Preparation method and application of phenylacetaldehyde

Abstract

The present invention addresses the problem of establishing a safe, environmentally friendly, and highly efficient method for producing phenylacetaldehyde. The present invention relates to a method for producing phenylacetaldehyde, comprising a step for causing a medium to act on phenylalanine or an analogue thereof, the medium being a compound containing a ring structure having at least one hydroxyl group. The present invention also relates to a phenylacetaldehyde-generating enzyme agent, an enzyme agent for improving the flavor of foods and drinks, and a method for improving the flavor of foods and drinks.

Description

Technical Field

[0001] The present invention relates to a method for producing phenylacetaldehyde and a phenylacetaldehyde-producing enzyme agent. Background Art

[0002] Phenylacetaldehyde has been used as a raw material for spices, medicines, pesticides, artificial sweeteners, etc. It is also an important substance as an intermediate of amino acids. Phenylacetaldehyde has a sweet, rose, and fresh aroma, so it is mainly used as a flavor blending raw material in spices and foods.

[0003] In addition, phenylacetaldehyde is known to be one of the components that increases when food is heated, and is also known to be a component that makes people feel rich flavor. For example, Patent Document 1 discloses that heating cruciferous vegetables increases phenylacetaldehyde, which contributes to increasing rich flavor.

[0004] As a method for producing phenylacetaldehyde, various methods have been studied in addition to heat treatment of cruciferous vegetables, etc. For example, Patent Document 2 discloses a method for producing aromatic acetaldehyde by organic synthesis. In addition, Patent Document 3 discloses a method for producing phenylacetaldehyde by allowing tyrosinase to act on phenylalanine, and Patent Document 4 discloses a method for producing phenylacetaldehyde from phenylalanine by reacting pyridoxal phosphate (PLP)-dependent aromatic decarboxylase from rose in the presence of PLP.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2018 / 207818

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 5-229981

[0009] Patent Document 3: Japanese Patent Application Publication No. 2003-310289

[0010] Patent Document 4: Japanese Patent Application Publication No. 2007-189943 Summary of the invention

[0011] Since phenylacetaldehyde shows a flowery aroma, it is expected to mask odor and improve flavor by adding it to food, but there is still room for improvement in the flavor of food. In addition, it is necessary to establish a new method for producing phenylacetaldehyde that is safe, environmentally friendly and efficient.

[0012] Therefore, the object of the present invention is to establish a safe, environmentally friendly and efficient method for producing phenylacetaldehyde.

[0013] As a result of intensive studies to solve the above problems, the present inventors have found that by providing a step of allowing a mediator having a predetermined structure to act on phenylalanine or an analog thereof, phenylacetaldehyde is generated and a floral aroma is exhibited, thereby completing the present invention.

[0014] Specifically, the present invention has the following configurations.

[0015] [1] A method for producing phenylacetaldehyde, comprising the steps of allowing a mediator to act on phenylalanine or an analog thereof,

[0016] The medium is a compound including a ring structure having at least one hydroxyl group.

[0017] [2] The method for producing phenylacetaldehyde according to [1], wherein the step of allowing the mediator to act is performed in the presence of a multi-copper oxidase.

[0018] [3] The method for producing phenylacetaldehyde according to [1] or [2], wherein the medium is a compound containing a ring structure having at least one hydroxyl group and an alkoxy group, or a compound containing a ring structure having two or more hydroxyl groups.

[0019] [4] The method for producing phenylacetaldehyde according to any one of [1] to [3], wherein the ring structure is a benzene ring or a dihydrofuran ring.

[0020] [5] The method for producing phenylacetaldehyde according to any one of [1] to [4], wherein the medium is at least one selected from the group consisting of DL-catechin, tannic acid, chlorogenic acid, caffeic acid, curcumin, hesperidin, L-DOPA, pyrogallol, vanillin, eugenol, gallic acid monohydrate, ferulic acid, isoeugenol, α-glucosylrutin and rutin.

[0021] [6] The method for producing phenylacetaldehyde according to any one of [1] to [5], wherein the medium is a compound contained in at least one selected from apple extract, pectin, green coffee bean extract, and tea extract.

[0022] [7] The method for producing phenylacetaldehyde according to any one of [1] to [6], wherein the medium is a polyphenol.

[0023] [8] The method for producing phenylacetaldehyde according to any one of [1] to [7], wherein the medium is at least one selected from the group consisting of α-glucosylrutin, chlorogenic acid, caffeic acid, tannic acid and rutin.

[0024] [9] The method for producing phenylacetaldehyde according to any one of [2] to [8], wherein the multi-copper oxidase is at least one selected from the group consisting of phenoloxidase and bilirubin oxidase.

[0025]

[10] The method for producing phenylacetaldehyde according to any one of [2] to [9], wherein the multi-copper oxidase is laccase.

[0026]

[11] The method for producing phenylacetaldehyde according to any one of [1] to

[10] , wherein the reaction temperature in the step of allowing the medium to act is 60° C. or less.

[0027]

[12] A phenylacetaldehyde generating enzyme agent comprising a mediator and a multi-copper oxidase,

[0028] The medium is a compound including a ring structure having at least one hydroxyl group.

[0029] In addition, the present invention has the following configurations.

[0030] [A] Phenylacetaldehyde produced by the method for producing phenylacetaldehyde according to any one of [1] to

[11] .

[0031] [B] A method for improving the flavor of a food or beverage, comprising the step of allowing a mediator having a ring structure having at least one hydroxyl group and a multi-copper oxidase to act on phenylalanine or an analog thereof.

[0032] [C] An enzyme agent for improving the flavor of food and drink, comprising: a mediator having a ring structure having at least one hydroxyl group and a multi-copper oxidase.

[0033] [D] Use of an enzyme agent comprising a mediator having a ring structure having at least one hydroxyl group and a multi-copper oxidase for improving the flavor of a food or drink.

[0034] [E] An enzyme agent for improving the flavor of food and beverage, comprising a mediator and a multi-copper oxidase,

[0035] The medium is a compound including a ring structure having at least one hydroxyl group.

[0036] [F] A method for improving the flavor of food and drink, comprising the steps of allowing a mediator and a multi-copper oxidase to act on phenylalanine or an analog thereof,

[0037] The medium is a compound including a ring structure having at least one hydroxyl group.

[0038] According to the present invention, a safe, environmentally friendly and efficient method for producing phenylacetaldehyde can be provided. In addition, according to the present invention, a phenylacetaldehyde generating enzyme agent, an enzyme agent for improving the flavor of food and drink, and a method for improving the flavor of food and drink can also be provided. DETAILED DESCRIPTION

[0039] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below is sometimes based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. It should be noted that the numerical range represented by "to" in this specification represents a range including the numerical values ​​described before and after "to" as the lower limit and the upper limit.

[0040] (Method for producing phenylacetaldehyde)

[0041] The present invention relates to a method for producing phenylacetaldehyde, which comprises a step of allowing a medium to act on phenylalanine or an analog thereof. The medium is a compound containing a ring structure having at least one hydroxyl group. Due to the above-mentioned structure, the present invention can produce phenylacetaldehyde safely, environmentally friendly and efficiently. The phenylacetaldehyde obtained by the production method of the present invention is highly safe and can be added to food, so that the odor in the food can be masked and the flavor can be improved.

[0042] In the present invention, the step of allowing a mediator to act on phenylalanine or an analog thereof is preferably a step performed in the presence of a multi-copper oxidase. That is, the present invention preferably relates to a method for producing phenylacetaldehyde, comprising the step of allowing a mediator to act on phenylalanine or an analog thereof in the presence of a multi-copper oxidase. By performing the step of allowing a mediator to act on phenylalanine or an analog thereof in the presence of a multi-copper oxidase, the yield of phenylacetaldehyde can be increased, and phenylacetaldehyde can be produced more efficiently.

[0043] The process of making the medium act on phenylalanine or its analogue in the present invention can be a process of making the medium with the above-mentioned prescribed structure play a role in the food and beverage containing phenylalanine or the food and beverage added with phenylalanine. In addition, the above-mentioned process can also be a process of making the medium with the above-mentioned prescribed structure and multi-copper oxidase play a role in the food and beverage containing phenylalanine or the food and beverage added with phenylalanine. It is generally believed that phenylacetaldehyde is not stable, but by making the medium play a role in the food and beverage containing phenylalanine or the food and beverage added with phenylalanine, it is also possible to directly obtain the food and beverage containing phenylacetaldehyde without separating phenylacetaldehyde.

[0044] It should be noted that the manufacturing process of phenylacetaldehyde of the present invention can be included in the manufacturing method of the food and drink containing phenylacetaldehyde. In this case, the manufacturing method of the food and drink containing phenylacetaldehyde can include the process of making the medium with the above-mentioned prescribed structure play a role in the food and drink material containing phenylalanine, the food and drink material with phenylalanine added. In addition, the above-mentioned process can also be the process of making the medium with the above-mentioned prescribed structure and multi-copper oxidase play a role in the food and drink material containing phenylalanine, the food and drink material with phenylalanine added.

[0045] Phenylalanine is an amino acid that is present in many foods. In the method for producing phenylacetaldehyde of the present invention, phenylalanine is used as a raw material, and a medium and a multi-copper oxidase are allowed to act on phenylalanine or an analog thereof to produce phenylacetaldehyde. Since food-derived compounds and highly safe compounds can be used as mediums and multi-copper oxidases, the obtained phenylacetaldehyde is highly safe and can be used in pharmaceuticals and foods. In addition, the method for producing phenylacetaldehyde of the present invention is not obtained by organic synthesis, and the reaction can be carried out in an aqueous solvent, so the load on the environment can also be reduced.

[0046] In addition, in the present invention, by allowing the mediator and the multi-copper oxidase to act on phenylalanine or its analogs, in addition to generating phenylacetaldehyde, benzaldehyde can also be generated. Benzaldehyde exhibits a sweet fruity aroma, so it is preferably used in seasonings and various processed foods. In addition, it is also known that benzaldehyde exerts antibacterial activity against Staphylococcus aureus and the like, and is also useful as an antibacterial component.

[0047] <Phenylalanine>

[0048] In the method for producing phenylacetaldehyde of the present invention, phenylalanine or an analog thereof is used as a reaction substrate (oxidation substrate). As the phenylalanine analog, it is preferred that at least one hydrogen atom of the side chain of phenylalanine is substituted by a substituent, and it is more preferred that the hydrogen atom of the benzene ring of the side chain of phenylalanine is substituted. As the substituent, for example, halogen atoms such as fluorine, chlorine, bromine, and iodine; acetylene; cyano; azido and the like substituents can be cited.

[0049] The phenylalanine used in the method for producing phenylacetaldehyde of the present invention is preferably L-phenylalanine (LPA), a naturally occurring compound, or D-phenylalanine (DPA), an artificially produced compound. In addition, a food containing L-phenylalanine and added with D-phenylalanine may be used, or a food containing neither L-phenylalanine and added with L-phenylalanine and D-phenylalanine may be used.

[0050] The concentration of phenylalanine used in the method for producing phenylacetaldehyde of the present invention is preferably appropriately adjusted according to the type and concentration of the medium, multi-copper oxidase, etc. that acts. For example, the initial concentration of phenylalanine in the step of allowing the medium to act on phenylalanine or its analog is preferably 0.01mM or more, more preferably 0.1mM or more, further preferably 1mM or more, further preferably 5mM or more, and particularly preferably 10mM or more. In addition, the initial concentration of phenylalanine is preferably 1000mM or less, more preferably 900mM or less, further preferably 800mM or less, further preferably 700mM or less, and particularly preferably 500mM or less. In addition, the initial concentration of phenylalanine may be 200mM or less, 150mM or less, 100mM or less, 50mM or less, 30mM or less, or 20mM or less. By making the initial concentration of phenylalanine within the above range, phenylacetaldehyde can be efficiently generated.

[0051] <Medium>

[0052] The method for producing phenylacetaldehyde of the present invention comprises a step of allowing a mediator to act on phenylalanine or an analog thereof. Here, the mediator is a compound containing a ring structure having at least one hydroxyl group. It should be noted that the mediator in this specification may also be a reduction substrate. In addition, the mediator is preferably a component or compound that promotes or mediates the production of phenylacetaldehyde from phenylalanine. It is believed that in the production step of phenylacetaldehyde of the present invention, the hydroxyl group possessed by the ring structure of the mediator is oxidized, and the oxidized compound acts on phenylalanine or an analog thereof, resulting in Strecker decomposition, thereby producing phenylacetaldehyde.

[0053] The medium may be a compound containing one ring structure having at least one hydroxyl group in one molecule, or may be a compound containing two or more such ring structures.

[0054] The medium is preferably a compound containing a ring structure having at least one hydroxyl group and an alkoxy group, or a compound containing a ring structure having two or more hydroxyl groups. For example, the medium is preferably a compound having the following structure.

[0055]

[0056] In formula (1), X represents a ring structure, R represents a substituent, n is a natural number including 0, and represents a natural number with the number of substitutable groups as the upper limit, and R 1 represents a hydrogen atom or an alkyl group.

[0057] The ring structure represented by X is preferably a heterocycle or a cyclic hydrocarbon. The ring structure represented by X is preferably a three-membered ring to a ten-membered ring, more preferably a four-membered ring to an eight-membered ring, and further preferably a five-membered ring or a six-membered ring. When the ring structure represented by X is a heterocycle, as heteroatoms constituting the heterocycle, for example, oxygen atoms, nitrogen atoms, sulfur atoms, etc. can be cited. Among them, the heteroatom is preferably an oxygen atom. When the ring structure represented by X is a cyclic hydrocarbon, as cyclic hydrocarbons, cycloalkanes, cycloolefins, aromatic hydrocarbons, etc. can be cited. In this case, as the ring structure represented by X, for example, cyclobutane, cyclopentane, cyclohexane, cyclopentene, cyclohexene, benzene ring, naphthalene ring, anthracene ring, etc. can be cited.

[0058] R 1 The alkyl group represented by is preferably an alkyl group having 1 to 5 carbon atoms, more preferably an alkyl group having 1 to 3 carbon atoms, further preferably an alkyl group having 1 or 2 carbon atoms, and particularly preferably a methyl group. 1 Preferred is a hydrogen atom or a methyl group.

[0059] As the substituent represented by R, there can be mentioned aliphatic hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, cycloalkyl), aromatic hydrocarbon groups (e.g., phenyl, naphthyl, anthracenyl, phenanthryl, biphenyl), heterocyclic groups, acyl groups, alkoxy groups, amino groups, hydroxyl groups, mercapto groups, halogen atoms (e.g., fluorine atoms, chlorine atoms, bromine atoms, iodine atoms) and the like. In addition, two Rs can form an oxo group together. The substituent can also be substituted by other substituents, and the same substituent can also be mentioned as other substituents. As the substituent represented by R, there can be mentioned, for example, a hydrocarbon group, a halogen atom, or a monovalent group composed of one or more -O-, -C(=O)- and a hydrocarbon group. The substituent represented by R can be connected to a monosaccharide structure, and when the medium contains two or more of the above-mentioned ring structures in one molecule, the substituent represented by R can be connected to the above-mentioned ring structure. In addition, when n is 2 or more, the substituents represented by R can be connected to each other to form a ring.

[0060] Among them, the medium is preferably a compound having the following structure.

[0061]

[0062] In formula (2), R represents a substituent, n is an integer from 0 to 3, and R 1 represents a hydrogen atom or an alkyl group, R 2 In formulas (3) and (3'), R represents a substituent, n is an integer from 0 to 2, and R 1represents a hydrogen atom or an alkyl group, and Y represents an oxygen atom, a nitrogen atom or a sulfur atom. In formulas (3) and (3'), Y is particularly preferably an oxygen atom, and the ring structure possessed by the medium is particularly preferably a benzene ring or a dihydrofuran ring. Among them, when the ring structure possessed by the medium is a dihydrofuran ring, it is preferably a structure represented by formula (3). In addition, when the ring structure possessed by the medium is a dihydrofuran ring, it is preferred that the 3-position and 4-position have a hydroxyl group or an alkoxy group, respectively.

[0063] The mediator is particularly preferably a compound having the following structure.

[0064]

[0065] In formula (4), R represents a substituent, n is an integer from 0 to 3, and R 1 represents a hydrogen atom or an alkyl group, R 2 represents a hydrogen atom or a hydroxyl group. In formula (4), n is more preferably an integer of 0 to 2, and particularly preferably an integer of 0 or 1. In formula (5), R represents a substituent, n is an integer of 0 or 1, and R 1 In the formula (5), n is particularly preferably 1.

[0066] R in formulas (2) to (5) 1 The specific examples and preferred ranges of R and R are the same as those of R in formula (1). 1 The specific examples and preferred ranges are the same as those for R.

[0067] Specific examples of the medium include, for example, DL-catechin, tannic acid, chlorogenic acid, caffeic acid, curcumin, hesperidin, L-DOPA, pyrogallol, vanillin, eugenol, gallic acid monohydrate, ferulic acid, isoeugenol, α-glucosylrutin, rutin, lecithin, beet pectin, resveratrol, anthocyanin, anthocyanidin, proanthocyanidin, phloridzin, quercetin, vanillic acid, apple polyphenol, etc. Among them, the medium is preferably at least one selected from the group consisting of DL-catechin, tannic acid, chlorogenic acid, caffeic acid, curcumin, hesperidin, L-DOPA, pyrogallol, vanillin, eugenol, gallic acid monohydrate, ferulic acid, isoeugenol, α-glucosylrutin, and rutin.

[0068] Among them, the medium is preferably a polyphenol. The polyphenols in this specification refer to compounds having a structure in which multiple hydroxyl groups are bonded to a benzene ring or a naphthalene ring. As polyphenols, for example, DL-catechin, tannic acid, chlorogenic acid, caffeic acid, L-DOPA, pyrogallol, gallic acid monohydrate, α-glucosylrutin, rutin, proanthocyanidins, quercetin glycosides, verbascoside, iso-verbascoside, diosmetin, diglucuronide, apigenin 7-diglucuronide, luteolin 7-diglucuronide, etc. can be cited. Among these polyphenols, the medium is particularly preferably at least one selected from α-glucosylrutin, chlorogenic acid, caffeic acid, tannic acid and rutin. By using the above compounds as the medium, the yield of phenylacetaldehyde can be more effectively increased.

[0069] In addition, the medium may be a compound contained in a plant extract, for example, a compound contained in pectin, green coffee bean extract, tea extract, apple extract, lemon verbena extract, etc. Among them, the medium is preferably a compound contained in at least one selected from apple extract, pectin, green coffee bean extract and tea extract. For example, pectin contains ferulic acid, green coffee bean extract and tea extract contain DL-catechin, chlorogenic acid, and caffeic acid, apple extract contains proanthocyanidins (a structure formed by bonding 2 to 15 catechins), catechins, chlorogenic acid, phloridzin, quercetin glycoside and other apple polyphenols, and lemon verbena extract contains verbascoside, isovalascoside, diosmin, diglucuronide, apigenin 7-diglucuronide, luteolin 7-diglucuronide and other polyphenols. These components and extracts may contain analogs of the above compounds. As analogs, compounds having the basic skeleton of the above compounds and having at least one or more hydrogen atoms of the side chain substituted by a substituent can be cited. For example, analogs of chlorogenic acid include 4,5-dicaffeoylquinic acid, 4-caffeoylquinic acid, 4-feruloylquinic acid, 3,5-dicaffeoylquinic acid, 3-caffeoylquinic acid, 3-feruloylquinic acid, 3,4-dicaffeoylquinic acid, 5-caffeoylquinic acid, 5-feruloylquinic acid, etc., analogs of catechin include epicatechin, epigallocatechin, epigallocatechin gallate, etc., and analogs of proanthocyanidins include procyanidin B1, procyanidin B2, procyanidin A2, etc. In addition, pectin and the above extracts may contain a plurality of the above compounds and analogs.

[0070] As the extract, commercially available products may be used, for example, Sunfood 100 manufactured by Mitsubishi Chemical Corporation, tea extract (food material) manufactured by SANCT Co., Ltd., apple polyphenol powder manufactured by Frontierfoods Co., Ltd., etc. may be used.

[0071] When the medium is allowed to act on phenylalanine or its analog, the amount of the medium to be acted on is not particularly limited, and the concentration of the medium in the reaction solution is preferably 0.01mM or more, more preferably 0.05mM or more, further preferably 0.1mM or more, and particularly preferably 0.5mM or more. The concentration of the medium in the reaction solution is preferably 500mM or less, more preferably 300mM or less, and further preferably 100mM or less. In addition, the concentration of the medium in the reaction solution can be 50mM or less, or 25mM or less, or 10mM or less, or 5mM or less. It should be noted that the amount added is preferably appropriately adjusted corresponding to the type of the medium.

[0072] In addition, the concentration of the medium in the reaction solution is preferably 0.001 mass % or more, more preferably 0.005 mass % or more, further preferably 0.01 mass % or more, particularly preferably 0.05 mass % or more. The concentration of the medium in the reaction solution is preferably 50 mass % or less, more preferably 30 mass % or less, further preferably 10 mass % or less. In addition, the concentration of the medium in the reaction solution can be 5 mass % or less, can also be 2.5 mass % or less, can also be 1 mass % or less, can also be 0.5 mass % or less. It should be noted that the addition amount is preferably appropriately adjusted corresponding to the type of the medium.

[0073] <Multi-copper oxidase>

[0074] The method for producing phenylacetaldehyde of the present invention preferably includes a step of allowing a mediator to act on phenylalanine or an analog thereof in the presence of a multi-copper oxidase. The multi-copper oxidase is an enzyme that contains 2 to 8 atoms of copper required for enzyme activity in the molecule and is an enzyme that generates water molecules by electron reduction of oxygen. Examples of the multi-copper oxidase include laccase, bilirubin oxidase, ascorbate oxidase, ceruloplasmin, Fet3p, CueO, CotA, Stellacyanin, tyrosinase, catechol oxidase, nitrite reductase, and the like.

[0075] In the production process of phenylacetaldehyde of the present invention, it is considered that multicopper oxidase acts on a mediator containing a hydroxyl group, oxidizes the hydroxyl group in the ring structure of the mediator, and the oxidized compound acts on phenylalanine or an analog thereof to cause Strecker decomposition, thereby generating phenylacetaldehyde.

[0076] Among them, the multi-copper oxidase is preferably at least one selected from the group consisting of phenol oxidase and bilirubin oxidase. The multi-copper oxidase is more preferably phenol oxidase, and the phenol oxidase is preferably polyphenol oxidase, and among them, laccase is particularly preferred.

[0077] Laccase is an enzyme having phenol oxidase activity (EC1.10.3.2), and can be used for any purpose utilizing the oxidation reaction of the substrate and / or the various chemical reactions caused by the free radical substances of the reaction intermediates generated by the oxidation reaction. Examples of such oxidation reactions or chemical reactions caused by them include oxidation of phenolic compounds such as catechol, hydroquinone, urushiol, and laccol; oxidation of aromatic amines such as p-phenylenediamine; decomposition of lignin; cross-linking of proteins having easily oxidizable functional groups such as tyrosine side chains (phenolic hydroxyl groups), cysteine ​​side chains (thiol groups), lysine side chains (ε-amino groups), and histidine side chains (imidazolyl groups). Such a phenomenon in which the substrate undergoes chemical changes through the oxidation reaction of laccase and the chemical reactions caused by them can also be referred to as "oxidative modification".

[0078] Laccase exists in plants, fungi, bacteria, animals, etc. Among them, the laccase used in the present invention is preferably a laccase derived from fungi or bacteria. Specific examples include those derived from the genera Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia, Rigidoporus, Coprinus, Psatyrella, Myceliophtera, and Schtalidium. spp.), Polyporus spp., Phlebia spp., Coriolus spp., Bacillus spp., etc., preferably, a laccase derived from Trametes spp. By using a laccase derived from Trametes spp., phenylacetaldehyde can be efficiently produced.

[0079] Laccase can be prepared from the culture solution of the microorganism as the source of laccase. As a specific preparation method, a method for recovering laccase from the culture solution or bacterial cell of the above-mentioned microorganism can be cited. For example, in the case of using a laccase secretory microorganism, after recovering the bacterial cell in advance by filtering, centrifugation, etc. from the culture solution as needed, the enzyme is separated and / or purified. In addition, in the case of using a laccase non-secreting microorganism, after recovering the bacterial cell in advance from the culture solution as needed, the bacterial cell is crushed by pressurization, ultrasonic treatment, etc. to expose the enzyme, and the enzyme is separated and / or purified. As the separation and / or purification method of the enzyme, there is no particular limitation, and a known protein separation and / or purification method can be used, for example, centrifugation, UF concentration method, salting-out method, various chromatography methods using ion exchange resins, etc. can be cited. The separated and / or purified enzyme can be powdered by drying methods such as freeze drying and reduced pressure drying, and in addition, it can also be powdered by using a suitable excipient and / or drying aid in the drying method. Alternatively, the isolated and / or purified enzyme may be liquefied by adding appropriate additives and sterilizing by filtration.

[0080] As laccase, commercially available products may be used. Examples of preferred commercially available products include Trametes-derived laccase (LC) manufactured by AMANO ENZYME CO., LTD.

[0081] Bilirubin oxidase is an enzyme having bilirubin oxidase activity (EC1.3.3.5). For example, bilirubin oxidase derived from microorganisms belonging to the genera Myrothecium spp., Coprinus spp., Penicillium spp., and Bacillus spp. can be used. Examples of the genus Myrothecium include Myrothecium verrucaria MT-1, FERM-BP 653 (see Agricultural and biological chemistry, Vol. 45, pp. 2383-2384 (1981)), Myrothecium verrucaria IFO 6113, Myrothecium verrucaria IFO 6133, Myrothecium verrucaria IFO 6351, Myrothecium verrucaria IFO 9056, Myrothecium cinctum IFO 9950, and Myrothecium verrucaria IFO 9531. Examples of the genus Coprinus include Coprinus cinereus IFO 8371, Coprinus lagopides IFO 30120, and other deposited strains. Examples of Penicillium include Penicillium janthinellum (see Japanese Unexamined Patent Application No. 63-309187). Examples of Bacillus include Bacillus licheniformis (see Japanese Unexamined Patent Application No. 61-209587). These strains can be cultured in liquid or solid form by conventional methods, and the culture solution can be subjected to extraction, salting out, dialysis, ion exchange, gel filtration, etc. to obtain a purified standard product of the enzyme bilirubin oxidase. On the other hand, bilirubin oxidase derived from Schizophyllum commune (see Japanese Unexamined Patent Application No. 59-135886), derived from Asteraceae plants (see Japanese Unexamined Patent Application No. 62-285782), and derived from alfalfa (see Japanese Unexamined Patent Application No. 6-319536), and recombinant products (see Japanese Unexamined Patent Application No. 5-199882) are also known, and these bilirubin oxidases can also be used.

[0082] As bilirubin oxidase, commercial products may be used. Preferred examples of commercial products include those from Amano Enzyme Co., Ltd., Takara Bio Co., Ltd., Asahi Kasei Corporation, Sigma-Aldrich Corporation, and the like.

[0083] When the multi-copper oxidase is allowed to act on the medium in the manufacturing process of phenylacetaldehyde, the amount of the multi-copper oxidase that acts is not particularly limited, and it is preferably added in a manner such that the enzyme activity is 1U / mL or more relative to 1mM of the medium. From the viewpoint of further increasing the yield of phenylacetaldehyde, the enzyme activity is more preferably 5U / mL or more relative to 1mM of the medium, more preferably 10U / mL or more, more preferably 12U / mL or more, and particularly preferably 20U / mL or more. In addition, the enzyme activity can be 40U / ml or more, 60U / ml or more, 80U / ml or more, 100U / ml or more, or 110U / ml or more. The upper limit of the enzyme activity is not particularly limited, for example, relative to 1mM of the medium, preferably 2000U / mL or less, more preferably 1500U / mL or less, more preferably 1200U / mL or less, and particularly preferably 1000U / mL or less. In addition, the upper limit of the enzyme activity may be 800U / ml or less, 600U / ml or less, 400U / ml or less, 300U / ml or less, or 200U / ml or less. When laccase is used as the multi-copper oxidase, the amount of laccase added is preferably within the above range. It should be noted that the amount added is preferably appropriately adjusted according to the type of the multi-copper oxidase.

[0084] In addition, the amount of the multi-copper oxidase that plays a role in the manufacturing process of phenylacetaldehyde is not particularly limited, and it is preferably added in a manner such that the enzyme activity is 1U / mL or more relative to 10mM of phenylalanine. From the viewpoint of further improving the yield of phenylacetaldehyde, the enzyme activity is more preferably 5U / mL or more relative to 10mM of phenylalanine, more preferably 10U / mL or more, more preferably 12U / mL or more, and particularly preferably 20U / mL or more. In addition, the enzyme activity can be more than 40U / ml, more than 60U / ml, more than 80U / ml, more than 100U / ml, or more than 110U / ml. The upper limit of the enzyme activity is not particularly limited, for example, relative to 10mM of phenylalanine, preferably less than 2000U / mL, more preferably less than 1500U / mL, more preferably less than 1200U / mL, and particularly preferably less than 1000U / mL. In addition, the upper limit of the enzyme activity may be 800U / ml or less, 6000U / ml or less, 400U / ml or less, 300U / ml or less, or 200U / ml or less. When laccase is used as the multi-copper oxidase, the amount of laccase added is preferably within the above range. It should be noted that the amount added is preferably appropriately adjusted according to the type of the multi-copper oxidase.

[0085] The enzyme activity of multi-copper oxidase was determined by the following method. First, a solution prepared by mixing 0.25 mol / L phenol test solution, 0.009 mol / L 4-aminoantipyrine test solution, and 1 mol / L acetic acid·sodium acetate buffer (pH 4.5) in a ratio of 1:1:0.5 was used as the substrate solution. 2.5 ml of the substrate solution was placed in a cuvette, preheated at 30°C, and then 0.5 ml of the enzyme solution was added, stirred, and incubated at 30°C. The absorbance at 505 nm was measured after 10 seconds and 40 seconds. Then, when the absorbance increased by 0.1 within 1 minute, the amount of enzyme contained in 1 mL of the reaction solution was defined as 1 unit (U).

[0086] <Optional ingredients>

[0087] In the production process of phenylacetaldehyde, in addition to the above-mentioned components, an optional component may be appropriately present. For example, a pH adjuster (hydrochloric acid, sodium hydroxide, citric acid, acetic acid, etc.) may be added as an optional component.

[0088] <Manufacturing conditions>

[0089] The reaction time, temperature, pH of the reaction solution, etc. for allowing the mediator to act on phenylalanine or its analogs are not particularly limited. The reaction temperature is, for example, preferably 10°C or more, more preferably 15°C or more, further preferably 20°C or more, and particularly preferably 25°C or more. In addition, the reaction temperature is preferably 80°C or less, more preferably 70°C or less, and further preferably 60°C or less. In addition, the reaction temperature may be 50°C or less, or 40°C or less. The pH of the reaction solution is, for example, preferably 4 or more, and more preferably 5 or more. In addition, the pH of the reaction solution is preferably 10 or less, more preferably 9 or less, and further preferably 8 or less. The reaction time is, for example, preferably 1 minute or more, more preferably 10 minutes or more, and further preferably 15 minutes or more. The reaction time is preferably 240 hours or less, more preferably 96 hours or less, further preferably 48 hours or less, and particularly preferably 24 hours or less. In addition, the reaction time may be 12 hours or less, 6 hours or less, or 3 hours or less. It should be noted that the above reaction conditions are preferably adopted even in the presence of multi-copper oxidase when the mediator is allowed to act. By adopting the above reaction conditions, phenylacetaldehyde can be efficiently produced. It should be noted that the optimal reaction conditions may be determined by preliminary experiments or the like.

[0090] In the step of allowing the medium to act on phenylalanine or an analog thereof, shaking or stirring may be performed for at least a portion of the reaction time. In the shaking or stirring, shaking or stirring is preferably performed under conditions in which the components are sufficiently mixed.

[0091] When both the multi-copper oxidase and the mediator are allowed to act on phenylalanine or an analog thereof, the multi-copper oxidase and the mediator are preferably added to a solution containing phenylalanine. The order of addition is not particularly limited, and the mediator may be added after the multi-copper oxidase is added to the solution containing phenylalanine, or the multi-copper oxidase may be added after the mediator is added to the solution containing phenylalanine, or the multi-copper oxidase and the mediator may be added simultaneously to the solution containing phenylalanine.

[0092] As the solvent for allowing the multi-copper oxidase and the mediator to act on phenylalanine or its analogue, water, an organic solvent, or a mixed solvent thereof is preferably used. Examples of the organic solvent include alcohols (aliphatic saturated alcohols, aliphatic unsaturated alcohols, alicyclic alcohols, aromatic alcohols, heterocyclic alcohols, etc. such as methanol, ethanol, propanol, isopropanol, butanol, isobutyl alcohol, amyl alcohol, hexanol, heptanol, octanol, n-octanol, allyl alcohol, cyclopentanol, benzyl alcohol, etc.), ethers (methyl ether, ethyl ether, propyl ether, methyl ethyl ether, methyl propyl ether, methyl butyl ether, vinyl ether, etc.), esters (esters of formic acid, acetic acid, propionic acid, butyric acid, valeric acid, phthalic acid and other carboxylic acids with alcohols such as methanol, ethanol, propanol, isobutyl alcohol, butanol, isobutyl alcohol, n-pentanol, isopentanol, etc.), ketones (aliphatic saturated ketones, aliphatic unsaturated ketones, alicyclic ketones, aromatic ketones, etc. such as acetone, methyl ethyl ketone, methyl acetone, methyl vinyl ketone, cyclobutanone, acetophenone, etc.), and the like. Among them, from the viewpoint of reducing the load on the environment, the reaction solvent is preferably an aqueous solvent.

[0093] After the step of allowing the multi-copper oxidase and the mediator to act on phenylalanine or its analogue, a step of inactivating the multi-copper oxidase may be included. In the step of inactivating the multi-copper oxidase, for example, the reaction solution containing the multi-copper oxidase may be heated to 80° C. or higher and the enzyme solution may be separated by centrifugation or the like.

[0094] The amount of phenylacetaldehyde produced can be analyzed by, for example, GC-MS. Alternatively, the degree of phenylacetaldehyde production can be evaluated by sensory evaluation of the presence or absence of floral aroma.

[0095] (phenylacetaldehyde)

[0096] The present invention may also relate to phenylacetaldehyde produced by the above-mentioned production method. Since the phenylacetaldehyde obtained by the above-mentioned production method is safe, it can be added to medicines and food and beverages. That is, the present invention may also relate to phenylacetaldehyde for medicines and food and beverages, and may also relate to medicines and food and beverages containing the phenylacetaldehyde obtained by the above-mentioned production method.

[0097] The phenylacetaldehyde produced by the above production method can be isolated by, for example, dissolving it in an organic solvent that dissolves phenylacetaldehyde, separating the organic solvent layer, and then distilling off the solvent.

[0098] (Phenylacetaldehyde generating enzyme / enzyme for improving food and beverage flavor)

[0099] The present invention may also relate to a phenylacetaldehyde generating enzyme comprising a mediator and a multi-copper oxidase. In addition, the present invention may also relate to an enzyme for improving the flavor of food and drink comprising a mediator and a multi-copper oxidase. Here, the mediator is a compound comprising a ring structure having at least one hydroxyl group. By adding the phenylacetaldehyde obtained by the above-mentioned manufacturing method to food and drink, food and drink materials, it is possible to mask peculiar smell, give a rich flavor, fragrance to the food and drink, and improve the flavor.

[0100] Phenylacetaldehyde generating enzyme agent and food flavor improving enzyme agent can be any form in powder, solid, gel, liquid.Phenylacetaldehyde can be generated by adding phenylacetaldehyde generating enzyme agent and food flavor improving enzyme agent to the solution containing phenylalanine.In addition, phenylacetaldehyde generating enzyme agent and food flavor improving enzyme agent can be added to food and beverage containing phenylalanine, food and beverage material, in this case, phenylacetaldehyde can be generated by the phenylalanine contained in food and beverage, food and beverage material.It should be noted that when food and beverage, food and beverage material originally contain the above-mentioned medium, the medium may not be added sometimes.

[0101] (Method for improving the flavor of food and beverage)

[0102] The present invention may also relate to a method for improving the flavor of a food and drink comprising a process of causing a medium and a multi-copper oxidase to act on phenylalanine or its analog. Here, the medium is a compound containing a ring structure having at least one hydroxyl group. It should be noted that, in the case where food and drink or food and drink materials contain phenylalanine, the method for improving the flavor of a food and drink may be a method for improving the flavor of a food and drink comprising a process of causing a medium and a multi-copper oxidase to act on food and drink or food and drink materials, or may be a method for improving the flavor of a food and drink comprising a process of further adding phenylalanine to food and drink or food and drink materials and causing the medium and the multi-copper oxidase to play a role. By causing the medium and the multi-copper oxidase to act on phenylalanine or its analog, phenylacetaldehyde is generated, and the generated phenylacetaldehyde can mask the peculiar smell of food and drink, and give the food and drink a rich flavor and fragrance to improve the flavor of the food and drink.

[0103] As food and beverages, for example, dairy products, coffee drinks, black tea drinks, green tea drinks, beer, plant-based drinks (soy milk, almond milk, oat milk, rice juice, coconut milk, pea milk, potato milk), wine, cocoa and other beverages, plant-based meat substitutes and other foods. In addition, food and beverage materials are raw materials (materials) used to make food and beverages. As food and beverage materials, for example, cereals such as barley, rice, wheat, rye, oats, buckwheat, sorghum, barnyard grass, millet, teff, quinoa, and corn can be cited; beans and cereals such as soybeans, lentils, hollow beans, peas, chickpeas, mung beans, lupines, and kidney beans; seeds such as hemp (industrial hemp), canary seeds, flax seeds, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, ovate olive kernels, chestnuts, sesame seeds, and pine nuts can be cited; potatoes such as potatoes, sweet potatoes, and taro can be cited.

[0104] Example

[0105] Below, enumerate embodiment and comparative example and feature of the present invention is described in more detail.As long as the material, usage amount, ratio, processing content, processing sequence etc. shown in the following embodiment do not depart from the gist of the present invention, then can be changed appropriately.Therefore, the scope of the present invention should not be interpreted restrictively by the specific example shown below.

[0106] (Enzyme activity assay method)

[0107] The reagents described in this assay are prepared according to the polyphenol oxidase activity assay method in the 9th edition of the official manual of food additives. Specifically, 0.25 mol / L phenol test solution, 0.009 mol / L 4-aminoantipyrine test solution and 1 mol / L acetic acid·sodium acetate buffer (pH 4.5) are mixed in a ratio of 1:1:0.5 to prepare a substrate solution. 2.5 ml of the substrate solution is placed in a cuvette, preheated at 30°C, and 0.5 ml of the enzyme solution is added, stirred, incubated at 30°C, and the absorbance at 505 nm is measured after 10 seconds and 40 seconds. When the absorbance increases by 0.1 within 1 minute under this condition, the amount of enzyme contained in 1 mL of the reaction solution is defined as 1 unit (U).

[0108] (Materials used)

[0109] As a substrate (oxidation substrate), L-phenylalanine (Phe) (Fuji Film Wako Pure Chemical Industries, Ltd.) was used. In addition, as enzymes, Trametes-derived laccase (LC) (Amano Enzyme Co., Ltd.) and Myroculitis-derived bilirubin oxidase (BO) (Amano Enzyme Co., Ltd.) were used. It should be noted that as mediators (reduction substrates), the compounds described in the following table were used.

[0110] [Table 1]

[0111]

[0112] The substances in the medium* were added to a final concentration of 0.1% by mass, and the other media were added to a final concentration of 1 mM.

[0113] (Test Example 1)

[0114] 1 ml of 250 mM Tris-HCl (pH 7.0) buffer, 2.5 ml of 20 mM L-phenylalanine, 0.5 ml of various medium solutions of 10 mM or 1 mass %, and 1 ml of 0.5 mass % laccase solution were added to a 15 ml centrifuge tube, and the mixture was shaken and reacted at 30°C for 2 hours. After the reaction, the enzyme was inactivated by boiling for 10 minutes. It should be noted that the medium was dissolved in water or ethanol and added in a manner to a final concentration of 1 mM or 0.1 mass %.

[0115] [Table 2]

[0116]

[0117] After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and the generation of phenylacetaldehyde (PAA) was analyzed by GC-MS.

[0118] The analysis conditions of PAA were as follows, and the concentration of PAA was calculated using a calibration curve.

[0119] SPME Arrow (Solid Phase Extraction)

[0120] Fiber used: DVB / PDMS / CAR, 1cm

[0121] Extraction conditions: 30 minutes of heat preservation at 50°C → 30 minutes of adsorption at 50°C → 1 minute of desorption at 240°C

[0122] Baking conditions: 250℃, 30 minutes

[0123] GC-MS QP-2020NX

[0124] Column: InertCap pure-WAX (30m, 0.25mm, 0.25μm)

[0125] GC conditions: Heating: 60℃ for 2 min, 10℃ / min to 170℃, 20℃ / min to 240℃, 240℃ for 10 min Inlet: 250℃

[0126] MS conditions: SIM mode, monitoring ions m / z 65, 91, 120

[0127] The sensory evaluation in Table 3 confirmed the presence or absence of a floral aroma peculiar to phenylacetaldehyde after the reaction, and the odor was evaluated according to the following indexes.

[0128] ○: There is a flowery smell

[0129] △: There is a faint floral smell

[0130] ×: No fancy smell

[0131] [Table 3]

[0132]

[0133]

[0134] [Table 4]

[0135]

[0136] The above results show that compounds containing a ring structure having at least one hydroxyl group tend to easily generate PAA. In particular, compounds containing a catechol structure tend to easily generate PAA.

[0137] (Test Example 2)

[0138] The test was conducted using bilirubin oxidase (BO) derived from Myrocera (Amano Enzyme Co., Ltd.) as an enzyme. The reaction composition is as described in Table 5 below. Specifically, 1 ml of 250 mM Tris-HCl (pH 7.0) buffer, 2.5 ml of 20 mM L-phenylalanine, 0.5 ml of 10 mM DL-catechin, and 1 ml of 0.5 mass% enzyme (laccase (LC) derived from Trametes or bilirubin oxidase (BO) derived from Myrocera) were added to a 15 ml centrifuge tube, and the reaction was shaken at 30°C for 2 hours. After the reaction, an equal amount (5 ml) of 0.8 M formic acid solution was added to stop the reaction. After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and the generation of phenylacetaldehyde (PAA) was analyzed by GC-MS. In addition, the presence or absence of the floral aroma unique to phenylacetaldehyde was sensory evaluated using the same method as in Test Example 1.

[0139] [Table 5]

[0140]

[0141] [Table 6]

[0142] result

[0143]

[0144] From the above results, it was confirmed that when the bilirubin oxidase derived from Myroculina was used as the enzyme, PAA was produced in an amount similar to that when laccase was used.

[0145] (Test Example 3)

[0146] The effect of the pH of the reaction solution was confirmed. The reaction composition is as described in Table 7 below. Specifically, 250 mM Tris-HCl (pH 7.0) buffer or phosphate buffer (pH 6.0) or citric acid buffer (pH 5.0) 2 ml, 20 mM L-phenylalanine 5 ml, 10 mM tannic acid solution 1 ml, 0.5 mass % laccase solution 2 ml were added to a 15 ml centrifuge tube, and the reaction was shaken at 30°C or 50°C for 0.5 to 4 hours. Then, the enzyme was inactivated by boiling for 10 minutes.

[0147] After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and the generation of phenylacetaldehyde (PAA) was analyzed by GC-MS.

[0148] [Table 7]

[0149]

[0150] [Table 8]

[0151] result

[0152]

[0153] The reaction time was 0.5 hours, and PAA was produced at a level that could be detected in sensory evaluation. The production of PAA was also observed at a reaction temperature of 30°C, but the PAA yield was higher at 50°C. Regarding pH, the production of PAA was observed under all conditions, but the preferred condition was pH 6 or less.

[0154] (Test Example 4)

[0155] The effect of using green coffee bean extract, α-glucosylrutin, tannic acid, and no enzyme was confirmed. The reaction composition is described in Table 9 below. Specifically, 1 ml of 250 mM Tris-HCl (pH 7.0) buffer or phosphate buffer (pH 6.0), 2.5 ml of 20 mM L-phenylalanine, 0.5 ml of 10 mM or 1 mass % medium, and 1 ml of 0.5 mass % laccase solution were added to a 15 ml centrifuge tube, and the reaction was shaken at 30°C or 50°C for 2 hours. Then, the enzyme was inactivated by boiling for 10 minutes. For the medium here, a green coffee bean extract was added to a final concentration of 0.1 mass %, and tannic acid and α-glucosylrutin were added to a final concentration of 1 mM.

[0156] After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and the generation of phenylacetaldehyde (PAA) was analyzed by GC-MS.

[0157] [Table 9]

[0158]

[0159] [Table 10]

[0160] result

[0161]

[0162] Although PAA was generated even without using an enzyme, a trend that the generation of PAA further increased in the presence of an enzyme was confirmed.

[0163] (Test Example 5)

[0164] In the above-mentioned test example, the same test was conducted except that soy milk was used instead of the Tris-HCl buffer. The generation of PAA was also observed when soy milk was used.

[0165] (Test Example 6)

[0166] 1 ml of 250 mM phosphate buffer (pH 6.0), 2.5 ml of 20 mM L-phenylalanine, 0.5 ml of 1 mass % apple polyphenol solution, and 1 ml of 0.5 mass % laccase solution were added to a 15 ml centrifuge tube and reacted with shaking at 50° C. for 1 hour. After the reaction, the enzyme was inactivated by boiling for 10 minutes.

[0167] [Table 11]

[0168]

[0169] After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and the product was analyzed by GC-MS. The analysis conditions were as follows.

[0170] SPME Arrow (Solid Phase Extraction) General

[0171] Fiber used: DVB / PDMS / CAR, 1cm

[0172] Extraction conditions: 30 minutes of heat preservation at 50°C → 30 minutes of adsorption at 50°C → 1 minute of desorption at 240°C

[0173] Baking conditions: 250℃, 30 minutes

[0174] GC-MSQP-2020NX

[0175] Column: InertCap pure-WAX (30m, 0.25mm, 0.25μm)

[0176] GC conditions: Heating: 40℃ for 4 min, 10℃ / min to 170℃, 20℃ / min to 240℃, 240℃ for 10 min Inlet: 250℃

[0177] MS conditions: Measurement mode SCAN

[0178] <Sensory Evaluation>

[0179] The product contained in the reaction solution was evaluated for its odor. As a result, a strong floral aroma was perceived.

[0180] <Analysis by GC-MS>

[0181] The products were identified using the standards shown in Table 12 below.

[0182] [Table 12]

[0183]

[0184] [Table 13]

[0185]

[0186] As a result of the analysis based on GC-MS, as shown in Table 13, it was confirmed that benzaldehyde was generated as a product in addition to phenylacetaldehyde from L-phenylalanine.

[0187] (Test Example 7)

[0188] Confirm the amount of phenylacetaldehyde and benzaldehyde products.

[0189] 1 ml of 250 mM phosphate buffer (pH 6.0), 2.5 ml of 20 mM L-phenylalanine, 1 ml of 1 mass % apple polyphenol solution, and 1 ml of 0.5 mass % laccase solution were added to a 15 ml centrifuge tube and reacted with shaking at 50° C. for 1 hour. After the reaction, the enzyme was inactivated by boiling for 10 minutes.

[0190] [Table 14]

[0191]

[0192] After dissolving 0.8 g of NaCl in 4 ml of the reaction solution, solid phase extraction was performed using SPME Arrow, and analysis was performed using GC-MS.

[0193] [Table 15]

[0194]

[0195] (+ indicates added, - indicates no added (※ water is added instead))

[0196] When L-phenylalanine was used as the substrate, BA (benzaldehyde) increased by about 70 times and PAA (phenylacetaldehyde) increased by about 8 times compared to when only apple polyphenol was used. It is predicted that benzaldehyde will be produced as a by-product after the production of phenylacetaldehyde.

Claims

1. A method for producing phenylacetaldehyde, comprising the step of allowing a medium to act on phenylalanine or an analog thereof, The medium is a compound including a ring structure having at least one hydroxyl group.

2. The method for producing phenylacetaldehyde according to claim 1, wherein The step of allowing the mediator to act is a step performed in the presence of a multi-copper oxidase.

3. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The medium is a compound including a ring structure having at least one hydroxyl group and an alkoxy group, or a compound including a ring structure having two or more hydroxyl groups.

4. The method for producing phenylacetaldehyde according to claim 3, wherein The ring structure is a benzene ring or a dihydrofuran ring.

5. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The medium is at least one selected from DL-catechin, tannic acid, chlorogenic acid, caffeic acid, curcumin, hesperidin, L-DOPA, pyrogallol, vanillin, eugenol, gallic acid monohydrate, ferulic acid, isoeugenol, α-glucosylrutin and rutin.

6. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The medium is a compound contained in at least one selected from apple extract, pectin, green coffee bean extract and tea extract.

7. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The medium is polyphenols.

8. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The medium is at least one selected from α-glucosylrutin, chlorogenic acid, caffeic acid, tannic acid and rutin.

9. The method for producing phenylacetaldehyde according to claim 2, wherein The multi-copper oxidase is at least one selected from the group consisting of phenol oxidase and bilirubin oxidase.

10. The method for producing phenylacetaldehyde according to claim 9, wherein The multi-copper oxidase is laccase.

11. The method for producing phenylacetaldehyde according to claim 1 or 2, wherein The reaction temperature in the step of allowing the medium to act is 60° C. or lower.

12. A phenylacetaldehyde generating enzyme agent comprising a mediator and a multi-copper oxidase, The medium is a compound including a ring structure having at least one hydroxyl group.

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