can be efficiently fermented by anaerobic microorganisms and that can be used in the production of fermented foods. [Means for solving the problem]

[0005] As a result of extensive investigations, the present inventors have come up with the idea that such a substance is L-cystine, and have thus completed the present invention.

[0006] [1] A method for producing a functional substance, comprising a step of producing a functional substance from a solution containing a material for the functional substance using an anaerobic microorganism capable of producing the functional substance from the material, The production method, wherein the solution containing the functional substance material contains L-cystine. [2] The manufacturing method described in [1], wherein the material of the functional substance is daidzein and the functional substance is equol. [3] The production method described in [2], wherein the anaerobic microorganism is a microorganism belonging to Adlercreutzia equolifaciens subsp. celatus and / or a microorganism belonging to Adlercreutzia equolifaciens subsp. equolifaciens. [4] A step of producing equol from daidzein using an anaerobic microorganism capable of producing equol from daidzein in a solution containing daidzein, the solution containing daidzein containing L-cystine; and A method for producing a food or drink containing equol, comprising the step of blending the produced equol with ingredients for the food or drink. [5] The manufacturing method described in [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 a microorganism belonging to Clostridium bolteae. Effect of the Invention

[0007] According to the present invention, a technology for producing a functional substance can be provided by efficiently carrying out fermentation with anaerobic microorganisms using L-cystine as a substance that can also be used in the production of fermented foods. Since L-cystine is safe for food, the obtained functional substance can be used to produce safe fermented foods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In this specification, the accession numbers of strains beginning with the word JCM are those of the Japan Collection of Microorganisms (RIKEN BioResource Center, Microbial Materials Research Center). Microorganisms stored in the Laboratory of Microorganisms (Hokkaido University, Tsukuba City, Ibaraki Prefecture, Japan, Postal Code: 305-0074, Address: 3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture) The number is assigned to the organization and is available from the organization. In addition, in this specification, the accession number of the strain beginning with the word FERM is the accession number of the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Technology and Evaluation, Patent Organism Depositary Center, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). ) and can be obtained from the institution. In addition, in this specification, the accession numbers of strains beginning with the word DSM are given to microorganisms stored at DSMZ (DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH). This is the identification number given to the agency and is available from the agency. In addition, in this specification, the accession numbers of the strains beginning with the wording KCCM refer to the microbial strains preserved in the Korean Culture Center of Microorganisms (KCCM). The number is assigned to the organization and is available from the organization.

[0009] The present invention relates to a method for producing a functional substance, the method comprising the step of causing an anaerobic microorganism capable of producing a functional substance from a material in a solution containing the material for the functional substance to produce the functional substance from the material, wherein the solution containing the material for the functional substance contains L-cystine.

[0010] (Functional substance) The functional substance of the present invention is not particularly limited as long as it can cause an anaerobic microorganism capable of producing 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, dihydrogossippetin, dihydrokaempferol, dihydromyricetin, 5-hydroxyequol, enterolactone, enterodiol, and tetrahydrocurcumin.

[0011] (Functional Materials) The material for the functional substance of the present invention is not particularly limited as long as it can cause the anaerobic microorganism to produce the functional substance of the present invention from the material, and may be a raw material, a product between the raw material and the functional substance, or a precursor of the functional substance. When the functional substance of the present invention is equol, an example of the material of the functional substance is daidzein. When the functional substance of the present invention is urolithin A, an example of the material of the functional substance is ellagic acid.

[0012] (Anaerobic microorganisms capable of producing functional substances from raw materials for functional substances) In the present invention, the anaerobic microorganism capable of producing a functional substance from a functional substance material 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, Collinsella, Cryptobacterium, Denitrobacteria Microorganisms belonging to the genus Dennitrobacterium, Microorganisms belonging to the genus Eggerthella microorganisms belonging to the genus Enterorhabdus, microorganisms belonging to the genus Gordonibacter, microorganisms belonging to the genus Olsenella, Paraegasella Microorganisms belonging to the genus Paraeggerthella, Slackia, Lactococcus, Bacteroides, Eubacterium, Ruminococcus Examples of microorganisms that belong to the genus Streptococcus include microorganisms that belong to the genus Streptococcus.

[0014] Preferably, the microorganism is selected from the group consisting of microorganisms belonging to Adlercreutzia equolifaciens subsp. equolifaciens, microorganisms belonging to Adlercreutzia equolifaciens subsp. celatus, and microorganisms belonging to Eggerthella sp. ), microorganisms belonging to Paraeggerthella sp., Microorganisms belonging to Slackia isoflavoniconvertens, Slackia equolifaciens, Microorganisms belonging to Slackia sp., Lactococcus garvieae, Bacteroides ovatus Microorganisms belonging to Eubacterium sp., Microorganisms belonging to Lu They are microorganisms belonging to the genus Ruminococcus productus and the genus Streptococcus intermedius.

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

[0016] In a preferred embodiment of the present invention, when the functional substance of the present invention is equol, the material of the functional substance is daidzein, and the anaerobic microorganism capable of producing the functional substance from the material of the functional substance is Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain and / or Adlercreutzia equolifaciens subsp. The strain is Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450.

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

[0018] Preferably, the microorganism belongs to Eggerthella sp., Gordonibacillus subtilis, Microorganisms belonging to the species Gordonibacter pamelaeae Microorganisms belonging to Gordonibacter urolithinfaciens, Gordonibacter faecihominis, Clostridium bolteae, Clostridium aeruginosa, Clostridium asparagiforme is a microorganism belonging to the family Clostridium. A microorganism belonging to the genus Clostridium citroniae, Clostridium sp. It is a microorganism belonging to the genus Clostridium.

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

[0020] A preferred embodiment when the functional substance of the present invention is urolithin A is, for example, that the material of the functional substance is ellagic acid, and the anaerobic microorganism capable of producing 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 capable of producing a functional substance from a material of a functional substance in the present invention is not limited to the same strain as the deposited strain, but may be a strain substantially equivalent to the deposited strain. A substantially equivalent strain is a microorganism whose 16S rRNA gene base sequence has a homology of 97.5% or more, preferably 98% or more, more preferably 99% to the 16S rRNA gene base sequence of the deposited strain. Furthermore, the anaerobic microorganism capable of producing a functional substance from a material of a functional substance may be a strain bred from the deposited strain or a substantially equivalent strain by mutation treatment, genetic recombination, selection of natural mutants, or the like, as long as the effect of the present invention is not impaired.

[0022] (An anaerobic microorganism capable of producing functional substances from functional substance raw materials) In the present invention, the anaerobic microorganism capable of producing a functional substance from a material for the functional substance includes its resting bacterial body. The resting bacterial body refers to a bacterial body that is not growing, which is obtained by removing medium components from a cultured microorganism by an operation such as centrifugation, washing with a salt solution such as water or physiological saline, or a buffer solution, and then suspended in the same liquid as the washing liquid. In the present invention, the resting bacterial body refers to a bacterial body that has at least a metabolic system capable of producing a functional substance from a material for the functional substance. As the buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citrate-phosphate buffer solution, citrate buffer solution, MOPS buffer solution, acetate buffer solution, glycine buffer solution, etc. are preferable. The pH and concentration of the buffer solution can be appropriately adjusted according to a conventional method.

[0023] (Solution containing functional material) The solution containing the material of the functional substance in the present invention is not particularly limited as long as it can cause the anaerobic microorganism capable of producing a functional substance from the material of the functional substance to produce the functional substance from the material of the functional substance. A culture medium is preferable, and a culture medium described in the section "Culture medium and production of functional substances by culture" described later is more preferable. In addition, when the anaerobic microorganism capable of producing a functional substance from the material of the functional substance is a resting cell, the above-mentioned water, salt solution, or buffer solution is preferable. In addition, the term "culture medium" as used in this specification refers to a solution in which microorganisms can grow, including a minimal medium, and does not include solutions in which microorganisms cannot grow, such as the above-mentioned water, salt solutions, buffer solutions, etc.

[0024] When the material for the functional substance is added to the solution, it may be added before or during the production of the functional substance, and may be added all at once, gradually, 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, and usually 100 g / L or less, preferably 20 g / L or less, more preferably 10 g / L or less.

[0025] The solution containing the functional material of 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, the content is usually 5.0 g / L or less, preferably 3.0 g / L or less, more preferably 1.0 g / L or less.

[0026] (Culture medium and production of functional substances through cultivation) In the step of producing a functional substance from a solution containing a material of a functional substance by an anaerobic microorganism having the ability to produce the functional substance from the material, the solution is preferably a culture medium. The culture medium is not particularly limited, but for example, when the functional substance is equol, examples of the culture medium include BHI medium (manufactured by Difco) and the culture medium used in the Examples, and when the functional substance is urolithin A, examples of the culture medium include Wilkins-Chalgren Anaerobe Broth and Brain Anaerobe Broth. Examples of the medium include Heart Infusion Broth and the medium used in the Examples. Below, additives and conditions will be described when the solution containing the functional substance material is a culture medium, but the same applies when the solution containing the functional substance material is not a culture medium.

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

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

[0029] In addition to the above carbon sources, a nitrogen source can be added to the medium, which may be any of various nitrogen compounds that can be used in normal fermentation. Preferred inorganic nitrogen sources include ammonium salts and nitrates, and more preferred are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. Examples of organic nitrogen sources include amino acids, yeast extract, peptones (e.g., polypeptone N, soybean peptone, etc.), meat extracts (e.g., Ehrlich bonito extract, Lab-Remco powder, bouillon, etc.), seafood extracts, liver extracts, digested serum powders, fish oils, etc. More preferred are arginine, cysteine, citrulline, lysine, yeast extract, and peptones (e.g., polypeptone N, etc.).

[0030] Furthermore, in addition to carbon and nitrogen sources, the growth and activity of bacteria can be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following are examples of microbial growth cofactors derived from animals and plants, such as inorganic compounds, vitamins, and fatty acids. The following can be mentioned.

[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 Selenite Ferrous Ammonium Sulfate Sodium Acetate Trihydrate Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate

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

[0033] The medium of the present invention may also contain dextrins. By culturing anaerobic microorganisms in a medium containing dextrins, a solution containing a functional substance and dextrins can be obtained without adding dextrins even if dextrins are required in the culture solution after the culture. Dextrins can be added to the medium before and during the culture of the microorganisms.

[0034] The anaerobic microorganism of the present invention can be cultured according to a known method for culturing a microorganism. In industrial production, a continuous fermentation system capable of continuously supplying the medium and substrate gas and having a mechanism for recovering the culture product can be used.

[0035] In the production of the functional material of the present invention, it is necessary to prevent oxygen from being mixed into the continuous culture system. A commonly used culture tank can be used as it is as a culture vessel. Culture tanks that can also be used for culturing the anaerobic microorganism of the present invention are commercially available. An anaerobic atmosphere can be created by replacing the oxygen mixed into the culture tank with an inert gas such as nitrogen or a substrate gas.

[0036] Depending on the shape of the culture tank, a stirrer or the like can be used to sufficiently stir the medium. By stirring the culture in the culture tank, the opportunities for contacting the medium components and substrate gas with the anaerobic microorganism of the present invention can be increased, thereby optimizing the production efficiency of the functional substance.

[0037] The culture system may be a closed system such as a bottle or test tube sealed with a rubber stopper without ventilation, but it is preferable that the system does not contain air or oxygen. For example, nitrogen and / or hydrogen may be used in any suitable system. It may contain nitrogen and / or carbon dioxide in any ratio, and is preferably a gas phase or aqueous phase containing hydrogen. The method for creating such an environment in the gas phase and aqueous phase during culture is not particularly limited, but examples of the method include replacing the gas phase with the gas before culture, supplying the gas from the bottom of the culture vessel and / or to the gas phase of the culture vessel during culture, and bubbling the aqueous phase with the gas before culture. As the hydrogen, hydrogen gas may be used as it is. Alternatively, a source of hydrogen such as formic acid and / or a salt thereof may be added to the culture medium, and hydrogen may be generated during culture by the action of the microorganism.

[0038] For sufficient growth of the anaerobic microorganisms of the present invention, the pH of the culture is preferably 5.0 or higher, more preferably 6.0 or higher, and even more preferably 6.5 or higher, while it is preferably 8.0 or lower, and more preferably 7.5 or lower. 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 higher, more preferably 33°C or higher, while it is preferably 40°C or lower, more preferably 38°C or lower. The culture time can be appropriately set depending on the amount of the functional substance produced, the remaining amount of the functional substance material, etc. It is usually 8 hours or more, preferably 12 hours or more, more preferably 16 hours or more, while it is usually 120 hours or less, preferably 72 hours or less, more preferably 60 hours or less. In addition, in order to efficiently produce a functional substance, the medium can be continuously supplied. The amount of ventilation is preferably 0.01 to 2.0 vvm. The mixed gas can also be supplied in the form of nanobubbles. The pressurization conditions for the culture vessel are not particularly limited as long as they allow the growth of the anaerobic microorganism of the present invention, but are preferably 0.02 to 0.2 MPa.

[0039] (Production of functional substances by resting cells) When the anaerobic microorganism capable of producing a functional substance from a functional substance material is in the form of a resting cell, the solution is preferably water, a salt solution, or a buffer solution as described in the above-mentioned section "Resting cell of anaerobic microorganism capable of producing a functional substance from a functional substance material" instead of the medium. For other conditions, the description in the above section "Culture medium and production of functional substances by culture" is used.

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

[0041] Other Aspects of the Invention Another aspect of the present invention is a method for producing a food or beverage containing a functional substance, comprising the steps of: causing an anaerobic microorganism capable of producing a functional substance from a material in a solution containing the material of the functional substance to produce the functional substance from the material, the solution containing the material of the functional substance containing L-cystine; and blending the produced functional substance with a raw material for the food or beverage. In this specification, even when the term "food" is used, it refers to "food and drink products" including "beverages."

[0042] For details of the step of producing a functional substance from a material in a solution containing the material of a functional substance by an anaerobic microorganism having the ability to produce the functional substance from the material, the solution containing the material of the functional substance containing L-cystine, the explanation of the method for producing a functional substance mentioned above is incorporated herein by reference.

[0043] The method for producing food and drink of this embodiment also includes a step of blending the functional substance produced in the above step with food and drink ingredients. The food and drink is produced by blending the functional substance produced in the above step with a commonly used food and drink ingredient in a conventional manner, and the blending time is not particularly limited. The food and drink ingredients also include food additives. Furthermore, the food and drink ingredients can be sealed in a suitable container such as a bottle, bag, can, box, or pack, as necessary.

[0044] The main components of the food and drink may be water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. Examples of proteins include animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as hydrolysates thereof, and butter. Examples of carbohydrates include sugars, modified starches (dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fibers. Examples of lipids include vegetable oils and fats such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils, and interesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, 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. These components may be used in combination of two or more kinds, or may be synthetic products.

[0045] The content of the functional substance produced in the above steps relative to the total amount of the food or drink is not particularly limited, but is preferably a content that allows the desired effect of the functional substance to be obtained when the food or drink is ingested. The content of the functional substance relative to the total amount of the food or drink varies depending on the type of functional substance and the desired effect, but is, for example, 10 -6 % by mass to 50% by mass or less.

[0046] When the food or drink is a supplement, the form may be any of solid, gel, or liquid, for example, various processed food or drink, powder, tablet, pill, capsule, jelly, granule, etc. Furthermore, if necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, box, pack, etc. The supplement may contain additives such as excipients such as dextrin, preservatives such as vitamin C, flavorings such as vanillin, colorants such as safflower color, monosaccharides, oligosaccharides and polysaccharides (e.g., glucose, fructose, sucrose, saccharose, and carbohydrates containing these), acidulants, flavorings, oils and fats, emulsifiers, whole milk powder, or agar. Two or more of these ingredients may be used in combination, or 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 The medium, with the composition shown in Table 1 and adjusted to pH 6.9, was dispensed into 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.) in 10 mL portions, and sterilized at 115°C for 15 minutes with butyl rubber stoppers and plastic caps attached while replacing the gas phase with nitrogen. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain, which had been frozen and stored at -80°C, was inoculated into this medium, and the gas phase was sterilized with hydrogen gas passed through a sterile filter. After replacing the medium with water for 2 minutes or more, the mixture was cultured at 37°C and 200 spm for 18 hours with shaking to prepare a preculture solution.

[0049] [Table 1]

[0050] Example 2 The composition of the medium shown in Table 1 was adjusted to pH 6.9 by adding 0.5 g / L of daidzein. The mixture was then dispensed into 18 mm test tubes (manufactured by Sanshin Kogyo) for culturing aerobic microorganisms, and the gas phase was replaced with nitrogen while the tubes were inflated. The container was then fitted with a rubber stopper and a plastic cap and sterilized at 115°C for 15 minutes. Inoculate 0.1 mL of the preculture solution prepared in step 1, and infuse the gas phase with sterile filtered hydrogen gas for more than 2 minutes. After the medium replacement, the mixture was cultured at 37°C for 28 hours with shaking at 200 spm. After 28 hours, 0.45 g / L of equol was produced in the culture medium.

[0051] Example 3 The same procedure was repeated as in Examples 1 to 4 above, except that the concentration of L-cystine added in Example 2 was changed to 0.34 g / L. Cultivation and reaction were carried out in the same manner as in Example 2. As a result, 0.45 g / L of equol was produced in the culture medium.

[0052] Example 4 Cultivation and reaction were carried out in the same manner as in Examples 1 and 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 medium.

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

[0054] Comparative Example 1 Cultivation and reaction were carried out in the same manner as in Examples 1 and 2, except that the 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 medium. In other words, even when L-cystine was used, the product was obtained in the same amount as when L-cysteine ​​hydrochloride was used.

[0055] Example 6 The medium, with the composition shown in Table 2 and adjusted to pH 7.3, was dispensed into 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.) in 10 mL portions, and sterilized at 115°C for 15 minutes with butyl rubber stoppers and plastic caps attached while replacing the gas phase with nitrogen. Gordonibacter pamelaeae DSM 19378 strain and Clostridium bolteae JCM 12243 strain, which had been frozen and stored at -80°C, were inoculated into this medium, and the gas phase was replaced with nitrogen gas passed through a sterile filter for at least 2 minutes. After that, the medium was shake-cultured at 37°C at 200 spm for 2 days, and the pre-cultured medium was used. A solution was prepared.

[0056] [Table 2]

[0057] Example 7 The composition of the medium shown in Table 2 was adjusted to a concentration of 1.0 g / L of ellagic acid and a pH of 7.3. The mixture was then dispensed into 18 mm test tubes (manufactured by Sanshin Kogyo) for culturing aerobic microorganisms, and the gas phase was replaced with nitrogen while the tubes were inflated. The container was then fitted with a rubber stopper and a plastic cap and sterilized at 115°C for 15 minutes. Inoculate 0.5 mL of the preculture solution prepared in step 6, and infuse the gas phase with sterile filtered nitrogen gas for at least 2 minutes. After replacement of the medium, the mixture was cultured at 37°C with shaking at 200 spm for 5 days. After 5 days, 0.25 g / L of urolithin A was produced in the culture medium.

[0058] Comparative Example 2 Cultivation and reaction were carried out in the same manner as in Examples 6 and 7, except that the L-cystine added in Examples 6 and 7 was changed to 0.3 g / L of L-cysteine ​​hydrochloride. The amount of produced lolitin A was 0.37 g / L. In other words, the amount of the product obtained when L-cystine was used was equal to that obtained when L-cysteine ​​hydrochloride was used.

[0059] Comparative Example 3 Cultivation and reaction were carried out in the same manner as in Examples 6 and 7, except that the L-cystine added in Examples 6 and 7 was changed to 0.3 g / L sodium thioglycolate. As a result, 0.33 g / L of urolithin A was produced in the culture medium. In other words, even when L-cystine was used, the product was obtained in the same amount as when sodium thioglycolate was used. [Industrial Applicability]

[0060] According to the present invention, a method for producing a functional substance using a reducing agent that can also be used in the production of fermented foods can be provided. The functional substances obtained by the method of the present invention can be used according to the intended 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. The anaerobic microorganism is Gordonibacter pamelaeae. and / or Clostridium bolteae. The method according to claim 3, wherein the microorganism is a microorganism belonging to the genus Bacillus.

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