Use of cruciferae eutrema and wasabi plants

Abstract

To provide a method for producing conjugates in which an isothiocyanate compound derived from one or more plants selected from the group consisting of plants of the genus Eutrema and plants of the genus Wasabi and a thiol group-containing compound are bonded by a dithiocarbamic acid structure.SOLUTION: The production method includes a production step of storing a paste-like mixture containing a crushed product of one or more plants selected from the group consisting of Eutrema plants and Japanese horseradish plants or a part thereof and a thiol group-containing compound at 0 °C or higher and 20 °C or lower for 12 hours or more to produce a conjugation product, in which the paste-like mixture in the production step contains the thiol group-containing compound such that the number of moles of thiol groups is 0. 100g or more and 1mmol or less with respect to 50mmol of the crushed product.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present specification relates to the use of plants belonging to the genus Wasabi and the genus Horseradish of the Brassicaceae family. [Background technology]

[0002] Plants of the genus Wasabi and the genus Horseradish in the Brassicaceae family are useful as edible plants. These plants are known to contain glucosinolates, precursors of isothiocyanate compounds that induce detoxification and immune activation. When glucosinolates are crushed, the glucose is released by the plant's own myrosinase, producing allyl isothiocyanate (AITC), 6-methylsulfinylhexyl isothiocyanate (6-MSITC), and other compounds.

[0003] Isothiocyanate compounds are unstable compounds. Patent Document 1 describes the production of a certain type of conjugate by adding water to ground arugula seeds, which belong to the Brassicaceae family, and then adding yeast extract powder or yeast extract solution and allowing to stand at 25°C for 3 hours. The conjugate is an isothiocyanate compound-thiol group-containing compound in which an isothiocyanate compound and a thiol group-containing compound are linked via a dithiocarbamate structure. Patent Document 1 also describes that supplying this conjugate to B16 melanoma cells and culturing them showed a promoting effect on glutathione production in the cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 002279 Summary of the Invention [Problem to be solved by the invention]

[0005] Even those skilled in the art could not predict whether isothiocyanate compound-thiol group-containing compound conjugates could be obtained in the same way from plants of the genus Wasabi and plants of the genus Horseradish. This is because the composition of the reaction product obtained using plants of the genus Wasabi is different from that obtained using arugula seeds. Furthermore, a method for efficiently producing conjugates from plants of the genus Wasabi was not clear.

[0006] The present specification provides techniques for more effective utilization of plants of the genus Wasabi and Horseradish. [Means for solving the problem]

[0007] The present inventors focused on 6-MSITC, AITC, and other compounds obtained from wasabi, a plant of the Wasabi genus. After investigating various methods for converting glucosinolates, precursors of these isothiocyanate compounds, into isothiocyanate compound-thiol group-containing conjugates, they discovered a method for efficiently obtaining these conjugates. It was found that this method can also be applied to plants of the Wasabi genus and Horseradish genus. Furthermore, they discovered a method for producing processed products containing such conjugates. Based on these findings, the present specification provides the following means.

[0008] [1] A method for producing a conjugate in which an isothiocyanate compound derived from one or more plants selected from the group consisting of plants of the genus Wasabi and plants of the genus Horseradish and a thiol group-containing compound are bonded via a dithiocarbamate structure, the method comprising: a production step of storing a paste-like mixture containing crushed material of the plant or a part thereof and the thiol group-containing compound at a temperature of 0°C to 20°C for 12 hours or more to produce the conjugate; and the paste-like mixture in the production step contains the thiol group-containing compound in an amount such that the number of moles of thiol groups is 0.1 mmol to 50 mmol per 100 g of the crushed material. [2] The method of producing a paste-like mixture according to [1], wherein the producing step is a step of storing the paste-like mixture at a temperature of 0°C or higher and 15°C or lower. [3] The method of producing a thiol-containing compound according to [1], wherein the generating step is a step of storing the crushed material and the thiol-containing compound at a temperature of 0°C or higher and 10°C or lower for 20 hours or longer. [4] A manufacturing method according to any one of [1] to [3], wherein the production step uses a mixture of more than 0 parts by mass and 20 parts by mass or less of water mixed with 100 parts by mass of the crushed material and the thiol group-containing compound. [5] The method of producing a thiol group-containing compound according to [3], wherein the production step uses a mixture of 100 parts by mass of the crushed material and more than 0 parts by mass and 20 parts by mass or more and 200 parts by mass or less of water. [6] The manufacturing method according to any one of [1] to [5], further comprising a mixture preparation step of mixing and crushing the plant or a part thereof, water, and the thiol group-containing compound to prepare the paste-like mixture prior to the production step. [7] The manufacturing method according to [6], wherein the mixture preparation step is a step of preparing the paste-like mixture without using any solvent other than water. [8] The method according to any one of [1] to [7], wherein the thiol group-containing compound is a cysteine-containing peptide. [9] The method according to any one of [1] to [8], wherein the isothiocyanate compound is allyl isothiocyanate or 6-methylsulfinylhexyl isothiocyanate.

[10] The method of any one of [1] to [9], further comprising a freezing step of freezing the paste mixture after the producing step. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present specification discloses a method for efficiently producing a functional ingredient derived from one or more plants selected from plants of the genus Wasabi and plants of the genus Horseradish (hereinafter simply referred to as the plant).The method disclosed herein makes it possible to efficiently obtain an isothiocyanate compound-thiol group-containing conjugate (hereinafter simply referred to as the conjugate) derived from such a plant or a part thereof, thereby enabling the effective use of the plant or a part thereof.

[0010] According to the production method disclosed herein, by crushing the plant or a part thereof and preparing a paste-like mixture containing a certain amount of the thiol group-containing compound under certain conditions, it is inferred that the production of isothiocyanate compounds from the glucosinolates contained in the plant or a part thereof is promoted, and furthermore, the bonding reaction between the produced isothiocyanate compounds and the thiol group-containing compound is promoted, further promoting the production of isothiocyanate compounds. Therefore, it is believed that conjugates can be obtained efficiently.

[0011] Furthermore, although not intended to be limiting to the disclosure of this specification, it is presumed that the conjugate is taken up into the body and decomposed to produce an isothiocyanate compound and a thiol group-containing compound, each of which exhibits a function useful to the body.

[0012] In this specification, Wasabi plants include Wasabia sp. plants in the Brassicaceae family, typically Eutrema japonicum, and Horseradish plants include Armoracia sp. plants in the Brassicaceae family, typically Armoracia rusticana (also known as horseradish, lefort, or mountain wasabi).

[0013] The method for producing the conjugate disclosed in this specification (hereinafter also simply referred to as the present production method) will be described below.

[0014] This production method is a method for producing a conjugate. The production method includes a production step of storing a paste-like mixture containing the crushed plant or a part thereof and the thiol group-containing compound at a temperature between 0°C and 20°C for 12 hours or more to produce the conjugate. The conjugate is formed by bonding an isothiocyanate compound and a thiol group-containing compound via a dithiocarbamate structure. The structure of the conjugate is represented by the following formula (1):

[0015] [ka]

[0016] In formula (1), R 1 indicates the structural portion of the isothiocyanate compound excluding NCS, and R 2 is a thiol group (SH or S) in a thiol group-containing compound. - ) is shown.

[0017] (Isothiocyanate compounds) The isothiocyanate compound is not particularly limited as long as it is a compound derived from the plant and has an NCS group, and examples thereof include 6-MSITC, AITC, and 2-phenylethyl isothiocyanate (PEITC), as shown below.

[0018] [ka] [ka] [ka]

[0019] (Thiol group-containing compounds) The thiol group-containing compound is a compound containing a thiol group (SH or S - ) and is not particularly limited as long as it can be bonded to an isothiocyanate compound to form a conjugate. As the thiol group-containing compound, for example, in terms of reactivity and application, an amino acid having a thiol group or a derivative thereof, a peptide containing a natural amino acid having a thiol group, a protein containing an amino acid having a thiol group, as well as a thiolated peptide and protein can be used. More specifically, cysteine ​​or a derivative thereof, or a peptide containing cysteine ​​can be used.

[0020] Such thiol group-containing compounds may be natural products, extracts, or chemically or microbially synthesized products. Peptides may be used in the L-form, D-form, or a mixture thereof (e.g., racemic form), although the L-form may be preferred.

[0021] Examples of thiol group-containing compounds include one or more cysteine-containing peptides selected from glutathione, cysteine, cysteinylglycine, N-acetylcysteine, and γ-glutamylcysteine, or salts thereof. Glutathione may be reduced or oxidized. In this specification, the cysteine-containing peptide includes cysteine ​​and peptides containing one or more cysteines.

[0022] The thiol group-containing compound may be a composition containing the thiol group-containing compound. Examples include those derived from fermentation products of the genera Saccharomyces, Schizosaccharomyces, and Candida. These fermentation product-derived compositions may contain, for example, one or more cysteine-containing peptides selected from glutathione, cysteine, and γ-glutamylcysteine, or salts thereof. Glutathione may be preferred as the cysteine-containing peptide.

[0023] As a composition derived from a fermentation product, commercially available yeast extracts are derived from yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, the genus Schizosaccharomyces, such as Schizosaccharomyces pombe, and the genus Candida, such as Candida utilis. For example, yeast extracts derived from yeasts belonging to the genus Saccharomyces or Candida may be suitable in terms of the composition and content of thiol group-containing compounds. Yeast extracts are available as commercial products containing, for example, 5 to 20% by mass of glutathione.

[0024] The salt of the thiol group-containing compound is not particularly limited. Examples of salts include salts with inorganic bases, salts with inorganic acids, and salts with organic acids, and it is preferable to select a salt that is acceptable for use in medicines, foods, beverages, etc. Examples of salts with inorganic bases include alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as calcium and magnesium, and ammonium salts. Examples of salts with inorganic acids include salts with hydrohalic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, etc. Examples of salts with organic acids include salts with formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, maleic acid, fumaric acid, and citric acid.

[0025] (Conjugate production process) In the conjugate production step of this production method, a paste-like mixture containing crushed material of the plant or a part thereof (hereinafter also referred to as the raw material) and a thiol group-containing compound is used. "Paste-like" refers to a viscous fluid. Use of the paste-like mixture in the conjugate production step can promote the production of the isothiocyanate compound and the conjugate.

[0026] The raw material contains glucosinolates that produce isothiocyanate compounds, as well as myrosinase that dissociates glucose from the glucosinolates to produce isothiocyanate compounds.

[0027] The raw material of a plant of the genus Wasabi is not particularly limited, and may be, for example, one or a suitable combination of two or more parts selected from the group consisting of rhizomes, fine roots, thick roots, tillers, flowers, leaves, petioles (stems), etc. Furthermore, the raw material of a plant of the genus Horseradish is not particularly limited, and may be, for example, one or a suitable combination of two or more parts selected from the group consisting of taproots, lateral roots, etc.

[0028] For example, the main edible part of wasabi is the rhizome (underground stem). The rhizome is sometimes used as a seasoning. Other edible parts include the flowers, leaves, tillers, petioles, and roots (thin roots and thick roots). These edible parts may be used as is or in processed foods.

[0029] Ingredients can include fresh produce (those that have not undergone any special processing after harvesting), as well as refrigerated (above 0°C and below 10°C), frozen (below -18°C), thawed frozen products, and other generally undried ingredients (hereinafter also referred to as raw ingredients) that are equivalent to raw. Dried ingredients, such as freeze-dried raw ingredients, can also be used.

[0030] The paste-like mixture used in the conjugate production step may be prepared in any manner. It may be prepared as a mixture of a disrupted material (single disrupted material) obtained by disrupting the raw material and a thiol group-containing compound. Alternatively, it may be prepared as a simultaneous disrupted material obtained by disrupting the raw material and the thiol group-containing compound together. Furthermore, water may be added to such a single disrupted material, mixture, or simultaneous disrupted material before and / or after disruption, as necessary.

[0031] The crushed material is not particularly limited, but generally, the crushed material has a size of 0.1 mm to 2 mm, for example, 50% or more, for example, 60% or more, for example, 70% or more, for example, 80% or more, or for example, 90% or more. The crushed material is not particularly limited, but can be obtained, for example, by using a mixer, grinder, shredding, crushing, pulverizing, grinding, or grating the raw material.

[0032] When raw raw materials are used as raw materials, these raw materials are not intended to be actively dried, and therefore retain a certain amount of moisture before harvesting, regardless of the form they are in. Therefore, there are cases where water is not used during crushing, and external water does not need to be added to the final paste-like mixture.

[0033] On the other hand, even if the raw materials are used, they can be crushed using water or water can be added after crushing, if necessary. For example, more than 0 parts by mass but not more than 20 parts by mass of water can be used for crushing per 100 parts by mass of the raw materials. The amount of water used is such that when the raw materials are mixed with the thiol group-containing compound, they become paste-like. In this case, the amount of water used can be, for example, 1 part by mass or more and 16 parts by mass or less, 2 parts by mass or more and 14 parts by mass or less, or 4 parts by mass or more and 12 parts by mass or less, per 100 parts by mass of the raw materials.

[0034] When using dry raw materials as raw materials, in order to obtain a paste-like mixture, water can be used in an amount of 10 to 200 parts by weight per 100 parts by weight of the dry materials, depending on the amount of moisture in the dry materials.

[0035] In the paste-like mixture, it is preferable not to use any solvent other than water, even lower alcohols such as ethanol, because such solvents reduce the activity of myrosinase and inhibit the formation of conjugates.

[0036] The paste-like mixture may contain, in addition to the raw materials and the thiol group-containing compound, various salts, and sugars such as sucrose, starch syrup, and sugar alcohols.

[0037] The paste-like mixture can typically be prepared prior to the production step by mixing and crushing the raw material, the thiol group-containing compound, and, if necessary, water, etc. In this way, it is expected that the isothiocyanate compound is produced from the glucosinolate simultaneously with the crushing and mixing of the raw material, and the conjugate production reaction between the isothiocyanate compound and the thiol group-containing compound proceeds all at once.

[0038] (Amount of thiol group-containing compound used, etc.) The conjugate is produced by the equimolar reaction of the NCS group in the isothiocyanate compound with the SH group in the thiol group-containing compound. The present inventors have found that the conjugate can be stably obtained by using a predetermined amount of the thiol group-containing compound relative to the amount of the raw material, without quantifying the amount of the thiol group-containing compound in the raw material.

[0039] According to the present inventors, when various raw materials (various parts or combinations thereof) in the present specification are stored for a relatively long time in a low temperature range as described below to produce a conjugate, the number of thiol groups (S - A stable conjugate can be obtained by using a thiol group-containing compound so that the number of moles of thiol group (or SH group) is 0.1 mmol to 50 mmol. If the amount is less than 0.1 mmol, it is difficult to promote the formation of the conjugate, and if it is 50 mmol or more, it is difficult to obtain the effect of promoting the formation of the conjugate.

[0040] The lower limit of the amount of the thiol group-containing compound used per 100 g of raw material (e.g., crushed material) is, for example, 0.5 mmol, 1.0 mmol, 1.2 mmol, 1.4 mmol, 1.6 mmol, 1.8 mmol, 2.0 mmol, or 2.2 mmol. The upper limit of the amount used is, for example, 10 mmol, 8.0 mmol, 6.0 mmol, 5.8 mmol, 5.6 mmol, 5.4 mmol, 5.2 mmol, 5.0 mmol, 4.8 mmol, 4.6 mmol, 4.4 mmol, 4.0 mmol, 3.8 mmol, 3.6 mmol, or 3.0 mmol. The range of the amount of the thiol group-containing compound used can be set by appropriately combining the above-mentioned upper and lower limits, and is, for example, 0.5 mmol to 10 mmol, 0.5 mmol to 6.0 mmol, 2.0 mmol to 4.0 mmol, or 2.0 mmol to 3.0 mmol.

[0041] The combination of the isothiocyanate compound and the thiol group-containing compound that constitute the conjugate is not particularly limited. Examples of the conjugate include the following: 6-MSITC-cysteine ​​conjugate, 6-MSITC-glutathione conjugate, 6-MSITC-γ-glutamylcysteine ​​conjugate, AITC-cysteine ​​conjugate, AITC-glutathione conjugate, AITC-γ-glutamylcysteine ​​conjugate, PEITC-cysteine ​​conjugate, PEITC-glutathione conjugate, and PEITC-γ-glutamylcysteine ​​conjugate.

[0042] For example, when using a yeast extract containing a certain concentration of glutathione, the amount of yeast extract used is determined so that the molar equivalent of the thiol group in this glutathione falls within the above range.

[0043] (Temperature and time for obtaining conjugate) The present production method can include a production step of producing the conjugate by storing the paste-like mixture at a temperature of 0° C. or higher and 20° C. or lower for 12 hours or longer. The production step is carried out while the paste-like mixture is left to stand.

[0044] With regard to temperature, temperatures below 0°C inhibit the production of isothiocyanate compounds, while temperatures above 20°C increase the proliferation of microorganisms derived from the raw materials, which tends to cause fluctuations in the amount of conjugates produced, and in many cases inhibit the production of isothiocyanate compounds, which in turn inhibits the production of conjugates.Storage at such low temperatures ensures that isothiocyanate compounds are produced from glucosinolates by the action of myrosinase in the paste-like mixture, and the produced isothiocyanate compounds can be rapidly converted into conjugates with thiol group-containing compounds while suppressing volatilization.

[0045] The upper limit of the temperature can be, for example, less than 20°C, 19°C or less, 15°C or less, 10°C or less, or 9°C or less. The lower limit of the storage temperature can be 1°C or more, 2°C or more, 3°C or more, 4°C or more, or 5°C or more. The temperature range can be set by appropriately combining these upper and lower limit temperatures. For example, the range can be 1°C or more and 19°C or less, 1°C or more and 15°C or less, 1°C or more and 10°C or less, or 2°C or more and 9°C or less.

[0046] With regard to the storage time, if it is less than 12 hours, the production of the isothiocyanate compound may be insufficient. The upper limit of the storage time can be, for example, 60 hours or less, 55 hours or less, 50 hours or less, 48 ​​hours or less, 36 hours or less, 30 hours or less, 24 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, etc. The lower limit of the storage time can be, for example, 14 hours or more, 16 hours or more, 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, etc. The range of the storage time can be set by appropriately combining these upper and lower limit temperatures. For example, the storage time can be 12 hours or more to 60 hours or less, 12 hours or more to 48 hours or less, 12 hours or more to 36 hours or less, 12 hours or more to 30 hours or less, 24 hours or more to 48 hours or less, etc.

[0047] The conjugate production step can typically be carried out by storing a paste mixture using raw raw materials at a temperature of 1°C to 15°C, 1°C to 10°C, or 2°C to 9°C for 12 to 36 hours, 12 to 24 hours, or 12 to 18 hours.

[0048] The paste-like mixture after the production step contains the produced conjugate. The paste-like mixture is processed into an effective form for foods, supplements, etc., either as is or after appropriate drying and freezing. Since the conjugate suppresses the pungency due to the isothiocyanate compound, oral ingestion also suppresses the pungency while still allowing the functionality of the isothiocyanate compound to be exhibited.

[0049] For example, after the production step, a freezing step may be performed in which the paste-like mixture is frozen. Even if the freezing step is performed, the conjugate can be stably maintained. Furthermore, freeze-drying may be performed simultaneously with or after the freezing step. This allows the conjugate to be maintained even more stably.

[0050] For example, after the production step, a solid-liquid separation step may be carried out to remove solid impurities derived from raw materials, yeast extract, etc. Furthermore, after the production step, a concentration step may be carried out to reduce the water content of the paste-like mixture.

[0051] These various steps after the production step can be combined as appropriate.

[0052] The resulting conjugate can be used for various purposes. In the conjugate state, an unstable isothiocyanate compound can be stabilized as a conjugate with a thiol group-containing compound. Therefore, various forms of conjugate-containing compositions (foods (including seasonings), supplements, food additives, quasi-drugs, pharmaceuticals, etc.) can be produced.

[0053] Furthermore, when in the conjugated state, it is expected that isothiocyanate compounds can be delivered stably to the body through oral ingestion by humans and other animals. The conjugates are decomposed by metabolism in intestinal bacteria and the action of gamma-glutamyl transferase in the body, and the isothiocyanate compounds can contribute to detoxification and immunostimulation in the body.

[0054] The formulation form of supplements, quasi-drugs, pharmaceuticals, etc. intended for oral intake is not particularly limited, but examples include tablets, granules, powders, capsules, etc. [Example]

[0055] Examples will be described below as specific examples to more specifically explain the disclosure of this specification. The following examples are intended to illustrate the disclosure of this specification, but are not intended to limit the scope thereof. [Example]

[0056] In this example, the rhizome, leaves, stems, and white root of Japanese wasabi (whole frozen and stored) were each chopped (to about 10 mm to 20 mm), mixed with cysteine-containing yeast extract (containing 15% by mass of glutathione), and water in the mixing ratio (mass ratio) shown in the table below, stirred and chopped in a mixer for 5 to 10 minutes, and made into paste form, producing pastes of Production Examples 1 to 7.

[0057] [Table 1]

[0058] These pastes were stored in a refrigerator at 5°C for 20 hours. After refrigeration, these pastes were cut into plates approximately 30 mm thick and frozen at -18°C for 72 hours. The frozen pastes of Examples 1 to 7 were analyzed by LC / MS to measure the amounts of 6-MSITC-glutathione (GSH) conjugates and AITC-glutathione conjugates produced. LC / MS was performed under the following conditions. Specifically, the reaction solution was introduced into an LC-MS system (Waters Japan, Acquity UPLC high-performance liquid chromatograph mass spectrometer), the cone voltage was set to 30 eV, and the compounds were detected by specifying their molecular weights in SIR mode. The analytical conditions were as shown in Table 1. The results are also shown in Table 1. The sample solution used was a filtrate obtained by dissolving 1 g of the paste material in 10 ml of 80% acetonitrile, adding 1 ml of the solution to 9 ml of 80% acetonitrile, and filtering the resulting solution.

[0059] As shown in Table 1, for mixtures of various parts of Japanese wasabi, large amounts of glutathione conjugates could be obtained when the cysteine-containing yeast extract was used in a range of 0.294 to 100 parts by mass (0.1 mmol to 50 mmol, particularly 0.5 mmol to 10 mmol, 0.5 mmol to 6.0 mmol in terms of the number of moles of thiol groups) per 100 parts by mass of the mixture.

[0060] With reference to Production Examples 1 to 3, it was found that the production of conjugates was ensured when a thiol group-containing compound was used in a range of 0.1 to 50 mmol of thiol groups per 100 g of mixture. Furthermore, with reference to Production Examples 4 to 7, which differ from Production Examples 1 to 3 in that water was used, it was found that the amount of conjugate produced increased when the number of moles of thiol groups per 100 g of mixture was in a range of 0.5 to 6 mmol. From the above, it was found that the production of conjugates was ensured by using a thiol group-containing compound in a range of 0.1 to 50 mmol of thiol groups per 100 g of raw material.

[0061] It is generally believed that an isothiocyanate compound and a thiol group-containing compound react in equimolar amounts to form a conjugate. However, it is difficult to estimate the extent to which the isothiocyanate compound is produced and volatilized in the paste-like mixture during the production process, and furthermore, whether conjugation has occurred. Therefore, it has been found useful to define the amount of the thiol group-containing compound used in terms of the number of moles of thiol groups relative to the mass of the raw materials.

[0062] From the above results, it was found that conjugates can be obtained efficiently by using 0.1 mmol to 50 mmol, 0.5 mmol to 10 mmol, or even 1.0 mmol to 6.0 mmol of a thiol group-containing compound per 100 mass parts of a plant of the genus Wasabi or a closely related plant of the genus Horseradish or a part thereof, and storing it at low temperature for a long period of time. [Example]

[0063] In this example, shredded material (approximately 5mm to 20mm) of various parts of Japanese wasabi (rhizome, leaves, stem, white root, all frozen) was mixed with cysteine-containing yeast extract (containing 15% by mass of glutathione) and water in the mixing ratio (mass ratio) shown in the table below, stirred and shredded using a mixer for 5 to 10 minutes, and made into a paste form, producing the pastes of Production Examples 8 to 12.

[0064] [Table 2]

[0065] These pastes were stored in a refrigerator at 5°C for 48 hours, except for Preparation Example 9, which was stored at 5°C for 24 hours.

[0066] After refrigerated storage, the pastes of Production Examples 8 and 10 to 12 were cut into plates with a thickness of approximately 30 mm and frozen for 24 hours at −18° C. Production Example 9 was similarly frozen for 48 hours at −18° C. For the frozen pastes of Examples 8 to 12, the amounts of 6-MSITC-glutathione conjugate and AITC-glutathione conjugate produced were measured in the same manner as in Example 1.

[0067] As shown in Table 2, when 100 parts by mass of crushed material from each part of Japanese wasabi was used in combination with cysteine-containing yeast extract (containing 15% by mass of glutathione) (0.75 parts by mass of glutathione, 2.44 mmol as the molar equivalent of the thiol group-containing compound), a large amount of MSITC-glutathione conjugate and AITC-glutathione conjugate was obtained.

[0068] From the above, it was found that regardless of the part of wasabi, a conjugate can be efficiently obtained by adding a thiol group-containing compound in a predetermined thiol group molar equivalent to 100 g of wasabi-derived part and storing it at low temperature for a long period of time.

Claims

1. A method for producing a conjugate in which an isothiocyanate compound derived from one or more plants selected from the group consisting of plants of the genus Wasabi and plants of the genus Horseradish and a thiol group-containing compound are bonded via a dithiocarbamate structure, the method comprising: and a production step of storing a paste-like mixture containing the crushed plant or part thereof and the thiol group-containing compound at a temperature of 0°C to 20°C for 12 hours or longer to produce the conjugate, wherein the paste-like mixture in the production step contains the thiol group-containing compound so that the number of moles of thiol groups is 0.1 mmol to 50 mmol per 100 g of the crushed plant or part thereof.

2. The method according to claim 1 , wherein the producing step is a step of storing the paste-like mixture at a temperature of 0° C. or higher and 15° C. or lower.

3. The method according to claim 1 , wherein the generating step comprises storing the crushed material and the thiol group-containing compound at a temperature of 0° C. or higher and 10° C. or lower for 20 hours or longer.

4. The method according to claim 1 , wherein the producing step uses a mixture of more than 0 parts by mass and 20 parts by mass or less of water per 100 parts by mass of the crushed material and the thiol group-containing compound.

5. The production process uses a mixture of more than 0 parts by mass and 20 parts by mass or more than 10 parts by mass and 200 parts by mass or less of water per 100 parts by mass of the crushed material and the thiol group-containing compound.

6. The manufacturing method according to claim 1, further comprising a mixture preparation step of mixing and crushing the plant or part thereof, water, and the thiol group-containing compound to prepare the paste-like mixture prior to the production step.

7. The method according to claim 6 , wherein the mixture preparation step is a step of preparing the paste-like mixture without using any solvent other than water.

8. The method according to claim 1 , wherein the thiol group-containing compound is a cysteine-containing peptide.

9. The method according to claim 1, wherein the isothiocyanate compound is allyl isothiocyanate or 6-methylsulfinylhexyl isothiocyanate.

10. The method according to claim 1 , further comprising a freezing step of freezing the paste-like mixture after the producing step.

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