Methionine γ-lyase activity inhibitor and method for screening the same

Abstract

To provide a methionine γ-lyase activity inhibitor that exerts excellent enzyme activity inhibitory performance by vapor phase reaction to methionine γ-lyase, and a method for screening the inhibitor.SOLUTION: Provided is a methionine γ-lyase activity inhibitor which consists of any compound selected from the following (i) to (v): (i) a specific monoterpene compound; (ii) a specific aliphatic ester compound; (iii) a specific carbonyl compound; (iv) a specific aldehyde group or hydroxyl group-containing unsaturated linear aliphatic compound; and (v) a specific aromatic alcohol compound.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of methionine γ-lyase activity and a screening method therefor. [Background technology]

[0002] Volatile sulfur compounds are components contained in the putrid odor that occurs when food waste and other materials decay. Volatile sulfur compounds are one of the unpleasant odor components in the home environment, where the putrid odor can be felt even at low concentrations, and there is a demand for effective deodorizing or preventing technologies for these compounds. In particular, because volatile sulfur compounds are relatively stable neutral compounds, it is difficult to deodorize them by chemical decomposition or adsorption, and there is a great demand for effective deodorizing or preventing technologies for them.

[0003] Food waste emits volatile sulfur compounds, which cause a putrid odor, when they are decomposed by bacteria to produce volatile sulfur compounds such as methyl mercaptan and hydrogen sulfide, which are foul-smelling gases. For example, among volatile sulfur compounds, hydrogen sulfide is produced from the sulfur-containing amino acid cysteine ​​by the action of cysteine ​​desulfurase, and methyl mercaptan is produced from methionine by the action of methionine γ-lyase (methioninase). Furthermore, the production of volatile sulfur compounds in the domestic environment can be caused by Pseudomonas aeruginosa, a type of aerobic gram-negative bacterium, or Escherichia coli, a type of facultative anaerobic gram-negative bacterium (see, for example, Patent Document 1).

[0004] Patent Document 1 reports that various fragrance ingredients have an inhibitory effect on the production of volatile sulfur compounds. Specifically, decanal, hexanal, octanal, benzaldehyde, citral, citronellal, hydroxycitronellal, β-ionone, methyl dihydrojasmonate, cis-jasmone, lyral, nootkatone, globanone, nerolidol, heliotropin, phenylpropylaldehyde, anisaldehyde, vanillin, cyclamen aldehyde, ethyl vanillin, helional, phenylacetaldehyde, cinnamic aldehyde, amylcinnamic aldehyde, hexylcinnamic aldehyde, methylnonylacetaldehyde, undecanoic acid, and the like. It has been reported that methylal, undecanal, dodecanal, nonanal, dupical, triplal, myrac aldehyde, β-damascone, γ-methylionone, α-ionone, α-damascone, α-methylionone, 8-cyclohexadecen-1-one, 9-cycloheptadecen-1-one, 4-cyclopentadecene, 7-cyclohexadecenolide, 10-oxa-16-hexadecenolide, and phenylacetaldehyde dimethyl acetal inhibit the production of volatile sulfur compounds from cysteine ​​and methionine.

[0005] The invention disclosed in Patent Document 1 relates to a preparation that suppresses the generation of volatile sulfur compounds, which have unpleasant odors, by topically applying it to areas in a domestic environment where these compounds are likely to be generated (e.g., paragraphs 0009, 0023, etc.). The preparations disclosed therein are specifically spray-type deodorants, dishwashing detergents, and fabric softeners (e.g., Examples 5 to 7 in paragraphs 0044 to 0049, etc.). In other words, the preparations disclosed in Patent Document 1 are agents in which the fragrance in liquid form is applied to a system containing a source of volatile sulfur compounds (hereinafter, this mode of action is referred to as a "liquid-phase reaction").

[0006] For this reason, in Patent Document 1, in the screening test for the above-mentioned fragrance ingredients, a substrate solution containing methionine or cysteine ​​is contacted with a solution containing a microorganism or an enzyme derived from a microorganism that produces volatile sulfur compounds and the above-mentioned fragrance, causing a reaction, thereby evaluating the ability of the above-mentioned fragrance ingredients to inhibit the production of volatile sulfur compounds (e.g., paragraphs 0032 to 0042, etc.). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-173441 Summary of the Invention [Problem to be solved by the invention]

[0008] There are liquid deodorizers housed in containers that can be attached to the inside of the lid of a lidded trash can or hung from it. This type of liquid deodorizer is volatile (volatile deodorizer). The container that houses this volatile deodorizer is partially equipped with, for example, a microporous sheet that is permeable to gas but not liquid, and the active ingredient vaporized from the liquid deodorizer volatilizes to the outside of the container through the microporous sheet. In this case, the active ingredient vaporized to the outside of the container is introduced to and acts in a reaction system in which volatile sulfur compounds are produced from a source of volatile sulfur compounds in the gas phase (hereinafter, this mode of action is referred to as a "gas-phase reaction").

[0009] Through intensive research by the present inventors, it has been found that the mechanism of action of the above-mentioned active ingredient, i.e., a compound (catalytic inhibitor) that inhibits the activity of a biocatalyst such as a microorganism or enzyme having volatile sulfur compound-producing activity, on a biocatalyst differs depending on whether the system containing the source of volatile sulfur compounds and the biocatalyst undergoes a liquid-phase or gas-phase reaction. In other words, the present inventors have discovered that a catalytic inhibitor that has excellent catalytic activity inhibition ability in a liquid-phase reaction may not be able to exhibit the same performance in a gas-phase reaction, and further that there are compounds with novel attributes that exhibit particularly excellent catalytic activity inhibition ability in a gas-phase reaction.

[0010] In addition, in order to suppress the production of volatile sulfur compounds, it is conceivable to use a disinfectant or antibacterial agent to remove microorganisms that have the activity of producing volatile sulfur compounds. However, in this case, caution may be required in handling from a safety standpoint. There is also a risk of the development of bacteria resistant to the disinfectant or antibacterial agent. Furthermore, there is a possibility that there may be bacteria that cannot be disinfected. On the other hand, if the activity of an enzyme that has the activity of producing volatile sulfur compounds can be inhibited without disinfection, it is possible to suppress the generation of bad odors. Specifically, if the enzymatic activity of the above-mentioned methionine γ-lyase can be inhibited, the production of methyl mercaptan from methionine can be suppressed, and the generation of bad odors can be suppressed. In this case, the above risks associated with disinfection can also be avoided.

[0011] An object of the present invention is to provide a methionine γ-lyase activity inhibitor that exhibits excellent inhibitory activity against methionine γ-lyase through a gas-phase reaction, and a screening method for the same. [Means for solving the problem]

[0012] According to one aspect of the present invention, there is provided an inhibitor of methionine γ-lyase activity, comprising any one of compounds selected from the following (i) to (v): (i) a monoterpene compound selected from the following: open-chain monoterpenes containing an ester bond, open-chain monoterpenes having a hydroxyl group at at least one end, cyclic monoterpenes containing a ring structure and composed only of hydrocarbons, cyclic monoterpenes containing saturated cyclic ketones, and cyclic monoterpenes containing a ring structure and an ether bond; (ii) an aliphatic ester compound represented by formula I: [ka] In the formula, R 11 represents an alkyl group having 1 to 4 carbon atoms, a cyclic saturated aliphatic group, or an unsaturated aliphatic group, and R 12 represents an alkyl group or an unsaturated aliphatic group, and L 11 represents a single bond or an ether bond, L 12 represents a single bond, an alkylene group, or an alkynylene group, and R 11 and L 11 and L 12 The total number of carbon atoms in R is 7 or less. 11 and R 12 at least one of represents an unsaturated aliphatic group; (iii) Carbonyl compounds represented by formula II: [ka] In the formula, R 21 represents a cycloalkadiene group or a phenyl group substituted with an amino group, and R 22 represents an alkoxy group or an unsaturated aliphatic group; (iv) Unsaturated chain aliphatic compounds having at least one end substituted with an aldehyde group or a hydroxyl group; provided that when only one end is substituted with an aldehyde group, the number of carbon atoms contained in the molecule is 11 or less, and when only one end is substituted with a hydroxyl group, the number of carbon atoms contained in the molecule is 10 or less; (v) an aromatic alcohol compound represented by formula III: [ka] In the formula, R 31 represents a single bond or a linking group, R 32represents a hydrogen atom or an alkyl group.

[0013] According to another aspect of the present invention, there is provided a methyl mercaptan production inhibitor containing, as an active ingredient, one or more of the methionine γ-lyase activity inhibitors according to the above aspect.

[0014] According to yet another aspect of the present invention, there is provided a methyl mercaptan production inhibitor according to the above aspect, which is a volatile agent.

[0015] According to yet another aspect of the present invention, there is provided a drug volatilization device comprising a container at least partially made of a microporous sheet and a methyl mercaptan production inhibitor according to any of the above aspects contained in the container, wherein the methionine gamma-lyase activity inhibitor volatilizes through the microporous sheet.

[0016] According to yet another aspect of the present invention, there is provided a method for screening for a substance that inhibits methionine γ-lyase activity, comprising the steps of: a primary screening step of selecting from test substances substances that inhibit the metabolic activity of a microorganism that produces methyl mercaptan from methionine in a gas-phase reaction; and a second screening step of selecting, from the substances selected in the first screening step, substances that inhibit the enzymatic activity of methionine γ-lyase, which produces methyl mercaptan from methionine, in a liquid phase reaction; A screening method is provided, comprising:

[0017] According to yet another aspect of the present invention, there is provided a screening method according to the above aspect, in which the gas-phase reaction in the primary screening step is carried out by vaporizing the test substance and introducing it into the reaction system in the gas phase.

[0018] According to yet another aspect of the present invention, the selection of the substance in the primary screening step involves selecting a substance that inhibits the activity of the microorganism using as an index a difference between the amount of methyl mercaptan produced in the presence of the test substance and the amount of methyl mercaptan produced in the absence of the test substance; The screening method according to any of the above aspects is provided, wherein the selection of the substance in the second screening step is performed by selecting a substance that inhibits the activity of methionine γ-lyase using as an index the difference between the amount of methyl mercaptan produced in the presence of the test substance and the amount of methyl mercaptan produced in the absence of the test substance.

[0019] According to yet another aspect of the present invention, there is provided a screening method according to any one of the above aspects, wherein the microorganism comprises one or more microorganisms selected from the genera Bacillus, Geotrichum, and Galactomyces.

[0020] According to yet another aspect of the present invention, there is provided a screening method according to the above aspect, wherein the microorganism comprises one or more microorganisms selected from Bacillus cereus, Bacillus mycoides, Geotrichum cadidum, and Galactomyces candidum.

[0021] According to yet another aspect of the present invention, there is provided a screening method according to any one of the above aspects, which is used for screening for an active ingredient of a methyl mercaptan production inhibitor.

[0022] According to yet another aspect of the present invention, there is provided the screening method according to the above aspect, wherein the methyl mercaptan production inhibitor is a volatile deodorant.

[0023] According to yet another aspect of the present invention, there is provided an inhibitor of methionine γ-lyase activity screened by the screening method according to any one of the above aspects. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide methionine γ-lyase activity inhibitors that exhibit excellent enzyme activity inhibitory performance through a gas-phase reaction with methionine γ-lyase, and a screening method for the same. DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, an embodiment of the present invention will be described. <Methionine γ-lyase activity inhibitor> The methionine gamma-lyase activity inhibitor according to the first embodiment of the present invention effectively inhibits the production of methyl mercaptan from methionine by inhibiting methionine gamma-lyase activity. Here, "methionine gamma-lyase activity" refers to the activity of catalyzing the reaction that produces methyl mercaptan using methionine, the source of methyl mercaptan, as a substrate. As described above, the methionine gamma-lyase activity inhibitor according to this embodiment inhibits the production of methyl mercaptan by enzyme inhibition rather than by sterilization. Therefore, it is highly safe, does not pose a problem of the development of resistant bacteria, and can effectively act against bacteria that cannot be sterilized. It is also highly useful in situations where sterilization is not permitted, such as compost.

[0026] Furthermore, the methionine γ-lyase activity inhibitor of this embodiment exhibits particularly effective inhibitory effect on the enzyme activity of methionine γ-lyase in gas-phase reactions, and is therefore particularly useful as an active ingredient in volatile deodorants.

[0027] The methionine γ-lyase activity inhibitor according to this embodiment is composed of any compound selected from the group consisting of (i) monoterpene compounds, (ii) aliphatic ester compounds, (iii) carbonyl compounds, (iv) aldehyde- or hydroxyl-containing unsaturated chain aliphatic compounds, and (v) aromatic alcohol compounds, which are described below. These compounds were found to exhibit excellent enzyme activity inhibitory properties in gas-phase reactions against methionine γ-lyase and to possess the novel attribute of effectively suppressing the production of methyl mercaptan, using a screening method according to the second embodiment of the present invention, which will be described later. These compounds will be explained below.

[0028] (i) Monoterpene compounds In one aspect, the methionine γ-lyase activity inhibitor according to this embodiment is a monoterpene compound. The monoterpene compound may be a chain monoterpene or a cyclic monoterpene. The monoterpene preferably contains at least one bond selected from the group consisting of a hydroxyl group, an ester bond, a carbonyl group, a cycloalkenyl group, and an ether bond. However, those containing an aldehyde group are excluded.

[0029] Specifically, the monoterpene compound is selected from a chain monoterpene containing an ester bond, a chain monoterpene having a hydroxyl group at at least one end, a cyclic monoterpene containing a ring structure and composed only of hydrocarbons, a cyclic monoterpene containing a saturated cyclic ketone, and a cyclic monoterpene containing a ring structure and an ether bond. In this specification, "chain" means either a straight chain or a branched chain, and may be either. Preferred specific examples of the monoterpene compound include the compounds shown in Table 1.

[0030] [Table 1]

[0031] (ii) Aliphatic ester compounds In another aspect, the methionine γ-lyase activity inhibitor according to this embodiment is an aliphatic ester compound represented by formula I. [ka]

[0032] In the formula, R 11 represents an alkyl group having 1 to 4 carbon atoms, a cyclic saturated aliphatic group, or an unsaturated aliphatic group, and R 12 represents an alkyl group or an unsaturated aliphatic group, and L 11 represents a single bond or an ether bond, L 12 represents a single bond, an alkylene group, or an alkynylene group, and R 11 and L 11 and L 12 The total number of carbon atoms in R is 7 or less. 11 and R 12 At least one of these represents an unsaturated aliphatic group.

[0033] R 11 The alkyl group having 1 to 4 carbon atoms, represented by the formula (I) may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, a sec-butyl group, and a tert-butyl group.

[0034] R 11 The cyclic saturated aliphatic group represented by the formula (R) may be monocyclic or polycyclic, but is preferably monocyclic. Examples of the monocyclic saturated aliphatic group include a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group. 11 may be a combination of the above alkyl group and a cyclic saturated aliphatic group.

[0035] R 11Specific examples of the unsaturated aliphatic group represented by the formula (I) include an alkenyl group, an alkynyl group, and a monocyclic or polycyclic unsaturated aliphatic group. The alkenyl group may be linear or branched, and has, for example, 2 to 7 carbon atoms. The alkynyl group may be linear or branched, and has, for example, 2 to 7 carbon atoms. Examples of the monocyclic unsaturated aliphatic group include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and a cycloheptenyl group. R 11 may be a combination of two or more of the above unsaturated aliphatic groups.

[0036] R 12 The alkyl group represented by the formula (I) may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, and an n-pentyl group.

[0037] R 12 Specific examples of the unsaturated aliphatic group represented by the formula (I) include an alkenyl group, an alkynyl group, and a monocyclic or polycyclic unsaturated aliphatic group. The alkenyl group may be linear or branched, and may have, for example, 2 to 12 carbon atoms, or 5 to 10 carbon atoms. The alkynyl group may be linear or branched, and may have, for example, 2 to 12 carbon atoms, or 5 to 10 carbon atoms. Examples of the monocyclic unsaturated aliphatic group include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, a cycloheptenyl group, a cyclooctenyl group, a cyclononenyl group, and a cyclodecenyl group. Examples of the polycyclic unsaturated aliphatic group include an adamantenyl group, a norbornenyl group, an isobornenyl group, a tricyclodecenyl group, and a tetracyclododecenyl group. R 12 may be a combination of two or more of the above unsaturated aliphatic groups.

[0038] L 12 The alkylene group represented by the formula (I) may be linear or branched, and has, for example, 1 to 7 carbon atoms, or 1 to 5 carbon atoms. L 12 The alkynylene group represented by the formula (I) may be linear or branched, and has, for example, 2 to 7 carbon atoms, or 2 to 5 carbon atoms.

[0039] Preferred specific examples of the aliphatic ester compound represented by formula I include the compounds shown in Table 2.

[0040] [Table 2]

[0041] (iii) Carbonyl compounds In another aspect, the methionine γ-lyase activity inhibitor according to this embodiment is a carbonyl compound represented by formula II. [ka]

[0042] In the formula, R 21 represents a cycloalkadiene group or a phenyl group substituted with an amino group, and R 22 represents an alkoxy group or an unsaturated aliphatic group;

[0043] R 21 is a cyclic alkenyl group containing two unsaturated bonds (alkenyl groups) in a cyclic structure, and preferably has 4 to 8 carbon atoms. Specific examples of the cycloalkadiene group include cyclobutadiene, cyclopentadiene, cyclohexadiene, cycloheptadiene, and cyclooctadiene.

[0044] R 22 The alkyl group contained in the alkoxy group represented by the formula (I) may be linear or branched, and may have, for example, 1 or more and 10 or less, or 1 or more and 6 or less, carbon atoms.

[0045] R 22Specific examples of the unsaturated aliphatic group represented by the formula (I) include an alkenyl group, an alkynyl group, and a monocyclic or polycyclic unsaturated aliphatic group. The alkenyl group may be linear or branched, and may have, for example, 2 to 10 carbon atoms, or 3 to 6 carbon atoms. The alkynyl group may be linear or branched, and may have, for example, 2 to 10 carbon atoms, or 3 to 6 carbon atoms. Examples of the monocyclic unsaturated aliphatic group include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, a cycloheptenyl group, a cyclooctenyl group, a cyclononenyl group, and a cyclodecenyl group. Examples of the polycyclic unsaturated aliphatic group include an adamantenyl group, a norbornenyl group, an isobornenyl group, a tricyclodecenyl group, and a tetracyclododecenyl group. R 22 The unsaturated aliphatic group represented by may be a combination of two or more of the above listed unsaturated aliphatic groups.

[0046] Preferred specific examples of the carbonyl compound represented by formula II include the compounds shown in Table 3.

[0047] [Table 3]

[0048] (iv) Aldehyde group- or hydroxyl group-containing unsaturated chain aliphatic compounds In another embodiment, the methionine γ-lyase activity inhibitor according to this embodiment is an unsaturated chain aliphatic compound having at least one end substituted with an aldehyde group or a hydroxyl group (an aldehyde group- or hydroxyl group-containing unsaturated chain aliphatic compound). The unsaturated chain aliphatic group contained in this compound can be an alkenyl group or an alkynyl group. The alkenyl group can be linear or branched, and has, for example, 2 to 10 carbon atoms, or 3 to 6 carbon atoms. The alkynyl group can be linear or branched, and has, for example, 2 to 10 carbon atoms, or 3 to 6 carbon atoms.

[0049] However, when only one end of this unsaturated chain aliphatic compound is substituted with an aldehyde group, the number of carbon atoms contained in the molecule is 11 or less, preferably 8 or less. When only one end is substituted with a hydroxyl group, the number of carbon atoms contained in the molecule is 10 or less.

[0050] Preferred specific examples of the aldehyde group- or hydroxyl group-containing unsaturated chain aliphatic compound include the compounds shown in Table 4.

[0051] [Table 4]

[0052] (v) Aromatic alcohol compounds In another aspect, the methionine γ-lyase activity inhibitor according to this embodiment is an aromatic alcohol compound represented by formula III. [ka]

[0053] In the formula, R 31 represents a single bond or a linking group, R 32 represents a hydrogen atom or an alkyl group. R 31 Specific examples of the linking group represented by the formula include an alkylene group or an alkenylene group. The alkylene group may be linear or branched, and for example, has 1 to 7 carbon atoms, or 1 to 5 carbon atoms. The alkenylene group may be linear or branched, and for example, has 2 to 7 carbon atoms, or 2 to 5 carbon atoms.

[0054] R 32 The alkyl group represented by the formula (I) may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, and an n-pentyl group.

[0055] Preferred specific examples of the aromatic alcohol compound represented by formula III include the compounds shown in Table 5.

[0056] [Table 5]

[0057] The methionine γ-lyase activity inhibitor according to the present embodiment is a volatile compound conventionally known as a fragrance ingredient. These compounds may be isolated from natural products or chemically synthesized. Essential oils containing the compounds may also be used, such as lemongrass oil, verbena oil, lemon myrtle oil, lemon oil, orange oil, thyme oil, clove oil, pimento oil, bay leaf oil, cinnamon oil, and ylang-ylang oil.

[0058] <Screening method> The screening method according to the second embodiment of the present invention is a method for screening for substances that inhibit methionine γ-lyase activity, and the methionine γ-lyase activity inhibitor according to the first embodiment described above is a compound selected by the screening method according to this embodiment. The substance selected by the screening method according to this embodiment is a methionine γ-lyase activity inhibitor that effectively suppresses the production of methyl mercaptan by inhibiting the enzyme methionine γ-lyase rather than by eliminating bacteria. Furthermore, this methionine γ-lyase activity inhibitor can be suitably used, in particular, as an active ingredient in a volatile methyl mercaptan production inhibitor. The screening method according to this embodiment is described below.

[0059] The screening method according to this embodiment includes the following steps: a primary screening step of selecting from test substances substances that inhibit the metabolic activity of a microorganism that produces methyl mercaptan from methionine in a gas-phase reaction; and The method includes a second screening step of selecting, from the substances selected in the first screening step, substances that inhibit the catalytic activity of methionine γ-lyase, which produces methyl mercaptan from methionine, in a liquid-phase reaction.

[0060] The test substance screened by the screening method according to this embodiment may or may not be a fragrance, and may be a synthetic or natural substance. The test substance may be, for example, a natural or synthetic fragrance, deodorant, antibacterial agent, or insecticide, or may be, for example, a terpene compound, an aliphatic ester compound, an aromatic ester compound, an aliphatic aldehyde compound, an aromatic aldehyde compound, an aliphatic alcohol compound, an aromatic alcohol compound, a carbonyl compound, an alicyclic compound, an aromatic compound, or a nitrogen-containing aliphatic compound. In one embodiment, the test substance is preferably a volatile substance. Here, "volatile substance" refers to, for example, a component having a boiling point in the range of 50 to 400°C that volatilizes at room temperature and normal pressure (e.g., 20°C and 1 atmosphere) and becomes gaseous in the air.

[0061] In the primary screening step, methyl mercaptan is produced from methionine by a metabolic reaction of a microorganism having methyl mercaptan-producing activity against methionine in the presence and absence of a test substance. The metabolic reaction in the presence of the test substance is carried out by vaporizing the test substance and introducing it in the gas phase into a reaction system of methionine and the microorganism. Specifically, the gaseous test substance is brought into contact with a medium containing methionine and the microorganism and allowed to stand for a predetermined time at a predetermined temperature. The medium used in the reaction system can be appropriately selected from media suitable for the microorganism, and a standard agar medium can be used, for example. The temperature required for the metabolic reaction in the primary screening step may be, for example, within the range of 25 to 37°C. The time required for the metabolic reaction may be, for example, 8 to 72 hours.

[0062] The microorganism may be, for example, a bacterium, yeast, or mold, and any microorganism known to have volatile sulfur compound-producing activity can be used. The present inventors have also confirmed that bacteria belonging to the genera Bacillus, Geotrichum, and Galactomyces produce methyl mercaptan from methionine (see Test Example 1 below). Specific examples of these bacteria include Bacillus cereus, Bacillus mycoides, Geotrichum candidum, and Galactomyces candidum.

[0063] In the primary screening step, substances that inhibit metabolic activity are selected using the difference between the amount of methyl mercaptan produced by the metabolic reaction in the presence of the test substance and the amount of methyl mercaptan produced by the catalytic reaction in the absence of the test substance as an indicator.

[0064] The difference in the amount of methyl mercaptan produced by the catalytic reaction in the presence and absence of a test substance can be quantified using, for example, Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid); DTNB). The SH groups contained in methyl mercaptan cleave the S—S bonds contained in an amount of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) equivalent to the amount of SH groups, producing 5-mercapto-2-nitrobenzoic acid (TNB). Methyl mercaptan can be quantified by measuring the absorbance (λmax = 412 nm) of the SH groups contained in TNB.

[0065] [ka]

[0066] The percentage calculated using the absorbance at 412 nm in the presence of the test substance and the absorbance at 412 nm in the absence of the test substance (blank) using the following formula (1) is the "malodor suppression rate," which indicates the percentage of suppression of methyl mercaptan generation. This malodor suppression rate is due to the inhibition of the metabolic activity of microorganisms that produce methyl mercaptan from methionine, so it is not only due to enzyme inhibition, but also includes suppression due to sterilization.

[0067]

number

[0068] In the screening method according to this embodiment, test substances with a malodor suppression rate of, for example, 50% or more in the first screening step are selected as test substances for the subsequent second screening step.

[0069] In the secondary screening step, methionine is catalytically reacted with methionine γ-lyase in the presence and absence of a test substance to produce methyl mercaptan from methionine. In the secondary screening step, the catalytic reaction in the presence of a test substance can be carried out by a known liquid-phase reaction. For example, a mixed aqueous solution of a methionine solution, a phosphate buffer, and a pyridoxal phosphate solution is prepared, and the test substance dissolved in an organic solvent and a methionine γ-lyase solution are mixed therewith, followed by standing in a thermostatic bath at a predetermined temperature.

[0070] In the second screening step, selection of substances that inhibit methionine γ-lyase activity is performed, as in the first screening step, using the difference between the amount of methyl mercaptan produced by the catalytic reaction in the presence of the test substance and the amount of methyl mercaptan produced by the catalytic reaction in the absence of the test substance as an index. The quantification of the difference in the amount of methyl mercaptan produced by the catalytic reaction in the presence and absence of the test substance is performed in the same manner as in the first screening step. In this case, the "malodor suppression rate" calculated by the above formula (1) is due only to enzyme inhibition in the second screening, and therefore may be distinguished by being called the "enzyme inhibition rate."

[0071]

number

[0072] As described above, the screening method according to this embodiment involves two steps: a primary screening step and a secondary screening step. In the primary screening step, substances that inhibit the metabolic activity of microorganisms in a gas-phase reaction are screened, and in the subsequent secondary screening step, substances that inhibit methionine γ-lyase activity in a liquid-phase reaction are screened for. This is for the following reasons.

[0073] Specifically, in the primary screening step, for example, if a suspension containing microorganisms and methionine is added to an agar medium, the microorganisms will grow primarily on the surface of the agar medium. Therefore, by introducing the test substance into this reaction system in the gas phase, it is possible to bring the microorganisms and the test substance into contact. In contrast, if, instead of the secondary screening step, the microorganism is replaced with methionine γ-lyase in the primary screening step and a mixture of methionine γ-lyase and methionine is added to the agar medium, the methionine γ-lyase may not remain on the agar surface but may penetrate into the agar. In such cases, it is impossible to confirm whether the methionine γ-lyase and the gas-phase test substance are in contact with each other, making it impossible to properly select substances with the ability to inhibit methionine γ-lyase activity.

[0074] On the other hand, if, instead of the primary screening step, a gas-phase reaction is carried out in the secondary screening step by contacting a gas-phase test substance with a mixed solution containing methionine, phosphate buffer, pyridoxal phosphate, and methionine γ-lyase, the difference in the solubility of methionine γ-lyase in the mixed solution will have an impact, making it impossible to properly select substances that have the ability to inhibit methionine γ-lyase activity.

[0075] Therefore, the screening method of this embodiment selects, in a primary screening step, substances that have good methyl mercaptan production inhibitory ability in a gas-phase reaction against a microorganism that has methyl mercaptan production activity, and then, in a subsequent secondary screening step, selects, from the substances selected in the primary screening step, substances that have the ability to inhibit methionine γ-lyase activity in a liquid-phase reaction.Methionine γ-lyase activity inhibitors selected by such a screening method of this embodiment can be suitably used, in particular, as the active ingredient of a volatile methyl mercaptan production inhibitor.

[0076] <Methyl mercaptan production inhibitor> The methyl mercaptan production inhibitor according to the third embodiment of the present invention contains one or more of the methionine gamma-lyase activity inhibitors according to the first embodiment described above. Therefore, the methyl mercaptan production inhibitor according to this embodiment is particularly useful as a volatile deodorizer.

[0077] The form of use of the methyl mercaptan production inhibitor according to this embodiment is not particularly limited. The methyl mercaptan production inhibitor according to this embodiment may be an agent consisting of only one or more methionine γ-lyase activity inhibitors, or may further contain other components as necessary. Examples of other components include solvents, surfactants, deodorants, insect repellents, repellents, antibacterial agents, fungicides, dyes, pigments, antioxidants, ultraviolet absorbers, and fragrance components.

[0078] The methyl mercaptan production inhibitor according to this embodiment can be formulated into various dosage forms by conventionally known methods by blending optional ingredients with a methionine γ-lyase activity inhibitor, as needed. Specific examples of dosage forms include liquid, gel, sheet, and solid forms. Of these dosage forms, the liquid form of the methyl mercaptan production inhibitor is preferred.

[0079] When a carrier is used to carry the methionine γ-lyase activity inhibitor in the methyl mercaptan production inhibitor, examples of the carrier include wood, paper, cloth, nonwoven fabric, silica, talc, activated carbon, silica gel, zeolite, fluorite, cellulose beads, activated carbon, and ceramics.

[0080] Furthermore, in the methyl mercaptan production inhibitor according to the present embodiment, when the methionine γ-lyase activity inhibitor is gelled using a gelling agent, any known gelling agent can be used, such as carrageenan, gellan gum, agar, gelatin, guar gum, pectin, locust bean gum, xanthan gum, sodium alginate, cellulose derivatives, superabsorbent resins such as sodium acrylate, dibenzylidene-D-sorbitol, and hydroxypropylated polysaccharides.

[0081] The methyl mercaptan production inhibitor according to the present embodiment can be used for, for example, food waste, which is a source of methyl mercaptan. By placing the methyl mercaptan production inhibitor in trash cans, trash corners, etc. installed in ordinary homes, restaurants, campsites, barbecue areas, etc. where food waste is generated, the generation of methyl mercaptan, a putrid odor produced by food waste, can be effectively suppressed.

[0082] Examples of methods for applying the methyl mercaptan production inhibitor according to the present embodiment include placing a liquid, gel, sheet, or solid methyl mercaptan production inhibitor near a source of methyl mercaptan (methionine), such as food waste, and allowing it to volatilize naturally at room temperature, and directly spraying a liquid methyl mercaptan production inhibitor on a source of volatile sulfur compounds. As described above, the compound contained as an active ingredient in the methyl mercaptan production inhibitor according to the present embodiment reacts with methionine γ-lyase to particularly effectively suppress the generation of methyl mercaptan from methionine. Therefore, a preferred method for applying the methyl mercaptan production inhibitor according to the present embodiment is to place the inhibitor in a closed space containing food waste, such as a lidded trash can, and allow it to volatilize naturally at room temperature. The amount of methionine γ-lyase according to the first embodiment volatilized into the closed space in this manner is preferably such that the concentration of methionine γ-lyase in the space is 0.01% by mass or more.

[0083] <Drug volatilizer> The chemical volatilizer according to the fourth embodiment of the present invention contains the methyl mercaptan production inhibitor according to the third embodiment. It is particularly preferable that the methyl mercaptan production inhibitor according to the third embodiment be naturally volatilized at room temperature using a chemical volatilizer.

[0084] An example of a chemical volatilizer according to this embodiment is a chemical volatilizer in which a liquid methyl mercaptan production inhibitor according to the third embodiment is contained in a container at least partially made of a microporous sheet. The chemical volatilizer according to this example may be, for example, a bag-shaped container made entirely of a microporous sheet, with a liquid methyl mercaptan production inhibitor contained in this bag-shaped container. Alternatively, the chemical volatilizer according to this embodiment may be a container with an open top, in which a liquid methyl mercaptan production inhibitor is contained, and the opening is blocked with a microporous sheet.

[0085] The microporous sheet provided in the medicine volatilizer according to this embodiment is a sheet having fine pores that are permeable to gas but not to liquid. Examples of microporous sheets include a sheet containing a filler, a sheet made of a physically foamed resin film, and a sheet made of a resin to which a foaming agent has been added. The diameter of the pores in the microporous sheet is not particularly limited, but a diameter of about 0.1 to 10 μm is generally preferred from the viewpoints of suppressing leakage of the medicine and efficiently volatilizing the medicine. These sheets may be a single film or a composite film made by laminating two or more films.

[0086] The material of the microporous sheet is not particularly limited, and examples include thermoplastic resins such as polyolefin resins and those that have been treated with fluorine, silicone, or the like to be water-repellent. The microporous sheet is preferably a drug-permeable sheet obtained by stretching a resin composition in which a filler is blended with a polyolefin resin to provide fine pores. In the drug volatilizer according to this embodiment, the methionine gamma-lyase activity inhibitor according to the first embodiment contained in the methyl mercaptan production inhibitor according to the third embodiment volatilizes through the microporous sheet and reacts with and acts on bacteria and enzymes in a gaseous state.

[0087] Another example of a chemical volatilizer is one that has a volatilization port and an openable container that contains a chemical impregnated body impregnated with the methyl mercaptan production inhibitor according to the third embodiment. Examples of chemical impregnated bodies that can be used include filter paper and impregnated pulp paper. In this example of a chemical volatilizer, the methionine gamma-lyase activity inhibitor contained in the methyl mercaptan production inhibitor volatilizes from the volatilization port and reacts and acts on bacteria and enzymes in a gaseous state.

[0088] The chemical volatilization device may be provided with a member for hanging the chemical volatilization device or a member for attaching the chemical volatilization device. This allows the chemical volatilization device to be attached or hung, for example, on the backside of a trash can lid. For the chemical volatilization device according to the present embodiment described here, see, for example, Figures 1 to 4 in Japanese Patent No. 5871746. [Example]

[0089] Test examples carried out in connection with the present invention are described below. (Test Example 1) Identification of methyl mercaptan-producing bacteria A simulated food waste was prepared according to Table 6. After leaving it at 25°C for one week, 40 g of the simulated food waste was suspended in 30 ml of physiological saline and shaken for 30 minutes to obtain a liquid used as a food waste extract. The extracted food waste extract was spread on a standard agar medium and cultured for isolation, yielding six isolated bacteria (test bacteria 1 to 6) as described below. Test bacteria 1 to 6 were evaluated for their ability to produce methyl mercaptan from methionine using the following method.

[0090] [Table 6]

[0091] One colony was picked from each of the test bacteria 1 to 6 and cultured in normal bouillon medium for 24 hours to prepare a culture solution for each of the test bacteria 1 to 6.

[0092] The food waste extract or each culture medium prepared above was placed in a 10 L plastic bag on a standard agar medium supplemented with methionine, and the bag was filled with odorless air and allowed to stand. The initial concentration of the malodorous component (methyl mercaptan) in the plastic bag at room temperature (25°C) and the concentration of the malodorous component (methyl mercaptan) after a certain period of time (24 hours, 48 ​​hours) were measured using a detector tube (Gastec Corporation, Methyl Mercaptan Detector Tube No. 71). The measurement results are shown in Table 7.

[0093] "Blank" in Table 7 refers to the measurement results of the concentration of the malodorous component (methyl mercaptan) when normal bouillon medium was added instead of the culture medium and a standard agar medium to which methionine was added was used.

[0094] [Table 7]

[0095] Homology analysis using the 16S rRNA base sequence showed that the target bacteria 2, 3, and 6, which were confirmed to produce methyl mercaptan in Table 7, were likely to be the following bacterial species. Test bacterium 2: Bacillus cereus Test bacterium 3: Bacillus mycoides Test fungi 6: Geoerichum candidum, Galactomyces candidum

[0096] As shown in Table 7, among the target organisms 1 to 6, the production of methyl mercaptan was confirmed when target organisms 2 (Bacillus cereus), 3 (Bacillus mycoides), and 6 (Geotrichum candidum, Galactomyces candidum) were used. These microorganisms were found to have the ability to generate methyl mercaptan from methionine.

[0097] Furthermore, the measurement results in Table 7 show that among specimens 2, 3, and 6, the Bacillus cereus in specimen 2 exhibits methyl mercaptan production behavior similar to that of food waste extract. Furthermore, specimens 2 and 6 are known to be microorganisms detected in food. Based on these results, we selected this Bacillus cereus as the bacterial cell to be used in the primary screening step in the screening test for the compounds described below. By using Bacillus cereus, which exhibits methyl mercaptan production behavior similar to that of the food waste culture solution isolated from simulated food waste, it is possible to screen for agents that are particularly effective in suppressing odors emitted from food waste.

[0098] (Test Example 2) Evaluation of the performance of substances that inhibit methionine γ-lyase activity 1. Primary screening A centrifugal ultrafiltration filter unit (Amicon® Ultra-15 (Merck)) consisting of a lower container (centrifugation test tube) and an upper container (filter cup) was prepared. A suspension of 25 μl of the Bacillus cereus culture solution prepared in Test Example 1 and 25 μl of a 200 mM methionine aqueous solution was mixed and added to 0.5 g of standard agar medium in the lower container to prepare a lower layer.

[0099] 148 types of fragrances with unknown inhibitory activity against methyl mercaptan generation were prepared as screening targets (test substances) in the primary screening process. 10 μl of each compound was impregnated into filter paper. The filter paper impregnated with each compound was attached to the bottom of the upper container of the filter unit. This upper container was attached to the lower container equipped with the lower layer described above.

[0100] Next, 1 ml of 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) diluted with 0.25 M phosphate buffer (pH 7.2) was added to the upper container, forming the upper layer. The resulting sample was allowed to stand at 25°C for 24 hours. A blank was prepared by replacing the filter paper with one not impregnated with the compound.

[0101] After 24 hours, the absorbance at 412 nm of 1 mL of the upper layer of each sample was measured using a V-650 spectrophotometer. From the measured values, the malodor suppression rate (%), i.e., the methyl mercaptan generation suppression rate (%), was calculated according to the following formula (1).

[0102]

number

[0103] As a result, 97 of the 148 tested substances were confirmed to have a malodor suppression rate of 50% or more (methyl mercaptan suppression rate). Of these, 50 showed a malodor suppression rate of 80% or more, and 47 showed a malodor suppression rate of 50% or more but less than 80%. These 97 test substances that showed a malodor suppression rate of 50% or more were selected as test substances for the second screening. As mentioned above, the malodor suppression rate measured here includes not only that due to enzyme inhibition but also that due to sterilization.

[0104] 2. Secondary screening The same centrifugal ultrafiltration filter unit (Amicon (registered trademark) Ultra-15 (Merck)) used in the primary screening was prepared. A substrate solution was prepared by mixing 1 mL of 1 M phosphate buffer (pH 8.0), 0.02 mL of 10 mL of pyridoxal phosphate solution, 2 mL of 50 mM methionine solution, and 6.5 mL of sterile ion-exchanged water. 0.95 mL of the substrate solution, 0.05 mL of a 10 mass% solution of the test substance dissolved in an organic solvent, and 0.05 mL of a 1 g / L methionine γ-lyase solution were added to the lower container of the filter unit and mixed to prepare a lower layer. After attaching the upper container, 0.25 M phosphate buffer (pH 7.0) was added to the upper container. 1.0 mL of 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) solution prepared in 2) was added, and the mixture was placed in a constant temperature bath at 30°C. The final flavoring concentration was 0.5% by mass. A control was prepared by adding 0.05 mL of the organic solvent instead of the test substance, and a blank was prepared by adding 100 mM phosphate buffer (pH 7.2) instead of the methionine γ-lyase solution. After the reaction, the absorbance at 412 nm of 1 mL of the upper layer of each sample was measured using a V-650 spectrophotometer. From the measured values, the enzyme inhibition rate (%), i.e., the percentage (%) of methyl mercaptan generation suppressed by enzyme inhibition, was calculated according to equation (1).

[0105]

number

[0106] As a result, 17 out of 97 tested substances were confirmed to have enzyme inhibition rates of 50% or more, with 8 showing enzyme inhibition rates of 80% or more and 9 showing enzyme inhibition rates of 50% to less than 80%.

[0107] Seventeen compounds (Examples 1 to 17) that showed a malodor inhibition rate of 50% or more in the first screening and an enzyme inhibition rate of 50% or more in the second screening are summarized in Table 8. Additionally, 10 compounds (Comparative Examples 1 to 10) that showed a malodor inhibition rate of 50% or more in the first screening but an enzyme inhibition rate of less than 30% confirmed in the second screening are summarized in Table 9. Note that negative values ​​for the enzyme inhibition rates shown in Table 9 indicate that the production of methyl mercaptan is promoted compared to the blank.

[0108] [Table 8-1]

[0109] [Table 8-2]

[0110] [Table 9]

Claims

1. A methionine γ-lyase activity inhibitor comprising any one of the following compounds (i) to (v): (i) a monoterpene compound selected from the following: open-chain monoterpenes containing an ester bond, open-chain monoterpenes having a hydroxyl group at at least one end, cyclic monoterpenes containing a ring structure and composed only of hydrocarbons, cyclic monoterpenes containing saturated cyclic ketones, and cyclic monoterpenes containing a ring structure and an ether bond; (ii) Aliphatic ester compounds represented by formula I: 【Chemistry 1】 In the formula, R １１ represents an alkyl group having 1 to 4 carbon atoms, a cyclic saturated aliphatic group, or an unsaturated aliphatic group; R １２ represents an alkyl group or an unsaturated aliphatic group, L １１ represents a single bond or an ether bond, L １２ represents a single bond, an alkylene group, or an alkynylene group; R １１ and L １１ and L １２ The total number of carbon atoms contained in R is 7 or less, １１ and R １２ At least one of represents an unsaturated aliphatic group; (iii) Carbonyl compounds represented by formula II: 【Chemistry 2】 In the formula, R ２１ represents a cycloalkadiene group or a phenyl group substituted with an amino group, R ２２ represents an alkoxy group or an unsaturated aliphatic group; (iv) Unsaturated chain aliphatic compounds having at least one end substituted with an aldehyde group or a hydroxyl group; provided that when only one end is substituted with an aldehyde group, the number of carbon atoms contained in the molecule is 11 or less, and when only one end is substituted with a hydroxyl group, the number of carbon atoms contained in the molecule is 10 or less; (v) Aromatic alcohol compounds represented by formula III: 【Transformation 3】 In the formula, R ３１ represents a single bond or a linking group, R ３２ represents a hydrogen atom or an alkyl group.

2. A methyl mercaptan production inhibitor comprising one or more of the methionine γ-lyase activity inhibitors according to claim 1 as an active ingredient.

3. The methyl mercaptan formation inhibitor according to claim 2, which is a volatile agent.

4. A drug volatilization device comprising a container at least partially made of a microporous sheet and the methyl mercaptan production inhibitor described in claim 2 or 3 contained in the container, wherein the methionine gamma-lyase activity inhibitor volatilizes through the microporous sheet.

5. A method for screening for a substance that inhibits methionine γ-lyase activity, comprising: a primary screening step of selecting, from the test substances, a substance that inhibits the metabolic activity of a microorganism that produces methyl mercaptan from methionine in a gas-phase reaction; and a second screening step of selecting, from the substances selected in the first screening step, a substance that inhibits the enzymatic activity of methionine γ-lyase, which produces methyl mercaptan from methionine, in a liquid phase reaction; A screening method including:

6. 6. The screening method according to claim 5, wherein the gas phase reaction in the primary screening step is carried out by vaporizing the test substance and introducing the vapor into the reaction system.

7. the selection of the substance in the primary screening step involves selecting a substance that inhibits the activity of the microorganism using as an index the difference between the amount of methyl mercaptan produced in the presence of the test substance and the amount of methyl mercaptan produced in the absence of the test substance; The screening method of claim 5, wherein the selection of the substance in the secondary screening step is performed by selecting a substance that inhibits the activity of methionine γ-lyase using the difference between the amount of methyl mercaptan produced in the presence of the test substance and the amount of methyl mercaptan produced in the absence of the test substance as an indicator.

8. The screening method according to claim 5 , wherein the microorganism comprises one or more microorganisms selected from the genera Bacillus, Geotrichum, and Galactomyces.

9. The screening method according to claim 5, wherein the microorganism comprises one or more microorganisms selected from the group consisting of Bacillus cereus, Bacillus mycoides, Geotrichum cadidum, and Galactomyces candidum.

10. The screening method according to claim 5, which is used to screen for an active ingredient of a methyl mercaptan production inhibitor.

11. The screening method according to claim 10, wherein the methyl mercaptan production inhibitor is a volatile deodorant.

12. A methionine γ-lyase activity inhibitor screened by the screening method according to any one of claims 5 to 11.

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