Method for producing ketooctadecadienoic acid

Abstract

To provide ketooctadecadienoic acid production methods that synthesizes ketooctadecadienoic acid safely, stably, and efficiently.SOLUTION: Provided is a ketooctadecadienoic acid production method comprising (A) allowing an enzyme material containing lipoxygenase to act on a raw material containing fatty acids, and (b) stopping the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in the step (a).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing ketooctadecadienoic acid. [Background technology]

[0002] In recent years, hydroxylated fatty acids and oxo fatty acids obtained by reacting linoleic acid or oleic acid with enzymes or other means have been attracting attention as so-called rare fatty acids. Along with the growing interest in people's health and medicine, these rare fatty acids are expected to be applied to various industrial uses, particularly in terms of their physiological activity. In particular, 13-oxo-octadecadienoic acid and 9-oxo-octadecadienoic acid, which are types of ketooctadecadienoic acid, which are oxo derivatives of linoleic acid, have been found to have activity in improving lifestyle-related diseases such as improving lipid metabolism, and are therefore being actively researched both domestically and internationally as functional ingredients that show a remarkable fat-burning effect.

[0003] It has been known for some time that ketooctadecadienoic acid is contained in tomatoes, but the amount is so small that it is only about 1 ng / mg, and it is not a sufficient source for use as an active ingredient in foods or medicines. Therefore, a source containing ketooctadecadienoic acid at a higher content and a more efficient synthesis of ketooctadienoic acid have been desired.

[0004] For example, Patent Document 1 describes a method for synthesizing ketooctadecadienoic acid, which includes a step of reacting an enzyme material containing lipoxygenase with a raw material containing linoleic acid in the presence of a metal such as manganese, zinc, iron, copper, or magnesium at a temperature of less than 20°C. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2016-178913 A Summary of the Invention [Problem to be solved by the invention]

[0006] The method for producing ketooctadecadienoic acid described in Patent Document 1 uses a metal as a catalyst, and when the synthesized ketooctadecadienoic acid is used for food or pharmaceutical purposes, there is a risk that the product will contain metal. In addition, in order to suppress the production of by-products and improve the yield, it is essential to set the reaction temperature to less than 20°C in the method for producing ketooctadecadienoic acid described in Patent Document 1, and the production process requires complicated equipment and machinery for temperature adjustment in the production facilities.

[0007] In addition, in the method for producing ketooctadecadienoic acid described in Patent Document 1, lipoxygenase is used as an enzyme. The addition of dehydrogenase together with lipoxygenase as the enzyme to be used is also suggested, but since lipoxygenase itself is an enzyme that significantly promotes the reaction of fatty acids, a large amount of peroxide is generated by lipoxygenase, particularly at a reaction temperature of 20°C or higher, which further oxidizes the generated ketooctadecanoic acid. This causes a problem that the final yield of the target substance, ketooctadecanoic acid, is reduced. There is a demand for a method for producing ketooctadecadienoic acid that uses an enzyme to synthesize ketooctadecadienoic acid safely, stably, and efficiently.

[0008] An object of the present invention is to provide a method for producing ketooctadecadienoic acid, which has been difficult to supply until now, in a safe, stable and efficient manner using an enzyme. [Means for solving the problem]

[0009] The present invention relates to a method for producing ketooctadecadienoic acid, comprising: (a) a step of reacting an enzyme material containing lipoxygenase with a raw material containing fatty acids; and (b) a step of terminating the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in step (a).

[0010] The present invention further relates to a method for producing ketooctadecadienoic acid, which comprises the step of (c) reacting the product obtained via the step (b) with a dehydrogenase.

[0011] The fatty acid-containing raw material is preferably a raw material containing an oil or fat containing linoleic acid as a constituent component, a raw material containing linoleic acid, or a method for producing ketooctadecadienoic acid, which is linoleic acid.

[0012] In a preferred method for producing ketooctadecadienoic acid, the step of reacting the enzyme material is carried out at a temperature of 10° C. or higher and 60° C. or lower.

[0013] In a preferred method for producing ketooctadecadienoic acid, the step of reacting with the enzyme material is carried out at a temperature of 20° C. or higher and 50° C. or lower.

[0014] In the method for producing ketooctadecadienoic acid, the enzyme material is preferably allowed to act for 0.5 hours or more and 72 hours or less in the step of allowing the enzyme material to act.

[0015] In a preferred method for producing ketooctadecadienoic acid, the step of terminating the enzymatic reaction is carried out by inactivating the enzymatic activity of lipoxygenase.

[0016] A preferred method for producing ketooctadecadienoic acid is the one in which the deactivation is carried out by heat treatment or pH adjustment.

[0017] In a preferred method for producing ketooctadecadienoic acid, the heat treatment is carried out by heating the reaction mixture to 70° C. or higher and 140° C. or lower.

[0018] A method for producing ketooctadecadienoic acid is preferred, wherein the pH adjustment is performed by adjusting the reaction mixture to a pH below 6 or above 12.

[0019] In a preferred method for producing ketooctadecadienoic acid, the step of terminating the enzymatic reaction is carried out by removing the lipoxygenase from the reaction mixture.

[0020] In a preferred method for producing ketooctadecadienoic acid, the step of terminating the enzymatic reaction is carried out by adding an inhibitor of the lipoxygenase to the reaction mixture to inhibit the enzymatic activity of the lipoxygenase.

[0021] The method for producing ketooctadecadienoic acid is preferably such that the dehydrogenase is an alcohol dehydrogenase.

[0022] In the method for producing ketooctadecadienoic acid, the step of reacting with a dehydrogenase is preferably carried out in the presence of a cofactor.

[0023] The cofactor is NAD + A method for producing ketooctadecadienoic acid is preferred,

[0024] In a preferred method for producing ketooctadecadienoic acid, the dehydrogenase reaction step is carried out at a temperature of 0° C. or higher and 50° C. or lower.

[0025] In a preferred method for producing ketooctadecadienoic acid, the dehydrogenase reaction step is carried out at a pH of 6 or more and 12 or less.

[0026] A method for producing ketooctadecadienoic acid is preferred, which comprises treating a raw material containing the fatty acid with an enzyme material containing the lipoxygenase, and then carrying out the heat treatment within 0.5 to 24 hours. Effect of the Invention

[0027] According to the method for producing ketooctadecadienoic acid of the present invention, two different enzymes can be reacted independently and sequentially, so that the amount of by-products, which has been a problem in the past in terms of reaction yield, can be significantly suppressed. Therefore, ketooctadecadienoic acid can be produced efficiently. In addition, the synthesis temperature can be room temperature, and ketooctadecadienoic acid can be produced with a sufficient yield even at a reaction temperature of 20°C or higher. Furthermore, the method for producing ketooctadecadienoic acid of the present invention does not require the use of special metals. Since only two types of enzymes are used, ketooctadecadienoic acid can be produced by a simpler method than the conventional technology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Ketooctadecadienoic acid is one of the metabolites obtained by the oxidative metabolism of linoleic acid in certain microorganisms and plants. Linoleic acid is enzymatically converted to lipid peroxide (hydroperoxyoctadecadienoic acid (HPODE)) by the action of lipoxygenase (LOX), one of the linoleic acid metabolic enzymes, and this lipid peroxide (HPODE) is converted nonenzymatically or enzymatically, for example by peroxidase, to hydroxyoctadecadienoic acid (HODE), a hydroxylated fatty acid, by releasing radicals. Ketooctadecadienoic acid, an oxo derivative of fatty acid, is produced by the action of dehydrogenases such as alcohol dehydrogenase (ADH) on hydroxyoctadecadienoic acid (HODE).

[0029] Specific examples of ketooctadecadienoic acids include 9-oxo-10,12-octadecadienoic acid (also abbreviated as 9-oxoODA in this specification), 13-oxo-9,11-octadecadienoic acid (also abbreviated as 13-oxoODA in this specification), 5-oxo-6,8-octadecadienoic acid, 6-oxo-9,12-octadecadienoic acid, 8-oxo-9,12-octadecadienoic acid, 10-oxo-8,12-octadecadienoic acid, 11-oxo-9,12-octadecadienoic acid, 12-oxo-9,13-octadecadienoic acid, and 14-oxo-9,12-octadecadienoic acid. In addition, when isomers exist in these compounds, all isomers and mixtures thereof are included.

[0030] The present invention provides a method for producing ketooctadecadienoic acid with high efficiency by enzymatically converting linoleic acid into hydroperoxyoctadecadienoic acid (HPODE) or hydroxyoctadecadienoic acid (HODE) using lipoxygenase (LOX), inactivating LOX, and then allowing a dehydrogenase such as ADH to act as necessary. LOX may be inactivated after HPODE is produced or after HODE is produced. Hereinafter, the embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0031] In some embodiments of the present invention, ketooctadecadienoic acid is produced from a raw material containing a fatty acid. Examples of the raw material containing a fatty acid include a raw material containing fats and oils containing fatty acids as a component, a raw material containing fatty acids, and fatty acids. Preferably, the raw material containing a fatty acid is a raw material containing fats and oils containing linoleic acid as a component, a raw material containing linoleic acid, or linoleic acid. Here, linoleic acid is not limited to linoleic acid, and may be conjugated linoleic acid or may include esters of linoleic acid.

[0032] The raw material containing fats and oils containing linoleic acid as a component or the raw material containing linoleic acid is not particularly limited, and for example, any raw material containing fats and oils containing linoleic acid as a component in a plant, animal or bacterial body or a raw material containing linoleic acid can be suitably used in the production method of this embodiment. If a plant body such as soybean, castor, safflower or corn, or an animal body such as egg, which is known to have a high content of fats and oils containing linoleic acid as a component or linoleic acid, is used, it is possible to produce ketooctadecadienoic acid in high yield, so it may be preferable to use such a raw material. In addition, high-purity linoleic acid extracted and / or purified from the above-mentioned raw material may be used as the raw material.

[0033] In some embodiments of the present invention, ketooctadecadienoic acid is produced by reacting an enzyme material containing lipoxygenase with a raw material containing the above-mentioned fatty acid. As the enzyme material containing lipoxygenase, a plant, animal or bacterial material containing lipoxygenase may be used. For example, the lipoxygenase contained in the above-mentioned raw material may be used as it is as a naturally occurring lipoxygenase contained in the enzyme material. From the viewpoint of efficiently promoting the oxidation reaction of fatty acids, a naturally occurring lipoxygenase, i.e., a plant-derived, animal-derived or bacterial-derived lipoxygenase purified by an appropriate isolation and purification technique from a plant body such as soybean, an animal body such as egg, or a bacterial body may be used. Furthermore, a lipoxygenase produced as a recombinant by genetic engineering techniques or a chemically synthesized lipoxygenase may be used. Furthermore, a commercially available product may be used. Furthermore, the enzyme material may contain other enzymes in addition to lipoxygenase.

[0034] The amount of the enzyme material to be used can be appropriately selected according to the raw material to be used. For example, an enzyme material containing 100 to 300,000 units of lipoxygenase in terms of purified enzyme is used for 10 g of linoleic acid contained in the raw material. 2,000 to 180,000 units of lipoxygenase may be used for 10 g of linoleic acid contained in the raw material. Also, 5,000 to 90,000 units of lipoxygenase may be used. In this specification, 1 unit is defined as the amount of enzyme that increases the absorbance of a substrate solution at a wavelength of 234 nm by 0.001 in 1 minute at pH 9 and temperature 25° C. in a quartz cell of 3 mL (1 cm wide) using linoleic acid as a substrate. Therefore, 1 unit of lipoxygenase activity here corresponds to the amount of oxidizing 0.12 μmol of linoleic acid per minute.

[0035] The reaction temperature at which the enzyme material acts on the raw material containing fatty acids can be appropriately set depending on the raw material used, but is preferably 0 to 90°C. If the temperature is lower than 0°C, the solvent may freeze and the reaction may not proceed. Furthermore, if the temperature is higher than 90°C, the LOX contained in the enzyme material is quickly deactivated. For example, the reaction temperature at which the enzyme material acts can be 10°C or higher and 60°C or lower. Preferably, the reaction temperature at which the enzyme material acts can be 15°C or higher and 50°C or lower. Preferably, the reaction temperature can be 20°C or higher and 50°C or lower. Preferably, the reaction temperature is 30°C or higher and 50°C or lower.

[0036] It should be noted that the reactions or steps described herein can be carried out in a suitable solvent. A suitable solvent can be easily selected by one skilled in the art. A suitable solvent is a solvent that is substantially unreactive with the reactants, intermediates and / or products of the reactions or steps described herein at the temperatures at which the reactions are carried out, as described above. The reactions or steps described herein can also be carried out in one solvent or in a mixture of two or more solvents.

[0037] The reaction time for which the enzyme material is allowed to act on the raw material containing fatty acids may be appropriately set depending on the content of LOX contained in the enzyme material and the reaction temperature, but is preferably, for example, 0.5 to 144 hours. If the reaction time is shorter than 0.5 hours, the oxidation reaction by LOX may not proceed. If the reaction time of the enzyme material is longer than 72 hours, side reactions are likely to occur. More preferably, the reaction time of the enzyme material is about 0.5 to 72 hours. Furthermore, it is preferably 24 to 72 hours.

[0038] The reaction in which the enzyme material containing LOX acts may be carried out in a pH range of about 6 to 12. At a pH lower than 6 or higher than 12, the LOX contained in the enzyme material may be inactivated and / or denatured in the reaction solution. In particular, the reaction in which the enzyme material containing LOX acts is preferably carried out under conditions of pH 9 to 12. Furthermore, it is preferable to inactivate LOX under conditions of pH 10 to 12. At a pH within this range, linoleic acid contained in the raw material may be easily emulsified, and the reaction may proceed easily. The pH in the reaction may be adjusted using a known pH adjuster, for example, potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate, or potassium hydrogen phosphate, sodium salts such as sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

[0039] By the reaction of the raw material containing the fatty acid with the enzyme material containing lipoxygenase, oxygen molecules are added to the fatty acid to generate lipid peroxides and / or hydroxylated fatty acids. In the method for producing ketooctadecadienoic acid of the present invention, the enzyme reaction of lipoxygenase is subsequently stopped in the reaction mixture obtained by the reaction of the raw material containing the fatty acid with the enzyme material containing lipoxygenase.

[0040] The enzyme reaction of lipoxygenase can be stopped, for example, by inactivating the enzyme activity of lipoxygenase, although there is no particular limitation thereto. Examples of methods for inactivating the enzyme activity of lipoxygenase include inactivation treatment of lipoxygenase activity by heat treatment and inactivation treatment of lipoxygenase activity by pH adjustment. The enzyme reaction of lipoxygenase can also be stopped by removing lipoxygenase from the reaction mixture, for example, by ultrafiltration or gel filtration, or by passing the reaction mixture through a reaction column such as a column using a so-called immobilized enzyme in which the enzyme is adsorbed or bound to the surface of a carrier or is encapsulated in a gel. Alternatively, the enzyme reaction of lipoxygenase can be stopped by adding a reagent such as an inhibitor that interacts with lipoxygenase to selectively inhibit the reaction of lipoxygenase to the reaction mixture. In other words, in the broad sense of the present specification, stopping the enzymatic reaction of lipoxygenase includes not only inactivating the enzymatic activity of lipoxygenase, but also inhibiting the enzymatic activity of lipoxygenase and removing lipoxygenase.

[0041] In some embodiments of the present invention, the enzyme reaction of lipoxygenase can be stopped by inactivating the lipoxygenase activity by heat-treating the reaction mixture. Inactivation by heat treatment is particularly preferred because it can be easily controlled and can be efficiently performed even in the case of large-scale production. The heating temperature and retention time of the heat treatment can be appropriately set under conditions that allow lipoxygenase to be sufficiently inactivated. For example, the heating temperature is preferably 70°C or higher. When it is preferable to inactivate the enzyme activity of lipoxygenase in a shorter time, it is preferable to set the heating temperature to 90°C or higher. The upper limit of the heating temperature is not particularly specified, but a temperature at which the solvent in the reaction mixture does not volatilize too much is preferred, and for example, it can be about the temperature (120 to 140°C) used in general autoclave treatment. By performing the step of stopping the enzyme reaction of lipoxygenase at such a temperature, for example, when the produced ketooctadecadienoic acid is used for food or pharmaceutical purposes, the product is heat-sterilized at the same time as the inactivation of lipoxygenase activity, which may be particularly preferred. The retention time, i.e., the time for inactivating lipoxygenase, is not particularly limited, but it can generally be 5 minutes or more, and preferably 10 minutes or more.

[0042] When inactivation of lipoxygenase activity is carried out by pH adjustment, the pH of the reaction mixture is adjusted to less than pH 6 or greater than pH 12. The pH adjustment can be carried out using known pH adjusters as described above.

[0043] In some embodiments of the present invention, the enzymatic reaction of lipoxygenase is stopped by inhibiting the enzymatic activity of lipoxygenase by adding a reagent such as an inhibitor to the reaction mixture. Lipoxygenase inhibitors include, for example, N-hydroxyurea derivatives, redox inhibitors, and non-redox inhibitors, including, but not limited to, N-[1-benzo[b]thien-2-ylethyl]-N-hydroxyurea (zileuton), 1-[4-[5-(4-fluorophenoxy)-2-furyl]but-3-yn-2-yl]-1-hydroxy-urea, tetrahydro-4-[3-[[4-(2- Methyl-1H-imidazol-1-yl)phenyl]thio]phenyl)-2H-pyran-4-carboxamide (CJ-13610), 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-cyclohexa-2,5-diene-1,4-dione (docebenone), 6-[[3-fluoro-5-(4-methoxyoxan-4-yl)phenoxy]methyl]-1-methyl-quinolin-2-one (ZD2138), and the like.

[0044] In some embodiments of the present invention, the enzymatic reaction of lipoxygenase is subsequently stopped, and then a dehydrogenase is applied to the resulting product in order to carry out a dehydrogenation reaction. As a result, ketooctadecadienoic acid is produced. The dehydrogenase used here is not particularly limited as long as it is an enzyme that can use the above-mentioned lipid peroxides or hydroxylated fatty acids as substrates and convert them into oxo derivatives of fatty acids, and is preferably alcohol dehydrogenase (ADH). The origin of the enzyme is also not particularly limited. ADH derived from plants such as cucumber or ADH derived from microorganisms such as yeast may be used, and ADH derived from animals may also be used. ADH produced as a recombinant by genetic engineering techniques or ADH chemically synthesized may also be used. Furthermore, commercially available ADH can also be used. In order to proceed with the reaction more efficiently, ADH purified from plants or yeast can be used.

[0045] In some embodiments of the present invention, the step of allowing the dehydrogenase to act is preferably carried out in the presence of a cofactor. The cofactor may be added to the product obtained through the step of stopping the enzymatic reaction of lipoxygenase. The cofactor may also be added to the product as a mixture together with the enzyme. The cofactor may be selected depending on the type of dehydrogenase. An example of the cofactor is nicotinamide adenine dinucleotide (NAD + ), Nicotinamide adenine dinucleotide phosphate (NADP + ), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), etc. Preferably, the cofactor is nicotinamide adenine dinucleotide (NAD + The concentration of the cofactor added may be any concentration that allows the dehydrogenation reaction to proceed efficiently.

[0046] The amount of ADH used can be appropriately selected depending on the raw material used, but for example, 10 to 15,000 units of ADH may be used in terms of purified enzyme for 10 g of linoleic acid contained in the raw material. Also, more than 15,000 units of ADH may be used. 20 to 5,000 units of ADH may be used, or 100 to 500 units of ADH may be used. In this specification, 1 unit is defined as the amount of enzyme that converts 1.0 μmol of ethanol to acetaldehyde in 1 minute under conditions of pH 8.8 and 25° C.

[0047] The temperature at which ADH is allowed to act can be appropriately set depending on the raw material used, but is preferably 0 to 50°C. If the temperature is lower than 0°C, the solvent may freeze and the reaction may not proceed. If the temperature is higher than 50°C, the ADH enzyme is quickly inactivated. More preferably, the temperature may be 10°C or higher and 40°C or lower.

[0048] The reaction time for ADH can be appropriately set depending on the reaction temperature, etc., but is preferably about 1 to 72 hours, for example. From the viewpoint of reaction efficiency, the reaction time for ADH is sufficient for about 24 hours.

[0049] The reaction in which ADH acts may preferably be carried out within a range of about pH 6 to 12. At a pH lower than 6 or exceeding pH 12, ADH may be inactivated and / or denatured. In particular, it is preferable to carry out the reaction under conditions of pH 8 to 12. Therefore, the pH conditions can be similar to those in the step in which an enzyme material containing LOX is allowed to act, and in this case, it is preferable because there is no need to adjust the pH again in the step in which ADH acts. The pH in the reaction can be adjusted using a known pH adjuster, for example, potassium salts such as potassium hydroxide, potassium carbonate, potassium hydrogen carbonate, or potassium hydrogen phosphate, sodium salts such as sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, or sodium hydrogen phosphate, amines such as triethylamine, or ammonia.

[0050] By allowing ADH to act on the product after the step in which the enzyme reaction of lipoxygenase is stopped, the lipid peroxides and hydroxylated fatty acids, which are lipoxygenase metabolites of fatty acids that were previously produced by the reaction with lipoxygenase, are converted to ketooctadecadienoic acid. Therefore, according to the production method of this embodiment, the production of by-products is significantly suppressed, and ketooctadecadienoic acid can be stably produced.

[0051] In an embodiment of the present invention, after HPODE is produced, LOX is inactivated by heat treatment, and ketooctadecadienoic acid can be obtained by subsequent heat treatment. In this case, the heat treatment is carried out within 0.5 to 24 hours after the enzyme material containing lipoxygenase is allowed to act on the raw material containing fatty acids, and the heat treatment time is 10 minutes or more, preferably 30 minutes or more.

[0052] The steps described herein can be monitored according to suitable analytical methods known in the art. The production of products can be monitored by various spectroscopic means or by chromatography. According to the production method of the present invention, among ketooctadecadienoic acids, in particular 9-oxo-10,12-octadecadienoic acid (9-oxoODA) and 13-oxo-9,11-octadecadienoic acid (13-oxoODA) are produced from a raw material containing linoleic acid. The production of 9-oxoODA, 13-oxoODA, etc. can be confirmed by analytical means such as LC-MS, and the concentration can also be quantitatively analyzed. Thus, the conversion yield of the product from linoleic acid, so-called yield, is calculated.

[0053] As mentioned above, ketooctadecadienoic acid exists in various isomers such as (E,E isomer), (Z,E isomer), (E,Z isomer), and (Z,Z isomer). However, their effects as functional ingredients are almost the same. Therefore, the yield of ketooctadecadienoic acid described in the following examples is the combined yield of these isomers.

[0054] The method for producing ketooctadecadienoic acid according to the above-mentioned embodiment may additionally include a step of purifying the produced ketooctadecadienoic acid. For example, the produced ketooctadecadienoic acid may be separated and purified by a general purification method, for example, by thin layer chromatography or liquid chromatography after solvent extraction. The purification method and conditions may vary depending on the experiment, and for example, the eluent, solvent, stationary phase, etc. in the chromatography and HPLC purification may be appropriately selected. However, the produced ketooctadecadienoic acid may be used as it is without purification, or as a crude product such as a product subjected to a crude purification treatment and dried after solvent extraction.

[0055] Among the ketooctadecadienoic acids produced by the method for producing ketooctadecadienoic acid according to the embodiment described above, 9-oxoODA and 13-oxoODA have been reported to have a PPARα activation effect, and are expected to have a fat burning effect (for example, Kim YI, et al., Mol. Nutr. Food. Res., 55 (2011) 585. and Young-il Kim, et al., PLoSONE, 7 (2012) 2), and can be used to prevent or improve, for example, obesity, lipid metabolism disorders, diabetes caused by insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. For example, for practical use, they can be added to supplements, foods, functional foods, cosmetics, and the like.

[0056] The inventors have also discovered that ketooctadecadienoic acid can be used as a plant activator that exhibits a strong resistance-inducing effect when applied to plants, and have found that it exhibits growth-promoting effects in various plants, fruit yield-increasing effects, and disease-suppressing effects. Specific examples of diseases that are effective in suppressing include gray mold, vine splitting, vine wilt, and downy mildew on the leaves of Cucurbitaceae plants such as cucumbers, watermelons, melons, and pumpkins; bacterial wilt, wilt, half-leaf wilt, damping-off, and brown root rot on Solanaceae plants such as tomatoes, eggplants, and potatoes; powdery mildew, black spot, gray mold, and anthracnose on Rosaceae plants such as roses and strawberries; downy mildew on Amaranthaceae plants such as spinach; black rot, soft rot, bacterial spot disease, Rhizoctonia disease, southern blight on Apiaceae plants such as carrots, and blast on Gramineae plants.

[0057] According to the method for producing ketooctadecadienoic acid according to the embodiment described above, ketooctadecadienoic acid can be produced simply, stably and efficiently using two different enzymes. Since ketooctadecadienoic acid, which has been difficult to supply, can be produced in a sufficient yield, it is expected that ketooctadecadienoic acid can be applied to physiological activity analysis and industrial use. Furthermore, since ketooctadecadienoic acid can be stably produced without the need for the addition of metals, the method for producing ketooctadecadienoic acid of the present invention is also useful in that the produced ketooctadecadienoic acid can be safely used in various fields, and the obtained ketooctadecadienoic acid is particularly suitable as a raw material for functional foods, pharmaceuticals, cosmetics, plant activators, etc.

[0058] Furthermore, according to the method for producing ketooctadecadienoic acid according to the embodiment described above, ketooctadecadienoic acid can be efficiently produced at room temperature by reacting two different enzymes independently and sequentially, and no special temperature control device or temperature control is required. Therefore, it is considered that labor, resource, and energy saving in the production, and ultimately reduction in production costs, can be achieved. EXAMPLES

[0059] The present invention will be described based on examples, but the present invention is not limited to only the examples.

[0060] Production of ketooctadecadienoic acid Example 1 As a raw material containing fatty acids, 2.8 g of 80% pure linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and 7.0 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) and 300 mL of distilled water were added to prepare a test solution. The pH of the test solution at this time was 11.

[0061] 0.2 mg of lipoxygenase (Sigma-Aldrich, derived from Glycine Max) was added to the test solution and reacted at 30°C for 36 hours, after which the reaction mixture was placed in a 90°C water bath for 5 minutes to inactivate the enzyme. After the reaction solution was returned to room temperature, 0.2 mg of alcohol dehydrogenase (Wako Pure Chemical Industries, Ltd., derived from Yeast) was added and reacted at 30°C for an additional 24 hours.

[0062] After the reaction, the products in the test solution were analyzed by MS using 13-oxoODA (Cayman Chemical) as the standard substance. 2 The compounds were identified by LC-MS using spectral analysis. Quantification was performed by the absolute calibration curve method at a UV detection wavelength of 272 nm. 9-oxoODA was quantified using the absorbance of 13-oxoODA as a reference.

[0063] As shown in Table 1, the combined yield of isomers such as (E,E isomer) and (E,Z isomer) was 5.4% for 13-oxoODA. The yield of 9-oxoODA was 10.9%. The yield (%) was calculated based on the following formula. Yield (%) = (wt% of 13-oxoODA or 9-oxoODA produced) / (initial wt% of raw linoleic acid used)

[0064] Example 2 The same operations and analyses were carried out as in Example 1, except that the reaction time of the test solution with lipoxygenase was changed from 36 hours to 24 hours.

[0065] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 3.5%, and the yield of 9-oxoODA was 3.7%.

[0066] Example 3 As a raw material containing fatty acids, 2.8 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) with a purity of 80% was used, to which 4.0 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), 23.3 g of dipotassium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), and 300 mL of distilled water were added to prepare a test solution. The pH of the test solution at this time was 9.0.

[0067] 0.4 mg of lipoxygenase (Sigma-Aldrich, derived from Glycine Max) was added to the test solution and reacted at 15°C for 3 hours, after which the reaction mixture was placed in a water bath at 90°C for 90 minutes to inactivate the enzyme. After completion of the reaction, the product was subjected to the same operations and analysis as in Example 1.

[0068] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 2.6%, while the yield of 9-oxoODA was 1.2%.

[0069] Comparative Example 1 As a raw material containing fatty acids, 0.1 g of linoleic acid with a purity of 80% (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and 0.23 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) and 10 mL of distilled water were added to prepare a test solution. The pH of the solution at this time was 11.

[0070] Lipoxygenase (Sigma-Aldrich, derived from Glycine max) and alcohol dehydrogenase (Wako Pure Chemical Industries, Ltd., derived from yeast) were added at 0.04 mg each to the test solution at the same time, and reacted for 24 hours at 5° C. After the reaction was completed, the products in the test solution were analyzed and quantified in the same manner as in Example 1.

[0071] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 2.2%, while the yield of 9-oxoODA was 0.02%.

[0072] Comparative Example 2 The operations and analyses were carried out in the same manner as in Comparative Example 1, except that the reaction temperature was changed from 5°C to 30°C.

[0073] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 2.2%, while the yield of 9-oxoODA was 0.37%.

[0074] Comparative Example 3 As a raw material containing fatty acids, 0.28 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) with a purity of 80% was used, to which 0.15 g of potassium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.05 g of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), and 10 mL of distilled water were added to prepare a test solution. The pH of the solution at this time was 10.2.

[0075] 0.2 g of soybean powder (variety: Fukuyutaka) and 0.1 g of Mn-containing yeast (5%, manufactured by Medience Corporation) were added to the test solution and reacted for 24 hours at 5° C. After the reaction was completed, the products in the test solution were analyzed and quantified in the same manner as in Example 1.

[0076] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 2.9%, while the yield of 9-oxoODA was 0.5%.

[0077] Comparative Example 4 The operations and analyses were carried out in the same manner as in Comparative Example 5, except that the reaction temperature was changed from 5°C to 30°C.

[0078] As shown in Table 1, the combined yield of 13-oxoODA including isomers such as (E,E isomer) and (E,Z isomer) was 1.9%, while the yield of 9-oxoODA was 1.1%.

[0079] [Table 1]

[0080] As shown in Table 1, the yields of ketooctadecadienoic acid in Examples 1 to 3 were much higher than the yields of ketooctadecadienoic acid in Comparative Examples 1 to 4. Therefore, it can be seen that the method for producing ketooctadecadienoic acid of the present invention has a significantly excellent effect of producing ketooctadecadienoic acid in high yield.

[0081] In addition, since the soybean powder used in Comparative Examples 3 and 4 is considered to contain both lipoxygenase and alcohol dehydrogenase, the results of Comparative Examples 3 and 4 are considered to be obtained under the same conditions as the simultaneous addition of lipoxygenase and alcohol dehydrogenase. In Comparative Examples 3 and 4, in addition to this soybean powder, Mn-containing yeast that supplies metals is further added to the test solution. That is, in Comparative Examples 3 and 4, although the effect of promoting the production reaction of ketooctadecadienoic acid contributed by the metal catalyst is also imparted, the yields of Comparative Examples 3 and 4 are merely lower than those of Examples 1 to 3. From these results, it can be seen that the production method of the present invention has an excellent effect on the conversion yield of ketooctadecadienoic acid from linoleic acid.

Claims

1. (a) subjecting a raw material containing linoleic acid to an enzyme material containing a plant-derived lipoxygenase isolated and purified from a plant; and (b) a step of inactivating and terminating the enzymatic reaction of the lipoxygenase contained in the reaction mixture obtained in step (a) by heating the reaction mixture to 70° C. or higher and 140° C. or lower. and does not include a step (c) of treating the product obtained via the step (b) with a dehydrogenase.

2. 2. The method for producing ketooctadecadienoic acid according to claim 1, wherein the raw material containing linoleic acid is a raw material containing fats and oils containing linoleic acid as a constituent component, a raw material containing linoleic acid, or linoleic acid itself.

3. The method for producing ketooctadecadienoic acid according to claim 1 or 2, wherein the step of reacting the enzyme material is carried out at a temperature of 10°C or higher and 60°C or lower.

4. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 3, wherein the step of reacting the enzyme material is carried out at a temperature of 20°C or higher and 50°C or lower.

5. The method for producing ketooctadecadienoic acid according to any one of claims 1 to 4, wherein in the step of reacting the enzyme material, the enzyme material is reacted for 0.5 hours or more and 72 hours or less.

6. 2. The method for producing ketooctadecadienoic acid according to claim 1, wherein the heating is carried out within 0.5 to 24 hours after the enzyme material containing lipoxygenase is allowed to act on the raw material containing linoleic acid.

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