Equol-rich fermentation product and method for producing the same

Abstract

To provide a method that enables the cost-effective production of an equol fermentation liquid with reduced soybean-derived impurities.SOLUTION: Anaerobic fermentation of equol is conducted using, as a fermentation substrate, a soybean hypocotyl extract product prepared by subjecting soybean hypocotyl extract to purification such as resin treatment or precipitation of isoflavones, while supplying necessary nutrients and a gas containing hydrogen.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a highly purified equol-containing fermented product obtained by using isoflavones purified from soybean hypocotyl extract as a raw material and by using anaerobic microorganisms capable of producing equol, and a method for producing the same. [Background technology]

[0002] Isoflavones, which are found in large quantities in legumes such as soybeans and kudzu, are a type of polyphenol and a flavonoid whose basic structure is isoflavone. Recent research has revealed that isoflavones have female hormone (estrogen) and antioxidant effects, and that ingesting isoflavones has a preventive effect against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, and other conditions. For example, in soybeans, isoflavones exist in the form of glycosides covalently bound to sugar, such as daidzin, glycitin, and genistin, and are aglycones. Daidzein is present in very small amounts in the form of malonylated glycosides. Some of these glycosides are further malonylated or acetylated. When these glycosides enter the human or animal body, they are converted into daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or β-glucosidase, an enzyme produced by intestinal bacteria. Furthermore, daidzein is converted into dihydrodaidzein by the action of intestinal bacteria. This is followed by O-desmethylangolensin (O-DMA) or equol, and genistein is converted to dihydrogenistein. It is known that it is converted to 5-hydroxyequol via acetylcholinesterase.

[0003] Of these metabolic products, equol is known to have the highest estrogenic activity. However, in humans, there are individual differences in isoflavone metabolism, and as mentioned above, only a few people have intestinal bacteria capable of fermenting daidzein to produce equol, with the prevalence being approximately 50% in Japanese and 30% in Westerners. Therefore, there was a problem in that people who do not have equol-producing bacteria are unable to produce equol in their bodies even if they ingest legume foods such as soybeans that contain isoflavones.

[0004] To overcome these problems, attempts have been made to produce equol ex vivo using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4), but it has not been confirmed whether equol can be produced in industrial quantities. In addition, fermented soybean hypocotyls and fermented soybean hypocotyl extracts, which use soybean hypocotyls or soybean hypocotyl extracts as raw materials and have higher equol contents and fewer impurities such as less genistein, an isoflavone, as well as methods for producing the same have been reported (Patent Documents 5 to 7), but specifically, only fermented products containing about 5.38 mg to 6.51 mg of equol per 1 g of soybean hypocotyl fermented product have been obtained.

[0005] When soybean hypocotyls or soybean hypocotyl extracts are used as fermentation raw materials, they contain many substances other than daidzein, which is the raw material for equol. Therefore, as long as soybean hypocotyls or soybean hypocotyl extracts are used as fermentation raw materials, it has been impossible to obtain high-purity equol unless the fermented product is further purified by a water-ethanol extraction method or a method of producing it by column chromatography, as shown in Patent Documents 5 to 7. However, a large amount of waste liquid is generated when fermentation liquid is purified. From the viewpoint of protecting the global environment, a method of discharging as little waste liquid as possible has been desired. In addition, the volatile organic compounds used when refining equol from the fermentation liquid also pose environmental problems. In addition, extraction and purification equipment that handles dangerous materials such as volatile organic compounds must be explosion-proof, and the construction costs of production plants are high.

[0006] When soybean hypocotyls or soybean hypocotyl extracts are used as fermentation raw materials, soybeans themselves contain allergens, and when powdered soybean hypocotyls or soybean hypocotyl extracts are put into a fermentation tank as fermentation raw materials, dust containing soybean allergens may fly up and come into contact with workers, causing soybean allergies. For this reason, special equipment such as local exhaust ventilation and sealed equipment was required to prevent workers from coming into contact with allergens. The fermentation method is a versatile method that can produce various products in one fermentation tank, but when allergens are used, special cleaning is required to make the equipment allergen-free in order to produce other allergen-free products, and the problem is that special cleaning significantly reduces productivity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2006-204296 [Patent Document 2] Special table 2006-504409 [Patent Document 3] Patent Publication No. 2008-61584 [Patent Document 4] Patent Publication 2010-104241 [Patent Document 5] Patent 7009043 [Patent Document 6] Patent 7009044 [Patent Document 7] Patent 7019279 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in view of these circumstances, and aims to provide an ecological method for producing a fermented product with a high equol content, which uses a purified soybean hypocotyl extract as a raw material and anaerobic microorganisms to produce equol, and which produces significantly less waste liquid and volatile organic compound emissions compared to conventional methods. Furthermore, in order to efficiently produce a fermented product with a high equol content that can be used as a food raw material, it is important to omit the purification step that uses organic solvents. As for the production equipment, since hazardous materials such as volatile organic compounds are not handled, there is no need to construct expensive explosion-proof facilities. We also want to eliminate the risk of production workers coming into contact with soy allergens and developing allergies. [Means for solving the problem]

[0009] The present inventors conducted extensive research to solve the above problems and discovered that by using a purified soybean hypocotyl extract obtained by purifying a soybean hypocotyl extract as the raw material, rather than soybean hypocotyls or soybean hypocotyl extracts, and supplementing it with the addition of necessary components, it is possible to produce equol by fermentation, and to obtain a fermented product containing a high content of equol and little soybean-derived impurities without refining the fermentation liquid.Soybean hypocotyl extract is extracted from soybean hypocotyls with ethanol, and this extract is used as the raw material and purified through one or more of a resin treatment step, a precipitation treatment step, or a crystallization treatment step to remove soybean-derived impurities from the target aqueous solution. A soybean hypocotyl extract purified product is obtained from which the impurities have been removed to a minimum level. If a soybean hypocotyl extract purified product with a low content of various impurities is used as a raw material, it is possible to reduce the content of soybean-derived components such as genisteins, glyciteins, and saponins that cause unpleasant tastes such as bitterness and astringency. The isoflavone content of the soybean hypocotyl extract purified product is preferably 85% or more in order to sufficiently reduce saponins and to make it allergen-free. However, an excessively high content purified product increases the production cost and the solubility of isoflavones themselves is low, so high-purity products with an isoflavone content of 98% or more, such as reagents, have poor dispersibility and poor reactivity unless they are crushed very finely. The higher the purity of isoflavones and the finer the particle size, the lower the minimum ignition energy, and the greater the risk of dust explosion, making it difficult to actually use high-purity products at the reagent level. Therefore, the upper limit of the isoflavone content is preferably 95%.

[0010] The purified soybean hypocotyl extract is a composition rich in isoflavones. When fermentation is carried out using anaerobic microorganisms capable of assimilating daidzein, a type of isoflavone, to produce equol, there are problems in that the raw material contains few nutrients for the growth of the anaerobic microorganisms, and growth is slower and fermentation is slower than when soybean hypocotyl extract is used. The inventors conducted extensive research and found that equol is produced when anaerobic fermentation is carried out by adding a substance containing at least one selected from the group consisting of a nitrogen source, a carbon source, inorganic salts, vitamins, and amino acids as a nutrient and supplying one or more gases including hydrogen to the gas phase. As the nitrogen source, preferred inorganic nitrogen sources include yeast extract, peptones, meat extracts, etc., which are components containing vitamins and amino acids, and more preferred components are yeast extract and peptones.In addition, as the carbon source, glucose is required, and as inorganic salts, salts containing sodium, potassium, magnesium, calcium, or phosphoric acid are required.As amino acids, a combination of arginine or citrulline and cysteine ​​or cystine is required.

[0011] More specifically, the present invention relates to the following <1> ~ <9> This provides: <1> The fermented product of the purified soybean hypocotyl extract is obtained by fermenting a purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine, and contains fewer soybean-derived impurities than fermentation using soybean hypocotyl or soybean hypocotyl extract as the raw material. <2> <1> In the fermentation product described above, impurities are genisteins, glyciteins, or saponins. A fermented product of a purified soybean hypocotyl extract containing one or more of the following allergens: <3> A fermented product obtained by fermenting a purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine, the fermented product containing 20 mg or more of equol per 1 g of dry weight, the equol content being 50% by weight or more of the total amount of isoflavones, the sum of genisteins being 10% by weight or less of the total amount of isoflavones, and the genisteins being genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof. <4> The total amount of the genisteins is within a range of 5.0% by weight or less based on the total amount of isoflavones. There is, the above <3> A fermented product of the purified soybean hypocotyl extract according to the present invention. <5> the above <1> or <2> or <3> or <4> An extract of the fermentation product described in <6> Contains 20-70 mg of equol per 1 g of dry solids. <5> The extract as described above, <7> the above <1> or <2> or <3> or <4> or <5> or <6> A supplement comprising the extract according to claim 1. <8> A method for producing a fermented product of a purified soybean hypocotyl extract containing equol and ornithine, comprising: fermenting a purified soybean hypocotyl extract as a fermentation raw material in the presence of nutritional components and arginine with a microorganism capable of producing equol and ornithine to produce a fermented product containing equol and ornithine. <1> also, <2> , <3> or <4> A method for producing a fermented product, comprising producing a fermented product of the purified soybean hypocotyl extract according to claim 1. <9> The nutritional components include at least one selected from the group consisting of a nitrogen source, a carbon source, inorganic salts, vitamins, and amino acids, and anaerobic fermentation is performed by supplying one or more types of gases including hydrogen to the gas phase. <8> The manufacturing method according to <10> The inorganic nitrogen source is ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate or sodium nitrate, the vitamins are biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid or vitamin B12, the organic nitrogen source is amino acids, yeast extract, peptones or meat extract, the carbon source is a sugar such as glucose, the inorganic salt is sodium, potassium, magnesium, calcium or an inorganic salt containing phosphoric acid, and / or the amino acid is arginine, citrulline, cysteine ​​or cystine. <9> The manufacturing method described above. <11> The isoflavone content of the purified soybean hypocotyl extract is 85% by weight or more. <8> Note The manufacturing method described above, <12> The isoflavone content of the purified soybean hypocotyl extract is 87% by weight or more. <11> The manufacturing method according to the present invention <13> The isoflavone content of the purified soybean hypocotyl extract is 90% by weight or more. <12> The manufacturing method according to the present invention <14> The upper limit of the isoflavone content of the soybean hypocotyl extract purified product is 95% by weight or less. 1>, <12> or <13> The manufacturing method according to the present invention <15> The total amount of genisteins in the purified soybean hypocotyl extract is 12% by weight or less based on the total amount of isoflavones. <8> The manufacturing method according to the present invention <16> The total amount of genisteins in the purified soybean hypocotyl extract is 6.4% by weight or less based on the total amount of isoflavones. <15> The manufacturing method according to the present invention <17> The minimum ignition energy of the purified soybean hypocotyl extract is 3 to 10 mJ. <8> Note The manufacturing method described above, <18> The method for producing the purified soybean hypocotyl extract is negative for soybean allergens. Effect of the Invention

[0012] By fermenting a purified soybean hypocotyl extract as a raw material, it has become possible to obtain a fermented product with reduced levels of impurities derived from soybean hypocotyls.

[0013] The present invention provides a method for producing equol inexpensively that produces far less waste liquid and volatile organic compound emissions than conventional methods and is useful from the perspective of protecting the global environment.

[0014] According to the present invention, allergens derived from soybeans can be removed in the purification process of soybean hypocotyl extract, so that fermentation raw materials that do not contain allergens derived from soybeans can be used. Since the fermentation raw materials do not contain soybean allergens, workers will not develop soybean allergies when putting the raw materials into the fermentation tank. Therefore, since soybean allergens are not brought into the fermentation process, it is possible to make the manufacturing plant allergen-free. Furthermore, even in soybean hypocotyl extract manufacturing plants, the risk of workers inhaling dust containing soybean allergens is significantly reduced because the soybean hypocotyl extract containing soybean allergens is dried, not the soybean hypocotyl extract containing soybean allergens, but the soybean hypocotyl extract purified product not containing soybean allergens, and the soybean allergens are transferred to the solution side in the purification process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the present invention, the soybean hypocotyl extract purified product refers to a composition rich in isoflavones purified from soybean hypocotyl extract. Soybean hypocotyl extract is extracted from soybean hypocotyl with ethanol. When this extract is used as a raw material and purified through one or more steps of a resin treatment step, a precipitation treatment step, or a crystallization treatment step, soybean-derived impurities are removed to a target level. A soybean hypocotyl extract purified product having a low content of various impurities can be obtained. If a soybean hypocotyl extract purified product having a low content of various impurities is used as a raw material, it is possible to reduce the content of soybean-derived components such as genisteins, glyciteins, and saponins that cause unpleasant tastes such as bitterness and astringency. The isoflavone content of the soybean hypocotyl extract purified product is 85% by weight or more, preferably 87% by weight or more, and more preferably 90% by weight or more in order to sufficiently reduce saponins and to make the product allergen-free. However, an excessively high content purified product increases the production cost and the solubility of isoflavones themselves is low, so high-purity products with an isoflavone content of 98% by weight or more, such as reagents, have poor dispersibility and poor reactivity unless they are crushed very finely. The higher the purity of isoflavones and the finer the particle size, the lower the minimum ignition energy, and the greater the risk of dust explosion, making it difficult to actually use high-purity products at the reagent level. Therefore, the upper limit of the isoflavone content is preferably 95% by weight. From soybean hypocotyl extract, resin treatment process, precipitation treatment process, or crystallization treatment process One or more purification steps can be carried out to obtain a purified soybean hypocotyl extract.

[0016] The resin used in the resin treatment step of the purification step of the soybean extract is not limited to any resin as long as it has hydrophobic adsorption ability, and may be a synthetic adsorption resin or an ion exchange resin. For example, if the base structure of the resin is styrene-based, examples include DIAION HP series (e.g., HP20, HP20SS, HP21, etc.; manufactured by Mitsubishi Chemical), SEPABEADS SP series (e.g., SP207, SP207SS, SP20SS, SP70, SP700, SP825, SP850, etc.; manufactured by Mitsubishi Chemical), and Amberlite XAD series (e.g., XAD4, XAD2000, XADFPX66, XAD1180N, XAD2, etc.; manufactured by Rohm and Haas). If the base structure of the resin is acrylic, examples include HP2MGL (manufactured by Mitsubishi Chemical), XAD7HP (manufactured by Rohm and Haas). In addition, a cation exchange resin whose base structure is styrene-based, for example, Na-type, K-type, or Ca-type of PK series (manufactured by Mitsubishi Chemical), UPK series (manufactured by Mitsubishi Chemical), etc. The above resin brands are not limited, and any resin having a similar base structure can be used without any restrictions.

[0017] The resin treatment step may be a method of elution after resin adsorption, or a method of chromatographic separation without adsorption.

[0018] The solvent used in the resin treatment process can be any solvent that can be used for food applications. For example, a mixed solvent of ethanol and water. By adjusting the water content of the mixed solvent according to the adsorption performance of the resin, it is possible to separate the impurities.

[0019] The resin treatment step may be performed by either a single column method or a simulated moving bed method without any limitation.

[0020] The precipitation process or crystallization process in the purification process of the soybean extract is not limited as to whether the solid obtained has a crystalline structure or not, and refers to a process of precipitating a solid from an extract and performing solid-liquid separation. Water may be added to the soybean hypocotyl extract dissolved in ethanol to reduce the solubility of isoflavones for precipitation, or the soybean hypocotyl extract dissolved in aqueous ethanol may be concentrated to precipitate isoflavones. There is no particular limitation as long as it is a commonly known crystallization method.

[0021] The fewer impurities in the soybean hypocotyl extract purified product obtained by the process of the present invention, the fewer impurities there will be in the soybean hypocotyl extract purified fermentation product obtained. A product with fewer impurities and an isoflavone content of 85% or more is used. In addition, a soybean hypocotyl extract product with an isoflavone content of 40% that is commonly available on the market can be dissolved in a solvent or aqueous solvent that can be used for food applications, and purified through one or more of the following processes: a resin treatment process, a precipitation process, and a crystallization process, to remove impurities and refine the product to a content of 85% or more for use.

[0022] In the present invention, the fermented product obtained by fermenting a purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine means a culture obtained by the steps of converting isoflavones in the purified soybean hypocotyl extract obtained by the above step into aglycones as necessary, culturing an anaerobic microorganism in a medium containing the aglycone, and fermenting the anaerobic microorganism in a gas phase consisting of one or more types of gases including hydrogen to convert the aglycone into equol. The culture contains 20 mg or more, preferably 20 to 200 mg, and particularly preferably 40 to 60 mg of equol per 1 g dry weight, equol is 50% by weight or more, preferably 50 to 70% by weight, and particularly preferably 50 to 60% by weight of the total amount of isoflavones, the sum of genisteins is 10% by weight or less, preferably 5.9% by weight or less, and particularly preferably 5% by weight or less of the total amount of isoflavones, and the genisteins are However, genistin, malonylgenistin, acetylgenistin, genistein, dihydrogen genistein, or a combination thereof. If necessary, the purified soybean hypocotyl extract obtained by the above process may be treated with an enzyme that converts the isoflavones in the purified soybean hypocotyl extract into aglycones. The enzyme for converting isoflavones into aglycones includes β-glucosidase, preferably aromatase, and the like. The enzyme is typically used at a temperature of 40 to 60° C., preferably 50° C., pH 6 to 8, and preferably The conversion reaction is preferably carried out at pH 7.0 for about 10 to 36 hours, preferably about 24 hours. . The process includes culturing anaerobic microorganisms in a medium containing the aglycone obtained in the above process, fermenting the mixture with the anaerobic microorganisms in a gas phase consisting of one or more types of gases including hydrogen, converting the aglycone into equol, and recovering the equol produced by the anaerobic microorganisms.

[0023] In the case of a microorganism having an enzyme that converts isoflavones to aglycone, the process of converting the isoflavones to aglycone is a common process to the fermentation process in which anaerobic microorganisms are cultured in a medium containing aglycone, and the aglycone is converted to equol by fermenting the anaerobic microorganisms in a gas phase consisting of one or more types of gases including hydrogen.

[0024] The combination of gases constituting the gas phase in the fermentation process is not particularly limited, and one or more types of gases including hydrogen selected from hydrogen, carbon dioxide, nitrogen, etc. can be used as constituent components.

[0025] The fermentation temperature in the fermentation step is not particularly limited as long as the anaerobic microorganism used is capable of producing equol from daidzein, but is preferably 30°C to 40°C, and more preferably 33°C to 38°C.

[0026] The microorganisms in the fermentation step can be cultured at normal pressure, but when pressurized, the pressurization conditions are not particularly limited as long as the microorganisms can grow under the pressurized conditions. Preferred pressurization conditions include, but are not limited to, a pressure of 1 MPa or less, more preferably 0.3 MPa or less.

[0027] The anaerobic microorganism used in the fermentation step is not particularly limited as long as it produces equol from at least one equol raw material selected from the group consisting of daidzein and dihydrodaidzein.

[0028] There are no particular limitations on the microorganisms used in the production method of the present invention, so long as they produce equol from the above-mentioned equol raw material. The equol-producing ability can be confirmed by quantifying daidzein, dihydrodaidzein, equol, etc. in the culture. These quantifications can be performed by those skilled in the art based on the descriptions in, for example, WO2012 / 033150, JP 2012-135217, JP 2012-135218, JP 2012-135219, etc. An example of these quantification methods is shown below.

[0029] For example, ethyl acetate is added to the culture solution, which is vigorously stirred and then centrifuged to remove the ethyl acetate layer. If necessary, the same procedure can be performed several times on the same culture solution, and the ethyl acetate layers can be combined to obtain an equol extract. This extract is concentrated and dried under reduced pressure using an evaporator, and dissolved in methanol. This is then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, which can then be used as a high-performance liquid chromatography measurement sample. Examples of high-performance liquid chromatography conditions include, but are not limited to, the following:

[0030] [High performance liquid chromatography conditions] Column: Phenomenox Luna 5uC18, 2.0mm x 150mm (Shimadzu GLC) Mobile phase: Water / methanol [55:45, v / v] Flow rate: 0.2mL / min Column temperature: 40℃ Detection: UV280nm Retention times: dihydrodaidzein 13.8 min, daidzein 19.6 min, glycitein 22.5 min, equol 25.6 min, genistein 35.0 min

[0031] Equol-producing microorganisms include, but are not limited to, those classified into the following genera: Genus Adlercreutzia Bacteroides genus Bifidobacterium Clostridium Genus Eggerthella Enterococcus Genus Enterorhabdus Eubacterium genus Genus Finegoldia Lactobacillus Lactococcus Genus Paraeggerthella Pediococcus genus Genus Proteus Genus Sharpea Genus Slackia Streptococcus Genus Veillonella

[0032] Specific examples of microorganisms that produce equol include, but are not limited to, the following microorganisms: Adlercreutzia aequorifaciens subsp. ceratus equolifaciens subsp. celatus) Adlercreutzia equolifaciens subsp. equolifaciens Bacteroides ovatus Bifidobacterium breve Bifidobacterium longum Clostridium sp. Eggerthella sp. Enterococcus faecalis Enterococcus faecium Enterorhabdus mucosicola Eubacterium sp. Finegoldia magna Lactobacillus fermentum Lactobacillus mucosae Lactobacillus paracasei Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus sp. Lactococcus garvieae Lactococcus sp. Paraeggerthella sp. Pediococcus pentosaceus Proteus mirabilis Sharpea azabuensis Slackia equolifaciens Slackia isoflavoniconvertens Slackia sp. Streptococcus constellatus Streptococcus intermedius Veillonella sp.

[0033] Among the above-mentioned microorganisms, for example, microorganisms classified into the family Eggerthellaceae, Microorganisms classified in the Bifidobacteriaceae family, microorganisms classified in the Clostridiaceae family, microorganisms classified in the Coriobacteriaceae family, microorganisms classified in the Enterococcaceae family, microorganisms classified in the Eubacteriaceae family, microorganisms classified in the Morganellaceae family, microorganisms classified in the Peptoniphilaceae family, microorganisms classified in the Lactobacillaceae family, microorganisms classified in the Streptococcus ceae family, microorganisms classified in the Veillonellaceae family, or microorganisms related to these. Preferred are the genus Adlercreutzia, the genus Bacteroides, The microorganism may be classified into the genus Bifidobacterium, Clostridium, Coriobacterium, Egasella, Enterococcus, Eubacterium, Finegordia, Lactobacillus, Lactococcus, Paraegasella, Pediococcus, Proteus, Chapaea, Slaakia, Streptococcus, Veillonea, or a microorganism related thereto. More preferably, Adrechlautia aequorifaciens subsp. ceratus, Adrechlautia aequorifaciens subsp. aequorifaciens, Bacteroides obatus, Bifidobacterium breve, Bifidobacterium longum, Clostridium sp., Egassella sp., Enterococcus faecalis, Enterococcus faecium, Enterohabdus mucosicola, Eubacterium sp., Finegordia magna, Lactobacillus fermentum, Lactobacillus intestina Lactobacillus subtilis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sp., Lactococcus garvieae, Lactococcus sp., Paraegasella sp., Pediococcus pentosaceus, Proteus mirabilis, Sharpea azabuensis, Slachia aequorifaciens, Slachia isoflavonic convertens, Slachia sp., Streptococcus constellatus, Streptococcus intermedius, Veillonea sp.

[0034] Among the above-mentioned microorganisms, any of the microorganisms described below or related microorganisms having similar species properties to these microorganisms can be mentioned as more preferred anaerobic microorganisms. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain Bacteroides ovatus strain E-23-15 Bifidibacterium breve ATCC 15700 strain Bifidobacterium longum BB536 strain Clostridium sp. HGH136 strain Eggerthella sp. Julong 732 strain Eggerthella sp. YY7918 strain Eggerthella sp. D1 strain Enterococcus faecalis INIA P333 strain Enterococcus faecium strain EPI1 Enterohabdus mucosicola Mt1B8 strain Eubacterium sp. strain D2 Finegoldia magna EPI3 strain Lactobacillus fermentum DPPMA114 strain Lactobacillus intestinalis KTCT13676BP strain Lactobacillus mucosae EPI2 strain Lactobacillus paracasei JS1 strain Lactobacillus plantarum DPPMA24W strain Lactobacillus plantarum DPPMASL33 strain Lactobacillus rhamnosus strain DPPMAAZ1 Lactobacillus rhamnosus INIA P540 strain Lactobacillus sp. Niu-O16 strain Lactococcus garvieae strain 20-92 Paraeggerthella sp. strain SNR40-432 Pediococcus pentosaceus strain CS1 Proteus mirabilis LH-52 strain Sharpea azabuensis ST18 strain Slackia equolifaciens strain DSM 24851 Slackia isoflavoniconvertens DSM 2200 6 stocks Slackia sp. FJK1 strain Slackia sp. NATTS strain Slackia sp. YIT11861 strain Slackia sp. TM-30 strain Streptococcus constellatus strain E-23-17 Streptococcus intermedius A6G-225 strain Veillonella sp. EP strain.

[0035] The above anaerobic microorganisms can be obtained from the depository institutions indicated by their accession numbers. Each accession number indicates that the anaerobic microorganism has been deposited at the following depository institutions: FERM International Patent Organism Depositary (IPOD) http: / / unit.aist.go.jp / pod / ci / index.html DSM German Collection of Microorganisms and Cell Cultures (DSMZ) http: / / www.dsmz.de / KCCM Korean Culture Center of Microorganisms

[0036] In the present invention, anaerobic microorganisms capable of producing equol are cultured under conditions suitable for the production of equol. In the present invention, conditions suitable for the production of equol refer to conditions under which the survival and activity of anaerobic microorganisms capable of producing equol can be maintained. More specifically, gas-phase conditions (anaerobic conditions) in which anaerobic microorganisms can survive are maintained, and the activity of the anaerobic microorganisms is maintained. "Equol-producing" refers to the provision of nutrients to support the activity and growth of anaerobic microorganisms. Various medium compositions suitable for the survival of anaerobic microorganisms are known. Therefore, those skilled in the art can select an appropriate medium composition for the anaerobic microorganisms capable of producing equol shown above. For example, BHI medium manufactured by Difco or the medium used in the examples can be used. Not limited to.

[0037] Nutrients for supporting growth, i.e., nutritional components, include at least one selected from the group consisting of nitrogen sources, carbon sources, inorganic salts, vitamins, and amino acids, with preferred inorganic nitrogen sources being ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, or sodium nitrate, preferred vitamins being biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, or vitamin B12, preferred organic nitrogen sources being amino acids, yeast extract, peptones, or meat extract, preferred carbon sources being sugars such as glucose, preferred inorganic salts being sodium, potassium, magnesium, calcium, or inorganic salts containing phosphoric acid, and preferred amino acids being arginine, citrulline, cysteine, or cystine. These nutrients exhibit their effects in combination with at least one other nutrient.

[0038] Water-soluble organic substances can be added to the medium used in the present invention as a carbon source. Examples of water-soluble organic substances include, but are not limited to, the following compounds: sugars such as sorbose, fructose, and glucose; Alcohols such as methanol; Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, etc., or their salts.

[0039] The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow anaerobic microorganisms in the medium.

[0040] A nitrogen source can be added to the medium. In the present invention, various nitrogen compounds that can be used in normal fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrates. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. On the other hand, preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder, and the like. More preferred organic nitrogen sources are arginine, cysteine, cystine, citrulline, lysine, yeast extract, and peptones.

[0041] Furthermore, in addition to the carbon and nitrogen sources, other organic or inorganic substances suitable for the production of equol can be added to the medium. For example, the growth and activity of anaerobic microorganisms can be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following inorganic compounds, vitamins, and microbial growth cofactors derived from animals and plants can be mentioned.

[0042] 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 Boric acid etc. Nickel chloride Sodium Tungstate Sodium Selenite Ferrous Ammonium Sulfate

[0043] A conventionally known method can be used to produce a culture solution by adding these inorganic compounds, vitamins, or growth cofactors. The medium can be liquid, semi-solid, or solid. In the present invention, the preferred form of the medium is a liquid medium.

[0044] The medium of the present invention can contain dextrins. By culturing anaerobic microorganisms in a medium containing dextrins, a liquid containing equol and dextrins can be prepared without contacting the culture with dextrins after the culture. Dextrins can be added to the medium before or during the culture of the microorganism.

[0045] The medium of the present invention contains an antifoaming agent, preferably soybean oil, more preferably soybean oil containing vitamin E. may include.

[0046] In the method of the present application, the anaerobic microorganism can be cultured according to a known method for culturing a microorganism. For industrial production, a continuous fermentation system capable of continuously supplying the medium and substrate gas and having a mechanism for recovering the culture can be used.

[0047] In the method of the present invention, when anaerobic microorganisms are used, it is advisable to prevent oxygen from entering the fermentation tank. A commonly used fermentation tank can be used as is. An anaerobic atmosphere can be created by replacing oxygen that enters the fermentation tank with an inert gas such as nitrogen.

[0048] When the method of the present application is carried out in a mixed gas phase, the combination of gases constituting the gas phase is not particularly limited, and in addition to hydrogen, one or more gases selected from carbon dioxide, nitrogen, etc. can be used as constituent components.

[0049] The fermentation time can be appropriately set depending on the amount of equol produced, the amount of remaining isoflavones, etc. Examples of the fermentation time include, but are not limited to, 8 to 120 hours, preferably 12 to 72 hours, and particularly preferably 16 to 60 hours. The fermentation liquid after the end of the culture contains a purified soybean hypocotyl extract fermentation product, and the material state of the soybean hypocotyl extract fermentation product of the present invention may be any of liquid, slurry, and solid states.

[0050] The fermented product cultured using the purified soybean hypocotyl extract of the present invention as a raw material, i.e., the purified soybean hypocotyl extract fermented product, can be solidified by heat drying, spray drying, or freeze drying as necessary. Heat drying can be performed using, for example, a rotary drum dryer, spray drying can be performed using, for example, a spray dryer, and freeze drying can be performed using a freeze dryer. Any dryer that can dry a liquid can be used for the drying method. The dried fermented product can be subjected to a powdering process as necessary.

[0051] The fermented product obtained by culturing the purified soybean hypocotyl extract of the present invention as a raw material, i.e., the fermented product of the purified soybean hypocotyl extract, can be optionally oxidized with β-cyclodextrin or γ-cyclodextrin. After entrapment solubilization, insoluble matter and bacterial cells may be removed by filtration, centrifugation, or membrane treatment. Furthermore, polymeric impurities can be removed by using a UF membrane. The liquid in which equol has been dissolved after these treatments can be solidified by heat drying, spray drying, or freeze drying. Heat drying can be performed using, for example, a rotary drum dryer, spray drying can be performed using, for example, a spray dryer, and freeze drying can be performed using, for example, a freeze dryer. Any dryer capable of drying liquids may be used for the drying method. The fermented product that has been dried may be subjected to powderization as necessary.

[0052] In the present invention, the extract of the fermented purified soybean hypocotyl extract refers to a product in which useful substances including equol are extracted from the fermented purified soybean hypocotyl extract with water or aqueous ethanol. The extract of the fermented purified soybean hypocotyl extract can be obtained by suspending the fermented purified soybean hypocotyl extract in water, removing impurities as necessary, and then extracting the components dissolved in water in the case of aqueous extraction, or with the aqueous ethanol extract in the case of aqueous ethanol extraction. The ethanol content of the aqueous ethanol extract is 30 to 95%, preferably 40 to 75%. The extract contains 20 to 70 mg of equol per gram in a dried and fixed state.

[0053] The supplement containing the fermented soybean hypocotyl extract purified product of the present invention may be any health food or nutritional supplement to which a fermented soybean hypocotyl extract purified product or an extract of a fermented soybean hypocotyl extract purified product has been added, and may also include those approved for functional claims such as functional foods and foods for specified health uses. The supplement may be in the form of a solid preparation such as a tablet, capsule, or granule, or in the form of a drink or jelly, but may be used as a solid supplement. In the case of a capsule, the fermented soybean hypocotyl extract purified product may be filled directly or diluted with an excipient into a food capsule formed of a plant base by a conventional method. In the case of a tablet, the desired capsule can be obtained by adding an excipient and one or more of a lubricant or a flow agent to the fermented soybean extract purified product by a conventional method to enhance moldability and tableting by a conventional method. In addition, when used as a chewable agent, flavorings or the like may be added to correct the taste. In the case of a granule, the desired supplement can be obtained by adding an excipient to the purified soybean extract and granulating it by a conventional method. The excipient is not particularly limited as long as it is one that is used in foods, and examples of the excipient that can be used include starch, crystalline cellulose, sugar alcohol, lactose, etc. Drinks, jellies, etc. can be produced by dissolving or suspending the fermented product of the purified soybean hypocotyl extract in water in a conventional manner.

[0054] The higher the isoflavone content of the soybean hypocotyl extract refined product, the smaller the minimum ignition energy. For example, if comparing products with the same moisture content and particle size, the minimum ignition energy of a commonly available soybean hypocotyl extract with an isoflavone content of 40% by weight is 100 mJ or more. The minimum ignition energy of a refined soybean hypocotyl extract with a soflavone content of 85 to 90% by weight is 10 mJ or less. Considering the risk of dust explosion, the minimum ignition energy is preferably 3 mJ or more. Soybean hypocotyl extract does not require any anti-static measures, but the soybean hypocotyl extract refined product requires grounding and anti-static measures for workers.

[0055] Daidzeins refer to daidzin, malonyldaidzin, acetyldaidzin, daidzein, and dihydrodaidzein. Genisteins refer to genistin, malonylgenistin, acetylgenistin, genistein, and dihydrogenistein. The glyciteins refer to glycitin, malonylglycitein, acetylglycitein, glycitein, and dihydroglycitein.

[0056] Allergens can be detected using commercially available kits such as FASKIT Slim Soybean (product name, manufactured by Nippon Ham Central Research Institute). For specific operation instructions, please refer to the manual that comes with the kit. For example, in the case of FASKIT Slim Soybean, 2 g of sample was used, 38 mL of the extraction buffer solution included in the kit was added, and after mixing for 30 to 60 minutes, extraction was performed three times. After that, the mixture was centrifuged at 3000 g, 4°C, and 20 minutes, and the supernatant was filtered through filter paper and diluted 10 times with the dilution buffer solution included in the kit to prepare the measurement sample. Measurements were performed by dropping 100 μL onto a test strip, developing it for 15 minutes, and then visually determining.

[0057] Measurement of minimum ignition energy can be performed by requesting a measurement organization such as the Environmental Health Research Institute Co., Ltd.

[0058] Saponin was analyzed by HPLC. The specimen (equivalent to 1 mg of saponin content) was extracted with a mixture of 1 mL of water and 18 mL of methanol. The insoluble matter was further extracted with 20 mL of methanol, and the extracts were mixed. Methanol was removed from this mixture by vacuum concentration. 10 mL of 10% hydrochloric acid in methanol was added, and the mixture was heated to reflux at 80°C for 2 hours, after which ethanol was added and this was used as a sample for HPLC analysis. 10 mg of each of the standards of soyasapogenol A and B (Tokiwa Plant Science Laboratory) was precisely weighed into a 100 mL measuring flask, dissolved in ethanol, and adjusted to the specified volume to prepare a standard solution. The soybean saponin A group value is calculated by multiplying the quantitative value of soyasapogenol A by the molecular weight ratio (1437.52 / 474.72). The soybean saponin B group value is calculated by multiplying the quantitative value of soyasapogenol B by the molecular weight ratio (943.12 / 458.73). The amount of saponin is the combined total of soybean saponin A group and soybean saponin B group. [High performance liquid chromatography conditions] Column: Inertsil ODS-2.5μm, φ2.0mm×150×150mm (GE (Manufactured by LuScience Co., Ltd.) Mobile phase: Acetonitrile / 1% acetic acid water = 7:3 Flow rate: 0,2mL / min Column temperature: 40℃ Detection: UV 210 nm Retention time: EXAMPLES

[0059] The present invention will be described below based on examples, but the scope of the present invention is not limited to the following examples. Example 1 <Various raw materials> A raw material containing soybean hypocotyl extract and daidzein purified from the soybean hypocotyl extract was prepared. Sample 1A: Soybean hypocotyl extract isoflavone P-40 (manufactured by Fujicco Co., Ltd.) Sample 1B: Soybean hypocotyl extract with 40% isoflavone content (made in China) Sample 1C: Soybean hypocotyl extract purified product 1 (recrystallized from aqueous ethanol) Sample 1D: Soybean hypocotyl extract purified product 2 (recrystallized from aqueous ethanol) Sample 1E: Soybean hypocotyl extract purified product 3 (recrystallized from aqueous ethanol) Sample 1F: Soybean hypocotyl extract purified product 4 (recrystallized from aqueous ethanol) Sample 1G: Soybean hypocotyl extract purified product 5 (resin chromatography separation product) The composition of the raw materials used is shown in Table 1.

[0060] [Table 1]

[0061] <Enzyme treatment> For each of Samples 1A to 1G, 5 g of each was weighed out in terms of isoflavone, and the samples were diluted with 50 mg of aromatase (Amano Pharmaceutical Co., Ltd.) as an enzyme and 900 g of deionized water to adjust the pH to 4 to 5. The enzyme treatment was carried out at 50°C for 24 hours with stirring.

[0062] <Fermentation by anaerobic microorganisms> (seed culture) The medium, with the composition shown in Table 2 and adjusted to pH 6.9, was dispensed into 10 mL of 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with a butyl rubber stopper and a plastic cap attached while replacing the gas phase with nitrogen. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain was inoculated, and the gas phase was replaced with hydrogen gas passed through a sterile filter for at least 2 minutes, after which the mixture was cultured with shaking at 37°C and 200 spm for 18 hours.

[0063] [Table 2]

[0064] (Main culture) To the enzyme treatment solution of each of the samples 1A to 1G, 3 g of L-arginine and each component shown in Table 2 were added to the respective concentrations shown in Table 2, and 1000 mL of each medium was prepared by adjusting the pH to 6.9. Then, the medium was heated at 121°C for 15 minutes and sterilized by high-pressure steam. Ten tubes of each of the above pre-culture solutions were inoculated into the medium, and the gas phase was aerated with hydrogen / nitrogen gas at a ratio of 6:4. Culture was carried out at 37°C for 48 hours, and the isoflavone and equol concentrations in the main culture solution were analyzed by HPLC.

[0065] <Recovery of equol-containing composition> The fermented liquid after the above culture was dried (spray dried) to obtain an equol-containing material.

[0066] The obtained fermentation liquid and the dried product of the fermentation liquid are shown in Table 3. By using isoflavones purified from soybean hypocotyl extract as the fermentation raw material, a fermentation product was obtained that had a high equol content, low genisteins, low glyciteins, and was negative for soy allergens. In addition, when a raw material with a high isoflavone content was used, the amount of saponin was low.

[0067] [Table 3]

[0068] Example 2 <Various raw materials> The raw materials used were the soybean hypocotyl extract and daidzeins purified from the soybean hypocotyl extract shown in Table 1 of Example 1.

[0069] <Enzyme treatment> For each of Samples 1A to 1G, 5 g of each was weighed out in terms of isoflavone, and the samples were diluted with 50 mg of aromatase (Amano Pharmaceutical Co., Ltd.) as an enzyme and 900 g of deionized water to adjust the pH to 4 to 5. The enzyme treatment was carried out at 50°C for 24 hours with stirring.

[0070] <Fermentation by anaerobic microorganisms> (seed culture) The medium, with the composition shown in Table 2 and adjusted to pH 6.9, was dispensed into 10 mL 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with butyl rubber stoppers and plastic caps attached while replacing the gas phase with nitrogen. Eggerthella sp. DC 3215 strain was inoculated into this medium, and the gas phase was sterilized with hydrogen gas passed through a sterile filter. After replacing the medium for 2 minutes or more, the mixture was cultured with shaking at 37°C and 200 spm for 18 hours.

[0071] (Main culture) To the enzyme treatment solution of each of the samples 1A to 1G, 3 g of L-arginine and each component shown in Table 2 were added to the respective concentrations shown in Table 2, and 1000 mL of each medium was prepared by adjusting the pH to 6.9. Then, the medium was heated at 121°C for 15 minutes and sterilized by high-pressure steam. Ten tubes of each of the above pre-culture solutions were inoculated into the medium, and the gas phase was aerated with hydrogen / nitrogen gas at a ratio of 6:4. Culture was carried out at 37°C for 48 hours, and the isoflavone and equol concentrations in the main culture solution were analyzed by HPLC.

[0072] <Recovery of equol-containing composition> The fermented liquid after the above culture was dried (spray dried) to obtain an equol-containing material.

[0073] The obtained fermentation liquid and the dried product of the fermentation liquid are shown in Table 4. By using isoflavones as fermentation raw materials, fermented products with high equol content, low genisteins and glyciteins, and negative for soy allergens were obtained. In addition, when raw materials with high isoflavone content were used, the amount of saponins was low.

[0074] [Table 4]

[0075] Example 3 <Various raw materials> The raw materials used were the soybean hypocotyl extract and daidzeins purified from the soybean hypocotyl extract shown in Table 1 of Example 1.

[0076] <Fermentation by anaerobic microorganisms> (seed culture) The medium, with the composition shown in Table 2 and adjusted to pH 6.9, was dispensed into 10 mL 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with a butyl rubber stopper and a plastic cap attached while replacing the gas phase with nitrogen. Lactococcus sp. DC 2021 strain was inoculated into this medium, and hydrogen gas passed through a sterile filter was added. After replacing the gas phase with for 2 minutes or more, the mixture was cultured with shaking at 37°C and 200 spm for 18 hours.

[0077] (Main culture) Each of Samples 1A to 1G was weighed out at 5 g in terms of isoflavone, and 3 g of L-arginine and each component shown in Table 2 were added to the concentrations shown in Table 2 to prepare 1000 mL of medium adjusted to pH 6.9, which was then heated at 121°C for 15 minutes and sterilized by high-pressure steam. Ten tubes of each of the above preculture solutions were inoculated, and the gas phase was aerated with hydrogen / nitrogen gas at a ratio of 6:4. Culture was carried out at 37°C for 48 hours, and the isoflavone and equol concentrations in the main culture solutions were analyzed by HPLC.

[0078] <Recovery of equol-containing composition> The fermented liquid after the above culture was dried (spray dried) to obtain an equol-containing material.

[0079] The obtained fermentation liquid and the dried product of the fermentation liquid are shown in Table 5. By using isoflavones purified from soybean hypocotyl extract as the fermentation raw material, a fermentation product was obtained that had a high equol content, low genisteins, low glyciteins, and was negative for soy allergens. In addition, when a raw material with a high isoflavone content was used, the amount of saponin was low.

[0080] [Table 5]

Claims

1. The fermented product of the purified soybean hypocotyl extract is obtained by fermenting a purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine, and contains fewer soybean-derived impurities than fermentation using soybean hypocotyl or soybean hypocotyl extract as the raw material.

2. 2. The fermented product of claim 1, wherein the impurities are any one or more of genisteins, glyciteins, saponin, and soybean allergens.

3. A fermented product obtained by fermenting a purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine, said fermented product containing 20 mg or more of equol per 1 g of dry weight, A fermented product of a purified soybean hypocotyl extract, in which quarts are 50% by weight or more based on the total amount of isoflavones, the sum of genisteins is 10% by weight or less based on the total amount of isoflavones, and the genisteins are genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof.

4. The total amount of the genisteins is in the range of 5.0% by weight or less based on the total amount of isoflavones. The fermented product of the purified soybean hypocotyl extract according to claim 3.

5. An extract of the fermentation product according to claim 1, claim 2, claim 3 or claim 4.

6. The extract according to claim 5, containing 20 to 70 mg of equol per gram of dry solids.

7. A supplement comprising the fermented product of claim 1, claim 2, claim 3 or claim 4, or the extract of claim 5 or claim 6.

8. A method for producing a fermented product of a purified soybean hypocotyl extract containing equol and ornithine, comprising: using a purified soybean hypocotyl extract as a fermentation raw material in the presence of nutritional components and arginine; and fermenting the purified soybean hypocotyl extract with a microorganism capable of producing equol and ornithine to produce a fermented product of a purified soybean hypocotyl extract according to claim 1, 2, 3, or 4 containing equol and ornithine.

9. The method according to claim 8, wherein the nutritional components include at least one selected from the group consisting of a nitrogen source, a carbon source, inorganic salts, vitamins, and amino acids, and anaerobic fermentation is carried out by supplying one or more types of gases including hydrogen to the gas phase.

10. The method according to claim 9, wherein the inorganic nitrogen source is ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate or sodium nitrate, the vitamins are biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid or vitamin B12, the organic nitrogen source is amino acids, yeast extract, peptones or meat extract, the carbon source is a sugar such as glucose, the inorganic salt is an inorganic salt containing sodium, potassium, magnesium, calcium or phosphoric acid, and / or the amino acid is arginine, citrulline, cysteine ​​or cystine.

11. 9. The method according to claim 8, wherein the isoflavone content of the purified soybean hypocotyl extract used as the fermentation raw material is 85% by weight or more.

12. The isoflavone content of the purified soybean hypocotyl extract used as the fermentation raw material is 87% by weight or more. The method according to claim 11 .

13. The method according to claim 12, wherein the isoflavone content of the purified soybean hypocotyl extract used as the fermentation raw material is 90% by weight or more.

14. The method according to claim 11, 12 or 13, wherein the upper limit of the isoflavone content in the purified soybean hypocotyl extract used as the fermentation raw material is 95% by weight.

15. 9. The method according to claim 8, wherein the total amount of genisteins in the purified soybean hypocotyl extract used as the fermentation raw material is 12% by weight or less based on the total amount of isoflavones.

16. The method according to claim 15, wherein the total amount of genisteins in the purified soybean hypocotyl extract used as the fermentation raw material is 6.4% by weight or less based on the total amount of isoflavones.

17. The method according to claim 8, wherein the minimum ignition energy of the purified soybean hypocotyl extract used as the fermentation raw material is 3 to 10 mJ.

18. The method according to claim 8, wherein the purified soybean hypocotyl extract used as the fermentation raw material is negative for soybean allergens.

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