JPWO2014088002A1 - Method for producing ketooctadecadienoic acid - Google Patents

Abstract

The present invention comprises a fermentation process in which a raw material containing linoleic acid or an ester of linoleic acid is used as a substrate to obtain a fermented product by fermentation with a filamentous fungus, and the filamentous fungus is a group consisting of filamentous fungi of the genus Penicillium A method for producing ketooctadecadienoic acid, which is at least one selected from the above, is provided.

Description

  The present invention relates to a method for producing ketooctadecadienoic acid, a fermented product and soy sauce moromi containing a high amount of ketooctadecadienoic acid, and a method for producing these. The present invention also relates to foods and functional foods containing the fermented products or soy sauce moromi.

  In recent years, due to excessive dietary intake around the world, diseases called lifestyle-related diseases such as diabetes, dyslipidemia, hypertension and obesity are increasing. A number of components targeting peroxisome proliferator-activated receptors (PPARs) have been developed as drugs for improving dyslipidemia.

  Conjugated linoleic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the like are known as components targeting PPAR that can be used by being contained in food. However, many conjugated linoleic acids have been chemically synthesized, and there are almost no cases where they are highly contained in foods with high food experience. Moreover, since EPA and DHA have a strong fishy odor, utilization has not expanded.

  In addition, 9-oxo-10,12-octadecadienoic acid contained in the tomato extract (also abbreviated as “9-oxo-ODA” in this specification. Unless otherwise specified, isomers are also included) and A component such as 13-oxo-9,11-octadecadienoic acid (abbreviated as “13-oxo-ODA” in the present specification, including isomers unless otherwise specified) has PPAR activation ability. It is known to have (Patent Document 1).

  It is known that 13-oxo-ODA has a higher PPAR activation ability than conjugated linoleic acid in an in vitro test (Non-patent Document 1).

  However, the content of 9-oxo-ODA contained in the fruit and vegetable tomato is around 1 μg / g per fruit and vegetable tomato although it varies depending on the variety. For this reason, 9-oxo-ODA may not be contained in the processed product depending on the tomato processing method (for example, Non-Patent Documents 2 and 3). Although 13-oxo-ODA has been detected in fruit and vegetable tomatoes and tomato juice, no content has been reported.

JP 2011-184411 A

PLoS ONE, Vol. 7 (2), e31317, 2012 Bioscience, Biotechnology, and Biochemistry, Vol. 75 (8), pp. 1621-1624, 2011 Bioscience, Biotechnology, and Biochemistry, Vol. 76 (3), P2012E2, 2012

  Ketooctadecadienoic acid, such as 9-oxo-ODA and 13-oxo-ODA, is promising as a component targeting PPAR that can be used in foods. It has been demanded.

  So far, methods for producing 9-oxo-ODA and 13-oxo-ODA by chemical synthesis have been known, but they have not been used by incorporating them in foods.

  Then, an object of this invention is to provide the manufacturing method which can manufacture 9-oxo-ODA and 13-oxo-ODA which can be contained and used for a foodstuff in large quantities. Another object of the present invention is to provide fermented products and soy sauce moromi that contain a high amount of 9-oxo-ODA and 13-oxo-ODA, and methods for producing them. Another object of the present invention is to provide a food and a functional food containing the fermented product or soy sauce moromi.

  As a result of intensive studies, the present inventors have found that filamentous fungi of the genus Penicillium and Penicillium have the ability to produce ketooctadecadienoic acids such as 9-oxo-ODA and 13-oxo-ODA. We obtained completely new knowledge that was not known until now. The present invention is based on this novel finding.

  That is, the present invention includes a fermentation process in which a raw material containing linoleic acid or an ester of linoleic acid is used as a substrate to ferment with a filamentous fungus to obtain a fermented product, wherein the filamentous fungus is a group consisting of Aspergillus and Penicillium sp. A method for producing ketooctadecadienoic acid, which is at least one selected from the above, is provided.

  According to the production method of the present invention, ketooctadecadienoic acid (e.g., fermented gonococcus or penicillium fungus) is produced in a fermented product (the koji mold fermented product or the Penicillium fungus product) by causing the filamentous fungus to act on linoleic acid or an ester of linoleic acid. 9-oxo-ODA and 13-oxo-ODA) can be produced in large quantities. Since the above-mentioned filamentous fungi have a high dietary experience, the ketooctadecadienoic acid obtained by the production method of the present invention can be used by containing it in food.

  In the fermentation process, the composition containing the raw material and the filamentous fungus may be stirred. Ketooctadecadienoic acid is produced by adding oxygen to linoleic acid or an ester of linoleic acid. By stirring, oxygen is sufficiently supplied to the composition, and the production efficiency of ketooctadecadienoic acid is further improved.

  Further, the composition may be stirred by ventilation (for example, bubbling). By stirring by aeration, the composition can be easily stirred, and oxygen and the composition come into contact with each other more efficiently, so that the production efficiency of ketooctadecadienoic acid is further improved. .

  In the said manufacturing method, fermentation by the yeast of the said fermented material does not need to progress substantially. The present inventors have also found that ketooctadecadienoic acid produced by Neisseria gonorrhoeae or Penicillium fungi decreases as yeast fermentation proceeds. Therefore, a large amount of ketooctadecadienoic acid can be obtained by collecting the fermented product before the fermentation of the fermented product by yeast substantially proceeds. Here, the yeast may enter the composition from the environment, or may be artificially added to the composition. In either case, the fermented material may be collected before the fermentation of the fermented material by yeast substantially proceeds.

  The raw material may be a raw material containing whole soybeans or a processed product of whole soybeans. Since round soybean contains a large amount of ester of linoleic acid, a large amount of ketooctadecadienoic acid can be obtained by using round soybean or a processed product of whole soybean as a raw material.

  The fermented product may be soy sauce moromi. Since soy sauce moromi contains high-concentration salt, the fermented product of the fermented product is improved and handling becomes easy.

  The present invention also includes a fermentation process in which a raw material containing linoleic acid or an ester of linoleic acid is used as a substrate to ferment with a filamentous fungus to obtain a fermented product, wherein the filamentous fungus is from a filamentous fungus of the genus Penicillium. Provided is a method for producing a fermented product containing a high amount of ketooctadecadienoic acid, which is at least one selected from the group consisting of, and stirs the composition containing the raw material and the filamentous fungus in the fermentation step. According to this production method, a fermentation product (Koji mold fermentation product or Penicillium fungus fermentation product) containing a high amount of ketooctadecadienoic acid can be obtained.

  In the method for producing a fermented product, the raw material may be a raw material containing whole soybeans or a processed product of whole soybeans. Since round soybean contains a large amount of linoleic acid and linoleic acid ester, it is possible to produce a fermented product containing ketooctadecadienoic acid at a higher level by using round soybean or a processed soybean product as a raw material.

  Further, the composition may be stirred by ventilation (for example, bubbling). By stirring by aeration, the above composition can be easily stirred, and oxygen and the above composition come into contact with each other more efficiently, so that a fermentation product containing a higher amount of ketooctadecadienoic acid. Can be manufactured.

  The method for producing the fermented product may further include fermenting the composition or the fermented product with lactic acid bacteria. Lactic acid fermentation improves the flavor of the resulting fermentation product.

  In the method for producing a fermented product, fermentation of the fermented product by yeast may not substantially proceed. Thereby, the fermentation product which contains ketooctadecadienoic acid still more highly can be manufactured.

  The fermentation product may be soy sauce moromi. Since soy sauce moromi contains high-concentration salt, the fermented product has improved antifungal properties and is easy to handle.

  The method for producing ketooctadecadienoic acid and the method for producing a fermentation product described above may further include adding an oxidizing agent containing a metal to the composition containing the raw material and the filamentous fungus, or the fermented product. Good. By adding an oxidizing agent containing a metal, the amount of ketooctadecadienoic acid produced increases.

  The fermented product and soy sauce moromi obtained by the method for producing the fermented product are high in ketooctadecadienoic acid and are fermented products by the above-mentioned filamentous fungi, so that they are included as food or in food. Can be used. Also, based on the ability to activate PPARα by ketooctadecadienoic acid, for example, the fermentation product itself can be used as a functional food to improve dyslipidemia, or the fermentation product can improve dyslipidemia. It can also be used in functional foods.

  The present invention also relates to the genus Penicillium comprising 9-oxo-10,12-octadecadienoic acid of 15 ng / mg or more, or 13-oxo-9,11-octadecadienoic acid of 5 ng / mg or more. Also provided is a fermentation product of at least one filamentous fungus selected from the group consisting of:

  According to the method for producing ketooctadecadienoic acid of the present invention, a large amount of ketooctadecadienoic acid (for example, 13-oxo-ODA and 9-oxo-ODA) can be produced by the action of Aspergillus or Penicillium fungi. . Moreover, since it is based on the action of Aspergillus or Penicillium filamentous fungi with a high dietary experience, it can be used by containing it in food.

  Since the fermented product or soy sauce moromi obtained by the method for producing a fermented product of the present invention contains a high amount of ketooctadecadienoic acid, it can be used, for example, for improving dyslipidemia. Moreover, since it is a fermented product of Aspergillus or Penicillium filamentous fungi with a high dietary experience, it can also be used as, for example, foods and functional foods.

It is the graph which compared 9-oxo-ODA and 13-oxo-ODA content. It is a graph which shows 9-oxo-ODA content in soy sauce moromi. It is a graph which shows 13-oxo-ODA content in soy sauce moromi. It is a graph which shows 9-oxo-ODA content in soy sauce moromi after high temperature fermentation. It is a graph which shows 13-oxo-ODA content in soy sauce moromi after high temperature fermentation.

  Hereinafter, the form for implementing this invention is demonstrated in detail. However, the present invention is not limited to the following embodiments.

  The method for producing ketooctadecadienoic acid of this embodiment includes a step (fermentation step) of fermenting a filamentous fungus with a raw material containing linoleic acid or an ester of linoleic acid as a substrate to obtain a fermented product. The filamentous fungus is at least one selected from the group consisting of Aspergillus and Penicillium.

The ketooctadecadienoic acid is not limited to its structural formula, and includes any ketooctadecadienoic acid having a PPAR activity. For example, 9-oxo-10 (E), 12 (E) -octadecadienoic acid (the following formula I), 9-oxo-10 (E), 12 (Z) -octadecadienoic acid (the following formula II), 9-oxo-10 (E), 12 (E) -octadecadienoic acid, 9-oxo-10 (E), 12 (Z) -octadecadienoic acid, 13-oxo-9 (E), 11 (E ) -Octadecadienoic acid (following formula III), 13-oxo-9 (Z), 11 (E) -octadecadienoic acid (following formula IV), 13-oxo-9 (E), 11 (Z)- Examples include octadecadienoic acid, 13-oxo-9 (Z), 11 (Z) -octadecadienoic acid, and the like. Further, 5-oxo-6,8-octadecadienoic acid, 6-oxo-9,12-octadecadienoic acid, 8-oxo-9,12-octadecadienoic acid, 10-oxo-8,12-octa Examples include decadienoic acid, 11-oxo-9,12-octadecadienoic acid, 12-oxo-9,13-octadecadienoic acid, 14-oxo-9,12-octadecadienoic acid, and the like ( Any of (E, E) body, (E, Z) body, (Z, E) body, and (Z, Z) body may be sufficient.

  As ketooctadecadienoic acid, 9-oxo-ODA and 13-oxo-ODA are preferable from the viewpoint of excellent PPAR activation ability, and the above formula (I), formula (II), formula (III) and formula (IV) are preferred. ) Is more preferable.

  The ketooctadecadienoic acid has PPAR activation ability. PPAR has three known isoforms of α, δ and γ in mammals, and the above-mentioned ketooctadecadienoic acid is known to have at least the ability to activate PPARα and PPARγ. It is known that PPARα regulates the expression of genes related to fatty acid transport and metabolism mainly in the liver and skeletal muscle. Since PPARα is deeply involved in lipid metabolism through such actions, the above-mentioned ketooctadecadienoic acid having PPARα activation ability is effective in improving lipid metabolism disorders. PPARγ is known to be a regulatory factor that governs the differentiation of adipocytes. Therefore, the above-mentioned ketooctadecadienoic acid having the ability to activate PPARγ promotes adipocyte differentiation, thereby reducing blood sugar and free fatty acids, and effective in reducing muscle free fatty acids and improving insulin resistance. It is.

The above-mentioned ketooctadecadienoic acid is considered to be produced from linoleic acid or an ester of linoleic acid by an oxygen addition reaction with lipoxygenase and dehydrogenase, for example, by the route shown in the following scheme I and scheme II. Further, it may be produced by an oxygen addition reaction with oxygenase such as cytochrome P450 or Neisseria gonorrhoeae.

  Therefore, the raw material used as the substrate of the filamentous fungi may contain linoleic acid or an ester of linoleic acid. Examples of linoleic acid include conjugated linoleic acid in addition to linoleic acid. The linoleic acid ester may be any linoleic acid carboxyl group esterified, such as methyl ester, ethyl ester, glycerin fatty acid ester (triacylglycerol, diglycerin fatty acid ester, monoglycerin fatty acid ester, etc. ), Esters with phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, phosphatidylserine, etc.), esters with glyceroglycolipids, and the like.

  As the above raw materials, raw materials including whole soybeans or processed whole soybeans, edible oils (sunflower oil, cottonseed oil, corn oil, soybean oil, sesame oil, walnut oil, grape seed oil, rice bran oil, peanut oil, rapeseed oil, olive oil, Linseed oil, perilla oil, sesame oil, fish oil, lard, palm oil, coconut oil, etc.) or raw materials containing processed products thereof, soy sauce oil or raw materials containing processed products thereof, wheat, peas, broad beans, red beans, lentils, Chickpeas, mung bean, coffee beans, beans, sesame, corn, walnuts, peanuts, buckwheat, poppy, pine nuts, pods, black beans, egoma, olive fruits, cashews, pistachios, lupine beans, sunflower seeds, grape seeds, Tomato seeds, olive seeds, rice bran, or other cereals, beans, nuts or seeds containing linoleic acid, or these It can be used a raw material containing a factory product. Since the content of linoleic acid and the ester of linoleic acid is high and there is a track record of fermentation by koji molds in brewing soy sauce and miso, raw materials containing whole soybeans or whole soybeans may be used. Processed round soybeans include defatted soybeans, soybeans or defatted soybeans that have been heat-denatured, those that have undergone modification treatment such as continuous expansion treatment, airflow expansion treatment, alcohol treatment or extruder, soymilk, soymilk manufacture Okara obtained in the process, soybean hypocotyl and the like can be used.

  Examples of koji molds include koji molds used in soy sauce koji, rice koji, miso, shochu, and red koji. More specifically, for example, Aspergillus oryzae, Aspergillus sojae, Aspergillus tamarii, Aspergillus niger, Aspergillus awamori, Aspergillus usamii, Aspergillus saitoi, Aspergillus glaucus, Aspergillus kawachii, Rhizopus oligosporus, Rhizopus oryzae, Monascus anka, Monascus pilosus, Monascus purpureus, Aspergillus genus, Rhizopus genus or Monascus such as Monascus vitreus, Monascus ruber Mention may be made of bacteria belonging to. Since the brewing aptitude is high, bacteria belonging to the genus Aspergillus may be used. Examples of such bacteria include Aspergillus oryzae and Aspergillus sojae.

  The fungus belonging to the genus Penicillium may be any fungus that has been used as an edible fungus or fermented product. Specific examples include Penicillium camemberti, Penicillium roqueforti, Penicillium candida, and Penicillium glaucum, which have been used in cheese production.

  The above-mentioned Aspergillus and Penicillium fungi may be used singly or in combination of two or more.

  In the production method, koji may be prepared using the koji mold. The koji may be either a solid koji obtained by culturing koji molds in a solid state or a liquid koji obtained by a liquid culture method.

  Solid rice bran includes, for example, heat-denatured protein raw materials such as soybeans, defatted soybeans, soybean cakes, soybean hulls, soybean hypocotyls, and okara, and wheat (wheat, barley, bare wheat, hato oats, wheat bran). 1) or 2 or more types of raw materials selected from those obtained by heating and denaturing starchy raw materials such as rice, and the water content of the mixture is 30-50% (w / After adjusting to w), it can be obtained by inoculating various koji mold seedlings (conidia, spores) and culturing at 20 to 40 ° C. for 1 to 4 days.

  The liquid koji is obtained, for example, by inoculating seed koji into a liquid medium in which water is mixed with the protein raw material, wheat, rice, cereal and other raw materials. There are no particular limitations on the shape of these raw materials, and unwhitened products, whitened products, granular products, powdered products, and the like can be used. The mixing ratio of the raw materials is prepared in a liquid medium in which 2 to 20% (w / v) of various raw materials are added to water. Organic substances and inorganic salts may be added as nutrient sources. Examples of inorganic salts include water-soluble compounds such as ammonium salts, nitrates, potassium salts, acidic phosphates, calcium salts, magnesium salts, iron, and zinc. Examples of organic substances include edible oil, soy sauce oil, yeast extract, peptone, and vitamins. You may add an antifoamer etc. as needed. The liquid medium thus obtained may be sterilized as necessary before inoculating the seed pods. For example, sterilization may be performed at 115 to 130 ° C. for about 5 to 60 minutes under high temperature and pressure.

  In addition, even when using Penicillium filamentous fungi, a cocoon-like material (solid cocoon-like material and liquid cocoon-like material) obtained according to the above-described method for preparing a solid cocoon and a liquid cocoon may be prepared.

  Add the above cocoon (solid cocoon, liquid cocoon) or cocoon-like material (solid cocoon-like material, liquid cocoon-like material) to the raw material that becomes the substrate of the filamentous fungus, or the filamentous material to the raw material that becomes the substrate of the filamentous fungus By incubating a composition (a composition containing the raw material and the filamentous fungus) obtained by directly adding the fungus, fermentation by the filamentous fungus proceeds and ketooctadecadienoic acid can be produced.

  The addition ratio of the raw material and the filamentous fungus in the composition may be appropriately set according to the type of raw material, the type of filamentous fungus, etc., for example, for 1 to 99% (w / w) of the raw material, You may mix | blend so that it may become 99-1% (w / w) of a cocoon or candy-like thing, and it may become 90-20% of cocoon or cocoon-like thing with respect to 10-80% (w / w) of a raw material You may mix | blend. In addition, when adding filamentous fungi directly, you may mix | blend so that it may become 99-0.01% (w / w) of filamentous fungi with respect to 1-99.99% (w / w) of raw materials, for example. The raw material may be blended to 95 to 0.1% with respect to 5 to 99.9% (w / w).

  In the said manufacturing method, you may further add the oxidizing agent containing a metal to the said composition or the said fermented material. The oxidizing agent containing a metal is not particularly limited as long as it promotes the production of ketooctadecadienoic acid. Examples of metal-containing oxidants include metalloproteins with metal ions at the active center, heme compounds such as heme iron, iron sulfate, iron chloride, iron chloride-cysteine, iron compounds, copper sulfate, copper compounds, etc. Can be mentioned. In order to reduce the manufacturing cost, an oxidizing agent containing iron as a metal may be used.

  The addition amount of the oxidizing agent containing a metal can be within a range of 0.01 mM to 1 M, for example, based on the total amount of the composition or the fermented product. Moreover, it may be within the range of 0.2 mM to 500 mM, and may be within the range of 2 mM to 200 mM.

  Moreover, you may add an antioxidant to the said composition or the said fermented product in order to prevent the decomposition | disassembly of ketooctadecadienoic acid by excessive oxidation, and the change of flavor. Examples of antioxidants include ascorbic acid and its derivatives, tocopheres and their derivatives, flavonoids, carotenoids, polyphenols such as lycopene, glutathione, metal chelate components (EDTA, citric acid, etc.), and other radical scavenging components be able to.

  Oxidizing agents and antioxidants containing metals may be added as appropriate in consideration of the production of target components and by-products such as fermentation start, during or after fermentation, and purification / concentration steps.

  The composition may contain ingredients other than the raw material and the filamentous fungus as long as the effects of the present invention are not impaired. Specifically, for example, water, salt, alcohols, saccharides (glucose, fructose), minerals (calcium, iron, zinc, magnesium, etc., and salts thereof), emulsifiers (glycerin fatty acid ester, acetic acid monoglyceride, lactic acid monoglyceride, Citric acid monoglyceride, diacetyltartaric acid monoglyceride, succinic acid monoglyceride, polyglycerin fatty acid ester, polyglycerin condensed linoleic acid ester, kiraya extract, soybean saponin, tea seed saponin, sucrose fatty acid ester, plant lecithin, egg yolk lecithin), pH adjuster (Sodium hydroxide, potassium hydroxide, lactic acid, citric acid, tartaric acid, malic acid, acetic acid, etc.), antifoaming agents (edible oil, soy sauce oil, etc.), nitrogen sources (wheat gluten, soybean-derived purified protein, yeast extract, peptone) ,amino , Enzymatic degradation liquid seasoning), food processing enzyme (protease, mention may be made of peptidase, cellulase, esterase, lipase).

  Examples of esterases include lipases, chlorogenic acid esterases, phytases, phosphorylases, phospholipases, and the like. Lipase produced by Neisseria gonorrhoeae or Penicillium spp. Also produces free linoleic acid and ketooctadecadienoic acid. However, the addition of an enzyme facilitates the release of linoleic acid in the ester form, which is free. The production efficiency of ketooctadecadienoic acid can be improved. As the lipase, commercially available products can be used. For example, lipase MY, lipase OF, lipase PL, lipase QLM, phospholipase D (all manufactured by Meika Sangyo Co., Ltd.), nuclease F, lipase A “Amano” 6, lipase AY “Amano” 30SD, lipase G “Amano” 50, lipase R “Amano”, lipase DF “Amano” 15 and lipase MER “Amano” (all manufactured by Amano Enzyme) may be used.

  The pH of the composition may be in the range of pH 2-12, may be in the range of pH 3-10, and may be in the range of pH 4-9. By making the pH of the composition within this range, the amount of ketooctadecadienoic acid produced is further increased.

  The temperature during fermentation may be in the range of 5 to 70 ° C, in the range of 10 to 60 ° C, or in the range of 15 to 55 ° C. When the temperature is in the range of 5 ° C. to 70 ° C., the metabolic activity of Aspergillus or Penicillium fungi is sufficient, and the amount of ketooctadecadienoic acid produced is further increased.

  The fermentation period can be appropriately set in consideration of the yield of ketooctadecadienoic acid. The fermentation period can be set to a period in which yeast fermentation does not substantially proceed. Moreover, from a viewpoint of the flavor production | generation by ageing | curing | ripening and the yield of ketooctadecadienoic acid, it can also be set to 1 to 240 days, may be 1 to 120 days, and may be 1 to 30 days. When the fermentation period is in this range, ketooctadecadienoic acid can be obtained in high yield.

  Moreover, since ketooctadecadienoic acid is produced from linoleic acid or an ester of linoleic acid by an oxygen addition reaction, it may be fermented under aerobic conditions. In order to maintain the aerobic condition, oxygen or air can be passed through the composition (for example, bubbling), and the composition can be stirred using a propeller stirrer. Aeration or stirring can be performed intermittently or continuously depending on the dissolved oxygen concentration. The dissolved oxygen concentration in the composition may be measured by a known oxygen electrode method or the like.

  In the said manufacturing method, from the viewpoint of the production amount of ketooctadecadienoic acid, the obtained fermented product (gonococcal fermented product or Penicillium fungus fermented product) may be one in which fermentation by yeast does not substantially proceed. .

  As specifically shown in the examples described below, the amount of ketooctadecadienoic acid produced decreases significantly as fermentation by yeast proceeds. Although this mechanism is not necessarily clear, the present inventors presume as follows. In other words, when yeast is vigorously grown by aeration fermentation, linoleic acid is assimilated by yeast metabolism, or an ethyl ester bond between alcohol components such as ethanol produced by yeast and linoleic acid or linoleic acid oxide is generated. The target component is considered to decrease. In conventional soy sauce moromi and miso, yeast fermentation is essential or strongly demanded from the viewpoint of flavor, and it inhabits the environment by adding cultured salt-tolerant yeast or without adding cultured yeast. It is considered that the target component disappeared or was significantly reduced by the growth of the wild salt-tolerant yeast.

  Here, the fact that the fermentation by yeast is not substantially progressing can be confirmed using the amount of ethanol, which is a typical metabolite of yeast, as an index. The amount of ethanol in the composition can be measured using, for example, a gas chromatographic method.

  When ethanol derived from yeast fermentation of a predetermined amount or more is detected in the composition, it may be determined that fermentation by yeast has substantially advanced and the fermentation may be terminated. For example, it can be determined that fermentation by yeast has substantially progressed when the ethanol concentration in the composition exceeds 3.0% (w / v). The ethanol concentration serving as an index may be appropriately set within a range of 3.0% w / v or less depending on the purpose and application, for example, 3.0% (w / v). It may be 5% (w / v) or 0.1% (w / v). In order to improve the antifungal property, ethanol can be added to the composition in advance. In such a case, the ethanol concentration as an index may be appropriately corrected according to the ethanol concentration.

  Alternatively, the ketooctadecadienoic acid content in the composition may be directly measured, and it may be determined that fermentation by yeast has substantially progressed using the ketooctadecadienoic acid content as an index. Ketooctadecadienoic acid content is obtained by, for example, uniformly pulverizing the composition or the fermented product, freeze-drying, and then extracting with an organic solvent (for example, chloroform-methanol (volume ratio 2: 1)). -Can be quantified by MS / MS analysis.

  The method for producing ketooctadecadienoic acid may further include a step of purifying ketooctadecadienoic acid from the fermented product. Purification of ketooctadecadienoic acid can be performed by a known method. For example, separation and purification can be performed after solvent extraction. As the solvent, alcohols such as methanol and ethanol, n-hexane, acetone, ethyl acetate, methyl acetate, and supercritical carbon dioxide can be used. The extraction can be performed by a known method such as physical stirring / crushing, solid-liquid extraction, ultrasonic treatment, extraction by reflux, immersion, leaching, decoction, microwave treatment, and the like. The solvent-treated one can be used as it is, but may be further purified by activated carbon treatment, chromatography, liquid-liquid distribution, distillation, gel filtration, microfiltration, or the like. Moreover, you may isolate | separate by collect | recovering the oil components which float by filtering and pressing a gonococcus fermented product, and leaving or centrifuging the obtained filtrate. More simply, ketooctadecadienoic acid is efficiently concentrated by, for example, a method in which the fermented product is allowed to stand and the floating oil layer is recovered, or a method in which the upper layer separated from the aqueous layer is recovered after low-temperature storage. Can do. You may perform said refinement | purification further from the isolate | separated oil.

  The method for producing ketooctadecadienoic acid described above can also be referred to as a method for producing a fermentation product containing a high amount of ketooctadecadienoic acid. Here, the fermented product may be the fermented product itself, or may be a product obtained by further processing the fermented product.

  Here, “fermented product containing a high amount of ketooctadecadienoic acid” means, for example, 9-oxo-10,12-octadecadienoic acid of 15 ng / mg or more or 13-oxo-per unit weight of the fermented product. It can be said to be a fermentation product containing 5 ng / mg or more of 9,11-octadecadienoic acid. As specifically shown in the below-mentioned Examples, 9-oxo-10,12-octadecadienoic acid and 13-oxo-9,11-octadecadienoic acid contained in conventional fermented koji molds are Below the numerical range.

  Therefore, the fermentation product according to one embodiment of the present invention is a fermentation product of at least one filamentous fungus selected from the group consisting of Aspergillus and Penicillium fungi, and per unit weight, 9-oxo-10, It contains 15 ng / mg or more of 12-octadecadienoic acid or 5 ng / mg or more of 13-oxo-9,11-octadecadienoic acid. The fermentation product according to one embodiment is a fermentation product of at least one filamentous fungus selected from the group consisting of Aspergillus and Penicillium fungi, and per unit weight, 9-oxo-10,12-octa It may contain 15 ng / mg or more of decadienoic acid and 5 ng / mg or more of 13-oxo-9,11-octadecadienoic acid.

  9-oxo-10,12-octadecadienoic acid content per unit weight is 20 ng / mg or more, 25 ng / mg or more, 30 ng / mg or more, 35 ng / mg or more, 40 ng / mg or more, 45 ng / mg or more, It may be 50 ng / mg or more. Although there is no restriction | limiting in particular in an upper limit, For example, 1,000,000 ng / mg may be sufficient and 500,000 ng / mg may be sufficient. Accordingly, the 9-oxo-10,12-octadecadienoic acid content per unit weight may be 15 ng / mg to 1,000,000 ng / mg, and 20 ng / mg to 1,000,000 ng / mg. 25 ng / mg to 1,000,000 ng / mg, 30 ng / mg to 1,000,000 ng / mg, 35 ng / mg to 1,000,000 ng / mg. 40 ng / mg to 1,000,000 ng / mg, 45 ng / mg to 1,000,000 ng / mg, or 50 ng / mg to 1,000,000 ng / mg. Good. Also, it may be 15 ng / mg to 500,000 ng / mg, 20 ng / mg to 500,000 ng / mg, 25 ng / mg to 500,000 ng / mg, 30 ng / mg to 500, 000 ng / mg, 35 ng / mg to 500,000 ng / mg, 40 ng / mg to 500,000 ng / mg, 45 ng / mg to 500,000 ng / mg, It may be 50 ng / mg to 500,000 ng / mg.

  The 13-oxo-9,11-octadecadienoic acid content per unit weight is 6 ng / mg or more, 7 ng / mg or more, 8 ng / mg or more, 9 ng / mg or more, 10 ng / mg or more, 15 ng / mg or more, It may be 20 ng / mg or more. The upper limit is not particularly limited, but may be, for example, 1,000,000 ng / mg, 500,000 ng / mg, 100,000 ng / mg, or 50,000 ng / mg. It may be. Accordingly, the 13-oxo-9,11-octadecadienoic acid content per unit weight may be 5 ng / mg to 1,000,000 ng / mg, and 6 ng / mg to 1,000,000 ng / mg. 7 ng / mg to 1,000,000 ng / mg, 8 ng / mg to 1,000,000 ng / mg, and 9 ng / mg to 1,000,000 ng / mg. 10 ng / mg to 1,000,000 ng / mg, 15 ng / mg to 1,000,000 ng / mg, or 20 ng / mg to 1,000,000 ng / mg. Good. Also, it may be 5 ng / mg to 500,000 ng / mg, 6 ng / mg to 500,000 ng / mg, 7 ng / mg to 500,000 ng / mg, 8 ng / mg to 500, 000 ng / mg, 9 ng / mg to 500,000 ng / mg, 10 ng / mg to 500,000 ng / mg, 15 ng / mg to 500,000 ng / mg, It may be 20 ng / mg to 500,000 ng / mg. Also, it may be 5 ng / mg to 100,000 ng / mg, 6 ng / mg to 100,000 ng / mg, 7 ng / mg to 100,000 ng / mg, 8 ng / mg to 100, 000 ng / mg, 9 ng / mg to 100,000 ng / mg, 10 ng / mg to 100,000 ng / mg, 15 ng / mg to 100,000 ng / mg, It may be 20 ng / mg to 100,000 ng / mg. Also, it may be 5 ng / mg to 50,000 ng / mg, 6 ng / mg to 50,000 ng / mg, 7 ng / mg to 50,000 ng / mg, 8 ng / mg to 50, 000 ng / mg, 9 ng / mg to 50,000 ng / mg, 10 ng / mg to 50,000 ng / mg, 15 ng / mg to 50,000 ng / mg, It may be 20 ng / mg to 50,000 ng / mg.

  In the method for producing ketooctadecadienoic acid and the method for producing the fermentation product, the obtained fermentation product may be soy sauce moromi. That is, using soybeans or raw materials containing whole soybeans as a substrate, soy sauce koji may be prepared and fermented with koji mold.

  The soy sauce cake should just be a soy sauce cake obtained according to a well-known soy sauce brewing method. Soy sauce lees are, for example, those obtained by heat-denaturing protein raw materials such as soybeans, defatted soybeans, and wheat (barley, barley, bare oats, hato oats) fried and cracked, or starches such as rice The raw material is heat-denatured and mixed, and the water content of the mixture is adjusted to 30-50% (w / w), and then seeded with Aspergillus oryzae, Aspergillus sojae, etc. It can be obtained by culturing at 1 to 4 days.

  The heat denaturation of the protein raw material may be performed by steaming, but is not limited to this, and denaturation treatment such as continuous expansion treatment, airflow expansion treatment, alcohol treatment, and extruder can be used.

  The heat denaturation of wheat may be carried out by fried rice or cracking, but is not limited to this, and denaturation treatment such as continuous expansion treatment, air flow type expansion treatment, alcohol treatment, and extruder can be used. . Heat denaturation of rice may be performed by steaming or cooking.

  The mixing ratio of protein raw materials and starch raw materials such as wheat and / or rice is not particularly limited, and is used in koikuchi soy sauce, thin soy sauce, tamari soy sauce, white soy sauce, etc., as defined by Japanese Agricultural and Forestry Standards. The mixing ratio can be used. For example, the ratio of protein raw material: starchy raw material = 30 to 70%: 70 to 30% (v / v) can be given as the blending ratio.

  Soy sauce mash (fermentation initial stage) can be obtained by adding saline to the soy sauce cake. The concentration of the salt solution to be added may be, for example, a concentration such that the salt concentration of soy sauce moromi is 1 to 20% (w / v), or a concentration that is 8 to 18% (w / v). The concentration may be 12 to 18% (w / v).

  Soy sauce moromi may be further fermented with Aspergillus in order to further increase the content of ketooctadecadienoic acid. Specific examples of the fermentation conditions are as described above.

  Soy sauce moromi may be added with or without lactic acid bacteria, and may be subjected to lactic acid fermentation as needed from the viewpoint of improving antifungal properties and flavor. Even when lactic acid bacteria are not added, lactic acid fermentation can be performed by growth of lactic acid bacteria in the environment. Examples of soy sauce lactic acid bacteria used for lactic acid fermentation include salt-tolerant lactic acid bacteria such as Tetragenococcus halophilus used in soy sauce brewing. The soy sauce moromi at the start of lactic acid fermentation is usually at pH 5.8 to 6.3, and the soy sauce moromi after completion of lactic acid fermentation is usually at pH 4.6 to 5.3. In addition, lactic acid fermentation can be implemented not only in the case of soy sauce moromi but also in the manufacturing method according to the above-described embodiment.

  The soy sauce moromi, which has finished the production of ketooctadecadienoic acid under aerobic conditions, may be further left still and aged under anaerobic conditions where salt-resistant yeast is not growing. If it is normal soy sauce moromi, it will be fermented and matured for 6 months to 1 year or more to produce the color and flavor of soy sauce. In the present embodiment, fermentation and ripening may be carried out in a state where the growth of salt-tolerant yeast and alcohol fermentation are prevented so that ketooctadecadienoic acid does not disappear. In addition to ketooctadecadienoic acid, in addition to ketooctadecadienoic acid, it is also possible to take into account components produced by Maillard reaction during fermentation and fermentation with soy sauce lactic acid bacteria and aging.

  Since the fermented product or soy sauce moromi obtained by the above production method contains a high amount of ketooctadecadienoic acid having PPAR activation ability, for example, diabetes, obesity, dyslipidemia, insulin resistance, hyperlipidemia Can be used to prevent or ameliorate symptom, arteriosclerosis and coronary artery disease. In addition, the fermented products or soy sauce moromi are fermented by koji mold or Penicillium filamentous fungi with a high dietary experience, so they are used as foods, functional foods, etc. or included in foods, functional foods, etc. can do.

  Examples of foods and functional foods include miso, moromi-flavored seasonings, soy sauce, soy sauce processed products, mirin, soy sauce, sauce, Japanese-style broth, Western-style broth, Chinese broth, dressing, ketchup, tomato sauce, pasta sauce, Worcester sauce and Other seasonings such as sauces, breads, cakes / confectionery, noodles, jellies, frozen foods, retort foods, freeze-dried foods, ice creams, dairy products, soups, tofu, milk fermented foods, fermented soy milk foods In addition to various processed foods such as soybean fermented food, examples of supplement forms include tablet tablets, tablets, granules, capsules, and syrups. Examples of the drink include soy milk, fruit juice drink, carbonated drink, tea drink, near water, sports drink, milk drink, alcoholic drink, soft drink and the like. Examples of edible oils include cooking oils, mayonnaise, margarine and other processed oils and fats.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

<Test Example 1>
[Preparation of various food samples]
As various food samples, commercially available tomato juice and various fermented products (miso and natto) were prepared. “Kagome Tomato Juice” (manufactured by Kagome), “Del Monte Tomato Juice” (manufactured by Nippon Del Monte), and “TOP VALUE Tomato Juice” (manufactured by AEON Co., Ltd.) were purchased as commercially available tomato juices. As for miso, “additive-free koji miso” (manufactured by Hanamaruki Co., Ltd.) as commercial miso 1, “additive-free kodama” (manufactured by Hikari Miso) as commercial miso 2, and “okame natto” (takano foods) as commercial natto 1 ), “Kume Natto” (manufactured by Mitsukan) as commercial natto 2 and “Hone Genki” (manufactured by Mitsukan) as commercial natto 3 were purchased. Reference Example 1 for Tomato Juice, Reference Example 4 for Commercial Miso 1, Reference Example 5 for Commercial Miso 2, Reference Example 6 for Commercial Natto 1, Reference Example 7 for Commercial Natto 2, and Reference Example 8 for Commercial Natto 3

[Preparation of soy sauce cake]
A soy sauce cake was prepared according to a conventional method. 10 kg of soybean (round soybean) was immersed in warm water to absorb water, and then steamed under pressure. After fried 10 kg of wheat, it was cracked. The obtained soybean and wheat were mixed to prepare a koji-making material having a water content of about 40% (w / w). The raw material for koji making was inoculated with Aspergillus sojae, and koji was made with a ventilating kneader, and a soy sauce koji was obtained after three days. When preparing soy sauce cake with defatted soybean, it was carried out under the same conditions to obtain defatted soybean soy sauce cake. Hereinafter, “soy sauce cake” is prepared from whole soybeans, and soy sauce cake prepared from defatted soybeans is referred to as “defatted soybean sauce”.

[Preparation of soy sauce moromi 1]
(Examples 1-3)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. Lactic acid bacteria and yeast were not added and mixed well by aeration. The moromi product temperature was maintained at 15 to 20 ° C., and fermentation with koji molds was performed. The soy sauce moromi just after preparation was set as Example 1, the soy sauce moromi after 14 days was set as Example 2, and the soy sauce moromi after 28 days was set as Example 3.

(Examples 4 to 5)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. According to a conventional method, Tetragenococcus halophilus was added as a lactic acid bacterium so that the initial concentration would be 1 × 10 ⁵ cells / ml, and mixed well by aeration. The moromi product temperature was maintained at 15 to 20 ° C., and fermentation with koji mold and lactic acid fermentation were performed. The soy sauce moromi which passed 14 days was set as Example 4, and the soy sauce moromi which passed 28 days was set as Example 5.

(Examples 6 to 7)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. According to a conventional method, Tetragenococcus halophilus as a lactic acid bacterium is added so that the initial concentration is 1 × 10 ⁵ cells / ml, and Zygosaccharomyces rouxii is added as a salt-resistant soy sauce yeast so that the initial concentration is 1 × 10 ⁶ cells / ml. Mix well by aeration. The moromi product temperature was maintained at 15 to 20 ° C., and fermentation with koji mold and lactic acid fermentation were performed. The soy sauce moromi which passed 14 days was set as Example 6, and the soy sauce moromi which passed 28 days was set as Example 7.

(Example 8 and Reference Example 2)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. According to a conventional method, Tetragenococcus halophilus was added as a lactic acid bacterium so that the initial concentration was 1 × 10 ⁵ cells / ml, and mixed well by aeration. The moromi product temperature was maintained at 15 to 20 ° C., and fermentation with koji mold and lactic acid fermentation were performed for 1 month. Subsequently, according to a conventional method, Zygosaccharomyces rouxii was added as a salt-resistant soy sauce yeast so that the initial concentration was 1 × 10 ⁶ pieces / ml, and aerated and stirred for 14 days while maintaining the moromi product temperature at 20 to 25 ° C., Yeast fermentation was performed (fermentation aging for a total of 45 days). Example 8 was soy sauce moromi after yeast fermentation. Further, the moromi product temperature was maintained at 25 to 30 ° C. and fermented and aged. The aging soy sauce moromi obtained after 2.5 months was used as Reference Example 2 (fermentation aging for a total of 4 months).

(Reference Example 3)
840 mL of 30% (w / v) saline and 280 mL of water were added to 800 g of defatted soybean soy sauce cake so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. . According to a conventional method, Tetragenococcus halophilus was added as a lactic acid bacterium so that the initial concentration was 1 × 10 ⁵ cells / ml, and mixed well by aeration. The moromi product temperature was maintained at 15 to 20 ° C., and fermentation with koji mold and lactic acid fermentation were performed for 1 month. Subsequently, according to a conventional method, Zygosaccharomyces rouxii was added as a salt-resistant soy sauce yeast so that the initial concentration was 1 × 10 ⁶ pieces / ml, and aerated and stirred for 14 days while maintaining the moromi product temperature at 20 to 25 ° C., Yeast fermentation was performed. Further, the moromi product temperature was maintained at 25 to 30 ° C. and fermented and aged. Aged soy sauce moromi obtained after 2.5 months was used as Reference Example 3 (fermented aging for a total of 4 months).

(Examples 9 to 10)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. Lactic acid bacteria and yeast were not added, they were mixed well by aeration, and immediately sterilized by heating at 80 ° C. for 1 hour. After the heat sterilization, the soy sauce moromi which was kept at 15-20 ° C. and allowed to stand for 14 days was designated as Example 9, and the soy sauce moromi which was allowed to stand for 28 days was designated as Example 10.

[Preparation of soy sauce moromi 2 (high-temperature fermentation)]
(Examples 11 to 12)
To 800 g of soy sauce cake, 840 mL of 30% (w / v) saline and 280 mL of water were added so that the salt concentration after fermentation and ripening was about 16% (w / v), and charged into a 5 L resin container. Lactic acid bacteria and yeast were not added and mixed well by aeration. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring. The soy sauce moromi obtained after 3 days was taken as Example 11. Also, after 3 days (the same day that the soy sauce moromi of Example 11 was obtained), the moromi product temperature was lowered to 25-30 ° C., lactic acid bacteria and yeast were not added, and the moromi product temperature was kept at 25-30 ° C. Example 12 was soy sauce moromi ripened for 1 month (31 days).

(Example 13)
In the same manner as in Examples 11 to 12, the moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring. Subsequently, after 3 days (the same day that the soy sauce moromi of Example 11 was obtained), the moromi product temperature was lowered to 25-30 ° C., and the initial concentration of Tetragenococcus halophilus as lactic acid bacteria was 1 × 10 ⁵ cells / ml. The soy sauce moromi which was added and aged for 1 month (31 days) while keeping the moromi product temperature at 25 to 30 ° C. was defined as Example 13.

(Reference Example 9)
In the same manner as in Examples 11 to 12, the moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring. Subsequently, after 3 days (the same day that the soy sauce moromi of Example 11 was obtained), the temperature of the moromi product was lowered to 30 ° C., and the salt concentration of Tetragenococcus halophilus as lactic acid bacteria was adjusted to 1 × 10 ⁵ / ml. Zygosaccharomyces rouxii as a soy soy yeast is added so that the initial concentration is 1 × 10 ⁶ / ml, and the yeast fermentation by aeration is carried out for 1 month (31 days) while keeping the temperature of the moromi product at 25-30 ° C. The soy sauce moromi taste was used as Reference Example 9.

(Reference Example 10)
In the same manner as in Examples 11 to 12, the moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring. Subsequently, after 3 days (the same day that the soy sauce moromi of Example 11 was obtained), the moromi product temperature was lowered to 25-30 ° C., and Zygosaccharomyces rouxii as the salt-resistant soy sauce yeast had an initial concentration of 1 × 10 ⁶ / ml. The soy sauce moromi | flavor which performed yeast fermentation by aeration for 1 month (31 days) was added as reference example 10 with adding so that it might become, and holding | maintaining moromi product temperature at 25-30 degreeC.

[Quantification of 9-oxo-ODA and 13-oxo-ODA]
9-oxo-ODA and 13-oxo-ODA in each sample were quantified by LC-MS / MS analysis. The specific procedure is as follows.

  9-oxo-10 (E), 12 (Z) -octadecadienoic acid and 13-oxo-9 (Z), 11 (E) -octadecadienoic acid manufactured by Cayman are used as preparations. As other reagents, special grade reagents manufactured by Wako Pure Chemical Industries, Ltd. were used.

  The fermented koji mold, soy sauce moromi, miso, and natto were crushed and homogenized with a mixer, and then lyophilized. 10 mg of the obtained sample was weighed into a 1.5 mL microtube. 0.5 mL chloroform-methanol (volume ratio 2: 1) was added to this, and it stirred vigorously for 10 to 20 seconds with the handy type homogenizer (T10 basic by IKA), and the sample was disperse | distributed. Subsequently, ultrasonic waves were irradiated for 7.5 minutes (interval 7.5 minutes (15.0 minutes in total)) with Cosmo Bio's BIORUPTOR. Centrifugation (15,000 rpm, 5 minutes), and the supernatant was transferred to a 2 mL microtube. A series of operations relating to extraction for collecting the supernatant from chloroform-methanol addition was performed twice to obtain about 1.0 mL of the supernatant. Using spo201U manufactured by Thermo, it was dried by concentration centrifugation. 1.0 mL of ethanol was added and redissolved. The redissolved sample was subjected to LC-MS / MS analysis.

  In addition, 9-oxo-ODA and 13-oxo-ODA in tomato juice and commercially available soymilk were lyophilized from commercial products, and 10 mg of a sample was weighed in the same manner as described above, and subjected to LC-MS / MS analysis in the same manner. Provided. In Reference Example 1, the average value of the commercially available tomato juice was calculated.

(LC-MS / MS analysis conditions)
LC conditions:
Mobile phase A; water (containing 0.1% formic acid)
Mobile phase B; acetonitrile (containing 0.1% formic acid)
Gradient conditions: Mobile phase A 50% (0 minutes) -80% (14 minutes) -99% (17-18 minutes) -50% (19 minutes)
Column temperature: 50 ° C
Column; YMC-Triart C18 (100 × 2.0 mm, 1.9 m)
Flow rate; 0.3 mL / min injection volume; 5 μL
1 analysis time; 38 minutes

MS conditions:
Scans in Period; 1069
Relative Start Time; 1100.00 msec
Scan Type; MRM
Polarity; Negative

MRM conditions:
・ 9-oxo-ODA
Q1mass Q3mass Dwell (msec)
293.10 185.20 500.00
・ 13-oxo-ODA
Q1mass Q3mass Dwell (msec)
293.30 113.00 500.00

[Quantification of ethanol]
The amount of ethanol contained in fermented koji mold, soy sauce moromi, and soy milk was determined by gas chromatography according to the method described in the soy sauce test method (founded by the Japan Soy Sauce Research Institute, published on March 1, 1985). Was quantified.

〔result〕
Tables 1 and 2 below show the results of quantification of 9-oxo-ODA and 13-oxo-ODA in various samples. In the table, “presence / absence of aeration” and “presence / absence of koji” mean “whether or not aeration was used during fermentation” and “whether or not koji mold was used” in the production process.

〔result〕
In tomato juice, the average 9-oxo-ODA content per unit mass of tomato juice was 7 ng / mg, and the average 13-oxo-ODA content was 2 ng / mg (Reference Example 1). The contents of 9-oxo-ODA and 13-oxo-ODA in soy sauce moromi using defatted soybeans were also comparable to tomato juice (Reference Example 3). On the other hand, in soy sauce moromi using whole soybeans, a very high content is shown immediately after the soy sauce moromi is charged (Example 1), and becomes maximum when fermented for 14 days after the charging. The oxo-ODA content was an average of 88 ng / mg, and the 13-oxo-ODA content was an average of 32 ng / mg (Example 6). Moreover, 9-oxo-ODA and 13-oxo-ODA content fell significantly as yeast fermentation progressed (Example 8). Furthermore, yeast fermentation progressed, and in soy sauce moromi which passed through the ripening process, the content of 9-oxo-ODA and 13-oxo-ODA was comparable to that of tomato juice (Reference Example 2).

  Until now, there was no idea to use soy sauce moromi before the initial stage of fermentation to the start of yeast fermentation. However, as is clear from the results of Test Example 1, 9-oxo-ODA and 13-oxo-ODA are contained in a high content in the soy sauce moromi from the initial charging stage to before the start of yeast fermentation in earnest. It has been revealed by the present invention for the first time that the utility value is high.

  FIG. 1 is a graph showing 9-oxo-ODA and 13-oxo-ODA contents of Reference Examples 1 to 3, Example 1, and Examples 6 to 8. In FIG. 1, “maru soybean moromi” means “round soybean soy sauce moromi” and “defatted moromi” means “defatted soybean soy moromi”.

  FIG. 2 is a graph showing the 9-oxo-ODA content in the soy sauce moromi of Examples 1 to 5, 9 and 10. FIG. 3 is a graph showing the 13-oxo-ODA content in the soy sauce moromi of Examples 1 to 5, 9 and 10. FIG. 4 is a graph showing the 9-oxo-ODA content in the soy sauce moromi of Examples 1, 11 to 13 and Reference Examples 9 to 10. FIG. 5 is a graph showing the 13-oxo-ODA content in the soy sauce moromi of Examples 1, 11 to 13 and Reference Examples 9 to 10.

  Examples 9 and 10 were sterilized immediately after the soy sauce moromi was charged, and it is considered that fermentation does not proceed after sterilization. In Examples 2 and 3 in which fermentation was performed only with Aspergillus or in Examples 4 and 5 where fermentation with Aspergillus or lactobacilli was performed, the contents of 9-oxo-ODA and 13-oxo-ODA were higher than those in Examples 9 and 10. Increased. As is clear from comparison with Examples 9 and 10, the increase in 9-oxo-ODA and 13-oxo-ODA content is due to the action of Aspergillus.

  In Examples 6 and 7 in which yeast was added in addition to koji molds and lactic acid bacteria, although the 9-oxo-ODA and 13-oxo-ODA contents on the 28th day of fermentation were slightly decreased, the contents were still high. On the other hand, 9-oxo-ODA and 13-oxo-ODA content decreased as fermentation by yeast further progressed (Example 8). In Reference Example 2 in which fermentation and ripening were performed over a period similar to that of normal soy sauce brewing, the contents of 9-oxo-ODA and 13-oxo-ODA were further reduced and were equivalent to the contents in tomato juice. became.

  Examples 11 to 13 and Reference Examples 9 to 10 are data on soy sauce moromi when high-temperature fermentation is performed. The fermentation period is shortened by high-temperature fermentation, and the antifungal property is enhanced, so that it is possible to prepare with low salt and no salt. As is clear from the comparison between Examples 11 to 13 and Reference Examples 9 to 10, 9-oxo-ODA and 13-oxo-ODA produced by Aspergillus were greatly reduced as yeast fermentation progressed.

  Reference Examples 4 to 8 are data of various fermented products containing soybean as a raw material. As shown in Reference Examples 4 to 5, 1.5 to 1.9% (w / v) of ethanol considered to be derived from yeast fermentation is generally contained. 9-oxo-ODA and 13-oxo-ODA were scarcely contained in miso that was supposed not to contain. This is thought to be due to the fact that the amount of ketooctadecadienoic acid produced was small in the first place due to the absence of aeration, and that linoleic acid was reduced to ethyl linoleate or metabolized by yeast fermentation. It is done.

  As in Reference Examples 6-8, 9-oxo-ODA and 13-oxo-ODA were scarcely contained in natto without using koji mold.

<Test Example 2>
[Preparation of various food samples]
As various food samples, commercially available soy milk “Raw Soy Milk” (manufactured by Sagamiya Shokusha) was purchased. Commercially available soymilk was designated as Reference Example 11.

(Preparation of liquid cake)
After sterilizing liquid medium (1.5 L) containing wheat bran 3% (w / v), soybean oil or soy sauce oil (antifoam) 0.1% (v / v), spores of Aspergillus sojae The mixture was added and cultured with stirring at 30 ° C. for 72 hours using a 2.5 L mini-jar fermenter to obtain a liquid koji.

(Examples 14 to 15)
To 800 g of soy sauce cake prepared in the same manner as in Test Example 1, 840 mL of 30% (w / v) saline, 280 mL of water, and linoleic acid (so that the salt concentration after fermentation and ripening is about 16% (w / v)) 420 g of Wako Pure Chemical Industries, Ltd. (special grade reagent) or commercially available rapeseed oil (Nisshin Oilio Co., Ltd.) was added, charged in a 5 L glass container, and mixed well by ventilation. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring. The gonococcal fermented product added with linoleic acid obtained 2 days later was designated as Example 14, and the gonococcal fermented product added with rapeseed oil was designated as Example 15.

(Example 16)
To 960 g of liquid koji, 640 g of 30% (w / v) saline solution and 480 g of expanded soybean (puff soybean, manufactured by Kikkoman Foods) were added. A 5 L glass container was charged. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring with aeration. The fermented bacilli obtained after 1 day was designated as Example 16.

(Example 17)
As in Example 16, 640 g of 30% (w / v) saline and 480 g of puffed soy beans (puff soy, manufactured by Kikkoman Foods) were added to 960 g of liquid koji, and linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added. , 105 g of special grade reagent) was added to a 5 L glass container. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring with aeration. The fermented bacilli obtained after 1 day was designated as Example 17.

(Example 18)
175 g of soy sauce cake and 190 g of sodium chloride prepared in the same manner as in Test Example 1 were added to 1600 mL of soy milk (manufactured by Kikkoman Soy Foods), and charged into a 5 L glass container. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring with aeration. The fermented bacilli obtained after 2 days was referred to as Example 18.

〔result〕
The results of quantification of 9-oxo-ODA and 13-oxo-ODA in various samples are shown in Table 3 below.

  In Example 14, the contents of 9-oxo-ODA and 13-oxo-ODA were greatly increased by adding linoleic acid to soy sauce koji (solid koji) and fermenting while stirring with aeration. Although the mechanism of action is not necessarily clear, it has been confirmed that ketooctadecadienoic acid is produced from linoleic acid by the action of soy sauce cake.

  In Example 15, the content of 9-oxo-ODA and 13-oxo-ODA was increased by adding rapeseed oil in the same manner as in Example 14. It was confirmed that ketooctadecadienoic acid can be produced from the ester of linoleic acid contained in edible oil, that is, triacylglycerol, by the action of koji mold. In the analysis method in this example, free octadecadienoic acid is measured, but linoleic acid in edible oil and whole soybeans is converted to ketooctadecadienoic acid while being bound to glycerol. There is a possibility that. When it is desired to obtain a free form, a higher content of free form can be obtained by using a koji mold having high lipase activity or by adding lipase.

  In Example 16, the total content of 9-oxo-ODA and 13-oxo-ODA was obtained by using liquid soybeans obtained by liquid culture using a whole soybean whose protein was denatured by swelling treatment as a substrate. , About 70 times that of tomato juice.

  In Example 17, 9-oxo-ODA and 13-oxo-ODA were obtained by using liquid soybeans obtained by liquid culture using swollen treated soybeans and free linole as substrates as in Example 16. The total content of was about 130 times that of tomato juice. It was confirmed that significant amounts of 9-oxo-ODA and 13-oxo-ODA can be obtained by allowing liquid koji to act on a substrate containing linoleic acid, such as whole soybeans or free linoleic acid.

  In the commercial soymilk of Reference Example 11, the contents of 9-oxo-ODA and 13-oxo-ODA were slight. In Example 18, it was confirmed that 9-oxo-ODA and 13-oxo-ODA are produced in soymilk by allowing soy sauce cake to act on soymilk (including linoleic acid or linoleic acid).

<Test Example 3>
(Preparation of liquid cake)
After sterilizing a liquid medium (1.5 L) containing 3% (w / v) wheat bran (w / v) and 0.1% (v / v) soybean oil or soy sauce oil (antifoam), various Aspergillus oryzae RIB40, Monascus purpureus IFO5965, Aspergillus sojae NBRC4239, Aspergillus usamii NISL1527, Aspergillus niger NISL7007) or added spores of Penicillium genus fungi (Penicillium camemberti NBRC32215, Penicillium roqueforti NBRC5459), 30 ℃ using 2.5L mini jar fermenter, 72 After stirring for a while, a liquid koji and a liquid koji-like product were obtained.

(Examples 19 to 25)
800g of liquid koji prepared by various koji molds or liquid koji-like material prepared by fungi of the genus Penicillium, 320ml of 30% (w / v) saline, 480ml of distilled water, puffed soybean (puff soybean, Kikkoman food) 480 g) and 86 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent) were added to a 5 L glass container. While maintaining the temperature of the moromi product at 40 to 45 ° C and stirring with aeration, fermentation was carried out using filamentous fungi or Penicillium sp. Various fermented bacilli obtained after 1 day were designated as Examples 19 to 23, and Penicillium fungi were designated as Examples 24 to 25.

〔result〕
Tables 4 and 5 below show the results of quantitative determination of 9-oxo-ODA and 13-oxo-ODA in various samples.

  As is clear from Examples 19 to 23 and Examples 24 to 25, even when various koji molds and various fungi of the genus Penicillium are used, 9-oxo-ODA in the koji mold fermentation product and the Penicillium genus fungus fermentation product is also used. And 13-oxo-ODA content increased.

<Test Example 4>
(Examples 26 to 31)
1.5 g of liquid koji prepared in the same manner as in Test Example 2 is dispensed into a disposable tube (manufactured by Japan BD, 15 mL capacity), 1.33 mL of 30% (w / v) saline, 1 g of soybean oil, lipase ( Lipase OF (manufactured by Meito Sangyo Co., Ltd.), appropriately ion-exchanged water added to make a total of 5 mL in Example 26, 0.1 M ferrous sulfate (food additive, manufactured by Kanto Chemical Co., Ltd.) 0 Example 27 with addition of 0.002 mL, Example 28 with addition of 0.02 mL, Example 29 with addition of 0.1 mL, Example 30 with addition of 0.2 mL, and Example 30 Example 31 was adopted. All were similarly prepared to be 5 mL. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring with aeration. The fermented bacilli obtained after 2 days were used as examples.

(Examples 32-37)
In accordance with the composition shown in Table 6 below, soybean oil (soybean white squeezed oil, manufactured by Showa Sangyo Co., Ltd.), distilled water, lipase (lipase OF, manufactured by Meika Sangyo Co., Ltd.) is charged into a 5 L glass container, and the product temperature is 40-45. The lipase reaction was carried out for 1 day while maintaining at ℃ and stirring with aeration. After the reaction, 30% (w / v) saline, liquid koji prepared in the same manner as in Test Example 2, soy sauce koji prepared in the same manner as in Test Example 1, puff soybeans (manufactured by Kikkoman Foods), ferrous sulfate ( A food additive (manufactured by Kanto Chemical Co., Inc.) was further added to the glass container according to the formulation shown in Table 6. The moromi product temperature was maintained at 40 to 45 ° C., and fermentation with koji mold was performed while stirring with aeration. The fermented bacilli obtained after 1 day was used as each example.

〔result〕
The results of quantitative determination of 9-oxo-ODA and 13-oxo-ODA in various samples are shown in Tables 7 and 8 below.

〔result〕
Addition of ferrous sulfate (metal-containing oxidizing agent) increased the 9-oxo-ODA and 13-oxo-ODA contents in the koji mold fermenter in a dose-dependent manner (Examples 26 to 31). Moreover, 9-oxo-ODA and 13-oxo-ODA content in the Aspergillus oryzae fermented material increased by carrying out the lipase process for the soybean oil used as a raw material previously (Examples 32-37).

Claims (21)

A fermentation process for obtaining a fermented product by fermenting a filamentous fungus with a raw material containing linoleic acid or an ester of linoleic acid as a substrate,
A method for producing ketooctadecadienoic acid, wherein the filamentous fungus is at least one selected from the group consisting of Aspergillus and Penicillium.   The manufacturing method of Claim 1 which stirs the composition containing the said raw material and the said filamentous fungus in the said fermentation process.   The manufacturing method of Claim 2 which stirs the said composition by ventilation | gas_flowing.   The manufacturing method as described in any one of Claims 1-3 with which fermentation by yeast of the said fermented material has not progressed substantially.   The manufacturing method as described in any one of Claims 1-4 with which the said raw material contains the processed material of whole soybeans or whole soybeans.   The manufacturing method as described in any one of Claims 1-5 which further includes adding the oxidizing agent containing a metal to the composition containing the said raw material and the said filamentous fungi, or the said fermented material.   The production method according to any one of claims 1 to 6, wherein the filamentous fungus is at least one selected from the group consisting of koji molds.   The manufacturing method of Claim 7 whose said fermented product is soy sauce moromi.   A fermentation process for obtaining a fermented product by fermenting a linoleic acid or a raw material containing an ester of linoleic acid with a filamentous fungus as a substrate, wherein the filamentous fungus is selected from the group consisting of Aspergillus and Penicillium A method for producing a fermented product containing a high amount of ketooctadecadienoic acid, in which the composition containing the raw material and the filamentous fungus is stirred in the fermentation step.   The manufacturing method of Claim 9 with which the said raw material contains the processed material of whole soybeans or whole soybeans.   The manufacturing method of Claim 9 or 10 which stirs the said composition by ventilation | gas_flowing.   The production method according to any one of claims 9 to 11, further comprising fermenting the composition or the fermented product with lactic acid bacteria.   The production method according to any one of claims 9 to 12, wherein fermentation of the fermentation product by yeast is not substantially advanced.   The manufacturing method as described in any one of Claims 9-13 which further includes adding the oxidizing agent containing a metal to the said composition or the said fermented material.   The manufacturing method according to any one of claims 9 to 14, wherein the filamentous fungus is at least one selected from the group consisting of koji molds.   The manufacturing method of Claim 15 whose said fermentation product is soy sauce moromi.   The fermentation product obtained by the manufacturing method as described in any one of Claims 9-15.   Soy sauce moromi obtained by the production method according to claim 16.   The foodstuff containing the fermented product of Claim 17, or the soy sauce moromi of Claim 18.   A functional food comprising the fermented product according to claim 17 or the soy sauce moromi according to claim 18.   It consists of filamentous fungi of the genus Penicillium and 9-oxo-10,12-octadecadienoic acid containing 15 ng / mg or more, or 13-oxo-9,11-octadecadienoic acid containing 5 ng / mg or more. A fermentation product of at least one filamentous fungus selected from the group.

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