WO2024106449A1 - Fenugreek seed processed product and method for producing same - Google Patents

Fenugreek seed processed product and method for producing same Download PDF

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Publication number
WO2024106449A1
WO2024106449A1 PCT/JP2023/041023 JP2023041023W WO2024106449A1 WO 2024106449 A1 WO2024106449 A1 WO 2024106449A1 JP 2023041023 W JP2023041023 W JP 2023041023W WO 2024106449 A1 WO2024106449 A1 WO 2024106449A1
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Prior art keywords
fenugreek
protodioscin
koji
ppm
seeds
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PCT/JP2023/041023
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French (fr)
Japanese (ja)
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圭子 武谷
守紘 青柳
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ハウス食品グループ本社株式会社
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Priority claimed from JP2023192499A external-priority patent/JP2024074259A/en
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Publication of WO2024106449A1 publication Critical patent/WO2024106449A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi

Definitions

  • the present invention relates to processed fenugreek seeds and their manufacturing method.
  • Fenugreek is an annual plant of the legume family. Fenugreek seeds have long been known as a spice, including being included in curry powder.
  • Non-Patent Document 1 reports that the main bitter component of fenugreek seeds is protodioscin, a furostanol-type saponin.
  • Patent Document 1 describes a method for producing fenugreek seeds with reduced bitterness, which is characterized by adding water to fenugreek seeds to elute components of the fenugreek seeds, adding ⁇ -glucosidase, and then allowing the components and the ⁇ -glucosidase to be absorbed by the fenugreek seeds after the addition of ⁇ -glucosidase.
  • saponin compounds such as protodioscin that have been eluted from fenugreek seeds into water are decomposed by ⁇ -glucosidase.
  • Patent Document 1 also describes the use of an enzyme preparation containing ⁇ -glucosidase as the ⁇ -glucosidase.
  • Non-Patent Document 2 discloses Aspergillus-fermented fenugreek (AFF) in which fenugreek seeds absorbed with Czapek-Dox Broth medium (CDB) are inoculated with spores of Aspergillus awamori and cultured at 25 ⁇ 2°C for 7 days.
  • AFF Aspergillus-fermented fenugreek
  • CDB Czapek-Dox Broth medium
  • Non-Patent Document 2 describes that on the 5th day of fermentation, Aspergillus-fermented fenugreek has increased total phenol content, condensed tannin content, and antioxidant activity compared to unfermented fenugreek.
  • Non-Patent Document 3 describes that the cytotoxicity of the product obtained by modifying fenugreek-derived saponin with microorganisms used in food manufacturing was confirmed. Specifically, Non-Patent Document 3 describes that Aspergillus niger KCTC6906, Aspergillus usamii KCTC6956, Bifidobacterium sp. Int57, Bifidobacterium infantis, Bifidobacterium breve YC2, Bifidobacterium sedocatenulatum SJ32, Bifidobacterium sp.
  • Non-Patent Document 3 describes that the reaction products of Aspergillus usamii and crude enzymes of Bifidobacterium genus bacteria with fenugreek seed extract have higher cytotoxicity than untreated fenugreek extract, whereas the reaction products of Aspergillus niger crude enzymes with fenugreek seed extract have lower cytotoxicity.
  • Non-Patent Document 3 further describes that the reaction products of Aspergillus usamii and crude enzymes of bacteria other than Bifidobacterium sedocatenulatum SJ32 among the above with fenugreek seed extract contain a lot of dioscin, whereas the reaction products of Aspergillus niger crude enzymes with fenugreek seed extract contain less dioscin and have increased other unspecified substances.
  • Non-Patent Document 4 describes that Aspergillus oryzae was cultured in a medium containing malt extract and Chinese yam water extract (enzyme inducer), and the crude enzyme obtained from the culture was reacted with total steroid saponin isolated from dried Chinese yam roots at 50°C for 24 hours.
  • Non-Patent Document 4 describes that the total steroid saponin of Chinese yam before treatment with the crude enzyme contains protodioscin, protogracillin, and dioscin, whereas the reaction product of the crude enzyme and Chinese yam total steroid saponin contains progenin III (prosapogenin A).
  • Non-Patent Document 4 also describes that the crude enzyme of Aspergillus oryzae cultured in a medium not containing Chinese yam water extract has almost no activity to generate progenin III from total steroid saponin.
  • One or more embodiments of the present invention aim to provide a processed fenugreek seed product with a reduced content of protodioscin, a bitter component of fenugreek seeds, and a method for producing the same.
  • a method for producing a processed fenugreek seed product comprising: (2) The method according to (1), wherein the carbohydrate content of the seeds of the plant and/or the processed products thereof is 65% by weight or less.
  • the weight content of protodioscin is PD
  • the weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is defined as DG
  • the fenugreek seed processed product described in (15), wherein the compound having a diosgenin skeleton other than protodioscin includes one or more selected from dioscin, prosapogenin and diosgenin.
  • a processed fenugreek seed product having a reduced protodioscin content and a method for producing the same are provided.
  • FIG. 1 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in fenugreek seed processed products made with okara koji containing added salt, fenugreek seed processed products made with okara koji without added salt, and fenugreek seeds without added okara koji (comparative example) after treatment at 40°C for 4, 8, 19 and 61 days in Test 1.
  • FIG. 2 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in the reaction solution obtained by reacting okara koji prepared using three types of commercially available Aspergillus seed koji with fenugreek seed extract at 55° C.
  • Figure 3 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in the reaction solutions obtained by reacting rice bran koji, soybean koji, fenugreek koji, wheat bran koji and sesame koji prepared using commercially available Aspergillus seed koji with fenugreek seed extract at 55°C for three days in Test 3.
  • Figure 4 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in fenugreek seed processed products made from fenugreek koji with added salt, and in fenugreek seed processed products made from fenugreek koji without added salt, after treatment at 40°C for 3 days, 12 days, 20 days, and 31 days in Test 4.
  • Fenugreek seeds used as a material in one or more embodiments of the present invention will be described.
  • Fenugreek seeds are seeds of fenugreek (scientific name Trigonella foenum-graecum).
  • the form of fenugreek seeds is not particularly limited, and may be in any form such as seeds that retain the form of the seeds themselves (whole seeds), crushed seeds, powdered seeds, etc.
  • the fenugreek seeds may be seeds that contain at least an embryo part, and may or may not further contain a seed coat and a galactomannan layer, but preferably contain an embryo part and a galactomannan layer, and more preferably contain an embryo part, a galactomannan layer, and a seed coat.
  • the fenugreek seeds may be fenugreek seeds whose moisture content has been adjusted by steaming and/or water absorption.
  • the plant seeds and/or processed products thereof used as a raw material for the fermentation product preferably have one or more characteristics among the following (A) to (E), more preferably have two or more characteristics among (A) to (E), more preferably have three or more characteristics among (A) to (E), more preferably have four or more characteristics among (A) to (E), more preferably have one or more characteristics among (A) to (E) including at least (A), more preferably have two or more characteristics among (A) to (E) including at least (A), more preferably have three or more characteristics among (A) to (E) including at least (A), more preferably have four or more characteristics among (A) to (E) including at least (A), and particularly preferably have all of the characteristics among (A) to (E).
  • the carbohydrate content is 65% by weight or less.
  • the protein content is 8% by weight or more.
  • the lipid content is 3% by weight or more.
  • the carbohydrate content is 75% by weight or less.
  • the dietary fiber content is 10% by weight or more.
  • the fermentation products of these raw materials by microorganisms have a particularly high activity in breaking down protodioscin derived from fenugreek seeds.
  • the carbohydrate content is more preferably 60% by weight or less, more preferably 55% by weight or less, and particularly preferably 40% by weight or less.
  • the carbohydrate content in the (A) above can be in the range of, for example, 0% by weight to 65% by weight, 2% by weight to 60% by weight, preferably 3% by weight to 55% by weight, and more preferably 5% by weight to 40% by weight.
  • the protein content is more preferably 10% by weight or more, particularly preferably 12% by weight or more.
  • the protein content in the above (B) can be in the range of, for example, 8% by weight to 50% by weight, preferably 10% by weight to 40% by weight, more preferably 12% by weight to 30% by weight.
  • the lipid content is more preferably 5% by weight or more.
  • the lipid content in the above (C) can be in the range of, for example, 3% by weight to 60% by weight, preferably 5% by weight to 30% by weight.
  • the carbohydrate content is more preferably 70% by weight or less, particularly preferably 65% by weight or less.
  • the carbohydrate content in the (D) above can be in the range of, for example, 10% by weight to 75% by weight, preferably 15% by weight to 70% by weight, more preferably 15% by weight to 65% by weight.
  • the dietary fiber content is more preferably 15% by weight or more, and particularly preferably 18% by weight or more.
  • the range of the dietary fiber content in the above (E) can be, for example, 10% by weight to 70% by weight, preferably 15% by weight to 50% by weight, and particularly preferably 18% by weight to 50% by weight.
  • each component here is the percentage relative to the weight of the plant seeds and/or processed products thereof before mixing with other materials, such as water and microorganisms, for producing a fermentation product.
  • examples of the plant seeds and/or processed products thereof having such characteristics include one or more seeds and/or processed products thereof selected from legumes, grasses, and sesame plants.
  • the plant is preferably one or more selected from the group consisting of legumes, rice plants, and sesame plants.
  • a fermentation product of seeds or processed products thereof by microorganisms of one or more plants selected from the group consisting of legumes, rice plants, and sesame plants has a particularly high activity of decomposing protodioscin derived from fenugreek seeds.
  • a plant of the subfamily Fabaceae is preferable, more preferably one or more selected from fenugreek, soybean, and peanut, and particularly preferably one or more selected from fenugreek and soybean.
  • grass family plant one or more selected from rice and wheat are particularly preferred.
  • Sesame is a particularly preferred plant of the Pedaliaceae family.
  • the plant seeds can be in any form, such as seeds that retain the shape of the seed itself (whole seeds), crushed seeds, powdered seeds, etc. Furthermore, the plant seeds can be seeds whose moisture content has been adjusted by steaming and/or absorbing water.
  • examples of processed plant seeds include processed soybean seeds such as okara, soy milk, and tofu, with okara being particularly preferred.
  • Another example of a processed plant seed is preferably rice bran when the plant is rice.
  • Rice bran generally includes the epidermis, pericarp, and germ of rice seeds.
  • Yet another example of a processed plant seed is preferably wheat bran when the plant is wheat.
  • Wheat bran generally includes the epidermis of wheat seeds.
  • the plant seeds and/or processed products thereof are preferably one or more selected from legume seeds, processed legume seeds, rice bran, wheat bran and sesame seeds, more preferably fenugreek seeds, soybean seeds, okara, rice bran, wheat bran or sesame seeds, and particularly preferably fenugreek seeds or okara. Fermentation products using these plant seeds and/or processed products thereof as substrates have particularly high activity in decomposing protodioscin.
  • the following table shows examples of the water, protein, lipid, carbohydrate, sugar and dietary fiber contents in these raw materials.
  • Carbohydrates consist of sugars and dietary fiber.
  • each ingredient here is based on information available from the Ministry of Education, Culture, Sports, Science and Technology's Food Composition Database (https://fooddb.mext.go.jp/) for rice bran, soybean seeds, dried okara, and sesame seeds, and from the United States Department of Agriculture's FoodData Central database (https://fdc.nal.usda.gov/fdc-app.html#/food-details/171324/nutrients and https://fdc.nal.usda.gov/fdc-app.html#/food-details/169722/nutrients) for fenugreek seeds and wheat bran.
  • the microorganism may be any microorganism capable of propagating using plant seeds and/or processed products thereof as a carbon source and nitrogen source (i.e., fermenting plant seeds and/or processed products thereof).
  • Preferred examples of the microorganism include microorganisms capable of producing an enzyme having activity of decomposing protodioscin, and in particular, microorganisms belonging to the genus Aspergillus, Bifidobacterium, or Leuconostoc are preferred, with microorganisms belonging to the genus Aspergillus (i.e., koji mold) being particularly preferred.
  • yellow koji such as Aspergillus oryzae and Aspergillus sojae
  • white koji such as Aspergillus luchensis and Aspergillus mut. kawachii
  • black koji such as Aspergillus var. awamori and Aspergillus niger
  • yellow koji Aspergillus oryzae is particularly preferred.
  • microorganism belonging to the genus Bifidobacterium microorganisms belonging to Bifidobacterium infantis, Bifidobacterium breve, or Bifidobacterium pseudocatenulatum are preferred, and for example, the microorganisms belonging to the genus Bifidobacterium described in Non-Patent Document 3 are preferred.
  • microorganism belonging to the genus Leuconostoc microorganisms belonging to Leuconostoc paramesenteroides are preferred, and for example, the microorganisms belonging to the genus Leuconostoc described in Non-Patent Document 3 are preferred.
  • the fermentation product containing the koji mold may be referred to as "koji.”
  • “koji” may be called “fenugreek koji,” when it is soybean, “okara koji” when it is bean lees, “rice bran koji” when it is rice bran, “wheat bran koji” when it is wheat bran, or “sesame koji” when it is sesame seed.
  • the fermentation product contains an enzyme having activity to decompose protodioscin, and can be used to manufacture fenugreek seed processed products with reduced bitterness.
  • the fermentation product contains the microorganism. Therefore, in an embodiment in which a mixture of the fermentation product and fenugreek seeds is kept under temperature conditions for the survival of the microorganism, the microorganism can penetrate the seed coat and galactomannan layer of the fenugreek seeds and enter the embryo part, and therefore can effectively decompose the protodioscin in the fenugreek seeds.
  • Patent Document 1 when using a ⁇ -glucosidase-containing enzyme preparation, in order to act on the protodioscin in the fenugreek seeds, it was necessary to soak the fenugreek seeds in an excess amount of water and dissolve the protodioscin into the water.
  • the fermentation product is, for example, A step of mixing the seeds of the plant and/or a processed product thereof with the microorganism to obtain a fermentation raw material (hereinafter sometimes referred to as "first step”); A step of culturing the microorganism in the fermentation raw material (hereinafter sometimes referred to as the "second step”);
  • the composition can be prepared by a method comprising the steps of:
  • the plant seeds and/or processed products thereof or the fermentation raw material does not contain a culture medium.
  • the fermentation raw material does not contain any culture medium components that can be used by the microorganism as a carbon source and nitrogen source in the culture in the second step.
  • the fermentation product obtained in this embodiment is highly safe as a food because it does not contain any artificially added culture medium components.
  • the microorganisms mixed in the first step may be microorganisms that have been propagated in advance on a culture substrate such as steamed rice, or microorganisms that have been pre-cultured (seed cultured).
  • a culture substrate such as steamed rice
  • the fermentation raw material can be obtained by mixing "seed koji," which is in the form of koji mold that has been propagated in advance on a culture substrate such as steamed rice, or in the form of koji mold spores, with the plant seeds and/or processed products thereof.
  • seed koji which is in the form of koji mold that has been propagated in advance on a culture substrate such as steamed rice, or in the form of koji mold spores
  • Commercially available products can be purchased and used as seed koji.
  • the fermentation raw material obtained in the first step may further contain water, inorganic salts, etc., in addition to the plant seeds and/or processed products thereof and the microorganisms.
  • inorganic salt is table salt.
  • the second step is a step of culturing the microorganism in the fermentation raw material.
  • the second step can be carried out under conditions that allow the microorganism to be cultured, and includes, for example, maintaining the fermentation raw material under temperature conditions, preferably between 10°C and 50°C, more preferably between 20°C and 45°C, more preferably between 30°C and 45°C, more preferably between 30°C and 40°C.
  • the second step more preferably includes maintaining the fermentation raw material under the temperature conditions, preferably for a period of between 30 hours and 120 hours, more preferably between 40 hours and 100 hours.
  • the fermentation product obtained by culturing the microorganism in the fermentation raw material in the second step can be subjected to the method for producing a fenugreek seed processed product while still containing the microorganism without removing it.
  • the fermentation product can contain dead or viable cells of the microorganism.
  • One or more embodiments of the present invention include A process for obtaining a mixture by mixing fenugreek seeds with a fermentation product containing a microorganism obtained by fermenting a plant seed and/or a processed product thereof with a microorganism (hereinafter, sometimes referred to as a "mixing process”); and A process for maintaining the mixture under temperature conditions at which protodioscin in the mixture is decomposed (hereinafter, sometimes referred to as a "reaction process”).
  • the present invention relates to a method for producing a processed fenugreek seed product comprising the steps of:
  • the method according to this embodiment makes it possible to efficiently produce a processed fenugreek seed product with a reduced protodioscin content and reduced bitterness compared to untreated fenugreek seeds, without the need for the use of a ⁇ -glucosidase-containing enzyme preparation.
  • the processed fenugreek seed product produced by the method according to this embodiment can be used as a food product such as a seasoning.
  • the protodioscin in the mixture contains at least protodioscin derived from the fenugreek seeds, and when the fermentation product contains protodioscin, it further contains protodioscin derived from the fermentation product.
  • the mixing ratio of the fenugreek seeds and the fermentation product is not particularly limited, but the fermentation product (the seeds of the plant used as the raw material for the fermentation product and/or the processed product thereof, calculated as the dry weight without adding water) can be mixed with 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water) in a ratio of, for example, 20 parts by weight to 500 parts by weight, preferably 50 parts by weight to 200 parts by weight.
  • salt can be mixed in a ratio of, for example, 5 parts by weight to 100 parts by weight, preferably 20 parts by weight to 60 parts by weight, per 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water).
  • Water can be mixed so that the water in the mixture (the total amount of water added, including the water from the fenugreek seeds and the fermentation product) is, for example, 200 parts by weight or more, preferably 300 parts by weight or more, per 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water).
  • the upper limit of the amount of water in the mixture is not particularly limited, and it may be adjusted to a range in which protodioscin can be decomposed by the reaction step. For example, 10,000 parts by weight or less of water can be mixed with 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water).
  • the fermentation product used in the mixing step can be purchased separately and used, but more preferably, the method according to this embodiment further includes a step of preparing a fermentation product including the first step and the second step, and the fermentation product prepared by this step can be used in the mixing step.
  • the temperature conditions in the reaction step may be any temperature conditions that allow protodioscin to be decomposed in the mixture by the enzyme activity of the fermentation product, and are preferably 10°C to 60°C, more preferably 15°C to 60°C, more preferably 20°C to 60°C, more preferably 30°C to 60°C, and more preferably 35°C to 57°C.
  • the survival of microorganisms from the fermentation product in the mixture is not essential, but if microorganisms are alive, as described above, the effect of decomposing protodioscin in fenugreek seeds is particularly high, and therefore it is preferable.
  • the temperature conditions in the reaction step are preferably 10°C to 50°C, more preferably 15°C to 50°C, more preferably 20°C to 50°C, more preferably 30°C to 50°C, more preferably 30°C to 45°C, and most preferably 35°C to 45°C.
  • the temperature conditions of the reaction step are preferably 50°C or higher and 60°C or lower, more preferably 50°C or higher and 57°C or lower.
  • the time for which the mixture is held under the temperature conditions can be appropriately adjusted depending on the desired degree of decomposition of protodioscin in the mixture.
  • the reaction process can be carried out so that the protodioscin content in the mixture is preferably 50% by weight or less, more preferably 20% by weight or less, more preferably 10% by weight or less, more preferably 5% by weight or less, and more preferably 1% by weight or less, when the protodioscin content immediately after the preparation of the mixture is 100% by weight.
  • the target value of the protodioscin content in the mixture after the reaction process is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, relative to the total weight of the mixture (based on dry weight), and may be below the detection limit (i.e., 0 ppm).
  • the time for which the mixture in the reaction step is held under the temperature conditions is, for example, 2 days or more, preferably 3 days or more, more preferably 10 days or more, and more preferably 15 days or more.
  • the time for which the mixture in the reaction step is held under the temperature conditions is preferably 2 years or less, and more preferably 1 year or less. That is, the time for which the mixture in the reaction step is held under the temperature conditions can be, for example, 2 days to 2 years, preferably 3 days to 2 years, more preferably 10 days to 1 year, and more preferably 15 days to 1 year.
  • the mixture that has undergone the reaction process can be a paste-like substance similar to miso, and can be used as a processed fenugreek seed product for food applications such as seasonings.
  • reaction step is carried out under conditions in which the microorganisms can survive, it is preferable to heat-treat the mixture after completion of the reaction step to kill the microorganisms.
  • the mixture that has undergone the reaction process can be further processed, such as by drying, concentrating, diluting, granulating, etc., and used as a processed fenugreek seed product.
  • One or more further embodiments of the present invention include The weight-based content of protodioscin is designated as PD.
  • PD weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin
  • DG weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin
  • PD/(PD+DG) is 0.78 or less; This relates to processed fenugreek seeds.
  • Unprocessed fenugreek seeds contain a lot of protodioscin, the substance that causes bitterness, and contain almost no compounds with a diosgenin skeleton other than protodioscin, so (PD/(PD+DG) is close to 1.0.
  • the fenugreek seed processed product of this embodiment which has a PD/(PD+DG) of 0.78 or less, has a smaller weight-based content of protodioscin compared to unprocessed fenugreek seeds, and a larger weight-based content of compounds with a diosgenin skeleton, a phytosteroid other than protodioscin that is expected to have physiological functions, making it particularly suitable for use in food products such as seasonings.
  • Compounds having a diosgenin skeleton other than protodioscin include one or more selected from diosgenin and compounds that produce diosgenin upon hydrolysis, typically one or more selected from diosgenin and diosgenin glycosides, specifically dioscin, prosapogenin, diosgenin, etc.
  • Compounds having a diosgenin skeleton other than protodioscin are preferably compounds that have a longer retention time than protodioscin in a chromatogram of reversed-phase liquid chromatography using an ODS column, and correspond to a peak up to diosgenin, and in which a fragment specific to the diosgenin skeleton is detected in mass spectrometry.
  • the fenugreek seed processed product according to this embodiment preferably has a physiological effect based on a compound having a diosgenin skeleton, such as dioscin or diosgenin, and in particular, one or more physiological effects selected from an antidiabetic effect, an effect of improving cognitive function, and an effect of improving menopausal disorders.
  • a diosgenin skeleton such as dioscin or diosgenin
  • dioscin include antiuricemia, antifungal and antiviral effects, antitumor, liver protection (fibrosis, acute liver damage, NAFLD, cholestasis, hepatic ischemia-reperfusion injury), lung protection, kidney protection, cardiopulmonary function protection, cerebral protection, anti-atherosclerosis, anti-inflammation, anti-arthritis, anti-obesity and anti-diabetes, anti-oxidative stress, anti-osteoporosis, inhibition of melanin production, growth hormone release, anti-breast cancer, anti-gastric cancer, anti-liver cancer, anti-myeloid leukemia, anti-lung cancer, anti-kidney cancer, anti-melanoma tumor, anti-prostate cancer, anti-chronic liver damage, liver regeneration, cardiovascular and cerebrovascular protection, and protection from gastric ischemia-reperfusion injury, as well as physiological effects to prevent or treat diseases such as non-alcoholic fatty liver disease and type II diabetes.
  • liver protection fibrosis,
  • the physiological effects of diosgenin are known to be anticancer, antifungal, antiviral, antithrombotic, antioxidant, neuroprotective, immunomodulatory, and other physiological effects, as well as physiological effects to prevent or treat diseases such as cardiovascular disease, myocardial disorder, vascular disorder, hyperlipidemia, type II diabetes, inflammation, menopausal disorders, skin aging, and osteoporosis, as well as physiological effects on cancer cell proliferation and apoptosis, tumor invasion, metastasis, and angiogenesis, and cortical neurons.
  • the fenugreek seed processed product according to this embodiment can preferably exert these physiological effects of compounds having a diosgenin skeleton, such as dioscin and diosgenin.
  • the PD/(PD+DG) of the processed fenugreek seed product according to this embodiment is more preferably 0.70 or less, more preferably 0.50 or less, more preferably 0.40 or less, more preferably 0.30 or less, more preferably 0.20 or less, more preferably 0.10 or less, more preferably 0.05 or less, more preferably 0.01 or less.
  • the processed fenugreek seed product according to this embodiment also includes cases where the PD is below the detection limit and the PD/(PD+DG) is 0.
  • the PD/(PD+DG) can be, for example, 0 to 0.78, preferably 0 to 0.70, more preferably 0 to 0.50, more preferably 0 to 0.40, more preferably 0 to 0.30, more preferably 0 to 0.20, more preferably 0 to 0.10, more preferably 0 to 0.05, more preferably 0 to 0.01.
  • PD and DG in processed fenugreek seed products can be measured using a liquid chromatograph mass spectrometer (LC-MS).
  • LC-MS liquid chromatograph mass spectrometer
  • a calibration curve for determining PD can be prepared using samples containing a known concentration of a protodioscin standard.
  • DG is the weight-based content of compounds other than protodioscin that produce fragments specific to the diosgenin skeleton in LC-MS measurement, converted into diosgenin.
  • a calibration curve for determining DG can be prepared using samples containing a known concentration of a diosgenin standard.
  • the value of the weight-based protodioscin content PD is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), relative to the total weight (dry weight) of the processed fenugreek seed product.
  • PD can be, for example, 0 ppm to 5000 ppm, preferably 0 ppm to 3000 ppm, more preferably 0 ppm to 1500 ppm, more preferably 0 ppm to 300 ppm, more preferably 0 ppm to 100 ppm, more preferably 0 ppm to 10 ppm.
  • the value of the weight-based content DG of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is, for example, 500 ppm or more, preferably 600 ppm or more, more preferably 900 ppm or more, more preferably 1200 ppm or more, more preferably 1500 ppm or more, and the upper limit is not particularly limited, but is, for example, 20000 ppm or less, relative to the total weight (dry weight basis) of the processed fenugreek seed product.
  • the DG can be, for example, 500 to 20000 ppm, preferably 600 ppm to 20000 ppm, more preferably 900 ppm to 20000 ppm, more preferably 1200 ppm to 20000 ppm, more preferably 1500 ppm to 20000 ppm.
  • the fenugreek seed processed product according to this embodiment preferably contains microbial cells, more preferably contains dead or viable microbial cells capable of producing an enzyme having protodioscin-degrading activity, and particularly preferably contains dead microbial cells capable of producing an enzyme having protodioscin-degrading activity.
  • the fenugreek seed processed product according to this embodiment containing microbial cells can be produced by the method according to the embodiment described in ⁇ 3. Production method of fenugreek seed processed product>. Specific examples of the microorganism include the microorganisms described in ⁇ 2. Fermentation products>.
  • the processed fenugreek seed product according to this embodiment can typically be a paste-like product like miso, and can be used as a food product such as a seasoning.
  • raw fenugreek seeds contain a large amount of protodioscin, which is a substance that causes bitterness.
  • a preferred embodiment of the processed fenugreek seeds according to this embodiment is characterized in that at least a part, preferably all, of the protodioscin in raw fenugreek is converted to one or more selected from dioscin, prosapogenin and diosgenin.
  • prosapogenin is a general term for prosapogenin A and prosapogenin B, and refers to one or both of prosapogenin A and prosapogenin B.
  • a further preferred embodiment of the processed fenugreek seeds according to this embodiment has the following characteristics (i), (ii) or (iii): (i) the dioscin content is greater than the protodioscin content by weight.
  • the dioscin content is greater than the prosapogenin content and the diosgenin content by weight; (ii) the prosapogenin content is greater than the protodioscin content, and preferably, the prosapogenin content is greater than the dioscin content and the diosgenin content, by weight; or (iii) the diosgenin content is greater than the protodioscin content, and preferably, the diosgenin content is greater than the dioscin content and the prosapogenin content, by weight.
  • Method for producing a fenugreek seed processed product> has the above characteristics (i), (ii) or (iii), and have completed the fenugreek seed processed product according to the preferred embodiment.
  • the weight-based content of prosapogenin is the total amount of prosapogenin A, prosapogenin B, and prosapogenin A and prosapogenin B, and is the equivalent amount of dioscin calculated using the dioscin calibration curve.
  • the content of protodioscin by weight is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm) relative to the total weight (dry weight) of the processed fenugreek seed product.
  • the content of protodioscin relative to the total weight (dry weight) of the processed fenugreek seed product can be, for example, 0 to 5000 ppm, preferably 0 to 3000 ppm, more preferably 0 to 1500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm.
  • the weight-based dioscin content, relative to the total weight (dry weight) of the fenugreek seed processed product can be, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 15000 ppm or less, for example, 300 ppm to 15000 ppm, preferably 1000 ppm to 15000 ppm, preferably 1500 ppm to 15000 ppm, more preferably 3000 ppm to 15000 ppm.
  • the weight-based content of prosapogenin and diosgenin is preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, more preferably 0 to 10 ppm, based on the total weight (dry weight) of the fenugreek seed processed product.
  • the content of prosapogenin by weight, relative to the total weight (dry weight) of the fenugreek seed processed product is, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 30,000 ppm or less, more preferably 15,000 ppm or less, for example, 300 ppm to 30,000 ppm, preferably 300 ppm to 15,000 ppm, preferably 1000 ppm to 15,000 ppm, preferably 1500 ppm to 15,000 ppm, more preferably 3000 ppm to 15,000 ppm.
  • the weight-based content of dioscin and diosgenin is preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm, based on the total weight (dry weight) of the processed fenugreek seed product.
  • the diosgenin content by weight, relative to the total weight (dry weight) of the fenugreek seed processed product is, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 30000 ppm or less, more preferably 15000 ppm or less, for example, 300 ppm to 30000 ppm, preferably 300 ppm to 15000 ppm, preferably 1000 ppm to 15000 ppm, preferably 1500 ppm to 15000 ppm, more preferably 3000 ppm to 15000 ppm.
  • the weight-based contents of dioscin and prosapogenin are preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm, based on the total weight (dry weight) of the processed fenugreek seed product.
  • the mixture was allowed to cool after autoclave sterilization, and then inoculated with 80 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain the okara koji raw material.
  • seed koji product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.
  • the container containing the okara koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the okara koji raw material, yielding a fermentation product of the koji mold in the okara (hereinafter referred to as "okara koji").
  • the okara koji raw material was stirred once during the fermentation.
  • the water-absorbed fenugreek seeds were sterilized in an autoclave and then cooled. Then, 200 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae was seeded and stirred to mix uniformly to obtain a fenugreek koji raw material.
  • seed koji product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.
  • the container containing the fenugreek koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the fenugreek koji raw material, yielding a fermentation product of fenugreek seeds by the koji mold (hereinafter referred to as "fenugreek koji").
  • the fenugreek koji raw material was stirred once during the fermentation.
  • okara koji Approximately 16 g of okara koji and 4 g of salt were mixed in advance. The resulting mixture was kneaded with approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been allowed to cool after autoclave sterilization and 20 g of water, and placed in a container. The container was left to ferment for up to 61 days in a 40°C incubator, yielding a fenugreek seed processed product made from salt-added okara koji. Samples were taken at each time point after 4 days, 8 days, 19 days, and 61 days.
  • the fenugreek seed processed product using salt-free okara koji was prepared by kneading approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been sterilized in an autoclave and then cooled with approximately 16 g of okara koji and 20 g of water, placing the mixture in a container, and leaving the container in an incubator at 40°C for up to 61 days to ferment. Samples were taken at each time point after 4 days, 8 days, 19 days, and 61 days.
  • the comparative sample (fenugreek seeds without the addition of okara koji) was prepared by mixing 10 g of dried okara (Yamami Co., Ltd.) with 6 g of water, sterilizing the resulting mixture in an autoclave, and then mixing this mixture with approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been allowed to cool after autoclave sterilization, and 20 g of water, in a kneaded manner.
  • samples of fenugreek seed processed products made with salt-added okara koji and fenugreek seed processed products made with salt-free okara koji were analyzed for compounds with protodioscin and diosgenin skeletons using the following procedure.
  • MS conditions spray voltage 3.5 kV, capillary temperature 270° C., MS scan range m/z 67-1005, ionization mode ESI positive, collision energy (CE) 50 eV, PRM measurement (precursor ions are shown in the table below).
  • a calibration curve was prepared using protodioscin and diosgenin samples. Based on the calibration curve, the concentrations (ppm) of compounds with protodioscin and diosgenin skeletons in the samples were calculated. Therefore, the concentrations of compounds with diosgenin skeletons are converted concentrations of diosgenin.
  • Fenugreek seeds contain protodioscin, which causes bitterness, but do not contain compounds with a diosgenin skeleton (Comparative Example). It was confirmed that the fermentation of fenugreek seeds with okara koji reduces the concentration of protodioscin and suppresses bitterness. In the fermentation of fenugreek seeds with okara koji, the concentration of compounds with a diosgenin skeleton increased with the reduction in the concentration of protodioscin. Compounds with a diosgenin skeleton are presumed to be dioscin, prosapogenin, diosgenin, etc., which are generated by the decomposition of protodioscin. It was confirmed that okara koji without added salt had a higher activity in decomposing protodioscin from fenugreek seeds than okara koji with added salt.
  • Dioscin, prosapogenin and diosgenin are all compounds with a diosgenin skeleton that are produced by the decomposition of protodioscin. Measurements were performed using the following procedure. Measurement samples were prepared from the samples using the procedure described in (1) above, and LCMS analysis was performed under the conditions described in (3). The precursor ions and product ions of protodioscin, dioscin, prosapogenin and diosgenin are shown in the table below.
  • the samples for the calibration curve were prepared by serially diluting Protodioscin (ChromaDex) standard, Dioscin (ChromaDex) standard, and Diosgenin (Fujifilm Wako Pure Chemical) standard with 80% methanol.
  • the concentrations (ppm) of protodioscin, dioscin and diosgenin in the samples were calculated based on the calibration curves of protodioscin, dioscin and diosgenin prepared using the calibration curve samples.
  • the concentration (ppm) of prosapogenin was calculated from the peak area value of the product ion of prosapogenin using the calibration curve of dioscin.
  • the obtained water-absorbed coarsely ground fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
  • concentrations of compounds with protodioscin and diosgenin skeletons were measured in fenugreek seed processed products using salt-added fenugreek koji after treatment for 3 days at 40°C using the procedure described above, and in fenugreek seed processed products using salt-added okara koji after treatment for 3 days at 40°C using the procedure described in 1.3 above.
  • the measurement method for each component is as described in 1.3 above.
  • concentrations of compounds with protodioscin and diosgenin skeletons in fenugreek koji were also measured in the same manner.
  • fenugreek koji contains protodioscin, which causes its bitter taste.
  • fenugreek seeds also contain protodioscin.
  • dioscin, prosapogenin and diosgenin in the fenugreek seed processed product using fenugreek koji after three days of treatment at 40°C were measured using the following procedure.
  • the results were 33 ppm dioscin, 707 ppm prosapogenin and 58 ppm diosgenin.
  • the protodioscin content was 462 ppm.
  • the difference in the content of each component in this test compared to the content of each component after three days of treatment in Test 4 may be due to the difference in the cooling temperature mentioned above.
  • dioscin, prosapogenin and diosgenin in the fenugreek seed processed product using salt-added okara koji after three days of treatment at 40°C were measured using the following procedure. The results were 591 ppm dioscin, 45 ppm prosapogenin and 33 ppm diosgenin. As mentioned above, the protodioscin content was 534 ppm.
  • the method for measuring dioscin, prosapogenin and diosgenin is as described in 1.3. (6) above.
  • the mixture which had been allowed to cool after autoclave sterilization, was inoculated with 80 mg of commercially available seed koji containing the koji mold Aspergillus oryzae and stirred to mix evenly to obtain the raw material for okara koji.
  • the commercially available seed koji containing the koji mold Aspergillus oryzae was either "Shirokoji No. 1" (product name, Akita Konno Shoten Co., Ltd.), "Yuki Komachi” (product name, Akita Konno Shoten Co., Ltd.), or "Aglycon” (product name, Akita Konno Shoten Co., Ltd.).
  • the container containing the okara koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the okara koji raw material, yielding a fermentation product of the koji mold in the okara (hereinafter referred to as "okara koji").
  • the okara koji raw material was stirred once during the fermentation.
  • a reaction solution was prepared by mixing 18.75 ⁇ L of a commercially available enzyme preparation containing ⁇ -glucosidase (product name "Cellulase SS", Nagase ChemteX Corporation) with 4.5 mL of the fenugreek extract prepared in 2.2 above, and the mixture was left to stand in an incubator at 55°C for 18 hours or 3 days.
  • a commercially available enzyme preparation containing ⁇ -glucosidase product name "Cellulase SS", Nagase ChemteX Corporation
  • Negative control test 4.5 mL of the fenugreek extract prepared in 2.2 above was left to stand in an incubator at 55°C for 18 hours or 3 days.
  • a 1 g sample of the reaction solution from each test group was placed in a 15 mL test tube, and 9 mL of methanol was added.
  • the test tube was agitated for 30 minutes on a shaker, and then diluted 1000-fold with 80% methanol.
  • the diluted solution was passed through a 0.2 ⁇ m filter and placed in a vial to prepare the measurement sample for LC-MS measurement.
  • the concentrations of protodioscin and compounds having a diosgenin skeleton in the measurement sample were measured according to the procedures described in 1.3. (2) to (4) above.
  • the water-absorbed fenugreek seeds were cooled after autoclave sterilization, and then 200 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae was seeded and stirred to mix uniformly to obtain a fenugreek koji raw material.
  • commercially available seed koji product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.
  • the container containing the fenugreek koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the fenugreek koji raw material, yielding a fermentation product of fenugreek seeds by the koji mold (hereinafter referred to as "fenugreek koji").
  • the fenugreek koji raw material was stirred once during the fermentation.
  • the soybeans were allowed to cool after autoclave sterilization, and then inoculated with 300 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and then stirred to mix uniformly to obtain the soybean koji raw material.
  • the container containing the soybean koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the soybean koji raw material, yielding a fermentation product of soybeans caused by the koji mold (hereafter referred to as "soybean koji").
  • the soybean koji raw material was stirred once during the fermentation process.
  • the mixture was allowed to cool after autoclave sterilization, and then seeded with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain the rice bran koji raw material.
  • the container containing the rice bran koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the rice bran koji raw material, resulting in a fermentation product of the rice bran koji mold (hereinafter referred to as "rice bran koji").
  • the rice bran koji raw material was stirred once during fermentation.
  • the mixture was allowed to cool after autoclave sterilization, and then seeded with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain wheat bran koji raw material.
  • the container containing the wheat bran koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the wheat bran raw material, resulting in a fermentation product of the wheat bran koji mold (hereinafter referred to as "wheat bran koji").
  • the wheat bran koji raw material was stirred once during fermentation.
  • sesame seeds which had been left to cool after autoclave sterilization, were inoculated with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and then stirred to mix uniformly to obtain the sesame koji raw material.
  • the container containing the sesame koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the sesame koji raw material, yielding a fermentation product of the sesame koji mold (hereinafter referred to as "sesame koji").
  • the sesame koji raw material was stirred once during fermentation.
  • a reaction solution was prepared by mixing 18.75 ⁇ L of a commercially available enzyme preparation containing ⁇ -glucosidase (product name "Cellulase SS", Nagase ChemteX Corporation) with 4.5 mL of the fenugreek extract prepared in 3.6 above, and allowed to stand in an incubator at 55°C for three days.
  • a commercially available enzyme preparation containing ⁇ -glucosidase product name "Cellulase SS", Nagase ChemteX Corporation
  • Negative control test 4.5 mL of the fenugreek extract prepared in 3.6 above was left to stand in an incubator at 55°C for 3 days.
  • the concentrations of protodioscin and compounds with diosgenin skeletons in the reaction solution of each test group were measured using the procedure described in 2.3 above.
  • dioscin was detected as a decomposition product of protodioscin, while prosapogenin and diosgenin were below the detection limit.
  • prosapogenin was detected as a decomposition product of protodioscin, while dioscin and diosgenin were below the detection limit.
  • Method for preparing fenugreek koji> A fermentation product of fenugreek seeds with Aspergillus oryzae (fenugreek koji) was prepared according to the procedure described in 1.2 above.
  • the obtained water-absorbed coarsely ground fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
  • fenugreek koji About 25 g of the fenugreek koji described in 4.1 above was mixed in advance with 4 g of salt. The resulting mixture was kneaded with about 25 g of the water-absorbed coarsely ground fenugreek seeds that had been cooled to about 60°C or less after autoclave sterilization, and 20 g of water, and placed in a container. The container was left to ferment for up to 31 days in a 40°C incubator, and a fenugreek seed processed product was obtained using fenugreek koji with added salt. Samples were taken at each time point after 3 days, 12 days, 20 days, and 31 days.
  • fenugreek koji described in 4.1 above was mixed with 25 g of the water-absorbed coarsely ground fenugreek seeds that had been cooled to about 60°C or less after autoclave sterilization and 20 g of water, and the mixture was placed in a container.
  • the container was left to stand in an incubator at 40°C for 31 days to allow fermentation, and a fenugreek seed processed product was obtained using salt-free fenugreek koji. Samples were taken at each time point after 3 days, 12 days, 20 days, and 31 days.
  • the concentrations of protodioscin, compounds with a diosgenin skeleton, dioscin, prosapogenin and diosgenin were measured for samples taken at each time point from the fenugreek seed processed product made with the above-mentioned fenugreek koji with added salt and the fenugreek seed processed product made with the fenugreek koji without added salt.
  • the measurement method for each component was as described in 1.3 above.
  • the measurement results for protodioscin, dioscin, prosapogenin and diosgenin are shown in the table below.
  • the measurement results for protodioscin are shown in the table above and Figure 4.
  • Test 4 it was shown that in the reaction of treating fenugreek seeds with fenugreek koji, the concentration of protodioscin decreased over time, and the concentration of compounds with a diosgenin skeleton increased.
  • the rate at which protodioscin was converted to compounds with a diosgenin skeleton tended to be higher when salt was not added than when salt was added.
  • dioscin was detected in the early stage of the 31-day reaction but was below the detection limit in the later stage, whereas the concentrations of prosapogenin and diosgenin increased in the early stage of the 31-day reaction and were maintained in the later stage.

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Abstract

One or more embodiments of the present invention provides: a fenugreek seed processed product having a reduced protodioscin content; and a method for producing the same. One more embodiments of the present invention relate to a method for producing a fenugreek seed processed product, the method comprising: a step of obtaining a mixture by mixing fenugreek seeds and a fermented product which is a seed of a plant and/or a processed product thereof that has been fermented by a microorganism, the fermented product including the microorganism; and a step of keeping the mixture under a temperature condition in which the protodioscin in the mixture is decomposed. One or more other embodiments of the present invention relate to a fenugreek seed processed product in which a weight-basis protodioscin content PD and a weight-basis dioscin skeleton compound content DG having a dioscin skeleton other than protodioscin satisfies the relationship: PD/(PD+DG) = 0.78 or less.

Description

フェヌグリーク種子加工品及びその製造方法Processed fenugreek seeds and their manufacturing method
　本発明はフェヌグリーク種子加工品及びその製造方法に関する。 The present invention relates to processed fenugreek seeds and their manufacturing method.
　フェヌグリークはマメ科の一年草である。フェヌグリーク種子はカレー粉に含まれるなど、香辛料として古くから知られている。 Fenugreek is an annual plant of the legume family. Fenugreek seeds have long been known as a spice, including being included in curry powder.
　フェヌグリーク種子は苦味成分を有することが知られている。非特許文献１には、フェヌグリーク種子の苦味の主成分がフロスタノール型サポニンであるプロトジオスシンであることが報告されている。 Fenugreek seeds are known to contain bitter components. Non-Patent Document 1 reports that the main bitter component of fenugreek seeds is protodioscin, a furostanol-type saponin.
　特許文献１では、苦味が低減されたフェヌグリーク種子の製造方法として、フェヌグリーク種子に水を加えて前記フェヌグリーク種子の成分を溶出させ、β－グルコシダーゼを添加して、β－グルコシダーゼ添加後に前記成分と前記β－グルコシダーゼとを前記フェヌグリーク種子に吸収させることを特徴とする方法が記載されている。特許文献１によれば、フェヌグリーク種子から水中に溶出したプロトジオスシン等のサポニン化合物が、β－グルコシダーゼにより分解される。特許文献１では、β－グルコシダーゼとして、β－グルコシダーゼを含む酵素製剤を用いることが記載されている。 Patent Document 1 describes a method for producing fenugreek seeds with reduced bitterness, which is characterized by adding water to fenugreek seeds to elute components of the fenugreek seeds, adding β-glucosidase, and then allowing the components and the β-glucosidase to be absorbed by the fenugreek seeds after the addition of β-glucosidase. According to Patent Document 1, saponin compounds such as protodioscin that have been eluted from fenugreek seeds into water are decomposed by β-glucosidase. Patent Document 1 also describes the use of an enzyme preparation containing β-glucosidase as the β-glucosidase.
　非特許文献２では、ツァペック・ドックス・ブロス培地（ＣＤＢ）を吸収させたフェヌグリーク種子に、アスペルギルス・アワモリ（Ａｓｐｅｒｇｉｌｌｕｓ　ａｗａｍｏｒｉ）の胞子を接種して、２５±２℃で７日間培養した、アスペルギルス発酵フェヌグリーク（ＡＦＦ）が開示されている。非特許文献２には、アスペルギルス発酵フェヌグリークは、発酵５日目に、未発酵フェヌグリークと比較して、総フェノール含量、縮合型タンニン含量及び抗酸化能が増加したことが記載されている。 Non-Patent Document 2 discloses Aspergillus-fermented fenugreek (AFF) in which fenugreek seeds absorbed with Czapek-Dox Broth medium (CDB) are inoculated with spores of Aspergillus awamori and cultured at 25±2°C for 7 days. Non-Patent Document 2 describes that on the 5th day of fermentation, Aspergillus-fermented fenugreek has increased total phenol content, condensed tannin content, and antioxidant activity compared to unfermented fenugreek.
　非特許文献３では、食品製造に用いられる微生物によりフェヌグリーク由来サポニンを改変して得られた生成物の細胞毒性を確認したことが記載されている。具体的には、非特許文献３では、食品製造に用いられる微生物であるアスペルギルス・ニガーＫＣＴＣ６９０６、アスペルギルス・ウサミＫＣＴＣ６９５６、ビフィドバクテリウムｓｐ．Ｉｎｔ５７、ビフィドバクテリウム・インファンティス、ビフィドバクテリウム・ブレベＹＣ２、ビフィドバクテリウム・セドカテヌラタムＳＪ３２、ビフィドバクテリウムｓｐ．ＳＨ５、及び、ロイコノストク・パラメセンテロイデスＰＲを培地中で培養し、細胞を破砕して分離した粗酵素と、フェヌグリーク種子の粉末の２５％（ｖ／ｖ）エタノールによる抽出物とを混合し、４５℃で２４時間反応させたことが記載されている。そして非特許文献３では、アスペルギルス・ウサミ及びビフィドバクテリウム属細菌の粗酵素とフェヌグリーク種子抽出物との反応生成物は、未処理フェヌグリーク抽出物と比較して細胞毒性が高いのに対して、アスペルギルス・ニガーの粗酵素とフェヌグリーク種子抽出物との反応生成物は細胞毒性が低いことが記載されている。非特許文献３では更に、アスペルギルス・ウサミ及び上記のうちビフィドバクテリウム・セドカテヌラタムＳＪ３２以外の細菌の粗酵素とフェヌグリーク種子抽出物との反応生成物はジオスシンを多く含むのに対して、アスペルギルス・ニガーの粗酵素とフェヌグリーク種子抽出物との反応生成物はジオスシンが少なく未特定の別の物質が増加したことが記載されている。 Non-Patent Document 3 describes that the cytotoxicity of the product obtained by modifying fenugreek-derived saponin with microorganisms used in food manufacturing was confirmed. Specifically, Non-Patent Document 3 describes that Aspergillus niger KCTC6906, Aspergillus usamii KCTC6956, Bifidobacterium sp. Int57, Bifidobacterium infantis, Bifidobacterium breve YC2, Bifidobacterium sedocatenulatum SJ32, Bifidobacterium sp. SH5, and Leuconostoc paramesenteroides PR, which are microorganisms used in food manufacturing, were cultured in a medium, and the cells were disrupted to separate the crude enzyme, which was mixed with an extract of fenugreek seed powder with 25% (v/v) ethanol, and reacted at 45°C for 24 hours. Non-Patent Document 3 describes that the reaction products of Aspergillus usamii and crude enzymes of Bifidobacterium genus bacteria with fenugreek seed extract have higher cytotoxicity than untreated fenugreek extract, whereas the reaction products of Aspergillus niger crude enzymes with fenugreek seed extract have lower cytotoxicity. Non-Patent Document 3 further describes that the reaction products of Aspergillus usamii and crude enzymes of bacteria other than Bifidobacterium sedocatenulatum SJ32 among the above with fenugreek seed extract contain a lot of dioscin, whereas the reaction products of Aspergillus niger crude enzymes with fenugreek seed extract contain less dioscin and have increased other unspecified substances.
　非特許文献４では、アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）を、麦芽抽出物及びヤマノイモ水抽出物（酵素誘導剤）を含む培地中で培養した培養物から得た粗酵素と、ヤマノイモの乾燥根から単離したトータルステロイドサポニンとを、５０℃で２４時間反応させたことが記載されている。非特許文献４では、前記粗酵素による処理前のヤマノイモのトータルステロイドサポニンは、プロトジオスシン、プロトグラシリン及びジオスシンを含むのに対して、前記粗酵素とヤマノイモのトータルステロイドサポニンとの反応物は、プロゲニンＩＩＩ（プロサポゲニンＡ）を含むことが記載されている。非特許文献４ではまた、ヤマノイモ水抽出物を含まない培地中で培養したアスペルギルス・オリゼの粗酵素は、トータルステロイドサポニンからプロゲニンＩＩＩを生成する活性がほとんどないことが記載されている。 Non-Patent Document 4 describes that Aspergillus oryzae was cultured in a medium containing malt extract and Chinese yam water extract (enzyme inducer), and the crude enzyme obtained from the culture was reacted with total steroid saponin isolated from dried Chinese yam roots at 50°C for 24 hours. Non-Patent Document 4 describes that the total steroid saponin of Chinese yam before treatment with the crude enzyme contains protodioscin, protogracillin, and dioscin, whereas the reaction product of the crude enzyme and Chinese yam total steroid saponin contains progenin III (prosapogenin A). Non-Patent Document 4 also describes that the crude enzyme of Aspergillus oryzae cultured in a medium not containing Chinese yam water extract has almost no activity to generate progenin III from total steroid saponin.
特開２００８－２８３９６０号公報JP 2008-283960 A
　本発明の一以上の実施形態は、フェヌグリーク種子の苦味成分であるプロトジオスシンの含有量が低減されたフェヌグリーク種子加工品及びその製造方法を提供することを目的とする。 One or more embodiments of the present invention aim to provide a processed fenugreek seed product with a reduced content of protodioscin, a bitter component of fenugreek seeds, and a method for producing the same.
　本明細書は、下記の本発明の一以上の実施形態を開示する。 This specification discloses one or more embodiments of the present invention as described below.
（１）フェヌグリーク種子と、植物の種子及び／又はその処理物が微生物により発酵された前記微生物を含む発酵産物とを混合して、混合物を得る工程、及び
　前記混合物を、前記混合物中のプロトジオスシンが分解される温度条件に保持する工程、
を含む、フェヌグリーク種子加工品の製造方法。
（２）前記植物の種子及び／又はその処理物の糖質の含有量が６５重量％以下、（１）に記載の方法。
（３）前記植物が、マメ科植物、イネ科植物及びゴマ科植物から選択される１以上である、（１）又は（２）に記載の方法。
（４）前記植物がフェヌグリーク及びダイズから選択される１以上である、（３）に記載の方法。
（５）前記植物がフェヌグリークである、（４）に記載の方法。
（６）前記植物がダイズであり、
　前記処理物がオカラである、（４）に記載の方法。
（７）前記植物がイネであり、
　前記処理物が米糠である、（３）に記載の方法。
（８）前記植物がコムギであり、
　前記処理物が小麦ふすまである、（３）に記載の方法。
（９）前記植物がゴマである、（３）に記載の方法。
（１０）前記微生物がアスペルギルス属に属する、（１）～（９）のいずれかに記載の方法。
（１１）前記温度条件が、１０℃以上６０℃以下の条件である、（１）～（１０）のいずれかに記載の方法。
（１２）前記植物の種子及び／又はその処理物と、前記微生物とを混合して、発酵原料を得る工程と、
　前記発酵原料中で前記微生物を培養する工程と、
を含む、前記発酵産物を調製する工程、
を更に含む、（１）～（１１）のいずれかに記載の方法。
（１３）前記マメ科植物の種子及び／又はその処理物が培地を含まない、（１２）に記載の方法。
（１４）前記発酵原料中で前記微生物を培養する工程が、前記発酵原料を１０℃以上５０℃以下に保持することを含む、（１２）又は（１３）に記載の方法。
（１５）プロトジオスシンの重量基準の含有量をＰＤとし、
　プロトジオスシン以外のジオスゲニン骨格を持つ化合物の、ジオスゲニンとして換算した重量基準の含有量をＤＧとしたとき、
　ＰＤ／（ＰＤ＋ＤＧ）が０．７８以下である、フェヌグリーク種子加工品。
（１６）プロトジオスシン以外のジオスゲニン骨格を持つ前記化合物が、ジオスシン、プロサポゲニン及びジオスゲニンから選択される１以上を含む、（１５）に記載のフェヌグリーク種子加工品。
（１７）プロトジオスシンを分解する活性を有する酵素を産生する能力を有する微生物の死滅細胞又は生存細胞を含有する、（１５）又は（１６）に記載のフェヌグリーク種子加工品。
（１８）重量基準での、ジオスシンの含有量が、プロトジオスシンの含有量よりも大きい、（１５）～（１７）のいずれかに記載のフェヌグリーク種子加工品。
（１９）重量基準での、ジオスシンの含有量が、プロサポゲニンの含有量及びジオスゲニンの含有量よりも大きい、（１８）に記載のフェヌグリーク種子加工品。
（２０）重量基準での、プロサポゲニンの含有量が、プロトジオスシンの含有量よりも大きい、（１５）～（１７）のいずれかに記載のフェヌグリーク種子加工品。
（２１）重量基準での、プロサポゲニンの含有量が、ジオスシンの含有量及びジオスゲニンの含有量よりも大きい、（２０）に記載のフェヌグリーク種子加工品。
（２２）重量基準での、ジオスゲニンの含有量が、プロトジオスシンの含有量よりも大きい、（１５）～（１７）のいずれかに記載のフェヌグリーク種子加工品。
（２３）重量基準での、ジオスゲニンの含有量が、ジオスシンの含有量及びプロサポゲニンの含有量よりも大きい、（２２）に記載のフェヌグリーク種子加工品。 (1) A step of mixing fenugreek seeds with a fermentation product containing a microorganism obtained by fermenting a plant seed and/or a processed product thereof with a microorganism to obtain a mixture; and a step of maintaining the mixture under temperature conditions at which protodioscin in the mixture is decomposed.
A method for producing a processed fenugreek seed product, comprising:
(2) The method according to (1), wherein the carbohydrate content of the seeds of the plant and/or the processed products thereof is 65% by weight or less.
(3) The method according to (1) or (2), wherein the plant is one or more selected from the group consisting of legumes, grasses, and sesame plants.
(4) The method according to (3), wherein the plant is one or more selected from fenugreek and soybean.
(5) The method according to (4), wherein the plant is fenugreek.
(6) The plant is soybean,
The method according to (4), wherein the processed material is bean curd refuse.
(7) The plant is rice,
The method according to (3), wherein the processed material is rice bran.
(8) The plant is wheat,
The method according to (3), wherein the processed material is wheat bran.
(9) The method according to (3), wherein the plant is sesame.
(10) The method according to any one of (1) to (9), wherein the microorganism belongs to the genus Aspergillus.
(11) The method according to any one of (1) to (10), wherein the temperature condition is 10° C. or higher and 60° C. or lower.
(12) Mixing the seeds of the plant and/or a processed product thereof with the microorganism to obtain a fermentation raw material;
Cultivating the microorganism in the fermentation feedstock;
preparing the fermentation product,
The method according to any one of (1) to (11), further comprising:
(13) The method according to (12), wherein the legume seeds and/or processed products thereof do not contain a culture medium.
(14) The method according to (12) or (13), wherein the step of culturing the microorganism in the fermentation raw material includes maintaining the fermentation raw material at a temperature of 10°C or higher and 50°C or lower.
(15) The weight content of protodioscin is PD,
When the weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is defined as DG,
A processed fenugreek seed product having a PD/(PD+DG) ratio of 0.78 or less.
(16) The fenugreek seed processed product described in (15), wherein the compound having a diosgenin skeleton other than protodioscin includes one or more selected from dioscin, prosapogenin and diosgenin.
(17) A processed fenugreek seed product according to (15) or (16), which contains dead or viable cells of a microorganism capable of producing an enzyme having an activity of decomposing protodioscin.
(18) A processed fenugreek seed product according to any one of (15) to (17), wherein the dioscin content by weight is greater than the protodioscin content.
(19) A processed fenugreek seed product according to (18), in which the dioscin content, by weight, is greater than the prosapogenin content and the diosgenin content.
(20) A processed fenugreek seed product according to any one of (15) to (17), in which the prosapogenin content, by weight, is greater than the protodioscin content.
(21) A processed fenugreek seed product according to (20), in which the prosapogenin content, by weight, is greater than the dioscin content and the diosgenin content.
(22) A processed fenugreek seed product according to any one of (15) to (17), having a diosgenin content, on a weight basis, greater than the protodioscin content.
(23) A processed fenugreek seed product according to (22), in which the diosgenin content, by weight, is greater than the dioscin content and the prosapogenin content.
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０２２－１８４７４３号及び２０２３－１９２４９９号の開示内容を包含する。
　また本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 This specification includes the disclosures of Japanese Patent Application Nos. 2022-184743 and 2023-192499, which are priority documents of this application.
Additionally, all publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
　本発明の一以上の実施形態によれば、プロトジオスシンの含有量が低減されたフェヌグリーク種子加工品及びその製造方法が提供される。 According to one or more embodiments of the present invention, a processed fenugreek seed product having a reduced protodioscin content and a method for producing the same are provided.
図１は、試験１における、４０℃で４日間、８日間、１９日間及び６１日間処理後の、食塩添加オカラ麹によるフェヌグリーク種子加工品、及び、食塩無添加オカラ麹によるフェヌグリーク種子加工品、並びに、オカラ麹を添加しないフェヌグリーク種子（比較例）の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の測定結果を示す。FIG. 1 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in fenugreek seed processed products made with okara koji containing added salt, fenugreek seed processed products made with okara koji without added salt, and fenugreek seeds without added okara koji (comparative example) after treatment at 40°C for 4, 8, 19 and 61 days in Test 1. 図２は、試験２における、３種類の市販のアスペルギルスの種麹を用いて調製したオカラ麹と、フェヌグリーク種子抽出液とを５５℃で１８時間及び３日間反応させた反応液の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の測定結果を示す。FIG. 2 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in the reaction solution obtained by reacting okara koji prepared using three types of commercially available Aspergillus seed koji with fenugreek seed extract at 55° C. for 18 hours and 3 days in Test 2. 図３は、試験３における、市販のアスペルギルスの種麹を用いて調製した米糠麹、豆麹、フェヌグリーク麹、小麦ふすま麹及びゴマ麹と、フェヌグリーク種子抽出液とを５５℃で３日間反応させた反応液の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の測定結果を示す。Figure 3 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in the reaction solutions obtained by reacting rice bran koji, soybean koji, fenugreek koji, wheat bran koji and sesame koji prepared using commercially available Aspergillus seed koji with fenugreek seed extract at 55°C for three days in Test 3. 図４は、試験４における、４０℃で３日間、１２日間、２０日間及び３１日間処理後の、食塩添加フェヌグリーク麹によるフェヌグリーク種子加工品、及び、食塩無添加フェヌグリーク麹によるフェヌグリーク種子加工品の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の測定結果を示す。Figure 4 shows the results of measurement of compounds having protodioscin and diosgenin skeletons in fenugreek seed processed products made from fenugreek koji with added salt, and in fenugreek seed processed products made from fenugreek koji without added salt, after treatment at 40°C for 3 days, 12 days, 20 days, and 31 days in Test 4.
＜１．フェヌグリーク種子＞
　本発明の一以上の実施形態において材料として使用するフェヌグリーク種子について説明する。フェヌグリーク種子は、フェヌグリーク（学名Ｔｒｉｇｏｎｅｌｌａ　ｆｏｅｎｕｍ－ｇｒａｅｃｕｍ）の種子である。フェヌグリーク種子の形態は特に限定されず、種子自体の形態を保持した種子（ホール種子）、破砕した種子、粉末化した種子等の任意の形態であることができる。フェヌグリーク種子は、胚部分を少なくとも含む種子であればよく、種皮及びガラクトマンナン層を更に含んでいても含んでいなくてもよいが、好ましくは、胚部分とガラクトマンナン層とを含み、より好ましくは、胚部分とガラクトマンナン層と種皮とを含む。フェヌグリーク種子は、蒸煮及び／又は吸水により水分が調整されたフェヌグリーク種子であることができる。 1. Fenugreek seeds
Fenugreek seeds used as a material in one or more embodiments of the present invention will be described. Fenugreek seeds are seeds of fenugreek (scientific name Trigonella foenum-graecum). The form of fenugreek seeds is not particularly limited, and may be in any form such as seeds that retain the form of the seeds themselves (whole seeds), crushed seeds, powdered seeds, etc. The fenugreek seeds may be seeds that contain at least an embryo part, and may or may not further contain a seed coat and a galactomannan layer, but preferably contain an embryo part and a galactomannan layer, and more preferably contain an embryo part, a galactomannan layer, and a seed coat. The fenugreek seeds may be fenugreek seeds whose moisture content has been adjusted by steaming and/or water absorption.
＜２．発酵産物＞
　続いて、本発明の一以上の実施形態において材料として使用する、植物の種子及び／又はその処理物が微生物により発酵された前記微生物を含む発酵産物について説明する。 2. Fermentation products
Next, a fermentation product containing a microorganism obtained by fermenting a plant seed and/or a processed product thereof with a microorganism, which is used as a material in one or more embodiments of the present invention, will be described.
　前記発酵産物の原料として用いる前記植物の種子及び／又はその処理物は、好ましくは、下記の（Ａ）～（Ｅ）のうち１以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち２以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち３以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち４以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち少なくとも（Ａ）を含む１以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち少なくとも（Ａ）を含む２以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち少なくとも（Ａ）を含む３以上の特徴を有し、より好ましくは（Ａ）～（Ｅ）のうち少なくとも（Ａ）を含む４以上の特徴を有し、特に好ましくは（Ａ）～（Ｅ）の全ての特徴を有する。
　（Ａ）糖質の含有量が６５重量％以下である。
　（Ｂ）タンパク質の含有量が８重量％以上である。
　（Ｃ）脂質の含有量が３重量％以上である。
　（Ｄ）炭水化物の含有量が７５重量％以下である。
　（Ｅ）食物繊維の含有量が１０重量％以上である。 The plant seeds and/or processed products thereof used as a raw material for the fermentation product preferably have one or more characteristics among the following (A) to (E), more preferably have two or more characteristics among (A) to (E), more preferably have three or more characteristics among (A) to (E), more preferably have four or more characteristics among (A) to (E), more preferably have one or more characteristics among (A) to (E) including at least (A), more preferably have two or more characteristics among (A) to (E) including at least (A), more preferably have three or more characteristics among (A) to (E) including at least (A), more preferably have four or more characteristics among (A) to (E) including at least (A), and particularly preferably have all of the characteristics among (A) to (E).
(A) The carbohydrate content is 65% by weight or less.
(B) The protein content is 8% by weight or more.
(C) The lipid content is 3% by weight or more.
(D) The carbohydrate content is 75% by weight or less.
(E) The dietary fiber content is 10% by weight or more.
　このような原料の微生物による発酵産物は、フェヌグリーク種子に由来するプロトジオスシンを分解する活性が特に高い。 The fermentation products of these raw materials by microorganisms have a particularly high activity in breaking down protodioscin derived from fenugreek seeds.
　前記（Ａ）において、糖質の含有量はより好ましくは６０重量％以下、より好ましくは５５重量％以下、特に好ましくは４０重量％以下である。前記（Ａ）における糖質の含有量の範囲は、例えば０重量％～６５重量％、２重量％～６０重量％、好ましくは３重量％～５５重量％、より好ましくは５重量％～４０重量％であることができる。
　前記（Ｂ）において、タンパク質の含有量はより好ましくは１０重量％以上、特に好ましくは１２重量％以上である。前記（Ｂ）におけるタンパク質の含有量の範囲は、例えば、８重量％～５０重量％、好ましくは１０重量％～４０重量％、より好ましくは１２重量％～３０重量％であることができる。
　前記（Ｃ）において、脂質の含有量はより好ましくは５重量％以上である。前記（Ｃ）における脂質の含有量の範囲は、例えば３重量％～６０重量％、好ましくは５重量％～３０重量％であることができる。
　前記（Ｄ）において、炭水化物の含有量はより好ましくは７０重量％以下、特に好ましくは６５重量％以下である。前記（Ｄ）における炭水化物の含有量の範囲は、例えば１０重量％～７５重量％、好ましくは１５重量％～７０重量％、より好ましくは１５重量％～６５重量％であることができる。
　前記（Ｅ）において、食物繊維の含有量はより好ましくは１５重量％以上、特に好ましくは１８重量％以上である。前記（Ｅ）における食物繊維の含有量の範囲は、例えば１０重量％～７０重量％、好ましくは１５重量％～５０重量％、特に好ましくは１８重量％～５０重量％であることができる。 In the (A) above, the carbohydrate content is more preferably 60% by weight or less, more preferably 55% by weight or less, and particularly preferably 40% by weight or less. The carbohydrate content in the (A) above can be in the range of, for example, 0% by weight to 65% by weight, 2% by weight to 60% by weight, preferably 3% by weight to 55% by weight, and more preferably 5% by weight to 40% by weight.
In the above (B), the protein content is more preferably 10% by weight or more, particularly preferably 12% by weight or more. The protein content in the above (B) can be in the range of, for example, 8% by weight to 50% by weight, preferably 10% by weight to 40% by weight, more preferably 12% by weight to 30% by weight.
In the above (C), the lipid content is more preferably 5% by weight or more. The lipid content in the above (C) can be in the range of, for example, 3% by weight to 60% by weight, preferably 5% by weight to 30% by weight.
In the (D) above, the carbohydrate content is more preferably 70% by weight or less, particularly preferably 65% by weight or less. The carbohydrate content in the (D) above can be in the range of, for example, 10% by weight to 75% by weight, preferably 15% by weight to 70% by weight, more preferably 15% by weight to 65% by weight.
In the above (E), the dietary fiber content is more preferably 15% by weight or more, and particularly preferably 18% by weight or more. The range of the dietary fiber content in the above (E) can be, for example, 10% by weight to 70% by weight, preferably 15% by weight to 50% by weight, and particularly preferably 18% by weight to 50% by weight.
　なおここで各成分の含有量は、植物の種子及び／又はその処理物の、水や微生物等の、発酵産物の製造のための他の材料と混合する前の状態の重量に対する割合である。このような特性を有する前記植物の種子及び／又はその処理物としては、マメ科植物、イネ科植物及びゴマ科植物から選択される１以上の種子及び／又はその処理物が例示できる。 The content of each component here is the percentage relative to the weight of the plant seeds and/or processed products thereof before mixing with other materials, such as water and microorganisms, for producing a fermentation product. Examples of the plant seeds and/or processed products thereof having such characteristics include one or more seeds and/or processed products thereof selected from legumes, grasses, and sesame plants.
　前記植物としては、マメ科植物、イネ科植物及びゴマ科植物から選択される１以上が好ましい。マメ科植物、イネ科植物及びゴマ科植物から選択される１以上の植物の種子又はその処理物の微生物による発酵産物は、フェヌグリーク種子に由来するプロトジオスシンを分解する活性が特に高い。 The plant is preferably one or more selected from the group consisting of legumes, rice plants, and sesame plants. A fermentation product of seeds or processed products thereof by microorganisms of one or more plants selected from the group consisting of legumes, rice plants, and sesame plants has a particularly high activity of decomposing protodioscin derived from fenugreek seeds.
　マメ科植物としては、マメ亜科植物が好ましく、フェヌグリーク、ダイズ、及び落花生から選択される１以上がより好ましく、フェヌグリーク及びダイズから選択される１以上が特に好ましい。 As the legume plant, a plant of the subfamily Fabaceae is preferable, more preferably one or more selected from fenugreek, soybean, and peanut, and particularly preferably one or more selected from fenugreek and soybean.
　イネ科植物としてはイネ及びコムギから選択される１以上が特に好ましい。 As the grass family plant, one or more selected from rice and wheat are particularly preferred.
　ゴマ科植物としてはゴマが特に好ましい。 Sesame is a particularly preferred plant of the Pedaliaceae family.
　植物の種子は、種子自体の形態を保持した種子（ホール種子）、破砕した種子、粉末化した種子等の任意の形態であることができる。また、植物の種子は、蒸煮及び／又は吸水により水分が調整された種子であることができる。 The plant seeds can be in any form, such as seeds that retain the shape of the seed itself (whole seeds), crushed seeds, powdered seeds, etc. Furthermore, the plant seeds can be seeds whose moisture content has been adjusted by steaming and/or absorbing water.
　植物の種子の処理物としては、植物がダイズである場合、オカラ、豆乳、豆腐等のダイズ種子処理物が例示でき、特にオカラが好ましい。植物の種子の処理物の別の例としては、植物がイネである場合、米糠が好ましい。米糠とは、一般的に、イネ種子の表皮、果皮及び胚芽を含む。植物の種子の処理物の更に別の例としては、植物がコムギである場合、小麦ふすまが好ましい。小麦ふすまとは、一般的にコムギ種子の表皮を含む。 When the plant is soybean, examples of processed plant seeds include processed soybean seeds such as okara, soy milk, and tofu, with okara being particularly preferred. Another example of a processed plant seed is preferably rice bran when the plant is rice. Rice bran generally includes the epidermis, pericarp, and germ of rice seeds. Yet another example of a processed plant seed is preferably wheat bran when the plant is wheat. Wheat bran generally includes the epidermis of wheat seeds.
　植物の種子及び／又はその処理物は、好ましくは、マメ科植物の種子、マメ科植物の種子の処理物、米糠、小麦ふすま及びゴマ科植物の種子から選択される１以上であり、より好ましくはフェヌグリーク種子、ダイズ種子、オカラ、米糠、小麦ふすま又はゴマ種子であり、特に好ましくは、フェヌグリーク種子又はオカラである。これらの植物の種子及び／又はその処理物を基質とする発酵産物は、プロトジオスシンを分解する活性が特に高い。なお、これらの原料における水、タンパク質、脂質、炭水化物、糖質、食物繊維の含有量の一例を下記表に示す。炭水化物は、糖質と食物繊維とからなる。ここで各成分の含有量は、米糠、ダイズ種子、乾燥オカラ、ゴマ種子については文部科学省の食品成分データベース（https://fooddb.mext.go.jp/）、フェヌグリーク種子、小麦ふすまについては米国農務省のデータベースＦｏｏｄＤａｔａ　Ｃｅｎｔｒａｌ（https://fdc.nal.usda.gov/fdc-app.html#/food-details/171324/nutrients、及び、https://fdc.nal.usda.gov/fdc-app.html#/food-details/169722/nutrients）から入手できる情報に基づく。 The plant seeds and/or processed products thereof are preferably one or more selected from legume seeds, processed legume seeds, rice bran, wheat bran and sesame seeds, more preferably fenugreek seeds, soybean seeds, okara, rice bran, wheat bran or sesame seeds, and particularly preferably fenugreek seeds or okara. Fermentation products using these plant seeds and/or processed products thereof as substrates have particularly high activity in decomposing protodioscin. The following table shows examples of the water, protein, lipid, carbohydrate, sugar and dietary fiber contents in these raw materials. Carbohydrates consist of sugars and dietary fiber. The content of each ingredient here is based on information available from the Ministry of Education, Culture, Sports, Science and Technology's Food Composition Database (https://fooddb.mext.go.jp/) for rice bran, soybean seeds, dried okara, and sesame seeds, and from the United States Department of Agriculture's FoodData Central database (https://fdc.nal.usda.gov/fdc-app.html#/food-details/171324/nutrients and https://fdc.nal.usda.gov/fdc-app.html#/food-details/169722/nutrients) for fenugreek seeds and wheat bran.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　前記微生物は、植物の種子及び／又はその処理物を炭素源及び窒素源として利用して繁殖する（すなわち、植物の種子及び／又はその処理物を発酵する）能力を有する微生物であればよい。前記微生物の好ましい例としては、プロトジオスシンを分解する活性を有する酵素を産生する能力を有する微生物が挙げられ、特に、アスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属、ビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属又はロイコノストク（Ｌｅｕｃｏｎｏｓｔｏｃ）属に属する微生物が好ましく、アスペルギルス属に属する微生物（すなわち麹菌）が特に好ましい。アスペルギルス属に属する微生物（麹菌）としては、アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）、アスペルギルス・ソーヤ（Ａｓｐｅｒｇｉｌｌｕｓ　ｓｏｊａｅ）等の黄麹、アスペルギルス・ルチェンシス（Ａｓｐｅｒｇｉｌｌｕｓ　ｌｕｃｈｅｎｓｉｓ）、アスペルギルス・カワチ（Ａｓｐｅｒｇｉｌｌｕｓ　ｍｕｔ．ｋａｗａｃｈｉｉ）等の白麹、アスペルギルス・アワモリ（Ａｓｐｅｒｇｉｌｌｕｓ　ｖａｒ．ａｗａｍｏｒｉ）、アスペルギルス・ニガー（Ａｓｐｅｒｇｉｌｌｕｓ　ｎｉｇｅｒ）等の黒麹を使用することができ、特に黄麹が好ましい。黄麹としてはアスペルギルス・オリゼが特に好ましい。ビフィドバクテリウム属に属する微生物としては、ビフィドバクテリウム・インファンティス（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｉｎｆａｎｔｉｓ）、ビフィドバクテリウム・ブレベ（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｒｅｖｅ）又はビフィドバクテリウム・セドカテヌラタム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｐｓｅｄｕｃａｔｅｎｕｌａｔｕｍ）に属する微生物が好ましく、例えば、非特許文献３に記載のビフィドバクテリウム属に属する微生物が好ましい。ロイコノストク属に属する微生物としては、ロイコノストク・パラメセンテロイデス（Ｌｅｕｃｏｎｏｓｔｏｃ　ｐａｒａｍｅｓｅｎｔｅｒｏｉｄｅｓ）に属する微生物が好ましく、例えば、非特許文献３に記載のロイコノストク属に属する微生物が好ましい。 The microorganism may be any microorganism capable of propagating using plant seeds and/or processed products thereof as a carbon source and nitrogen source (i.e., fermenting plant seeds and/or processed products thereof). Preferred examples of the microorganism include microorganisms capable of producing an enzyme having activity of decomposing protodioscin, and in particular, microorganisms belonging to the genus Aspergillus, Bifidobacterium, or Leuconostoc are preferred, with microorganisms belonging to the genus Aspergillus (i.e., koji mold) being particularly preferred. As microorganisms belonging to the genus Aspergillus (koji mold), yellow koji such as Aspergillus oryzae and Aspergillus sojae, white koji such as Aspergillus luchensis and Aspergillus mut. kawachii, and black koji such as Aspergillus var. awamori and Aspergillus niger can be used, and yellow koji is particularly preferred. As the yellow koji, Aspergillus oryzae is particularly preferred. As the microorganism belonging to the genus Bifidobacterium, microorganisms belonging to Bifidobacterium infantis, Bifidobacterium breve, or Bifidobacterium pseudocatenulatum are preferred, and for example, the microorganisms belonging to the genus Bifidobacterium described in Non-Patent Document 3 are preferred. As the microorganism belonging to the genus Leuconostoc, microorganisms belonging to Leuconostoc paramesenteroides are preferred, and for example, the microorganisms belonging to the genus Leuconostoc described in Non-Patent Document 3 are preferred.
　麹菌を含む前記発酵産物を「麹」と称する場合がある。「麹」は、基質となる植物の種子及び／又はその処理物がフェヌグリーク種子であるとき「フェヌグリーク麹」、ダイズであるとき「豆麹」、オカラであるとき「オカラ麹」、米糠であるとき「米糠麹」、小麦ふすまであるとき「小麦ふすま麹」、ゴマ種子であるとき「ゴマ麹」と称する場合がある。 The fermentation product containing the koji mold may be referred to as "koji." When the substrate plant seed and/or processed product is fenugreek seed, "koji" may be called "fenugreek koji," when it is soybean, "okara koji" when it is bean lees, "rice bran koji" when it is rice bran, "wheat bran koji" when it is wheat bran, or "sesame koji" when it is sesame seed.
　前記発酵産物は、プロトジオスシンを分解する活性を有する酵素を含み、苦味の低減されたフェヌグリーク種子加工品の製造に利用することができる。前記発酵産物は前記微生物を含む。このため前記発酵産物をフェヌグリーク種子と混合した混合物を、前記微生物の生存温度条件に保持する実施形態では、前記微生物がフェヌグリーク種子の種皮及びガラクトマンナン層を通過して胚部分に侵入できるため、フェヌグリーク種子内のプロトジオスシンを分解する作用を効果的に奏することができる。一方、特許文献１に記載されているように、β－グルコシダーゼ含有酵素製剤を用いる場合、フェヌグリーク種子内のプロトジオスシンに作用させるためには、フェヌグリーク種子を過剰量の水に浸漬して、プロトジオスシンを水に溶出させる必要があった。 The fermentation product contains an enzyme having activity to decompose protodioscin, and can be used to manufacture fenugreek seed processed products with reduced bitterness. The fermentation product contains the microorganism. Therefore, in an embodiment in which a mixture of the fermentation product and fenugreek seeds is kept under temperature conditions for the survival of the microorganism, the microorganism can penetrate the seed coat and galactomannan layer of the fenugreek seeds and enter the embryo part, and therefore can effectively decompose the protodioscin in the fenugreek seeds. On the other hand, as described in Patent Document 1, when using a β-glucosidase-containing enzyme preparation, in order to act on the protodioscin in the fenugreek seeds, it was necessary to soak the fenugreek seeds in an excess amount of water and dissolve the protodioscin into the water.
　前記発酵産物は、例えば、
　前記植物の種子及び／又はその処理物と、前記微生物とを混合して、発酵原料を得る工程（以下「第１工程」と称する場合がある）と、
　前記発酵原料中で前記微生物を培養する工程（以下「第２工程」と称する場合がある）と、
を含む方法により調製することができる。 The fermentation product is, for example,
A step of mixing the seeds of the plant and/or a processed product thereof with the microorganism to obtain a fermentation raw material (hereinafter sometimes referred to as "first step");
A step of culturing the microorganism in the fermentation raw material (hereinafter sometimes referred to as the "second step");
The composition can be prepared by a method comprising the steps of:
　前記第１工程において、前記植物の種子及び／又はその処理物或いは前記発酵原料は、培地を含まないことが好ましい。すなわち、前記発酵原料は、前記第２工程での培養において炭素源及び窒素源として前記微生物が利用することができる培地成分を含まないことが好ましい。この態様により得られる前記発酵産物は、人為的に添加された培地成分を含まないため食品としての安全性が高い。 In the first step, it is preferable that the plant seeds and/or processed products thereof or the fermentation raw material does not contain a culture medium. In other words, it is preferable that the fermentation raw material does not contain any culture medium components that can be used by the microorganism as a carbon source and nitrogen source in the culture in the second step. The fermentation product obtained in this embodiment is highly safe as a food because it does not contain any artificially added culture medium components.
　前記第１工程において混合する前記微生物は、予め蒸米等の培養基材上に繁殖させた微生物や、前培養（種培養）した微生物であってよい。例えば、前記微生物がアスペルギルス属に属する麹菌である態様では、予め蒸米等の培養基材上に麹菌を繁殖させた形態であるか或いは麹菌の胞子の形態である「種麹」を、前記植物の種子及び／又はその処理物と混合することで前記発酵原料を得ることができる。種麹としては、市販品を購入して使用することができる。 The microorganisms mixed in the first step may be microorganisms that have been propagated in advance on a culture substrate such as steamed rice, or microorganisms that have been pre-cultured (seed cultured). For example, in an embodiment in which the microorganism is a koji mold belonging to the genus Aspergillus, the fermentation raw material can be obtained by mixing "seed koji," which is in the form of koji mold that has been propagated in advance on a culture substrate such as steamed rice, or in the form of koji mold spores, with the plant seeds and/or processed products thereof. Commercially available products can be purchased and used as seed koji.
　前記第１工程において得られる前記発酵原料は、前記植物の種子及び／又はその処理物並びに前記微生物に加えて、水、無機塩等を更に含むことができる。無機塩の例としては食塩が挙げられる。 The fermentation raw material obtained in the first step may further contain water, inorganic salts, etc., in addition to the plant seeds and/or processed products thereof and the microorganisms. An example of an inorganic salt is table salt.
　前記第２工程は、前記発酵原料中で前記微生物を培養する工程である。前記第２工程は前記微生物の培養が可能な条件で行うことができ、例えば前記発酵原料を、好ましくは１０℃以上５０℃以下、より好ましくは２０℃以上４５℃以下、より好ましくは３０℃以上４５℃以下、より好ましくは３０℃以上４０℃以下の温度条件に保持することを含む。前記第２工程は、より好ましくは前記発酵原料を、前記温度条件に、好ましくは３０時間以上１２０時間、より好ましくは４０時間以上１００時間以下の期間、保持することを含む。 The second step is a step of culturing the microorganism in the fermentation raw material. The second step can be carried out under conditions that allow the microorganism to be cultured, and includes, for example, maintaining the fermentation raw material under temperature conditions, preferably between 10°C and 50°C, more preferably between 20°C and 45°C, more preferably between 30°C and 45°C, more preferably between 30°C and 40°C. The second step more preferably includes maintaining the fermentation raw material under the temperature conditions, preferably for a period of between 30 hours and 120 hours, more preferably between 40 hours and 100 hours.
　前記第２工程において前記発酵原料中で前記微生物を培養して得られた発酵産物は、前記微生物を除去せず含んだ状態で、フェヌグリーク種子加工品の製造方法に供することができる。フェヌグリーク種子加工品の製造方法に供する時点において、前記発酵産物は、前記微生物の死滅細胞又は生存細胞を含むことができる。 The fermentation product obtained by culturing the microorganism in the fermentation raw material in the second step can be subjected to the method for producing a fenugreek seed processed product while still containing the microorganism without removing it. At the time of subjecting the fermentation product to the method for producing a fenugreek seed processed product, the fermentation product can contain dead or viable cells of the microorganism.
＜３．フェヌグリーク種子加工品の製造方法＞
　本発明の一以上の実施形態は、
　フェヌグリーク種子と、植物の種子及び／又はその処理物が微生物により発酵された前記微生物を含む発酵産物とを混合して、混合物を得る工程（以下「混合工程」と称する場合がある）、及び
　前記混合物を、前記混合物中のプロトジオスシンが分解される温度条件に保持する工程（以下「反応工程」と称する場合がある）、
を含むフェヌグリーク種子加工品の製造方法に関する。 <3. Manufacturing method of processed fenugreek seeds>
One or more embodiments of the present invention include
A process for obtaining a mixture by mixing fenugreek seeds with a fermentation product containing a microorganism obtained by fermenting a plant seed and/or a processed product thereof with a microorganism (hereinafter, sometimes referred to as a "mixing process"); and A process for maintaining the mixture under temperature conditions at which protodioscin in the mixture is decomposed (hereinafter, sometimes referred to as a "reaction process").
The present invention relates to a method for producing a processed fenugreek seed product comprising the steps of:
　本実施形態に係る方法によれば、β－グルコシダーゼ含有酵素製剤の使用を必要とせずに、未処理のフェヌグリーク種子と比較してプロトジオスシン含有量が低減され苦味が低減されたフェヌグリーク種子加工品を効率良く製造することができる。本実施形態に係る方法により製造されたフェヌグリーク種子加工品は、調味料等の食品として利用することができる。なお、前記混合物中のプロトジオスシンは、前記フェヌグリーク種子に由来するプロトジオスシンを少なくとも含み、前記発酵産物がプロトジオスシンを含む場合は、前記発酵産物に由来するプロトジオスシンを更に含む。 The method according to this embodiment makes it possible to efficiently produce a processed fenugreek seed product with a reduced protodioscin content and reduced bitterness compared to untreated fenugreek seeds, without the need for the use of a β-glucosidase-containing enzyme preparation. The processed fenugreek seed product produced by the method according to this embodiment can be used as a food product such as a seasoning. The protodioscin in the mixture contains at least protodioscin derived from the fenugreek seeds, and when the fermentation product contains protodioscin, it further contains protodioscin derived from the fermentation product.
　前記混合工程において、前記フェヌグリーク種子と前記発酵産物との混合割合は特に限定されないが、前記フェヌグリーク種子（フェヌグリーク種子の、水を加えない状態での乾燥重量換算）１００重量部に対して、前記発酵産物（前記発酵産物の原料として用いた植物の種子及び／又はその処理物の、水を加えない状態での乾燥重量換算）を例えば２０重量部以上５００重量部以下、好ましくは５０重量部以上２００重量部以下の割合で混合することができる。 In the mixing step, the mixing ratio of the fenugreek seeds and the fermentation product is not particularly limited, but the fermentation product (the seeds of the plant used as the raw material for the fermentation product and/or the processed product thereof, calculated as the dry weight without adding water) can be mixed with 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water) in a ratio of, for example, 20 parts by weight to 500 parts by weight, preferably 50 parts by weight to 200 parts by weight.
　前記混合工程では、前記フェヌグリーク種子と前記発酵産物に加えて、水、無機塩等の他の成分を更に混合してもよく、特に水及び食塩を更に混合することが好ましい。食塩は、前記フェヌグリーク種子（フェヌグリーク種子の、水を加えない状態での乾燥重量換算）１００重量部に対して、例えば５重量部以上１００重量部以下、好ましくは２０重量部以上６０重量部以下の割合で混合することができる。前記混合物中の水（前記フェヌグリーク種子及び前記発酵産物からの水も含む、加えた水の総量）が、前記フェヌグリーク種子（フェヌグリーク種子の、水を加えない状態での乾燥重量換算）１００重量部に対して、例えば２００重量部以上、好ましくは３００重量部以上となるように水を混合することができる。前記混合物中の水の量の上限は特に限定されず、前記反応工程によるプロトジオスシンの分解が可能な範囲に調整すればよい。例えば、前記フェヌグリーク種子（フェヌグリーク種子の、水を加えない状態での乾燥重量換算）１００重量部に対して１００００重量部以下の水を混合することができる。 In the mixing step, in addition to the fenugreek seeds and the fermentation product, other components such as water and inorganic salts may be further mixed, and it is particularly preferable to further mix water and salt. Salt can be mixed in a ratio of, for example, 5 parts by weight to 100 parts by weight, preferably 20 parts by weight to 60 parts by weight, per 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water). Water can be mixed so that the water in the mixture (the total amount of water added, including the water from the fenugreek seeds and the fermentation product) is, for example, 200 parts by weight or more, preferably 300 parts by weight or more, per 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water). The upper limit of the amount of water in the mixture is not particularly limited, and it may be adjusted to a range in which protodioscin can be decomposed by the reaction step. For example, 10,000 parts by weight or less of water can be mixed with 100 parts by weight of the fenugreek seeds (calculated as the dry weight of the fenugreek seeds without adding water).
　本実施形態に係る方法において、前記混合工程に用いる前記発酵産物は別途購入して使用できるが、より好ましくは、本実施形態に係る方法は、前記第１工程及び前記第２工程を含む発酵産物を調製する工程を更に含み、この工程により調製した前記発酵産物を、前記混合工程に用いることができる。 In the method according to this embodiment, the fermentation product used in the mixing step can be purchased separately and used, but more preferably, the method according to this embodiment further includes a step of preparing a fermentation product including the first step and the second step, and the fermentation product prepared by this step can be used in the mixing step.
　前記反応工程における前記温度条件は、前記混合物中において、前記発酵産物が有する酵素活性により、プロトジオスシンが分解される温度条件であればよく、好ましくは１０℃以上６０℃以下、より好ましくは１５℃以上６０℃以下、より好ましくは２０℃以上６０℃以下、より好ましくは３０℃以上６０℃以下、より好ましくは３５℃以上５７℃以下である。前記反応工程において、前記混合物中の前記発酵産物からの微生物の生存は必須ではないが、微生物が生存している場合、上記の通り、フェヌグリーク種子内のプロトジオスシンを分解する作用が特に高いため好ましい。前記微生物を生存させて前記反応工程を行う場合、前記反応工程の前記温度条件は好ましくは１０℃以上５０℃未満、より好ましくは１５℃以上５０℃未満、より好ましくは２０℃以上５０℃未満、より好ましくは３０℃以上５０℃未満、より好ましくは３０℃以上４５℃以下、最も好ましくは３５℃以上４５℃以下の条件である。一方、前記微生物が生存できない条件で前記反応工程を行う場合、前記反応工程の前記温度条件は好ましく５０℃以上６０℃以下、より好ましくは５０℃以上５７℃以下の条件である。 The temperature conditions in the reaction step may be any temperature conditions that allow protodioscin to be decomposed in the mixture by the enzyme activity of the fermentation product, and are preferably 10°C to 60°C, more preferably 15°C to 60°C, more preferably 20°C to 60°C, more preferably 30°C to 60°C, and more preferably 35°C to 57°C. In the reaction step, the survival of microorganisms from the fermentation product in the mixture is not essential, but if microorganisms are alive, as described above, the effect of decomposing protodioscin in fenugreek seeds is particularly high, and therefore it is preferable. When the reaction step is performed while keeping the microorganisms alive, the temperature conditions in the reaction step are preferably 10°C to 50°C, more preferably 15°C to 50°C, more preferably 20°C to 50°C, more preferably 30°C to 50°C, more preferably 30°C to 45°C, and most preferably 35°C to 45°C. On the other hand, when the reaction step is carried out under conditions in which the microorganism cannot survive, the temperature conditions of the reaction step are preferably 50°C or higher and 60°C or lower, more preferably 50°C or higher and 57°C or lower.
　前記反応工程において、前記混合物を前記温度条件に保持する時間は、目的とする、前記混合物中のプロトジオスシンの分解の程度に応じて適宜調節することができる。例えば、前記混合物中のプロトジオスシンを、前記混合物の調製直後のプロトジオスシンの含有量を１００重量％とした場合に、好ましくは５０重量％以下、より好ましくは２０重量％以下、より好ましくは１０重量％以下、より好ましくは５重量％以下、より好ましくは１重量％以下となるように、前記反応工程を行うことができる。また、前記反応工程後の前記混合物中のプロトジオスシンの含有量の目標値としては、前記混合物の全重量（乾燥重量基準）に対して、例えば５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよい。このような水準までプロトジオスシンを分解するために、前記反応工程における前記混合物を前記温度条件に保持する時間は、例えば２日間以上、好ましくは３日間以上、より好ましくは１０日以上、より好ましくは１５日以上である。前記反応工程における前記混合物を前記温度条件に保持する時間の上限は特に限定されないが、好ましくは２年以下、より好ましくは１年以下であることができる。すなわち前記反応工程における前記混合物を前記温度条件に保持する時間は例えば２日間～２年間、好ましくは３日間～２年間、より好ましくは１０日間～１年間、より好ましくは１５日間～１年間であることができる。 In the reaction process, the time for which the mixture is held under the temperature conditions can be appropriately adjusted depending on the desired degree of decomposition of protodioscin in the mixture. For example, the reaction process can be carried out so that the protodioscin content in the mixture is preferably 50% by weight or less, more preferably 20% by weight or less, more preferably 10% by weight or less, more preferably 5% by weight or less, and more preferably 1% by weight or less, when the protodioscin content immediately after the preparation of the mixture is 100% by weight. In addition, the target value of the protodioscin content in the mixture after the reaction process is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, relative to the total weight of the mixture (based on dry weight), and may be below the detection limit (i.e., 0 ppm). In order to decompose protodioscin to such a level, the time for which the mixture in the reaction step is held under the temperature conditions is, for example, 2 days or more, preferably 3 days or more, more preferably 10 days or more, and more preferably 15 days or more. There is no particular upper limit to the time for which the mixture in the reaction step is held under the temperature conditions, but it is preferably 2 years or less, and more preferably 1 year or less. That is, the time for which the mixture in the reaction step is held under the temperature conditions can be, for example, 2 days to 2 years, preferably 3 days to 2 years, more preferably 10 days to 1 year, and more preferably 15 days to 1 year.
　前記反応工程を経た前記混合物は、味噌のようなペースト状物であることができ、それ自体をフェヌグリーク種子加工品として、調味料等の食品の用途に用いることができる。 The mixture that has undergone the reaction process can be a paste-like substance similar to miso, and can be used as a processed fenugreek seed product for food applications such as seasonings.
　前記反応工程を前記微生物が生存する条件で行う場合は、前記反応工程の終了後に前記混合物を加熱処理して前記微生物を死滅させることが好ましい。 If the reaction step is carried out under conditions in which the microorganisms can survive, it is preferable to heat-treat the mixture after completion of the reaction step to kill the microorganisms.
　前記反応工程を経た前記混合物に対して、更に乾燥、濃縮、希釈、造粒等の処理を施したものを、フェヌグリーク種子加工品として利用することもできる。 The mixture that has undergone the reaction process can be further processed, such as by drying, concentrating, diluting, granulating, etc., and used as a processed fenugreek seed product.
＜４．フェヌグリーク種子加工品＞
　本発明の別の一以上の実施形態は、
　プロトジオスシンの重量基準の含有量をＰＤとし、
　プロトジオスシン以外のジオスゲニン骨格を持つ化合物の、ジオスゲニンとして換算した重量基準の含有量をＤＧとしたとき、
　ＰＤ／（ＰＤ＋ＤＧ）が０．７８以下である、
フェヌグリーク種子加工品に関する。 <4. Processed Fenugreek Seed Products>
One or more further embodiments of the present invention include
The weight-based content of protodioscin is designated as PD.
When the weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is defined as DG,
PD/(PD+DG) is 0.78 or less;
This relates to processed fenugreek seeds.
　未加工のフェヌグリーク種子は、苦味の原因物質であるプロトジオスシンを多く含み、プロトジオスシン以外のジオスゲニン骨格を持つ化合物をほとんど含まないため、（ＰＤ／（ＰＤ＋ＤＧ）は１．０に近い。これに対し、ＰＤ／（ＰＤ＋ＤＧ）が０．７８以下である本実施形態に係るフェヌグリーク種子加工品は、未加工のフェヌグリーク種子と比較して、プロトジオスシンの重量基準の含有量が小さく、且つ、プロトジオスシン以外の、生理機能が期待されるフィトステロイドであるジオスゲニン骨格を持つ化合物の重量基準の含有量が大きいため、調味料等の食品の用途に特に適している。 Unprocessed fenugreek seeds contain a lot of protodioscin, the substance that causes bitterness, and contain almost no compounds with a diosgenin skeleton other than protodioscin, so (PD/(PD+DG) is close to 1.0. In contrast, the fenugreek seed processed product of this embodiment, which has a PD/(PD+DG) of 0.78 or less, has a smaller weight-based content of protodioscin compared to unprocessed fenugreek seeds, and a larger weight-based content of compounds with a diosgenin skeleton, a phytosteroid other than protodioscin that is expected to have physiological functions, making it particularly suitable for use in food products such as seasonings.
　プロトジオスシン以外のジオスゲニン骨格を持つ化合物としては、ジオスゲニン及び加水分解によりジオスゲニンを生じる化合物から選択される１以上が挙げられ、典型的には、ジオスゲニン及びジオスゲニン配糖体から選択される１以上が挙げられ、具体的には、ジオスシン、プロサポゲニン、ジオスゲニン等が例示できる。プロトジオスシン以外のジオスゲニン骨格を持つ化合物は、好ましくは、ＯＤＳカラムを用いた逆相液体クロマトグラフィーのクロマトグラムにおいて、プロトジオスシンよりも保持時間が長い、ジオスゲニンまでのピークに対応する化合物のうち、質量分析においてジオスゲニン骨格に特有のフラグメントが検出される化合物である。 Compounds having a diosgenin skeleton other than protodioscin include one or more selected from diosgenin and compounds that produce diosgenin upon hydrolysis, typically one or more selected from diosgenin and diosgenin glycosides, specifically dioscin, prosapogenin, diosgenin, etc. Compounds having a diosgenin skeleton other than protodioscin are preferably compounds that have a longer retention time than protodioscin in a chromatogram of reversed-phase liquid chromatography using an ODS column, and correspond to a peak up to diosgenin, and in which a fragment specific to the diosgenin skeleton is detected in mass spectrometry.
　本実施形態に係るフェヌグリーク種子加工品は、好ましくは、ジオスシン、ジオスゲニン等のジオスゲニン骨格を持つ化合物に基づく生理作用、特に、抗糖尿病作用、認知機能改善作用、及び、更年期障害改善作用から選択される１以上の生理作用、を有することができる。 The fenugreek seed processed product according to this embodiment preferably has a physiological effect based on a compound having a diosgenin skeleton, such as dioscin or diosgenin, and in particular, one or more physiological effects selected from an antidiabetic effect, an effect of improving cognitive function, and an effect of improving menopausal disorders.
　ジオスシンが有する生理作用としては、抗尿酸血症、抗真菌・ウィルス作用、抗腫瘍、肝保護（線維症、急性肝障害、ＮＡＦＬＤ、胆汁うっ滞、肝虚血再潅流障害）、肺保護、腎保護、心肺機能保護、大脳保護、抗アテローム性動脈硬化、抗炎症、抗関節炎、抗肥満・抗糖尿病、抗酸化ストレス、抗骨粗しょう症、メラニン生成抑制、成長ホルモン放出、抗乳がん、抗胃がん、抗肝がん、抗骨髄性白血病、抗肺がん、抗腎臓がん、抗黒色腫腫瘍、抗前立腺がん、抗慢性肝障害、肝臓再生、心血管・脳血管保護、胃虚血再潅流障害保護等の生理作用、並びに、非アルコール性脂肪性肝疾患、ＩＩ型糖尿病等の疾患を予防又は治療する生理作用が知られている。ジオスゲニンが有する生理作用としては、抗ガン、抗真菌、抗ウィルス、抗血栓、抗酸化、神経保護、免疫調節等の生理作用、心血管疾患、心筋障害、血管障害、高脂血症、ＩＩ型糖尿病、炎症、更年期障害、肌老化、骨粗しょう症等の疾患を予防又は治療する生理作用、並びに、ガン細胞増殖とアポトーシス、腫瘍侵入・転移・血管新生、皮質ニューロンに対する生理作用が知られている。本実施形態に係るフェヌグリーク種子加工品は、好ましくは、ジオスシン、ジオスゲニン等のジオスゲニン骨格を持つ化合物が持つこれらの生理作用を奏することができる。 The physiological effects of dioscin include antiuricemia, antifungal and antiviral effects, antitumor, liver protection (fibrosis, acute liver damage, NAFLD, cholestasis, hepatic ischemia-reperfusion injury), lung protection, kidney protection, cardiopulmonary function protection, cerebral protection, anti-atherosclerosis, anti-inflammation, anti-arthritis, anti-obesity and anti-diabetes, anti-oxidative stress, anti-osteoporosis, inhibition of melanin production, growth hormone release, anti-breast cancer, anti-gastric cancer, anti-liver cancer, anti-myeloid leukemia, anti-lung cancer, anti-kidney cancer, anti-melanoma tumor, anti-prostate cancer, anti-chronic liver damage, liver regeneration, cardiovascular and cerebrovascular protection, and protection from gastric ischemia-reperfusion injury, as well as physiological effects to prevent or treat diseases such as non-alcoholic fatty liver disease and type II diabetes. The physiological effects of diosgenin are known to be anticancer, antifungal, antiviral, antithrombotic, antioxidant, neuroprotective, immunomodulatory, and other physiological effects, as well as physiological effects to prevent or treat diseases such as cardiovascular disease, myocardial disorder, vascular disorder, hyperlipidemia, type II diabetes, inflammation, menopausal disorders, skin aging, and osteoporosis, as well as physiological effects on cancer cell proliferation and apoptosis, tumor invasion, metastasis, and angiogenesis, and cortical neurons. The fenugreek seed processed product according to this embodiment can preferably exert these physiological effects of compounds having a diosgenin skeleton, such as dioscin and diosgenin.
　本実施形態に係るフェヌグリーク種子加工品のＰＤ／（ＰＤ＋ＤＧ）は、より好ましくは０．７０以下、より好ましくは０．５０以下、より好ましくは０．４０以下、より好ましくは０．３０以下、より好ましくは０．２０以下、より好ましくは０．１０以下、より好ましくは０．０５以下、より好ましくは０．０１以下である。本実施形態に係るフェヌグリーク種子加工品は、ＰＤが検出限界以下でＰＤ／（ＰＤ＋ＤＧ）が０である場合も包含する。すなわちＰＤ／（ＰＤ＋ＤＧ）は、例えば０～０．７８、好ましくは０～０．７０、より好ましくは０～０．５０、より好ましくは０～０．４０、より好ましくは０～０．３０、より好ましくは０～０．２０、より好ましくは０～０．１０、より好ましくは０～０．０５、より好ましくは０～０．０１であることができる。 The PD/(PD+DG) of the processed fenugreek seed product according to this embodiment is more preferably 0.70 or less, more preferably 0.50 or less, more preferably 0.40 or less, more preferably 0.30 or less, more preferably 0.20 or less, more preferably 0.10 or less, more preferably 0.05 or less, more preferably 0.01 or less. The processed fenugreek seed product according to this embodiment also includes cases where the PD is below the detection limit and the PD/(PD+DG) is 0. That is, the PD/(PD+DG) can be, for example, 0 to 0.78, preferably 0 to 0.70, more preferably 0 to 0.50, more preferably 0 to 0.40, more preferably 0 to 0.30, more preferably 0 to 0.20, more preferably 0 to 0.10, more preferably 0 to 0.05, more preferably 0 to 0.01.
　フェヌグリーク種子加工品におけるＰＤ及びＤＧは、液体クロマトグラフ質量分析計（ＬＣ－ＭＳ）を用いて測定することができる。ＬＣ－ＭＳを用いた測定方法の具体例は実施例に記載の通りである。ＰＤの決定のための検量線は既知濃度のプロトジオスシン標品を含む試料を用いて作成することができる。ＤＧは、プロトジオスシン以外の、ＬＣ－ＭＳ測定においてジオスゲニン骨格に特有のフラグメントを生じる化合物を、ジオスゲニンとして換算した重量基準の含有量である。ＤＧの決定のための検量線は既知濃度のジオスゲニン標品を含む試料を用いて作成することができる。 PD and DG in processed fenugreek seed products can be measured using a liquid chromatograph mass spectrometer (LC-MS). A specific example of a measurement method using LC-MS is described in the Examples. A calibration curve for determining PD can be prepared using samples containing a known concentration of a protodioscin standard. DG is the weight-based content of compounds other than protodioscin that produce fragments specific to the diosgenin skeleton in LC-MS measurement, converted into diosgenin. A calibration curve for determining DG can be prepared using samples containing a known concentration of a diosgenin standard.
　本実施形態に係るフェヌグリーク種子加工品において、プロトジオスシンの重量基準の含有量ＰＤの値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよい。すなわちＰＤは、例えば０ｐｐｍ～５０００ｐｐｍ、好ましくは０ｐｐｍ～３０００ｐｐｍ、より好ましくは０ｐｐｍ～１５００ｐｐｍ、より好ましくは０ｐｐｍ～３００ｐｐｍ、より好ましくは０ｐｐｍ～１００ｐｐｍ、より好ましくは０ｐｐｍ～１０ｐｐｍであることができる。 In the processed fenugreek seed product according to this embodiment, the value of the weight-based protodioscin content PD is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), relative to the total weight (dry weight) of the processed fenugreek seed product. That is, PD can be, for example, 0 ppm to 5000 ppm, preferably 0 ppm to 3000 ppm, more preferably 0 ppm to 1500 ppm, more preferably 0 ppm to 300 ppm, more preferably 0 ppm to 100 ppm, more preferably 0 ppm to 10 ppm.
　本実施形態に係るフェヌグリーク種子加工品において、プロトジオスシン以外のジオスゲニン骨格を持つ化合物の、ジオスゲニンとして換算した重量基準の含有量ＤＧの値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば５００ｐｐｍ以上、好ましくは６００ｐｐｍ以上、より好ましくは９００ｐｐｍ以上、より好ましくは１２００ｐｐｍ以上、より好ましくは１５００ｐｐｍ以上であり、上限は特に限定されないが、例えば２００００ｐｐｍ以下である。すなわちＤＧは、例えば５００～２００００ｐｐｍ、好ましくは６００ｐｐｍ～２００００ｐｐｍ、より好ましくは９００ｐｐｍ～２００００ｐｐｍ、より好ましくは１２００ｐｐｍ～２００００ｐｐｍ、より好ましくは１５００ｐｐｍ～２００００ｐｐｍであることができる。 In the processed fenugreek seed product according to this embodiment, the value of the weight-based content DG of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is, for example, 500 ppm or more, preferably 600 ppm or more, more preferably 900 ppm or more, more preferably 1200 ppm or more, more preferably 1500 ppm or more, and the upper limit is not particularly limited, but is, for example, 20000 ppm or less, relative to the total weight (dry weight basis) of the processed fenugreek seed product. That is, the DG can be, for example, 500 to 20000 ppm, preferably 600 ppm to 20000 ppm, more preferably 900 ppm to 20000 ppm, more preferably 1200 ppm to 20000 ppm, more preferably 1500 ppm to 20000 ppm.
　本実施形態に係るフェヌグリーク種子加工品は、好ましくは、微生物の細胞を含み、より好ましくは、プロトジオスシンを分解する活性を有する酵素を産生する能力を有する微生物の死滅細胞又は生存細胞を含み、特に好ましくは、プロトジオスシンを分解する活性を有する酵素を産生する能力を有する微生物の死滅細胞を含む。微生物の細胞を含む本実施形態に係るフェヌグリーク種子加工品は、＜３．フェヌグリーク種子加工品の製造方法＞において説明した実施形態に係る方法により製造することができる。前記微生物の具体例としては、＜２．発酵産物＞に記載の微生物が挙げられる。 The fenugreek seed processed product according to this embodiment preferably contains microbial cells, more preferably contains dead or viable microbial cells capable of producing an enzyme having protodioscin-degrading activity, and particularly preferably contains dead microbial cells capable of producing an enzyme having protodioscin-degrading activity. The fenugreek seed processed product according to this embodiment containing microbial cells can be produced by the method according to the embodiment described in <3. Production method of fenugreek seed processed product>. Specific examples of the microorganism include the microorganisms described in <2. Fermentation products>.
　本実施形態に係るフェヌグリーク種子加工品は、典型的には、味噌のようなペースト状物であることができ、それ自体を調味料等の食品の用途に用いることができる。 The processed fenugreek seed product according to this embodiment can typically be a paste-like product like miso, and can be used as a food product such as a seasoning.
　上記の通り、未加工のフェヌグリーク種子は、苦味の原因物質であるプロトジオスシンを多く含む。本実施形態に係るフェヌグリーク種子加工品の好ましい実施形態は、未加工のフェヌグリーク中のプロトジオスシンの少なくとも一部、好ましくは全部が、ジオスシン、プロサポゲニン及びジオスゲニンから選択される１以上に変換されていることを特徴とする。ここでプロサポゲニンとは、プロサポゲニンＡとプロサポゲニンＢとの総称であり、プロサポゲニンＡとプロサポゲニンＢの一方又は両方を指す。本実施形態に係るフェヌグリーク種子加工品の更に好ましい実施形態は、下記（ｉ）、（ｉｉ）又は（ｉｉｉ）の特徴を有する：
（ｉ）重量基準での、ジオスシンの含有量が、プロトジオスシンの含有量よりも大きい。好ましくは更に、重量基準での、ジオスシンの含有量が、プロサポゲニンの含有量及びジオスゲニンの含有量よりも大きい；
（ｉｉ）重量基準での、プロサポゲニンの含有量が、プロトジオスシンの含有量よりも大きい。好ましくは更に、重量基準での、プロサポゲニンの含有量が、ジオスシンの含有量及びジオスゲニンの含有量よりも大きい；或いは
（ｉｉｉ）重量基準での、ジオスゲニンの含有量が、プロトジオスシンの含有量よりも大きい。好ましくは更に、重量基準での、ジオスゲニンの含有量が、ジオスシンの含有量及びプロサポゲニンの含有量よりも大きい。 As described above, raw fenugreek seeds contain a large amount of protodioscin, which is a substance that causes bitterness. A preferred embodiment of the processed fenugreek seeds according to this embodiment is characterized in that at least a part, preferably all, of the protodioscin in raw fenugreek is converted to one or more selected from dioscin, prosapogenin and diosgenin. Here, prosapogenin is a general term for prosapogenin A and prosapogenin B, and refers to one or both of prosapogenin A and prosapogenin B. A further preferred embodiment of the processed fenugreek seeds according to this embodiment has the following characteristics (i), (ii) or (iii):
(i) the dioscin content is greater than the protodioscin content by weight. Preferably, the dioscin content is greater than the prosapogenin content and the diosgenin content by weight;
(ii) the prosapogenin content is greater than the protodioscin content, and preferably, the prosapogenin content is greater than the dioscin content and the diosgenin content, by weight; or (iii) the diosgenin content is greater than the protodioscin content, and preferably, the diosgenin content is greater than the dioscin content and the prosapogenin content, by weight.
　本発明者らは、＜３．フェヌグリーク種子加工品の製造方法＞において説明した実施形態に係る方法により製造されたフェヌグリーク種子加工品が、前記（ｉ）、（ｉｉ）又は（ｉｉｉ）の特徴を有すること見出し、前記好ましい実施形態に係るフェヌグリーク種子加工品を完成させた。 The inventors have found that the fenugreek seed processed product produced by the method according to the embodiment described in <3. Method for producing a fenugreek seed processed product> has the above characteristics (i), (ii) or (iii), and have completed the fenugreek seed processed product according to the preferred embodiment.
　前記好ましい実施形態に係るフェヌグリーク種子加工品において、プロサポゲニンの重量基準の含有量は、プロサポゲニンＡ、プロサポゲニンＢ、プロサポゲニンＡとプロサポゲニンＢとの総量であり、且つ、ジオスシンの検量線を使用して算出したジオスシンとしての換算量である。 In the fenugreek seed processed product according to the preferred embodiment, the weight-based content of prosapogenin is the total amount of prosapogenin A, prosapogenin B, and prosapogenin A and prosapogenin B, and is the equivalent amount of dioscin calculated using the dioscin calibration curve.
　前記（ｉ）、（ｉｉ）及び（ｉｉｉ）のいずれかの特徴を有するフェヌグリーク種子加工品において、プロトジオスシンの重量基準の含有量の値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよい。すなわち、前記フェヌグリーク種子加工品において、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対するプロトジオスシンの含有量は、例えば０～５０００ｐｐｍ、好ましくは０～３０００ｐｐｍ、より好ましくは０～１５００ｐｐｍ、より好ましくは０～３００ｐｐｍ、より好ましくは０～１００ｐｐｍ、より好ましくは０～１０ｐｐｍであることができる。 In the processed fenugreek seed product having any of the characteristics (i), (ii), and (iii), the content of protodioscin by weight is, for example, 5000 ppm or less, preferably 3000 ppm or less, more preferably 1500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm) relative to the total weight (dry weight) of the processed fenugreek seed product. That is, in the processed fenugreek seed product, the content of protodioscin relative to the total weight (dry weight) of the processed fenugreek seed product can be, for example, 0 to 5000 ppm, preferably 0 to 3000 ppm, more preferably 0 to 1500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm.
　前記（ｉ）の特徴を有するフェヌグリーク種子加工品において、ジオスシンの重量基準の含有量の値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば３００ｐｐｍ以上、好ましくは１０００ｐｐｍ以上、好ましくは１５００ｐｐｍ以上、より好ましくは３０００ｐｐｍ以上であり、好ましくは１５０００ｐｐｍ以下であり、例えば３００ｐｐｍ～１５０００ｐｐｍ、好ましくは１０００ｐｐｍ～１５０００ｐｐｍ、好ましくは１５００ｐｐｍ～１５０００ｐｐｍ、より好ましくは３０００ｐｐｍ～１５０００ｐｐｍであることができる。前記（ｉ）の特徴を有するフェヌグリーク種子加工品では更に好ましくは、プロサポゲニン及びジオスゲニンの重量基準の含有量はそれぞれ、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば１００００ｐｐｍ以下、好ましくは５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよく、例えば０～１００００ｐｐｍ、好ましくは０～５０００ｐｐｍ、好ましくは０～３０００ｐｐｍ、より好ましくは０～１５００ｐｐｍ、より好ましくは０～３００ｐｐｍ、より好ましくは０～１００ｐｐｍ、より好ましくは０～１０ｐｐｍであることができる In a fenugreek seed processed product having the characteristic (i) above, the weight-based dioscin content, relative to the total weight (dry weight) of the fenugreek seed processed product, can be, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 15000 ppm or less, for example, 300 ppm to 15000 ppm, preferably 1000 ppm to 15000 ppm, preferably 1500 ppm to 15000 ppm, more preferably 3000 ppm to 15000 ppm. In the fenugreek seed processed product having the characteristic (i), the weight-based content of prosapogenin and diosgenin is preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, more preferably 0 to 10 ppm, based on the total weight (dry weight) of the fenugreek seed processed product.
　前記（ｉｉ）の特徴を有するフェヌグリーク種子加工品において、プロサポゲニンの重量基準の含有量の値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば３００ｐｐｍ以上、好ましくは１０００ｐｐｍ以上、好ましくは１５００ｐｐｍ以上、より好ましくは３０００ｐｐｍ以上であり、好ましくは３００００ｐｐｍ以下、より好ましくは１５０００ｐｐｍ以下であり、例えば３００ｐｐｍ～３００００ｐｐｍ、好ましくは３００ｐｐｍ～１５０００ｐｐｍ、好ましくは１０００ｐｐｍ～１５０００ｐｐｍ、好ましくは１５００ｐｐｍ～１５０００ｐｐｍ、より好ましくは３０００ｐｐｍ～１５０００ｐｐｍであるころができる。前記（ｉｉ）の特徴を有するフェヌグリーク種子加工品では更に好ましくは、ジオスシン及びジオスゲニンの重量基準の含有量はそれぞれ、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば１００００ｐｐｍ以下、好ましくは５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよく、例えば０～１００００ｐｐｍ、好ましくは０～５０００ｐｐｍ、好ましくは０～３０００ｐｐｍ、より好ましくは０～１５００ｐｐｍ、より好ましくは０～３００ｐｐｍ、より好ましくは０～１００ｐｐｍ、より好ましくは０～１０ｐｐｍであることができる。 In a fenugreek seed processed product having the characteristic (ii) above, the content of prosapogenin by weight, relative to the total weight (dry weight) of the fenugreek seed processed product, is, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 30,000 ppm or less, more preferably 15,000 ppm or less, for example, 300 ppm to 30,000 ppm, preferably 300 ppm to 15,000 ppm, preferably 1000 ppm to 15,000 ppm, preferably 1500 ppm to 15,000 ppm, more preferably 3000 ppm to 15,000 ppm. In the processed fenugreek seed product having the characteristic (ii), the weight-based content of dioscin and diosgenin is preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm, based on the total weight (dry weight) of the processed fenugreek seed product.
　前記（ｉｉｉ）の特徴を有するフェヌグリーク種子加工品において、ジオスゲニンの重量基準の含有量の値としては、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば３００ｐｐｍ以上、好ましくは１０００ｐｐｍ以上、好ましくは１５００ｐｐｍ以上、より好ましくは３０００ｐｐｍ以上であり、好ましくは３００００ｐｐｍ以下、より好ましくは１５０００ｐｐｍ以下であり、例えば３００ｐｐｍ～３００００ｐｐｍ、好ましくは３００ｐｐｍ～１５０００ｐｐｍ、好ましくは１０００ｐｐｍ～１５０００ｐｐｍ、好ましくは１５００ｐｐｍ～１５０００ｐｐｍ、より好ましくは３０００ｐｐｍ～１５０００ｐｐｍであるころができる。前記（ｉｉｉ）の特徴を有するフェヌグリーク種子加工品では更に好ましくは、ジオスシン及びプロサポゲニンの重量基準の含有量はそれぞれ、フェヌグリーク種子加工品の全重量（乾燥重量基準）に対して、例えば１００００ｐｐｍ以下、好ましくは５０００ｐｐｍ以下、好ましくは３０００ｐｐｍ以下、より好ましくは１５００ｐｐｍ以下、より好ましくは３００ｐｐｍ以下、より好ましくは１００ｐｐｍ以下、より好ましくは１０ｐｐｍ以下であり、検出限界以下（すなわち０ｐｐｍ）であってもよく、例えば０～１００００ｐｐｍ、好ましくは０～５０００ｐｐｍ、好ましくは０～３０００ｐｐｍ、より好ましくは０～１５００ｐｐｍ、より好ましくは０～３００ｐｐｍ、より好ましくは０～１００ｐｐｍ、より好ましくは０～１０ｐｐｍであることができる。 In a fenugreek seed processed product having the characteristic (iii) above, the diosgenin content by weight, relative to the total weight (dry weight) of the fenugreek seed processed product, is, for example, 300 ppm or more, preferably 1000 ppm or more, preferably 1500 ppm or more, more preferably 3000 ppm or more, and preferably 30000 ppm or less, more preferably 15000 ppm or less, for example, 300 ppm to 30000 ppm, preferably 300 ppm to 15000 ppm, preferably 1000 ppm to 15000 ppm, preferably 1500 ppm to 15000 ppm, more preferably 3000 ppm to 15000 ppm. In the processed fenugreek seed product having the characteristic (iii), the weight-based contents of dioscin and prosapogenin are preferably, for example, 10,000 ppm or less, preferably 5,000 ppm or less, preferably 3,000 ppm or less, more preferably 1,500 ppm or less, more preferably 300 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, and may be below the detection limit (i.e., 0 ppm), for example, 0 to 10,000 ppm, preferably 0 to 5,000 ppm, preferably 0 to 3,000 ppm, more preferably 0 to 1,500 ppm, more preferably 0 to 300 ppm, more preferably 0 to 100 ppm, and more preferably 0 to 10 ppm, based on the total weight (dry weight) of the processed fenugreek seed product.
＜１．試験１＞
＜１．１．オカラ麹の調製方法＞
　乾燥オカラ（株式会社やまみ）１０ｇと水６ｇとを均一に混合した。得られた混合物を１２１℃で１５分間オートクレーブ滅菌した。 <1. Test 1>
<1.1. Method for preparing okara koji>
10 g of dried soybean pulp (Yamami Co., Ltd.) and 6 g of water were mixed uniformly, and the mixture was sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記混合物に、麹菌アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）を含む市販の種麹（商品名「白麹１号菌」、株式会社秋田今野商店）８０ｍｇを接種し、撹拌して均一に混合して、オカラ麹原料を得た。 The mixture was allowed to cool after autoclave sterilization, and then inoculated with 80 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain the okara koji raw material.
　前記オカラ麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記オカラ麹原料の発酵を行い、オカラの麹菌による発酵産物（以下「オカラ麹」と称する）を得た。発酵の途中で一回、前記オカラ麹原料を撹拌した。 The container containing the okara koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the okara koji raw material, yielding a fermentation product of the koji mold in the okara (hereinafter referred to as "okara koji"). The okara koji raw material was stirred once during the fermentation.
＜１．２．フェヌグリーク麹の調製方法＞
　フェヌグリーク種子１０ｇと水１５ｇとを混合し、室温で１８時間静置し、フェヌグリーク種子に水を吸収させた。得られた吸水フェヌグリーク種子を１２１℃で１５分間オートクレーブ滅菌した。 <1.2. Method for preparing fenugreek koji>
10 g of fenugreek seeds and 15 g of water were mixed and left to stand at room temperature for 18 hours to allow the fenugreek seeds to absorb water. The obtained water-absorbed fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記吸水フェヌグリーク種子に、麹菌アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）を含む市販の種麹（商品名「白麹１号菌」、株式会社秋田今野商店）２００ｍｇを播種し撹拌して均一に混合して、フェヌグリーク麹原料を得た。 The water-absorbed fenugreek seeds were sterilized in an autoclave and then cooled. Then, 200 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae was seeded and stirred to mix uniformly to obtain a fenugreek koji raw material.
　前記フェヌグリーク麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記フェヌグリーク麹原料の発酵を行い、フェヌグリーク種子の麹菌による発酵産物（以下「フェヌグリーク麹」と称する）を得た。発酵の途中で一回、前記フェヌグリーク麹原料を撹拌した。 The container containing the fenugreek koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the fenugreek koji raw material, yielding a fermentation product of fenugreek seeds by the koji mold (hereinafter referred to as "fenugreek koji"). The fenugreek koji raw material was stirred once during the fermentation.
＜１．３．オカラ麹によるフェヌグリーク種子加工品＞
　粗粉砕したフェヌグリーク種子１０ｇと水１５ｇとを混合し、室温で２時間～１８時間静置して、粗粉砕したフェヌグリーク種子に水を吸収させた。得られた吸水粗粉砕フェヌグリーク種子を１２１℃で１５分間オートクレーブ滅菌した。 <1.3. Processed products from fenugreek seeds using okara koji>
10 g of coarsely ground fenugreek seeds and 15 g of water were mixed and left to stand at room temperature for 2 to 18 hours to allow the coarsely ground fenugreek seeds to absorb water. The obtained water-absorbed coarsely ground fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
　オカラ麹約１６ｇと食塩４ｇとを予め混合した。得られた混合物を、オートクレーブ滅菌後に放冷させた前記吸水粗粉砕フェヌグリーク種子約２５ｇ及び水２０ｇと練るように混ぜ、容器に収容した。前記容器を４０℃の恒温器中に、６１日後まで静置して発酵させ、食塩添加オカラ麹によるフェヌグリーク種子加工品を得た。４日後、８日後、１９日後及び６１日後の各時点でサンプル採取した。 Approximately 16 g of okara koji and 4 g of salt were mixed in advance. The resulting mixture was kneaded with approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been allowed to cool after autoclave sterilization and 20 g of water, and placed in a container. The container was left to ferment for up to 61 days in a 40°C incubator, yielding a fenugreek seed processed product made from salt-added okara koji. Samples were taken at each time point after 4 days, 8 days, 19 days, and 61 days.
　食塩無添加オカラ麹によるフェヌグリーク種子加工品は、オートクレーブ滅菌後に放冷させた前記吸水粗粉砕フェヌグリーク種子約２５ｇと、オカラ麹約１６ｇと、水２０ｇとを、練るように混ぜ、容器に収容し、前記容器を４０℃の恒温器中に、６１日後まで静置して発酵させることにより調製した。４日後、８日後、１９日後及び６１日後の各時点でサンプル採取した。 The fenugreek seed processed product using salt-free okara koji was prepared by kneading approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been sterilized in an autoclave and then cooled with approximately 16 g of okara koji and 20 g of water, placing the mixture in a container, and leaving the container in an incubator at 40°C for up to 61 days to ferment. Samples were taken at each time point after 4 days, 8 days, 19 days, and 61 days.
　比較例の試料（オカラ麹を添加しないフェヌグリーク種子）は、乾燥オカラ（株式会社やまみ）１０ｇと水６ｇとを混合し、得られた混合物をオートクレーブ滅菌し、続いてこの混合物に、オートクレーブ滅菌後に放冷させた前記吸水粗粉砕フェヌグリーク種子約２５ｇと、水２０ｇとを、練るように混ぜて調製した。 The comparative sample (fenugreek seeds without the addition of okara koji) was prepared by mixing 10 g of dried okara (Yamami Co., Ltd.) with 6 g of water, sterilizing the resulting mixture in an autoclave, and then mixing this mixture with approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been allowed to cool after autoclave sterilization, and 20 g of water, in a kneaded manner.
　上記手順により４０℃で４日間、８日間、１９日間及び６１日間処理後の、食塩添加オカラ麹によるフェヌグリーク種子加工品、及び、食塩無添加オカラ麹によるフェヌグリーク種子加工品の試料中の、プロトジオスシン及びジオスゲニン骨格を持つ化合物を、以下の手順により分析した。 　After treatment at 40°C for 4, 8, 19 and 61 days using the above procedure, samples of fenugreek seed processed products made with salt-added okara koji and fenugreek seed processed products made with salt-free okara koji were analyzed for compounds with protodioscin and diosgenin skeletons using the following procedure.
（１）測定試料の調製
　分析対象試料１ｇを１５ｍＬ容試験管に採取し、脱イオン水を９ｍＬ添加した。試験管を振とう器で３０分間撹拌した後、８０％メタノールにて１０００倍希釈した。希釈液を０．２μｍフィルターに通し、バイアルに入れ、測定試料とした。 (1) Preparation of measurement sample 1 g of the sample to be analyzed was placed in a 15 mL test tube, and 9 mL of deionized water was added. The test tube was stirred for 30 minutes with a shaker, and then diluted 1000 times with 80% methanol. The diluted solution was passed through a 0.2 μm filter and placed in a vial to prepare the measurement sample.
（２）検量線用試料の作成
　プロトジオスシン（Ｐｒｏｔｏｄｉｏｓｃｉｎ）（ＣｈｒｏｍａＤｅｘ）標品、ジオスゲニン（Ｄｉｏｓｇｅｎｉｎ）（富士フイルム和光純薬）標品を８０％メタノールで段階希釈し、検量線用試料を作成した。 (2) Preparation of Samples for Calibration Curve Protodioscin (ChromaDex) standard and diosgenin (Fujifilm Wako Pure Chemical Industries) standard were serially diluted with 80% methanol to prepare samples for the calibration curve.
（３）ＬＣＭＳ分析条件
ＬＣ：Ｕｌｔｉｍａｔｅ　３０００（サーモフィッシャーサイエンティフィック）
ＭＳ：Ｑ　Ｅｘａｃｔｉｖｅ　Ｆｏｃｕｓ（サーモフィッシャーサイエンティフィック）
分析カラム：Ｕｎｉｓｏｎ　ＵＫ－Ｃ１８，１５０ｍｍ×３ｍｍ，３μｍ（Ｉｍｔａｋｔ）
カラム温度：４０℃、注入量：２μＬ、流速０．５ｍＬ／ｍｉｎ
移動相Ａ：０．１％ギ酸水（ギ酸、富士フイルム和光純薬）
移動相Ｂ：メタノール（ＬＣＭＳグレード、関東化学） (3) LCMS analysis conditions LC: Ultimate 3000 (Thermo Fisher Scientific)
MS: Q Exactive Focus (Thermo Fisher Scientific)
Analytical column: Unison UK-C18, 150 mm x 3 mm, 3 μm (Imtakt)
Column temperature: 40°C, injection volume: 2 μL, flow rate: 0.5 mL/min
Mobile phase A: 0.1% formic acid water (formic acid, Fujifilm Wako Pure Chemical Industries, Ltd.)
Mobile phase B: Methanol (LCMS grade, Kanto Chemical)
Figure JPOXMLDOC01-appb-T000002
 ＭＳ条件：スプレー電圧３．５ｋＶ、キャピラリー温度２７０℃、ＭＳ　ｓｃａｎ　ｒａｎｇｅ　ｍ／ｚ　６７－１００５、イオン化モード　ＥＳＩポジティブ、コリジョンエネルギー（ＣＥ）５０ｅＶ、ＰＲＭ測定（プリカーサーイオンを下表に示す）
Figure JPOXMLDOC01-appb-T000002
 MS conditions: spray voltage 3.5 kV, capillary temperature 270° C., MS scan range m/z 67-1005, ionization mode ESI positive, collision energy (CE) 50 eV, PRM measurement (precursor ions are shown in the table below).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（４）ＬＣＭＳデータの解析
　ＬＣＭＳのＰＲＭ測定結果から、プロトジオスシン及びジオスゲニン骨格を持つ化合物のプロダクトイオンを抽出し、ピーク面積を算出した。各ピークは、異性体を含む。プロトジオスシンのＬＣでの保持時間は１５．７分、ジオスゲニンのＬＣでの保持時間は２４分であった。ジオスゲニン骨格を持つ化合物は、ＬＣでの保持時間がプロトジオスシンよりも長い、ジオスゲニンの保持時間までの成分のうち、ＬＣＭＳのＰＲＭ測定において、ジオスゲニン骨格に特有の下記のプロダクトイオンが検出された成分である。 (4) Analysis of LCMS data From the LCMS PRM measurement results, product ions of compounds having protodioscin and diosgenin skeletons were extracted and the peak areas were calculated. Each peak includes isomers. The LC retention time of protodioscin was 15.7 minutes, and the LC retention time of diosgenin was 24 minutes. Compounds having a diosgenin skeleton are components that have a longer LC retention time than protodioscin, up to the retention time of diosgenin, and in which the following product ions specific to the diosgenin skeleton were detected in the LCMS PRM measurement.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　プロトジオスシン標品及びジオスゲニン標品を用いて検量線を作成した。得られた検量線に基づき、試料中のプロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度（ｐｐｍ）を算出した。従って、ジオスゲニン骨格を持つ化合物の濃度は、ジオスゲニンとしての換算濃度である。 A calibration curve was prepared using protodioscin and diosgenin samples. Based on the calibration curve, the concentrations (ppm) of compounds with protodioscin and diosgenin skeletons in the samples were calculated. Therefore, the concentrations of compounds with diosgenin skeletons are converted concentrations of diosgenin.
　オカラ麹を添加しないフェヌグリーク種子（比較例）、及び、オカラ麹の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度も同様に測定した。 The concentrations of compounds with protodioscin and diosgenin skeletons in fenugreek seeds without added okara koji (comparative example) and in okara koji were also measured in the same manner.
（５）結果
　４０℃で４日間、８日間、１９日間及び６１日間処理後の、食塩添加オカラ麹によるフェヌグリーク種子加工品、及び、食塩無添加オカラ麹によるフェヌグリーク種子加工品、並びに、オカラ麹を添加しないフェヌグリーク種子（比較例）の試料中の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度（単位：ｐｐｍ）を下記表及び図１に示す。 (5) Results The concentrations (unit: ppm) of compounds having protodioscin and diosgenin skeletons in the samples of the fenugreek seed processed product made with okara koji added with salt, the fenugreek seed processed product made with okara koji without added salt, and the fenugreek seeds without added okara koji (comparative example) after treatment at 40°C for 4 days, 8 days, 19 days, and 61 days are shown in the table below and Figure 1.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　オカラ麹ではプロトジオスシン及びジオスゲニン骨格を持つ化合物は検出されなかった。 No compounds with protodioscin or diosgenin skeletons were detected in okara koji.
　フェヌグリーク種子には苦味の原因となるプロトジオスシンが含まれ、ジオスゲニン骨格を持つ化合物は含まれない（比較例）。オカラ麹によるフェヌグリーク種子の発酵により、プロトジオスシン濃度が低減し、苦味が抑制されることが確認された。オカラ麹によるフェヌグリーク種子の発酵では、プロトジオスシン濃度の低減に伴い、ジオスゲニン骨格を持つ化合物の濃度が上昇した。ジオスゲニン骨格を持つ化合物は、プロトジオスシンの分解により生じるジオスシン、プロサポゲニン、ジオスゲニン等であると推定される。
　食塩を添加しないオカラ麹は、食塩を添加したオカラ麹よりも、フェヌグリーク種子のプロトジオスシンを分解する活性が高いことが確認された。 Fenugreek seeds contain protodioscin, which causes bitterness, but do not contain compounds with a diosgenin skeleton (Comparative Example). It was confirmed that the fermentation of fenugreek seeds with okara koji reduces the concentration of protodioscin and suppresses bitterness. In the fermentation of fenugreek seeds with okara koji, the concentration of compounds with a diosgenin skeleton increased with the reduction in the concentration of protodioscin. Compounds with a diosgenin skeleton are presumed to be dioscin, prosapogenin, diosgenin, etc., which are generated by the decomposition of protodioscin.
It was confirmed that okara koji without added salt had a higher activity in decomposing protodioscin from fenugreek seeds than okara koji with added salt.
（６）ジオスシン、プロサポゲニン及びジオスゲニンの測定
　更に、上記のオカラ麹によるフェヌグリーク種子加工物の、ジオスシン、プロサポゲニン及びジオスゲニンの含有量を以下の手順で測定した。プロトジオスシンの測定方法及び測定結果は上記（５）と同じである。 (6) Measurement of dioscin, prosapogenin and diosgenin The contents of dioscin, prosapogenin and diosgenin in the above-mentioned processed fenugreek seed product using okara koji were measured by the following procedure. The method for measuring protodioscin and the results were the same as those in (5) above.
　ジオスシン、プロサポゲニン及びジオスゲニンはいずれもプロトジオスシンの分解に伴い生じる、ジオスゲニン骨格を持つ化合物である。測定は以下の手順で行った。試料から、上記（１）に記載の手順で測定試料を調製し、（３）に記載の条件でＬＣＭＳ分析を行った。プロトジオスシン、ジオスシン、プロサポゲニン及びジオスゲニンの前駆体イオン（プリカーサーイオン）及びプロダクトイオンを下記表に示す。 Dioscin, prosapogenin and diosgenin are all compounds with a diosgenin skeleton that are produced by the decomposition of protodioscin. Measurements were performed using the following procedure. Measurement samples were prepared from the samples using the procedure described in (1) above, and LCMS analysis was performed under the conditions described in (3). The precursor ions and product ions of protodioscin, dioscin, prosapogenin and diosgenin are shown in the table below.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　検量線用の試料は、プロトジオスシン（Ｐｒｏｔｏｄｉｏｓｃｉｎ）（ＣｈｒｏｍａＤｅｘ）標品、ジオスシン（Ｄｉｏｓｃｉｎ）（ＣｈｒｏｍａＤｅｘ）標品、ジオスゲニン（Ｄｉｏｓｇｅｎｉｎ）（富士フイルム和光純薬）標品を８０％メタノールで段階希釈し、調製した。 The samples for the calibration curve were prepared by serially diluting Protodioscin (ChromaDex) standard, Dioscin (ChromaDex) standard, and Diosgenin (Fujifilm Wako Pure Chemical) standard with 80% methanol.
　ＬＣＭＳのＰＲＭ測定結果から、プロトジオスシン、ジオスシン、プロサポゲニン及びジオスゲニンのプロダクトイオンを抽出し、ピーク面積を算出した。各ピークは、異性体を含む。プロサポゲニンはプロサポゲニンＡ及びプロサポゲニンＢの一方又は両方を含み、プロサポゲニン量はそれらの総量である。 From the LCMS PRM measurement results, product ions of protodioscin, dioscin, prosapogenin and diosgenin were extracted and the peak areas were calculated. Each peak contains isomers. Prosapogenin contains one or both of prosapogenin A and prosapogenin B, and the amount of prosapogenin is the total amount of these.
　前記検量線用試料を用いて作製したプロトジオスシン、ジオスシン及びジオスゲニンの検量線に基づき、試料中のプロトジオスシン、ジオスシン及びジオスゲニンの濃度（ｐｐｍ）を算出した。また、プロサポゲニンの濃度（ｐｐｍ）は、ジオスシンの検量線を用いて、プロサポゲニンのプロダクトイオンのピーク面積値から算出した。 The concentrations (ppm) of protodioscin, dioscin and diosgenin in the samples were calculated based on the calibration curves of protodioscin, dioscin and diosgenin prepared using the calibration curve samples. The concentration (ppm) of prosapogenin was calculated from the peak area value of the product ion of prosapogenin using the calibration curve of dioscin.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
＜１．４．フェヌグリーク麹によるフェヌグリーク種子加工品＞
　粗粉砕したフェヌグリーク種子１０ｇと水１５ｇとを混合し、室温で２時間～１８時間静置して、粗粉砕したフェヌグリーク種子に水を吸収させた。得られた吸水粗粉砕フェヌグリーク種子を１２１℃で１５分間オートクレーブ滅菌した。 <1.4. Fenugreek seed processed products using fenugreek koji>
10 g of coarsely ground fenugreek seeds and 15 g of water were mixed and left to stand at room temperature for 2 to 18 hours to allow the coarsely ground fenugreek seeds to absorb water. The obtained water-absorbed coarsely ground fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
　フェヌグリーク麹約２５ｇと食塩４ｇとを予め混合した。得られた混合物を、オートクレーブ滅菌後に放冷させた前記吸水粗粉砕フェヌグリーク種子約２５ｇ及び水２０ｇと練るように混ぜ、容器に収容した。前記容器を４０℃の恒温器中に３日間静置して発酵を行い、食塩添加フェヌグリーク麹によるフェヌグリーク種子加工品を得た。なお、後述する試験４では、前記吸水粗粉砕フェヌグリーク種子のオートクレーブ滅菌後に６０℃程度以下となるまで放冷したのに対し、本試験における放冷温度は試験４よりも高い。 Approximately 25 g of fenugreek koji and 4 g of salt were mixed in advance. The resulting mixture was kneaded with approximately 25 g of the water-absorbed coarsely ground fenugreek seeds that had been allowed to cool after autoclave sterilization and 20 g of water, and placed in a container. The container was left to stand in an incubator at 40°C for three days to allow fermentation, yielding a fenugreek seed processed product made from salt-added fenugreek koji. Note that in Test 4 described below, the water-absorbed coarsely ground fenugreek seeds were allowed to cool to approximately 60°C or less after autoclave sterilization, whereas the cooling temperature in this test was higher than that in Test 4.
　上記の手順により４０℃で３日間処理後の食塩添加フェヌグリーク麹によるフェヌグリーク種子加工品、及び、上記１．３．に記載の手順により４０℃で３日間処理後の食塩添加オカラ麹によるフェヌグリーク種子加工品の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度を測定した。各成分の測定方法は上記１．３．に記載の通りである。 The concentrations of compounds with protodioscin and diosgenin skeletons were measured in fenugreek seed processed products using salt-added fenugreek koji after treatment for 3 days at 40°C using the procedure described above, and in fenugreek seed processed products using salt-added okara koji after treatment for 3 days at 40°C using the procedure described in 1.3 above. The measurement method for each component is as described in 1.3 above.
　比較のため、フェヌグリーク麹の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度（麹中の濃度）も同様に測定した。 For comparison, the concentrations of compounds with protodioscin and diosgenin skeletons in fenugreek koji (concentration in koji) were also measured in the same manner.
　測定結果を下記表に示す。 The measurement results are shown in the table below.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　フェヌグリーク麹には、苦味の原因となるプロトジオスシンが含まれることが確認された。一方、上記１．３．の比較例で確認された通りフェヌグリーク種子にもプロトジオスシンが含まれる。 It has been confirmed that fenugreek koji contains protodioscin, which causes its bitter taste. On the other hand, as confirmed in the comparative example in 1.3 above, fenugreek seeds also contain protodioscin.
　食塩を添加したフェヌグリーク麹によるフェヌグリーク種子の発酵により、プロトジオスシン濃度が低減し、ジオスゲニン骨格を持つ化合物の濃度が上昇したことが確認された。３日間処理後の時点ではフェヌグリーク麹によるフェヌグリーク種子加工物ではプロトジオスシンは残存していたが、長期間発酵を行った場合は、上記１．３．でのオカラ麹を用いた試験と同様に、プロトジオスシン濃度は検出限界以下になることが、試験４において確認された。 It was confirmed that fermentation of fenugreek seeds with fenugreek koji containing added salt reduced the concentration of protodioscin and increased the concentration of compounds with a diosgenin skeleton. After three days of treatment, protodioscin remained in the fenugreek seed processed product with fenugreek koji, but in Test 4, it was confirmed that when fermentation was continued for a long period of time, the protodioscin concentration fell below the detection limit, as in the test using okara koji in 1.3 above.
　更に、上記の４０℃で３日間処理後のフェヌグリーク麹によるフェヌグリーク種子加工物の、ジオスシン、プロサポゲニン及びジオスゲニンの含有量を以下の手順で測定したところ、ジオスシンが３３ｐｐｍ、プロサポゲニンが７０７ｐｐｍ、ジオスゲニンが５８ｐｐｍであった。なお上記の通りプロトジオスシン含有量は４６２ｐｐｍであった。本試験での各成分の含有量の、試験４での３日間処理後の各成分の含有量との違いは、上記の放冷温度の違いによるものである可能性がある。 Furthermore, the contents of dioscin, prosapogenin and diosgenin in the fenugreek seed processed product using fenugreek koji after three days of treatment at 40°C were measured using the following procedure. The results were 33 ppm dioscin, 707 ppm prosapogenin and 58 ppm diosgenin. As mentioned above, the protodioscin content was 462 ppm. The difference in the content of each component in this test compared to the content of each component after three days of treatment in Test 4 may be due to the difference in the cooling temperature mentioned above.
　更に、上記の４０℃で３日間処理後の食塩添加オカラ麹によるフェヌグリーク種子加工物の、ジオスシン、プロサポゲニン及びジオスゲニンの含有量を以下の手順で測定したところ、ジオスシンが５９１ｐｐｍ、プロサポゲニンが４５ｐｐｍ、ジオスゲニンが３３ｐｐｍであった。なお上記の通りプロトジオスシン含有量は５３４ｐｐｍであった。 Furthermore, the contents of dioscin, prosapogenin and diosgenin in the fenugreek seed processed product using salt-added okara koji after three days of treatment at 40°C were measured using the following procedure. The results were 591 ppm dioscin, 45 ppm prosapogenin and 33 ppm diosgenin. As mentioned above, the protodioscin content was 534 ppm.
　ジオスシン、プロサポゲニン及びジオスゲニンの測定方法は、上記１．３．（６）に記載の通りである。 The method for measuring dioscin, prosapogenin and diosgenin is as described in 1.3. (6) above.
＜２．試験２＞
＜２．１．オカラ麹の調製方法＞
　乾燥オカラ（株式会社やまみ）１０ｇと水６ｇとを均一に混合した。得られた混合物を１２１℃で１５分間オートクレーブ滅菌した。 <2. Test 2>
2.1. Method for preparing okara koji
10 g of dried soybean pulp (Yamami Co., Ltd.) and 6 g of water were mixed uniformly, and the mixture was sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記混合物に、麹菌アスペルギルス・オリゼを含む市販の種麹８０ｍｇを接種し、撹拌して均一に混合して、オカラ麹原料を得た。ここで、麹菌アスペルギルス・オリゼを含む市販の種麹として、「白麹１号菌」（商品名、株式会社秋田今野商店）、「雪こまち」（商品名、株式会社秋田今野商店）、及び、「アグリコン」（商品名、株式会社秋田今野商店）のいずれかを用いた。 The mixture, which had been allowed to cool after autoclave sterilization, was inoculated with 80 mg of commercially available seed koji containing the koji mold Aspergillus oryzae and stirred to mix evenly to obtain the raw material for okara koji. Here, the commercially available seed koji containing the koji mold Aspergillus oryzae was either "Shirokoji No. 1" (product name, Akita Konno Shoten Co., Ltd.), "Yuki Komachi" (product name, Akita Konno Shoten Co., Ltd.), or "Aglycon" (product name, Akita Konno Shoten Co., Ltd.).
　前記オカラ麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記オカラ麹原料の発酵を行い、オカラの麹菌による発酵産物（以下「オカラ麹」と称する）を得た。発酵の途中で一回、前記オカラ麹原料を撹拌した。 The container containing the okara koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the okara koji raw material, yielding a fermentation product of the koji mold in the okara (hereinafter referred to as "okara koji"). The okara koji raw material was stirred once during the fermentation.
＜２．２．フェヌグリーク抽出液の調製方法＞
　フェヌグリーク種子を粗砕し、胚部分を選別し、得られた胚１０ｇに温水９０ｍＬを添加した混合液中で抽出を行った。前記混合液を遠心し、上清部分を回収し、フェヌグリーク抽出液を得た。 2.2. Method for preparing fenugreek extract
Fenugreek seeds were roughly crushed, and the embryo portion was selected. Extraction was performed in a mixture of 10 g of the resulting embryo and 90 mL of warm water. The mixture was centrifuged, and the supernatant was collected to obtain a fenugreek extract.
＜２．３．オカラ麹による、苦味成分プロトジオスシンの分解活性＞
　上記２．１．で調製したオカラ麹０．５ｇを１５ｍＬ容試験管に秤取り、上記２．２．で調製したフェヌグリーク抽出液４．５ｍＬを混合して、反応液とした。得られた反応液を、５５℃の恒温器内で１８時間もしくは３日間静置した。 2.3. Decomposition activity of bitter component protodioscin by okara koji
0.5 g of the okara koji prepared in 2.1 above was weighed into a 15 mL test tube and mixed with 4.5 mL of the fenugreek extract prepared in 2.2 above to prepare a reaction solution. The resulting reaction solution was left to stand in an incubator at 55° C. for 18 hours or 3 days.
　ポジティブコントロール試験：β－グルコシダーゼを含む市販の酵素製剤（商品名「セルラーゼＳＳ」、ナガセケムテックス株式会社）１８．７５μＬと、上記２．２．で調製したフェヌグリーク抽出液４．５ｍＬとを混合した反応液を、５５℃の恒温器内で１８時間もしくは３日間静置した。 Positive control test: A reaction solution was prepared by mixing 18.75 μL of a commercially available enzyme preparation containing β-glucosidase (product name "Cellulase SS", Nagase ChemteX Corporation) with 4.5 mL of the fenugreek extract prepared in 2.2 above, and the mixture was left to stand in an incubator at 55°C for 18 hours or 3 days.
　ネガティブコントロール試験：上記２．２．で調製したフェヌグリーク抽出液４．５ｍＬを、５５℃の恒温器内で１８時間もしくは３日間静置した。 Negative control test: 4.5 mL of the fenugreek extract prepared in 2.2 above was left to stand in an incubator at 55°C for 18 hours or 3 days.
　各試験区の反応液の試料１ｇを１５ｍＬ容試験管に採取し、メタノールを９ｍＬ添加した。試験管を振とう器で３０分間撹拌した後、８０％メタノールにて１０００倍希釈した。希釈液を０．２μｍフィルターに通し、バイアルに入れ、ＬＣ－ＭＳ測定のための測定試料とした。 A 1 g sample of the reaction solution from each test group was placed in a 15 mL test tube, and 9 mL of methanol was added. The test tube was agitated for 30 minutes on a shaker, and then diluted 1000-fold with 80% methanol. The diluted solution was passed through a 0.2 μm filter and placed in a vial to prepare the measurement sample for LC-MS measurement.
　上記１．３．の（２）～（４）に記載の手順により、前記測定試料中のプロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度を測定した。 The concentrations of protodioscin and compounds having a diosgenin skeleton in the measurement sample were measured according to the procedures described in 1.3. (2) to (4) above.
　各試験区の反応液中の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度の測定結果（単位：ｐｐｍ）を下記表及び図２に示す。 The measurement results (unit: ppm) of the concentrations of compounds with protodioscin and diosgenin skeletons in the reaction solution of each test group are shown in the table below and Figure 2.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　上記表及び図２の結果から、オカラ麹は、市販の３種の種麹のいずれを用いて調製した場合でも、フェヌグリーク抽出液中のプロトジオスシンを分解する活性が高いことが確認された。 The results in the table above and Figure 2 confirm that okara koji has high activity in breaking down protodioscin in fenugreek extract, regardless of whether it is prepared using any of the three commercially available types of seed koji.
＜３．試験３＞
＜３．１．フェヌグリーク麹の調製方法＞
　フェヌグリーク種子１０ｇと水１５ｇとを混合し、室温で１８時間静置し、フェヌグリーク種子に水を吸収させた。得られた吸水フェヌグリーク種子を１２１℃で１５分間オートクレーブ滅菌した。 <3. Test 3>
<3.1. Method for preparing fenugreek koji>
10 g of fenugreek seeds and 15 g of water were mixed and left to stand at room temperature for 18 hours to allow the fenugreek seeds to absorb water. The obtained water-absorbed fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記吸水フェヌグリーク種子に、麹菌アスペルギルス・オリゼを含む市販の種麹（商品名「白麹１号菌」、株式会社秋田今野商店）２００ｍｇを播種し撹拌して均一に混合して、フェヌグリーク麹原料を得た。 The water-absorbed fenugreek seeds were cooled after autoclave sterilization, and then 200 mg of commercially available seed koji (product name "Shirokoji No. 1", Akita Konno Shoten Co., Ltd.) containing the koji mold Aspergillus oryzae was seeded and stirred to mix uniformly to obtain a fenugreek koji raw material.
　前記フェヌグリーク麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記フェヌグリーク麹原料の発酵を行い、フェヌグリーク種子の麹菌による発酵産物（以下「フェヌグリーク麹」と称する）を得た。発酵の途中で一回、前記フェヌグリーク麹原料を撹拌した。 The container containing the fenugreek koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the fenugreek koji raw material, yielding a fermentation product of fenugreek seeds by the koji mold (hereinafter referred to as "fenugreek koji"). The fenugreek koji raw material was stirred once during the fermentation.
＜３．２．豆麹の調製方法＞
　一晩吸水させた大豆３０ｇを１２１℃で１５分間オートクレーブ滅菌した。 <3.2. Method for preparing soybean koji>
30 g of soybeans that had been soaked overnight were sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記大豆に、麹菌アスペルギルス・オリゼを含む、上記３．１．で用いた市販の種麹３００ｍｇを接種し、撹拌して均一に混合して、豆麹原料を得た。 The soybeans were allowed to cool after autoclave sterilization, and then inoculated with 300 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and then stirred to mix uniformly to obtain the soybean koji raw material.
　前記豆麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記豆麹原料の発酵を行い、大豆の麹菌による発酵産物（以下「豆麹」と称する）を得た。発酵の途中で一回、前記豆麹原料を撹拌した。 The container containing the soybean koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the soybean koji raw material, yielding a fermentation product of soybeans caused by the koji mold (hereafter referred to as "soybean koji"). The soybean koji raw material was stirred once during the fermentation process.
＜３．３．米糠麹の調製方法＞
　米糠１０ｇと水６ｇとを混合し、得られた混合物を１２１℃で１５分間オートクレーブ滅菌した。 <3.3. Method for preparing rice bran koji>
10 g of rice bran was mixed with 6 g of water, and the resulting mixture was sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記混合物に、麹菌アスペルギルス・オリゼを含む、上記３．１．で用いた市販の種麹８０ｍｇを播種し撹拌して均一に混合して、米糠麹原料を得た。 The mixture was allowed to cool after autoclave sterilization, and then seeded with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain the rice bran koji raw material.
　前記米糠麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記米糠麹原料の発酵を行い、米糠の麹菌による発酵産物（以下「米糠麹」と称する）を得た。発酵の途中で一回、前記米糠麹原料を撹拌した。 The container containing the rice bran koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the rice bran koji raw material, resulting in a fermentation product of the rice bran koji mold (hereinafter referred to as "rice bran koji"). The rice bran koji raw material was stirred once during fermentation.
＜３．４．小麦ふすま麹の調製方法＞
　小麦ふすま１０ｇと水６ｇとを混合し、得られた混合物を１２１℃で１５分間オートクレーブ滅菌した。 <3.4. Method for preparing wheat bran koji>
10 g of wheat bran was mixed with 6 g of water, and the resulting mixture was sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記混合物に、麹菌アスペルギルス・オリゼを含む、上記３．１．で用いた市販の種麹８０ｍｇを播種し撹拌して均一に混合して、小麦ふすま麹原料を得た。 The mixture was allowed to cool after autoclave sterilization, and then seeded with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and stirred to mix uniformly to obtain wheat bran koji raw material.
　前記小麦ふすま麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記小麦ふすま原料の発酵を行い、小麦ふすまの麹菌による発酵産物（以下「小麦ふすま麹」と称する）を得た。発酵の途中で一回、前記小麦ふすま麹原料を撹拌した。 The container containing the wheat bran koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the wheat bran raw material, resulting in a fermentation product of the wheat bran koji mold (hereinafter referred to as "wheat bran koji"). The wheat bran koji raw material was stirred once during fermentation.
＜３．５．ゴマ麹の調製方法＞
　ゴマ１０ｇと水６ｇとを混合し、得られた混合物を１２１℃で１５分間オートクレーブ滅菌した。 <3.5. Method for preparing sesame koji>
10 g of sesame seeds and 6 g of water were mixed, and the resulting mixture was sterilized in an autoclave at 121° C. for 15 minutes.
　オートクレーブ滅菌後に放冷させた前記ゴマに、麹菌アスペルギルス・オリゼを含む、上記３．１．で用いた市販の種麹８０ｍｇを接種し、撹拌して均一に混合して、ゴマ麹原料を得た。 The sesame seeds, which had been left to cool after autoclave sterilization, were inoculated with 80 mg of the commercially available seed koji used in 3.1 above, which contains the koji mold Aspergillus oryzae, and then stirred to mix uniformly to obtain the sesame koji raw material.
　前記ゴマ麹原料を収容した容器を、２日間、３５℃の恒温器内に置き、麹菌を培養して前記ゴマ麹原料の発酵を行い、ゴマの麹菌による発酵産物（以下「ゴマ麹」と称する）を得た。発酵の途中で一回、前記ゴマ麹原料を撹拌した。 The container containing the sesame koji raw material was placed in an incubator at 35°C for two days, and the koji mold was cultivated to ferment the sesame koji raw material, yielding a fermentation product of the sesame koji mold (hereinafter referred to as "sesame koji"). The sesame koji raw material was stirred once during fermentation.
＜３．６．フェヌグリーク抽出液の調製方法＞
　上記２．２．に記載の手順によりフェヌグリーク抽出液を調製した。 3.6. Method for preparing fenugreek extract
A fenugreek extract was prepared according to the procedure described in 2.2 above.
＜３．７．各種麹による、苦味成分プロトジオスシンの分解活性＞
　上記３．１．～３．５．で調製した麹０．５ｇを１５ｍＬ容試験管に秤取り、上記３．６．で調製したフェヌグリーク抽出液４．５ｍＬを混合して、反応液とした。得られた反応液を５５℃の恒温器内で３日間静置した。 <3.7. Decomposition activity of bitter component protodioscin by various koji>
0.5 g of the koji prepared in 3.1 to 3.5 above was weighed into a 15 mL test tube and mixed with 4.5 mL of the fenugreek extract prepared in 3.6 above to prepare a reaction solution. The resulting reaction solution was left to stand in an incubator at 55° C. for 3 days.
　ポジティブコントロール試験：β－グルコシダーゼを含む市販の酵素製剤（商品名「セルラーゼＳＳ」、ナガセケムテックス株式会社）１８．７５μＬと、上記３．６．で調製したフェヌグリーク抽出液４．５ｍＬとを混合した反応液を、５５℃の恒温器内で３日間静置した。 Positive control test: A reaction solution was prepared by mixing 18.75 μL of a commercially available enzyme preparation containing β-glucosidase (product name "Cellulase SS", Nagase ChemteX Corporation) with 4.5 mL of the fenugreek extract prepared in 3.6 above, and allowed to stand in an incubator at 55°C for three days.
　ネガティブコントロール試験：上記３．６．で調製したフェヌグリーク抽出液４．５ｍＬを、５５℃の恒温器内で３日間静置した。 Negative control test: 4.5 mL of the fenugreek extract prepared in 3.6 above was left to stand in an incubator at 55°C for 3 days.
　各試験区の反応液中のプロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度を、上記２．３．に記載の手順により測定した。 The concentrations of protodioscin and compounds with diosgenin skeletons in the reaction solution of each test group were measured using the procedure described in 2.3 above.
　各試験区の反応液中の、プロトジオスシン及びジオスゲニン骨格を持つ化合物の濃度の測定結果を下記表及び図３に示す。ポジティブコントロール試験の反応液と、フェヌグリーク麹とフェヌグリーク種子抽出液との反応液では、プロトジオスシンは検出限界以下であった。 The results of measuring the concentrations of protodioscin and compounds with a diosgenin skeleton in the reaction solutions of each test group are shown in the table below and Figure 3. Protodioscin was below the detection limit in the reaction solutions of the positive control test and in the reaction solutions of fenugreek koji and fenugreek seed extract.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　上記表及び図３の結果から、豆麹、米糠麹、フェヌグリーク麹、小麦ふすま麹及びゴマ麹は、フェヌグリーク抽出液中のプロトジオスシンを分解し、ジオスゲニン骨格を持つ化合物を生成する活性が高く、なかでもフェヌグリーク麹は前記活性が特に高いことが確認された。 The results in the table above and Figure 3 confirm that soybean koji, rice bran koji, fenugreek koji, wheat bran koji and sesame koji have high activity in breaking down protodioscin in fenugreek extract and generating compounds with a diosgenin skeleton, with fenugreek koji having particularly high activity.
　更に、上記の３日間処理後の各試験区の反応液の、プロトジオスシン、ジオスシン、プロサポゲニン及びジオスゲニンの含有量を上記１．３．（６）に記載の手順により測定した。結果を下記表に示す。 Furthermore, the protodioscin, dioscin, prosapogenin and diosgenin contents of the reaction solution of each test group after the above 3-day treatment were measured according to the procedure described in 1.3. (6) above. The results are shown in the table below.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　ポジティブコントロール試験の反応液、及び、米糠麹、豆麹、小麦ふすま麹又はゴマ麹とフェヌグリーク種子抽出液との反応液では、プロトジオスシンの分解物としてはジオスシンが検出され、プロサポゲニン及びジオスゲニンは検出限界以下であった。これに対し、フェヌグリーク麹とフェヌグリーク種子抽出液との反応液では、プロトジオスシンの分解物としてはプロサポゲニンが検出されジオスシン及びジオスゲニンは検出限界以下であった。 In the reaction solution of the positive control test and in the reaction solution of rice bran koji, soybean koji, wheat bran koji, or sesame koji with fenugreek seed extract, dioscin was detected as a decomposition product of protodioscin, while prosapogenin and diosgenin were below the detection limit. In contrast, in the reaction solution of fenugreek koji and fenugreek seed extract, prosapogenin was detected as a decomposition product of protodioscin, while dioscin and diosgenin were below the detection limit.
＜４．試験４＞
＜４．１．フェヌグリーク麹の調製方法＞
　上記１．２．に記載の手順により、フェヌグリーク種子の麹菌による発酵産物（フェヌグリーク麹）を調製した。 <4. Test 4>
<4.1. Method for preparing fenugreek koji>
A fermentation product of fenugreek seeds with Aspergillus oryzae (fenugreek koji) was prepared according to the procedure described in 1.2 above.
＜４．２．フェヌグリーク麹によるフェヌグリーク種子加工品＞
　粗粉砕したフェヌグリーク種子１０ｇと水１５ｇとを混合し、室温で２時間～１８時間静置して、粗粉砕したフェヌグリーク種子に水を吸収させた。得られた吸水粗粉砕フェヌグリーク種子を１２１℃で１５分間オートクレーブ滅菌した。 <4.2. Fenugreek seed processed products using fenugreek koji>
10 g of coarsely ground fenugreek seeds and 15 g of water were mixed and left to stand at room temperature for 2 to 18 hours to allow the coarsely ground fenugreek seeds to absorb water. The obtained water-absorbed coarsely ground fenugreek seeds were sterilized in an autoclave at 121° C. for 15 minutes.
　上記４．１．に記載のフェヌグリーク麹約２５ｇと食塩４ｇとを予め混合した。得られた混合物を、オートクレーブ滅菌後に６０℃程度以下まで放冷させた前記吸水粗粉砕フェヌグリーク種子約２５ｇ及び水２０ｇと練るように混ぜ、容器に収容した。前記容器を４０℃の恒温器中にて３１日後まで静置して発酵を行い、食塩添加フェヌグリーク麹によるフェヌグリーク種子加工品を得た。３日後、１２日後、２０日後及び３１日後の各時点でサンプル採取した。 About 25 g of the fenugreek koji described in 4.1 above was mixed in advance with 4 g of salt. The resulting mixture was kneaded with about 25 g of the water-absorbed coarsely ground fenugreek seeds that had been cooled to about 60°C or less after autoclave sterilization, and 20 g of water, and placed in a container. The container was left to ferment for up to 31 days in a 40°C incubator, and a fenugreek seed processed product was obtained using fenugreek koji with added salt. Samples were taken at each time point after 3 days, 12 days, 20 days, and 31 days.
　一方、上記４．１．に記載のフェヌグリーク麹２５ｇを、オートクレーブ滅菌後に６０℃程度以下まで放冷させた前記吸水粗粉砕フェヌグリーク種子２５ｇ及び水２０ｇと練るように混ぜ、容器に収容した。前記容器を４０℃の恒温器中にて３１日後まで静置して発酵を行い、食無塩添加フェヌグリーク麹によるフェヌグリーク種子加工品を得た。３日後、１２日後、２０日後及び３１日後の各時点でサンプル採取した。 Meanwhile, 25 g of the fenugreek koji described in 4.1 above was mixed with 25 g of the water-absorbed coarsely ground fenugreek seeds that had been cooled to about 60°C or less after autoclave sterilization and 20 g of water, and the mixture was placed in a container. The container was left to stand in an incubator at 40°C for 31 days to allow fermentation, and a fenugreek seed processed product was obtained using salt-free fenugreek koji. Samples were taken at each time point after 3 days, 12 days, 20 days, and 31 days.
　上記の食塩添加フェヌグリーク麹によるフェヌグリーク種子加工品、及び、食塩無添加フェヌグリーク麹によるフェヌグリーク種子加工品の、各時点で採取したサンプルの、プロトジオスシン、ジオスゲニン骨格を持つ化合物、ジオスシン、プロサポゲニン及びジオスゲニンの濃度を測定した。各成分の測定方法は上記１．３．に記載の通りである。 The concentrations of protodioscin, compounds with a diosgenin skeleton, dioscin, prosapogenin and diosgenin were measured for samples taken at each time point from the fenugreek seed processed product made with the above-mentioned fenugreek koji with added salt and the fenugreek seed processed product made with the fenugreek koji without added salt. The measurement method for each component was as described in 1.3 above.
　プロトジオスシン及びジオスゲニン骨格を持つ化合物の測定結果を下記表及び図４に示す。 The measurement results for compounds with protodioscin and diosgenin skeletons are shown in the table below and Figure 4.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　プロトジオスシン、ジオスシン、プロサポゲニン及びジオスゲニンの測定結果を下記表に示す。プロトジオスシンの測定結果は上記表及び図４に示したものである。 The measurement results for protodioscin, dioscin, prosapogenin and diosgenin are shown in the table below. The measurement results for protodioscin are shown in the table above and Figure 4.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　試験１では、フェヌグリーク種子にはプロトジオスシンが含まれ、ジオスゲニン骨格を有する化合物は検出限界以下であることが確認されている。 In Test 1, it was confirmed that fenugreek seeds contain protodioscin, and that compounds with a diosgenin skeleton are below the detection limit.
　試験４では、フェヌグリーク麹によりフェヌグリーク種子を処理する反応において、経時的にプロトジオスシン濃度が低減し、ジオスゲニン骨格を持つ化合物の濃度が上昇したことが示された。プロトジオスシンがジオスゲニン骨格を持つ化合物に転換される速度は、食塩を添加した場合と比較して、食塩を添加しない場合に高い傾向が見られた。また、ジオスゲニン骨格を持つ化合物のうちジオスシンは、３１日間の反応の初期に検出されたが、後期には検出限界以下となったのに対し、プロサポゲニン及びジオスゲニンの濃度は、３１日間の反応の初期に増加し、後期においても維持された。
  In Test 4, it was shown that in the reaction of treating fenugreek seeds with fenugreek koji, the concentration of protodioscin decreased over time, and the concentration of compounds with a diosgenin skeleton increased. The rate at which protodioscin was converted to compounds with a diosgenin skeleton tended to be higher when salt was not added than when salt was added. In addition, among the compounds with a diosgenin skeleton, dioscin was detected in the early stage of the 31-day reaction but was below the detection limit in the later stage, whereas the concentrations of prosapogenin and diosgenin increased in the early stage of the 31-day reaction and were maintained in the later stage.

Claims (17)

  1. 　フェヌグリーク種子と、植物の種子及び／又はその処理物が微生物により発酵された前記微生物を含む発酵産物とを混合して、混合物を得る工程、及び
    　前記混合物を、前記混合物中のプロトジオスシンが分解される温度条件に保持する工程、
    を含む、フェヌグリーク種子加工品の製造方法。     A step of mixing fenugreek seeds with a fermentation product containing the microorganism obtained by fermenting the seeds of a plant and/or a processed material thereof with the microorganism to obtain a mixture; and A step of maintaining the mixture under temperature conditions at which protodioscin in the mixture is decomposed.
    A method for producing a processed fenugreek seed product, comprising:
  2. 　前記植物の種子及び／又はその処理物の糖質の含有量が６５重量％以下である、請求項１に記載の方法。 The method according to claim 1, wherein the carbohydrate content of the plant seeds and/or the processed product thereof is 65% by weight or less.
  3. 　前記植物が、マメ科植物、イネ科植物及びゴマ科植物から選択される１以上である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the plant is one or more selected from the group consisting of legumes, grasses, and sesame plants.
  4. 　前記植物がフェヌグリーク及びダイズから選択される１以上である、請求項３に記載の方法。 The method according to claim 3, wherein the plant is one or more selected from fenugreek and soybean.
  5. 　前記植物がフェヌグリークである、請求項４に記載の方法。 The method of claim 4, wherein the plant is fenugreek.
  6. 　前記植物がダイズであり、
    　前記処理物がオカラである、請求項４に記載の方法。     the plant is soybean,
    The method according to claim 4, wherein the treated material is soybean lees.
  7. 　前記植物がイネであり、
    　前記処理物が米糠である、請求項３に記載の方法。     the plant is rice,
    The method of claim 3, wherein the treated material is rice bran.
  8. 　前記植物がコムギであり、
    　前記処理物が小麦ふすまである、請求項３に記載の方法。     the plant is wheat,
    The method of claim 3, wherein the treated material is wheat bran.
  9. 　前記植物がゴマである、請求項３に記載の方法。 The method of claim 3, wherein the plant is sesame.
  10. 　前記微生物がアスペルギルス属に属する、請求項１～９のいずれか１項に記載の方法。 The method according to any one of claims 1 to 9, wherein the microorganism belongs to the genus Aspergillus.
  11. 　前記温度条件が、１０℃以上６０℃以下の条件である、請求項１～１０のいずれか１項に記載の方法。 The method according to any one of claims 1 to 10, wherein the temperature conditions are 10°C or higher and 60°C or lower.
  12. 　前記植物の種子及び／又はその処理物と、前記微生物とを混合して、発酵原料を得る工程と、
    　前記発酵原料中で前記微生物を培養する工程と、
    を含む、前記発酵産物を調製する工程、
    を更に含む、請求項１～１１のいずれか１項に記載の方法。     Mixing the plant seeds and/or processed products thereof with the microorganism to obtain a fermentation raw material;
    Cultivating the microorganism in the fermentation feedstock;
    preparing the fermentation product,
    The method of any one of claims 1 to 11, further comprising:
  13. 　前記植物の種子及び／又はその処理物が培地を含まない、請求項１２に記載の方法。 The method according to claim 12, wherein the plant seeds and/or the processed products thereof do not contain a culture medium.
  14. 　前記発酵原料中で前記微生物を培養する工程が、前記発酵原料を１０℃以上５０℃以下に保持することを含む、請求項１２又は１３に記載の方法。 The method according to claim 12 or 13, wherein the step of culturing the microorganism in the fermentation raw material includes maintaining the fermentation raw material at a temperature of 10°C or higher and 50°C or lower.
  15. 　プロトジオスシンの重量基準の含有量をＰＤとし、
    　プロトジオスシン以外のジオスゲニン骨格を持つ化合物の、ジオスゲニンとして換算した重量基準の含有量をＤＧとしたとき、
    　ＰＤ／（ＰＤ＋ＤＧ）が０．７８以下である、フェヌグリーク種子加工品。     The weight-based content of protodioscin is designated as PD.
    When the weight-based content of compounds having a diosgenin skeleton other than protodioscin converted into diosgenin is defined as DG,
    A processed fenugreek seed product having a PD/(PD+DG) ratio of 0.78 or less.
  16. 　プロトジオスシン以外のジオスゲニン骨格を持つ前記化合物が、ジオスシン、プロサポゲニン及びジオスゲニンから選択される１以上を含む、請求項１５に記載のフェヌグリーク種子加工品。 The processed fenugreek seed product according to claim 15, wherein the compound having a diosgenin skeleton other than protodioscin includes one or more selected from dioscin, prosapogenin, and diosgenin.
  17. 　プロトジオスシンを分解する活性を有する酵素を産生する能力を有する微生物の死滅細胞又は生存細胞を含有する、請求項１５又は１６に記載のフェヌグリーク種子加工品。
     
          17. A processed fenugreek seed product according to claim 15 or 16, comprising dead or viable cells of a microorganism capable of producing an enzyme having an activity of decomposing protodioscin.
PCT/JP2023/041023 2022-11-18 2023-11-15 Fenugreek seed processed product and method for producing same WO2024106449A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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JP2005318880A (en) * 2004-05-08 2005-11-17 Nippon Medicine:Kk Isoflavone compound-containing solid food and method for producing the same
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Publication number Priority date Publication date Assignee Title
JP2005318880A (en) * 2004-05-08 2005-11-17 Nippon Medicine:Kk Isoflavone compound-containing solid food and method for producing the same
US20120039930A1 (en) * 2009-02-18 2012-02-16 Nestec S.A. Base, products containing the same, preparation methods and uses thereof

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DHULL SANJU BALA; PUNIA SNEH; KUMAR RAJESH; KUMAR MANOJ; NAIN KIRAN BALA; JANGRA KANCHAN; CHUDAMANI CHANCHAL: "Solid state fermentation of fenugreek (Trigonella foenum-graecum): implications on bioactive compounds, mineral content and in vitro bioavailability", JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, SPRINGER (INDIA) PRIVATE LTD., INDIA, vol. 58, no. 5, 1 January 1900 (1900-01-01), India , pages 1927 - 1936, XP037416476, ISSN: 0022-1155, DOI: 10.1007/s13197-020-04704-y *
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