Composition for inhibiting methane production in ruminants

Abstract

Disclosed is a composition for inhibiting methane production in ruminants, comprising a compound having chemical structural formula I, or a salt, a solvate, a hydrate, or an isomer thereof. The compound binds to a protein structure of methyl-coenzyme M reductase (MCR), which is a methanogen enzyme in the rumen of a ruminant, and inhibits enzymatic activity, thereby reducing methane production. In addition, a feed additive and a feed for ruminants comprising same are disclosed.

Description

Composition for inhibiting methane production in ruminant animals

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0132259 filed September 28, 2024 and Korean Patent Application No. 10-2024-0190635 filed December 18, 2024, and all contents disclosed in the documents of said Korean patent applications are incorporated herein as part of this specification.

[0003] The disclosure relates to a compound that effectively inhibits methane production by binding to the protein structure of methyl-coenzyme M reductase (MCR) of methanogenic bacteria in ruminants and inhibiting the methane production activity of the methanogenic bacteria, and a composition for inhibiting methane production containing the same. Additionally, a feed additive for ruminants and a feed containing the composition for inhibiting methane production are disclosed.

[0004] Greenhouse gases, known as one of the main causes of global warming, have been continuously increasing since industrialization. In particular, methane gas has a global warming potential about 25 times higher than carbon dioxide, making its impact on global warming very significant. Methane gas is one of the major greenhouse gases selected by the IPCC and is emitted from various environments, such as the combustion of fossil fuels, agriculture, and intestinal fermentation; it is known that more than 500 million tons are generated annually.

[0005] It is known that approximately 60% of global methane gas is generated in agriculture and livestock farming, with about 25% of this being emitted through fermentation during the digestive process of ruminants; this is cited as one of the major causes of methane emissions in the agricultural sector. In Korea as well, approximately 12.5 million tons of methane gas equivalent to CO2 are generated annually, with 56.8% of this attributed to the agricultural sector. In particular, methane gas emissions resulting from the digestive process, such as rumen fermentation, are known to reach approximately 4.3 million tons of CO2eq. Methane emissions from ruminants primarily occur in the anaerobic environment within the rumen, and this process is emerging as a significant issue in international discussions regarding global warming and greenhouse gas reduction.

[0006] Methane production in the rumen of ruminant animals is primarily carried out by methane-producing microorganisms, which produce methane using various substrates, such as hydrogen, carbon dioxide, and acetic acid. The methane production process is broadly divided into three stages: the first stage is the hydrolysis stage, in which anaerobic microorganisms decompose high-molecular-weight organic matter into low-molecular-weight organic matter; the second is the acid production stage, in which low-molecular-weight organic matter is converted into acetic acid, propionic acid, carbon dioxide, and hydrogen; and the third is the methane production stage, in which methane-producing microorganisms produce methane using carbon dioxide and hydrogen.

[0007] The methane production process is classified according to the substrates used. The hydrotrophic methanogenesis pathway utilizes hydrogen and carbon dioxide as primary substrates and is most commonly found in anaerobic environments such as the rumen. Carbon dioxide combines with methanofuran, is reduced to a methyl group by coenzymes, and finally, methane is produced through the action of Coenzyme B. Another pathway is acetoclastic methanogenesis, in which acetic acid is broken down into methane and carbon dioxide. This process requires less energy but is less common in rumen environments. Yet another pathway is methylotrophic methanogenesis, which converts methylated compounds such as methanol or methylamine into methane and is primarily found in marine or anaerobic digester environments.

[0008] In an attempt to reduce methane gas emissions in ruminants, various natural compounds have been studied for methane production reduction; however, existing studies based on natural compounds have had limitations in terms of the sustainability and extent of methane reduction, and their reduction and inhibition mechanisms were unclear. Furthermore, they often resulted in side effects because they failed to consider the characteristics of the diverse microbiomes within the digestive tract.

[0009] For example, although red algae extracts have been reported to be effective in inhibiting methane production, side effects such as reduced feed palatability and decreased dry matter intake occurred when the addition amount was increased, and there are limitations to their actual application due to the potential carcinogenicity of bromine compounds contained in red algae extracts.

[0010] Therefore, there is a high demand for compounds that can specifically inhibit methane-producing bacteria safely and effectively due to their clear mechanism of methane reduction.

[0011] To address these technical requirements, the present specification provides a natural compound capable of specifically inhibiting the activity of methane-producing bacteria present in the rumen of ruminants, and a methane production inhibiting composition containing the same. In particular, the present invention discloses a compound having an optimal inhibitory effect selected through molecular docking technology by targeting the methyl-coenzyme M reductase (MCR) protein structure of methane-producing bacteria. Specifically, the interaction between the MCR protein structure of methane-producing bacteria in the rumen of ruminants and the compound was analyzed, and considering the abundance of each type of methane-producing bacteria in the rumen of ruminants, the most effective methane production inhibiting compound was identified and its effect was experimentally confirmed. A composition containing the same is disclosed and applied as a feed additive or feed for ruminants.

[0012] The technical problems of this specification are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by a person skilled in the art from the description below.

[0013] The composition for inhibiting methane production in ruminants of this specification comprises a compound of chemical structure formula I, or a salt, solvate, hydrate, or isomer thereof.

[0014] Chemical structural formula I

[0015]

[0016] In the above formula, R1 is selected from the quinoline group of the following chemical structural formula R1a or the pyridine group of the following chemical structural formula R1b, and

[0017] Chemical structural formula R1a

[0018]

[0019] Chemical structural formula R1b

[0020]

[0021] The pyridine ring of the above quinoline group can be substituted with R2 at one or more of positions 2, 3, and 4, either simultaneously or independently, and

[0022] The benzene ring of the above quinoline group may be substituted with R3 at one or more of the 5, 6, 7, or 8 positions simultaneously or independently, and

[0023] The above pyridine group may be substituted with one or more R4s simultaneously or independently, and

[0024] The above R2 is selected from the group consisting of H, C1 to C6 alkyl groups, and

[0025] The above R3 is selected from the group consisting of H, C1 to C6 alkoxy groups, and

[0026] The above R4 is selected from the group consisting of H, C1 to C6 alkyl groups.

[0027] In one embodiment, the composition for inhibiting methane production may include a compound in which R1 in the chemical structure formula I is a quinoline group, the quinoline group is bonded to the chemical structure formula I at position 5, the pyridine ring of the quinoline group is substituted with R2 at position 2, and the benzene ring of the quinoline group is substituted with R3 at position 8.

[0028] In one embodiment, the composition for inhibiting methane production may include a compound of the following chemical structure formula II.

[0029] Chemical Structure Formula II

[0030]

[0031] In one embodiment, the composition for inhibiting methane production may include a compound in which R1 of the chemical structure I is a pyridine group and is bonded to the chemical structure I at the 4th position of the pyridine group.

[0032] In one embodiment, the composition for inhibiting methane production may include a compound of the following chemical structure formula III.

[0033] Chemical Structure Formula III

[0034]

[0035] In one embodiment, the compound included in the composition for inhibiting methane production binds to the protein structure of methyl-coenzyme M reductase (MCR) of methane-producing bacteria in the rumen of a ruminant animal, thereby inhibiting the activity of the methane-producing bacteria.

[0036] In one embodiment, the methanogenic bacteria are rumen methanogenic bacteria, specifically Methanobrevibacter smithii, Methanobrevibacter millerae, Methanobrevibacter thaurei, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanobrevibacter gottschalkii, Methanosarcina barkeri, and Methanobacterium It may be one or more selected from Methanobacterium bryantii, Methanobacterium formicicum, and Methanocaldococcus jannaschii.

[0037] In one embodiment, the composition for inhibiting methane production may reduce the relative abundance of methane-producing bacteria in the rumen.

[0038] In one embodiment, a composition for inhibiting methane production may contain a compound of chemical structure formula I at a concentration of 0.1 mg / mL or higher.

[0039] In one embodiment, a composition for inhibiting methane production may contain a compound of chemical structure formula I at a concentration of 0.4 mg / mL or higher.

[0040] In one embodiment, the pH of the methane production inhibiting composition may be in the range of about 6.0 to about 7.0.

[0041] In one embodiment, the composition for inhibiting methane production may additionally include one or more selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline solution, injectable solution, glycerol, methanol, and ethanol.

[0042] In one embodiment, the composition for inhibiting methane production may additionally include one or more selected from the group consisting of antioxidants, buffers, bacteriostatic agents, excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, flavoring agents, diluents, dispersants, pH adjusters, and surfactants.

[0043] In one embodiment, the composition for inhibiting methane production may have an aqueous solution, suspension, emulsion, injection, rumen formulation, powder, granule, tablet, capsule, pellet, block, or sustained-release block formulation.

[0044] In one embodiment, the composition for inhibiting methane production may be administered via one of the following routes: oral, rectal, intravenous, arterial, intraperitoneal, intramuscular, transdermal, nasal, inhalation, topical, ocular, or intradermal.

[0045] In one embodiment, the composition for inhibiting methane production may be one or more ruminant animals selected from cattle, goats, sheep, deer, reindeer, giraffes, camels, alpacas, llamas, and okapis.

[0046] In one embodiment, a feed additive for ruminants includes a composition for inhibiting methane production.

[0047] In one embodiment, when a feed additive for ruminants is added to the feed, the methane production rate can be reduced by more than 10% compared to when it is not added.

[0048] In one embodiment, the feed additive for ruminants may additionally include a protein-degrading enzyme or a cellulose-degrading enzyme to improve the digestive efficiency of ruminants.

[0049] In one embodiment, the feed for ruminants includes the methane production inhibiting composition.

[0050] Specific details of other embodiments are included in the detailed description and drawings.

[0051] The compounds of this specification and compositions for reducing methane production containing them are selected by identifying target proteins in the metabolic pathways of methane-producing bacteria and considering the abundance of each type of methane-producing bacteria in the rumen of ruminant animals, thereby maximizing the methane reduction effect while minimizing the possibility of side effects.

[0052] In addition, the compounds of this specification can be applied as feed additives and feed for ruminants. Therefore, when administered to ruminants, they can effectively reduce methane emissions and maintain or improve livestock productivity.

[0053] Figure 1 shows the 3D structure of a methyl-coenzyme M reductase (MCR) protein obtained from the genomes of 11 species of rumen methanogenic bacteria mainly found in the bovine rumen.

[0054] Figure 2 is a simplified diagram illustrating the process of identifying binding sites during the molecular docking process of a natural compound library for the exemplary rumen methanogenic bacterium Methanobrevibacter smithii.

[0055] Figure 3 shows the heatmap and the most frequent Murcko Scaffold Hash derived from the molecular docking process of a natural compound library for the exemplary rumen methanogenic bacterium Methanobrevibacter smithii.

[0056] Figure 4 is a table showing the average relative abundance of 11 types of methane-producing strains in the rumen for each collected sample.

[0057] Figure 5 is a photograph of the preparation for performing an in-vitro digestion experiment on 14 selected methane reduction candidate substances and a control group.

[0058] Figure 6 is a photograph of a gas bag prepared for gas sampling to measure the amount of gas generated during in-vitro digestion experiments on 14 selected methane reduction candidate substances and a control group.

[0059] Figure 7 shows the experimental results of measuring gas generation, methane production, ammonia nitrogen production, pH, dry matter digestibility (DMD), and methane yield for 14 selected methane reduction candidate substances.

[0060] Figure 8 shows the results of comparing the methane production (mL) of the methanol-added control group (MCON) and methane-reducing candidate substances H and X.

[0061] Figure 9 shows the results of comparing the methane yield (mL / g dDM) of the methanol-added control group (MCON) and methane-reducing candidate substances H and X.

[0062] Figure 10 shows the results of an in-vitro digestion experiment measuring gas generation, methane production, ammonia nitrogen production, pH, building digestion rate, and methane yield according to the concentration of compound X.

[0063] Figure 11 is a figure showing the difference between experimental data to evaluate the methane production reduction effect of the control group (CON) and the compound X treatment groups (1X, 2X, 4X) using a non-metric multidimensional scaling (NMDS) plot.

[0064] Figure 12 shows the results of a permutational multivariate analysis of variance test (PERMANOVA) performed to statistically verify the experimental results.

[0065] Embodiments of this specification are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, this specification may be implemented in various different forms and is not limited to the embodiments described herein.

[0066] Throughout the specification and claims, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this specification pertains. Any method and material similar or identical to that described herein may also be used in the practice or testing of this specification, but preferred methods and materials are described hereafter. All publications mentioned herein are incorporated by reference to disclose and describe methods and / or materials in connection with the reference of such publications.

[0068] In this specification, a composition for inhibiting methane production in ruminants is disclosed, comprising a compound having chemical structural formula I, or a salt, solvate, hydrate, or isomer thereof.

[0069] Chemical structural formula I

[0070]

[0071] In the above chemical structure I, R1 is selected from the quinoline group of the following chemical structure R1a or the pyridine group of the following chemical structure R1b, and

[0072] Chemical structural formula R1a

[0073]

[0074] Chemical structural formula R1b

[0075]

[0076] The pyridine ring of the above quinoline group can be substituted with R2 at one or more of positions 2, 3, and 4, either simultaneously or independently, and

[0077] The benzene ring of the above quinoline group may be substituted with R3 at one or more of the 5, 6, 7, or 8 positions simultaneously or independently, and

[0078] The above pyridine group may be substituted with one or more R4s simultaneously or independently, and

[0079] The above R2 is selected from the group consisting of H, C1 to C6 alkyl groups, and

[0080] The above R3 is selected from the group consisting of H, C1 to C6 alkoxy groups, and

[0081] The above R4 is selected from the group consisting of H, C1 to C6 alkyl groups.

[0082] The compound of chemical structure formula I can inhibit methane production by binding to the active site of the protein structure of methyl-coenzyme M reductase (MCR), a methanogenic enzyme in ruminants, and inhibiting the activity of the enzyme.

[0083] The compound of chemical structure formula I of this specification includes various substitution possibilities, and specific embodiments disclose the following substitution examples.

[0084] In one embodiment, the compound of chemical structure formula I may have R1 as a quinoline group, with the quinoline group bonded to chemical structure formula I at position 5. In this case, the 2nd position of the pyridine ring of the quinoline substituent may be substituted with R2 and / or the 8th position of the benzene ring may be substituted with R3. R2 may be selected from the group consisting of H, C1 to C6 alkyl groups, and R3 may be selected from the group consisting of H, C1 to C6 alkoxy groups. As a specific example of substitution included in chemical structure formula I, the compound of chemical structure formula II is disclosed exemplarily.

[0085] Chemical Structure Formula II

[0086]

[0087] In another embodiment, R1 in the compound of chemical structure formula I may be a pyridine group and may be bonded to chemical structure formula I at the 4th position of the pyridine group. In this case, the pyridine substituent may be substituted with R4 at a position other than the one bonded to chemical structure formula I. As a specific example of substitution included in chemical structure formula I, the compound of chemical structure formula III is disclosed as an example.

[0088] Chemical Structure Formula III

[0089]

[0090] Chemical structural formulas II and III can be represented as specific combinations of substitution groups defined in chemical structural formula I, each having a high binding affinity for the MCR enzymes of multiple methanogenic bacteria distributed in the rumen and exhibiting an inhibitory effect on methane production in the rumen.

[0091] Specific compounds of this specification may exist not only as compounds themselves but also in the form of acceptable salts. Such forms of salts are included within the scope of this specification, and the types of salts may include inorganic acid salts (e.g., hydrochlorides, sulfates) or organic acid salts (e.g., acetates, citrates), as well as metal salts (e.g., sodium salts, potassium salts) and non-metal salts (e.g., ammonium salts). Any form of salt used is also included within the scope of this specification insofar as it possesses equivalent technical validity.

[0092] Specific compounds of this specification may exist in a non-solvated form as well as in a solvated form including a hydrate. The hydrate form includes a state in which the compound is bonded to water molecules, and such a form is also included within the scope of this specification. Specific compounds may exist in a number of crystalline or amorphous forms, and all physical forms are included within the scope of this specification insofar as they have equivalent technical validity.

[0093] Specific compounds of this specification may have asymmetric carbon atoms (optical centers) or double bonds, and racemics, diastereomers, geometric isomers, positional isomers, and individual isomers (e.g., separated enantiomers) are included within the scope of this specification insofar as they have equivalent technical validity.

[0094] In this specification, "rumen methanogenic bacteria" refers to archaea that produce methane (CH4) using hydrogen (H2) and carbon dioxide (CO2) generated during the fermentation process in the anaerobic environment of the rumen of ruminant animals as the main substrates. Examples of rumen methanogenic bacteria include Methanobrevibacter smithii, Methanobrevibacter millerae, Methanobrevibacter thaurei, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanobrevibacter gottschalkii, Methanosarcina barkeri, and Methanobacterium bryanthi One or more selected from Methanobacterium bryantii, Methanobacterium formicicum, and Methanocaldococcus jannaschii may be cited as examples, but are not limited thereto.

[0095] A person skilled in the art will understand that these strains adapt to specific conditions within the rumen and play an important role in the methane production process, and that the composition of these strains may change depending on environmental factors. Accordingly, the rumen methane-producing bacteria of this specification are intended to include not only the strains exemplified but also all archaea involved in methane production within the rumen.

[0096] In this specification, methyl-coenzyme M reductase (MCR) refers to an enzyme involved in the methane production process of methanogenic bacteria, which catalyzes the reaction of reducing methyl-coenzyme M (CH3-S-CoM) to coenzyme B (HS-CoB) to produce methane (CH4) and heterodisulfide (CoM-SS-CoB), and is responsible for the final step of the methane production process.

[0097] The compound of the present specification can inhibit the activity of methanogenic bacteria by binding to the protein structure of the MCR enzyme of methanogenic bacteria in the rumen of ruminant animals. By binding to the MCR enzyme and blocking the ability to catalyze the methanogenic reaction, the compound of the present specification inhibits the process in which intermediate products such as methyl coenzyme M and coenzyme B are converted into methane, thereby reducing the amount of methane produced by methanogenic bacteria.

[0098] In one embodiment, the compound of this specification may reduce the relative abundance of methanogenic bacteria in the rumen. Although not bound by theory, the compound may bind to the MCR enzyme and interrupt the methane production pathway through MCR inhibition, thereby causing an accumulation of intermediate compounds, which depletes the energy supply of methanogenic bacteria and consequently reduces the viability of methanogenic bacteria.

[0099] In one embodiment, the compound of chemical formula I of this specification is added at a concentration necessary for inhibiting methane production and may be included in a composition for inhibiting methane production at a concentration of about 0.1 mg / mL or more, about preferably 0.2 mg / mL or more, more preferably about 0.4 mg / mL or more.

[0100] In one embodiment, the methane production inhibiting composition of this specification has a pH range that can act in the rumen of a ruminant animal. In one embodiment, the methane production inhibiting composition may have a pH in the range of about 5.0 to about 8.0, preferably in the range of about 6.0 to about 7.0, exemplarily in the range of about 6.3 to about 6.8, in the range of about 6.4 to 6.8, in the range of about 6.5 to about 6.8, in the range of about 6.3 to about 6.7, in the range of about 6.4 to 6.7, in the range of about 6.5 to about 6.7, in the range of about 6.3 to about 6.6, in the range of about 6.4 to 6.6, and in the range of about 6.5 to about 6.6, but is not limited thereto. Typically, the normal pH range of the rumen is between about 6.0 and 7.0, which is an environment necessary for the microbial community within the rumen to efficiently perform fermentation. The composition can effectively reduce methane production by inhibiting the activity of methane-producing bacteria while maintaining the fermentation process under pH conditions within the rumen of ruminant animals.

[0101] The composition for inhibiting methane production of the present specification may be formulated to include an additionally acceptable carrier in addition to the compound for administration. As a carrier, one or more of, for example, saline solution, sterile water, Ringer's solution, buffered saline solution, injectable solution, glycerol, and low-value alcohols, such as methanol and ethanol, may be used in a mixture or alone, but are not limited thereto. In one embodiment, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be additionally added.

[0102] The composition for inhibiting methane production of the present specification may include additionally acceptable adjuvants for administration in addition to the compound. As adjuvants, one or more of, for example, antioxidants, buffers, bacteriostatic agents, excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, flavoring agents, diluents, dispersants, pH adjusters, and surfactants may be used in combination or alone, but are not limited thereto.

[0103] The composition for inhibiting methane production of the present specification may be formulated into a solution, suspension, emulsion, injection, rumen formulation, powder, granule, tablet, capsule, pellet, block, or sustained-release block, etc.

[0104] In addition, the composition for inhibiting methane production of the present specification may be administered in a conventional manner through oral, rectal, intravenous, arterial, intraperitoneal, intramuscular, transdermal, nasal, inhalation, topical, ocular, or intradermal routes, etc.

[0105] In this specification, the term “administration” refers to, where applicable, to a subject, cell, tissue, organ, or biological fluid, for example, bringing a composition for inhibiting methane production into contact with a subject, cell, tissue, organ, or biological fluid. With respect to cells, administration includes contact of the reagent with the cell (e.g., in vitro or in vitro) as well as contact of the reagent with the fluid, wherein the fluid comes into contact with the cell.

[0106] In this specification, "ruminant" refers to an animal characterized by a rumen fermentation process in which the activity of methane-producing bacteria is observed, and is intended to be a target to which the composition of this specification for inhibiting methane production can be applied, and is not limited to a specific animal species. In one embodiment, the ruminant may be one or more selected from cattle, goats, sheep, deer, reindeer, giraffes, camels, alpacas, llamas, and okapis.

[0107] A feed additive for ruminants comprising the composition for inhibiting methane production of the present specification is disclosed.

[0108] In this specification, a feed additive for ruminants refers to a composition that is mixed into or added to the feed of ruminants to supplement the nutritional value of the feed, improve digestive efficiency, or induce specific physiological effects. The feed additive for ruminants described in this specification may be added to the feed for the purpose of providing the effect of reducing methane emissions by inhibiting the activity of methane-producing bacteria during the digestion process of ruminants or by regulating the efficiency of ruminal fermentation.

[0109] In one embodiment, the feed additive for ruminants of this specification may reduce the methane production rate by 5% or more, exemplarily by 8% or more, preferably by 10% or more, and more preferably by 12% or more when added compared to when not added.

[0110] In one embodiment, the feed additive for ruminants described herein may further include an auxiliary additive. The auxiliary additive may be added for purposes such as assisting in the breakdown and absorption of nutrients during the digestion process of ruminants. In one embodiment, the auxiliary additive may include a protein-degrading enzyme or a cellulose-degrading enzyme. The protein-degrading enzyme may break down proteins in the feed of ruminants into amino acids to promote absorption. Examples of protein-degrading enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, and alkaline protease. The cellulose-degrading enzyme may contribute to breaking down fiber or starch in the feed of ruminants and converting it into a form that can be utilized as energy. Examples of cellulose-degrading enzymes include, but are not limited to, cellulase, β-glucosidase, xylanase, amylase, and mannanase.

[0111] A feed for ruminants comprising the methane production inhibiting composition of the present specification is disclosed. The feed for ruminants is intended to reduce methane emissions by inhibiting the activity of methane-producing bacteria in the rumen when orally ingested by ruminants, while simultaneously maintaining or improving the digestive and fermentation efficiency of the ruminants.

[0112] Feed for ruminants can be provided in various formulations, for example, powder, granules, pellets, block form, liquid, etc. These feeds are administered orally and can be provided mixed with the daily feed of ruminants or as independent supplementary feeds.

[0113] The present specification will be explained in detail below through examples.

[0114] However, the following examples are merely illustrative of this specification, and the contents of this specification are not limited to the following examples.

[0115] <Example 1> In-silico screening

[0116] Using a protein structure-based molecular docking process, the binding energy between the methyl-coenzyme M reductase (MCR) protein and the ligand was analyzed, and methyl-coenzyme M reductase inhibitor candidates were screened in silico.

[0117] Using the sequence-structure prediction AI program (Pharmaco-Net, Republic of Korea, Calici Co., Ltd.), Methanobrevibacter millerae, Methanobrevibacter thaurei, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanobrevibacter gottschalkii, Methanosarcina barkeri, and Methanobacterium bryanthi 3D structural prediction of methyl-coenzyme M reductase (MCR) proteins obtained from the genomes of 11 species of rumen methanogenic bacteria, including Methanobacterium bryantii, Methanobacterium formicicum, and Methanocaldococcus jannaschii, was performed. This is presented in Figure 1.

[0118] Next, screening was performed to discover methyl-coenzyme M reductase inhibitors using a compound library consisting of approximately 37,000 natural compounds. The compound library was constructed based on a database of naturally derived substances. Specifically, it included compound structure files in the SDF (Structure Data File) format containing structural data of naturally derived compounds provided by ChemDiv and ChemBridge. Additionally, compound structure data obtained from the search results of ChEMBL, a database of biologically active compounds, were included by searching for phenolic and saponin series compounds using keywords, considering their chemical properties and potential interactions with the metabolic pathways of methanogenic bacteria in the rumen. By combining these data, a library containing a total of approximately 37,000 naturally derived compounds was constructed.

[0119] Ligand clustering and pharmacophore prediction were performed on a compound library. First, ligand clusters were formed based on the chemical and structural similarities between compounds within the library, classifying them into specific groups. Subsequently, the clustering of compounds according to chemical or structural characteristics was visualized using a heatmap. In the heatmap, closer proximity of compounds indicates higher structural or functional similarity, which is useful for predicting binding efficiency with target proteins.

[0120] Next, the most promising group of candidate compounds was screened in silico by calculating the binding affinities of ligands against the methyl-coenzyme M reductase protein structure using a sequence-structure prediction AI program (Pharmaco-Net, Republic of Korea, Calici Co., Ltd.). Binding affinities were calculated through a molecular docking process, which includes binding site identification, docking simulation, and weighting. Binding site identification refers to detecting the protein's active site or binding pocket where the ligand can bind. Docking simulation involves predicting how the ligand fits into the binding pocket by considering various factors such as shape complementarity, hydrogen bonding, hydrophobic interactions, and electrostatic forces. The weighting process is a step that quantitatively evaluates the binding affinity of the ligand to the protein. The lowest K d / K i Ligand structures with a value exhibit high binding affinity to protein structures, so it is expected that they will have high inhibitory efficacy.

[0121] First, a molecular docking process was performed for Methanobrevibacter smithii as an example, and the schematic diagram is illustrated in Fig. 2. Methanobrevibacter smithii produces methane (CH4) by utilizing hydrogen (H2) and carbon dioxide (CO2) generated during intestinal fermentation in the intestinal microbial communities of humans and animals, and is the most commonly found methane-producing bacterium in the human intestine.

[0122] As a result, it was confirmed that compounds in Group 1 of the heatmap had a high binding affinity for the structure of the methyl-coenzyme M reductase protein of Methanobrevibacter smithii, and the most frequent Murcko Scaffold Hash was found in the Group 1 compounds, which was analyzed as an important feature that significantly contributes to the binding affinity with the target protein. The Kd / Ki values ​​(uM) of each compound were sorted in descending order along with the structures of the Group 1 compounds. As a result, some of the derived heatmaps and the most frequent Murcko Scaffold Hash are shown in Figure 3.

[0123] The same process was performed on 11 types of rumen methane-producing bacteria selected earlier.

[0124] Next, considering the diversity of microbial strains within the rumen, the relative abundance of methane-producing bacteria was considered to select the most effective methyl-coenzyme M reductase inhibitor. To this end, rumen fluid samples were collected five times from the stomachs of cattle to analyze the abundance of each strain. Rumen fluid was collected via stomach tubing before the morning feed administration. The abundance of strains was measured for each sample, and the average relative abundance (W) of methane-producing strains i ) was calculated and is shown in Figure 4. Since the higher the density of methanogenic bacteria in the rumen, the higher the abundance of methyl-coenzyme M reductase (MCR) protein in the bacteria, this is taken into account.

[0125] Using the following formula, the previously measured average relative abundance (W i ) and binding affinity value (D ij The results were multiplied by ) and summed, sorted in descending order, and 14 ligand compounds were selected.

[0126]

[0127] The chemical structural formulas of the 14 types of compounds A to H, Q to T, X and Z selected in this first round are shown in Table 1 below.

[0128] Chemical Structure Formula Compound Name (English) A 2,2'-(1,4-phenylene)bis(3-amino-1H-inden-1-one) (2,2'-(1,4-phenylene)bis(3-amino-1H-inden-1-one))B 8-methyl-7-[(3-methyl-2-buten-1-yloxy)-4-phenyl-2H-chromen-2-one (8-methyl-7-[(3-methyl-2-buten-1-yloxy]-4-phenyl-2H-chromen-2-one)C 7-isopropoxy-4-(4-methoxyphenyl)-8-methyl-2H-chromen-2-one (7-isopropoxy-4-(4-methoxyphenyl)-8-methyl-2H-chromen-2-one)D 4-(4-methoxyphenyl)-8-methyl-7-[(3-phenyl-2-propen-1-yloxy)]-2H-chromen-2-one (4-(4-methoxyphenyl)-8-methyl-7-[(3-phenyl-2-propen-1-yloxy)]-2H-chromen-2-one)E 3-amino-2-(4-methoxyphenyl)-1H-inden-1-one (3-amino-2-(4-methoxyphenyl)-1H-inden-1-one)F 7-[(2-chloro-2-propen-1-yloxy)-4-(4-methoxyphenyl)-8-methyl-2H-chromen-2-one (7-[(2-chloro-2-propen-1-yloxy)-4-(4-methoxyphenyl)-8-methyl-2H-chromen-2-one)G rel-(1R,5S)-7-(pentacyclo[6.6.6.0 2 , 7 .0 9 , 14 .0 15 , 20]icosa-2,4,6,9,11,13,15,17,19-nonaen-4-ylmethyl)-7-azaspiro[4.5]decan-1-ol (rel-(1R,5S)-7-(pentacyclo[6.6.6.0 2 , 7 .0 9 , 14 .0 15 , 20 ]icosa-2,4,6,9,11,13,15,17,19-nonaen-4-ylmethyl)-7-azaspiro[4.5]decan-1-ol)H rac-(3R,4R)-7-pyridin-4-yl-3-pyrrolidin-1-ylchroman-4-ol (rac-(3R,4R)-7-pyridin-4-yl-3-pyrrolidin-1-ylchroman-4-ol)Q 7-[3-(5-methyl-2-furyl)benzyl]-2,7-diazaspiro[4.5]decane dihydrochloride (7-[3-(5-methyl-2-furyl)benzyl]-2,7-diazaspiro[4.5]decane dihydrochloride)R 4-(2-hydroxy-1-naphthyl)-N-[(3-hydroxy-3-piperidinyl)methyl]benzamide hydrochloride (4-(2-hydroxy-1-naphthyl)-N-[(3-hydroxy-3-piperidinyl)methyl]benzamide hydrochloride)S 4-[4-{[(3-hydroxy-3-piperidinyl)methyl]amino}carbonyl]phenyl]-2-thiophenecarboxamide hydrochloride (4-[4-{[(3-hydroxy-3-piperidinyl)methyl]amino}carbonyl]phenyl]-2-thiophenecarboxamide hydrochloride)T 4'-(difluoromethoxy)-N-[(3-hydroxy-3-piperidinyl)methyl]-4-biphenylcarboxamide hydrochloride (4'-(difluoromethoxy)-N-[(3-hydroxy-3-piperidinyl)methyl]-4-biphenylcarboxamide hydrochloride)X rac-(3R,4R)-7-(8-methoxy-2-methylquinolin-5-yl)-3-pyrrolidin-1-ylchroman-4-ol (rac-(3R,4R)-7-(8-methoxy-2-methylquinolin-5-yl)-3-pyrrolidin-1-ylchroman-4-ol)Z rac-(3R,4R)-7-(3,4-dimethoxyphenyl)-3-pyrrolidin-1-ylchroman-4-ol

[0129] <Example 2>

[0130] In vitro digestion experiments were performed using 14 compounds A to H, Q to T, X, and Z selected in Example 1 as candidate substances for methane reduction. First, an in vitro rumen buffer solution was prepared using the in vitro buffer preparation method of Goering and Van Soest (Goering, 1970). This is known as a method used in the preparation of in vitro buffer solutions for evaluating cellulose degradation and feed digestion in animals.

[0131] First, an in vitro rumen buffer solution was prepared by mixing 4 g of ammonium bicarbonate and 35 g of sodium bicarbonate in 1 L of distilled water. Next, an in vitro rumen macromineral solution was prepared by mixing 5.7 g of sodium hydrogen phosphate (dibasic), 6.0 g of potassium phosphate (monobasic), and 0.6 g of magnesium sulfate heptahydrate in 1 L of distilled water. Then, an in vitro rumen micromineral solution was prepared by mixing 6.6 g of calcium chloride dihydrate, 5.0 g of manganese chloride tetrahydrate, 0.5 g of cobalt chloride hexahydrate, and 4.0 g of iron chloride hexahydrate in 0.05 L of distilled water.

[0132] Subsequently, the final in vitro buffer was prepared by mixing 450 mL of the rumen buffer prepared above, 450 mL of the rumen macromineral solution, 0.2109 mL of the rumen micromineral solution, and 900 mL of distilled water. The in vitro buffer solution prepared in this way was used in the experiment.

[0133] Rumen fluid was collected via stomach tubing before the morning feed, and the reduction status was checked by continuously flushing oxygen-free carbon dioxide (O2-free CO2) gas into the in vitro buffer solution. Subsequently, the rumen fluid and the in vitro buffer solution were mixed in a 1:2 ratio, the pH was adjusted to 6.7, and the mixture was dispensed into culture bottles in appropriate volumes to be used as the inoculum.

[0134] A methane reduction candidate substance was dissolved in methanol at a concentration of 80 mg / mL, and then added to a culture medium to achieve a final concentration of approximately 0.1 mg / mL, specifically 0.0835 mg / mL.

[0135] The methanol-added control (MCON) was prepared by adding 100 μL of filtered sterilized methanol at a concentration of 0.5% (v / v) to 20 mL of culture medium (rumen fluid:in vitro buffer = 1:2) contained in a 60 mL culture bottle used for in vitro fermentation experiments to verify the effect of methanol addition. It was confirmed that the concentration of 0.5% (v / v) used in this experiment did not significantly affect the overall fermentation pattern. The control (CON) was treated only with the additive solvent. The prepared samples are shown in Figure 5.

[0136] Using the material prepared in this way, gas generation, methane production, ammonia nitrogen production, pH, dry matter digestibility (DMD), and methane yield were measured. First, to measure gas generation, a gas sampling gas bag was prepared by combining a gas bag, a silicone stopper, a needle, and a 3-way cock (Fig. 6). Gas pressure was measured in mbar using a pressure transducer, and pressure was measured by injecting air in mL into a 60 mL culture bottle containing the same amount of culture medium. Using the measurements obtained from this, the gas generation was converted from mbar to mL.

[0137] Methane gas measurements were performed using a gas chromatograph (YL6500 GC system, Youngin Chromass) equipped with a thermal conductivity detector and a packed GC column (G3591-80055, Agilent Technologies). A 2 mL volume from a gas sampling bag was manually collected using a gas-tight syringe (1010 TLL, Hamilton Company) and injected into the gas chromatograph. Gas was collected from a methane gas container (CH430%, CO2balance) in the same manner to perform calibration curve analysis (30%, 15%, 7.5%, 3.75%, 1.875%), and based on this, the methane concentration values ​​in the experimental samples were derived.

[0138] In addition, the amount of ammonia nitrogen (NH3-N) produced was measured. After the culture was finished, 1.8 mL of the contents of the culture bottle were collected and centrifuged in a microcentrifuge at 16,000×g at 4 ℃ for 15 minutes. 0.5 mL of the supernatant was mixed with 0.1 mL of 0.2 M sulfuric acid (H2SO4), and then 2 μL of the supernatant was mixed with 100 μL of a phenol dye reagent prepared by mixing 50 g of phenol, 0.25 g of sodium nitroferricyanide, and 1 L of distilled water, and 100 μL of an alkaline hypochlorite reagent prepared by mixing 25 g of sodium hydroxide, 16.8 mL of sodium hypochlorite, and 1 L of distilled water. After reacting this mixture at room temperature for 30 minutes, the absorbance was measured at 625 nm using a microplate spectrophotometer (INNO, LTEK).

[0139] To measure pH and dry matter digestibility (DMD), the pH of the contents of the culture bottle was measured using a pH meter after the culture was finished. Then, the contents were transferred using a funnel to a pre-weighed nylon bag (R510 nylon bag), dried at 65°C for 72 hours, cooled for 30 minutes, and the final weight was measured.

[0140] Methane yield (mL / g dDM) was expressed as the amount of methane gas produced per 1 g of degraded dry matter.

[0141] The experimental results are shown in Figure 7, and among the 14 candidate substances for methane reduction, compounds X and H showed particularly significant effects. The methane production and methane yield of compounds X and H compared to the methanol-added control group (MCON) are shown in Figures 8 and 9. In particular, regarding methane yield, candidate substance X showed a yield of approximately 17.58 mL / g dDM, which was confirmed to be reduced by more than 12.5% ​​compared to the methanol-added control group of approximately 20.14 mL / g dDM.

[0142] <Example 3>

[0143] To further verify the methane reduction effect, experiments were conducted using rumen fluid and an in vitro buffer solution.

[0144] An in vitro rumen buffer solution was prepared in the same manner as in Example 1.

[0145] First, an in vitro rumen buffer solution was prepared by mixing 4 g of ammonium bicarbonate and 35 g of sodium bicarbonate in 1 L of distilled water. Next, an in vitro rumen macromineral solution was prepared by mixing 5.7 g of sodium hydrogen phosphate (dibasic), 6.0 g of potassium phosphate (monobasic), and 0.6 g of magnesium sulfate heptahydrate in 1 L of distilled water. Then, an in vitro rumen micromineral solution was prepared by mixing 6.6 g of calcium chloride dihydrate, 5.0 g of manganese chloride tetrahydrate, 0.5 g of cobalt chloride hexahydrate, and 4.0 g of iron chloride hexahydrate in 0.05 L of distilled water.

[0146] Subsequently, the final in vitro buffer was prepared by mixing 450 mL of the rumen buffer prepared above, 450 mL of the rumen macromineral solution, 0.2109 mL of the rumen micromineral solution, and 900 mL of distilled water. The in vitro buffer solution prepared in this way was used in the experiment.

[0147] Rumen fluid was collected via stomach tubing before the morning feed, and the reduction status was checked by continuously flushing oxygen-free carbon dioxide (O2-free CO2) gas into the in vitro buffer solution. Subsequently, the rumen fluid and the in vitro buffer solution were mixed in a 1:2 ratio, the pH was adjusted to 6.7, and the mixture was dispensed into culture bottles in appropriate volumes to be used as the inoculum.

[0148] Compound X was dissolved in methanol at a concentration of 80 mg / mL and added to the culture medium to achieve final concentrations of 0.0835 mg / mL (1st dose), 0.167 mg / mL (2nd dose), and 0.334 mg / mL (4th dose). This corresponds to 1.67 mg (1st dose), 3.34 mg (2nd dose), and 6.68 mg (4th dose) per 0.2 g of feed. To confirm the effect of methanol addition, the methanol-added control (MCON) was prepared by adding 100 μL of filter-sterilized methanol at a concentration of 0.5% (v / v) to 20 mL of culture medium (rumen fluid:in vitro buffer = 1:2) contained in a 60 mL culture bottle used for in vitro fermentation experiments. It was confirmed that the concentration of 0.5% (v / v) used in this experiment did not significantly affect the overall fermentation pattern.

[0149] Five samples were prepared for each concentration and five samples were prepared for the methanol-added control group. Gas production, methane production, ammonia nitrogen production, pH, dry matter digestion, and methane yield were measured in the same manner as previously measured. The average of each experimental result was calculated, and the standard error (SEM) and p-value were calculated. The results are shown in Figure 10, where different superscripts (A, B, C) within the same row indicate statistically significant differences (p<0.05).

[0150] Experimental results confirmed a linear reduction in methane gas production with increasing concentration of compound X.

[0151] Figure 11 visually represents the differences between the experimental data to evaluate the methane production reduction effects of the control group (CON) and the compound X treatment groups (1X, 2X, 4X) using a non-metric multidimensional scaling (NMDS) plot. Each point represents the experimental result of each sample, and the distribution of points reflects the similarity or difference between the experimental groups. The confidence ellipse displayed on the plot indicates the variability between samples within a 95% confidence level centered around the mean position of each treatment group. Notably, a non-overlapping pattern was observed between the confidence ellipses of the control group and the 4X treatment group, suggesting that the 4X treatment group showed a significant methane production reduction effect compared to the control group.

[0152] To statistically verify the results shown in Figure 11, a permutational multivariate analysis of variance (PERMANOVA) test was performed. This analysis was used to evaluate whether the methane production reduction effect between treatment groups was significant, and additionally, Benjamini-Hochberg correction was applied to reduce errors caused by multiple comparisons. The PERMANOVA analysis results showed that the difference related to methane production between the CON and 4X treatment groups was statistically significant (P = 0.028). A table summarizing the results of the statistical verification is presented in Figure 12, which quantitatively shows the methane production reduction effect between each treatment group. Through this, it was confirmed that the methane production inhibitory effect according to the concentration change of compound X was effectively expressed not only in the treatment groups but also compared to the control group.

[0153] From experimental results, it was confirmed that the compound of the present specification effectively acts on the methyl-coenzyme M reductase (MCR) of methanogenic bacteria present in the rumen of ruminant animals, thereby inhibiting the activity of methanogenic bacteria and reducing the amount of methane produced.

[0154] Although embodiments and examples of the present specification have been described above with reference to the attached drawings, the present specification is not limited to the above embodiments and examples and can be manufactured in various different forms. A person skilled in the art to which the present specification pertains will understand that it can be implemented in other specific forms without changing the technical concept or essential features of the present specification. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for inhibiting methane production in ruminants comprising a compound of chemical structure formula I, or a salt, solvate, hydrate, or isomer thereof. Chemical structural formula I (In the above chemical structure I, R1 is selected from the quinoline group of the following chemical structure R1a or the pyridine group of the following chemical structure R1b, and Chemical structural formula R1a Chemical structural formula R1b The pyridine ring of the above quinoline group can be substituted with R2 at one or more of positions 2, 3, and 4, either simultaneously or independently, and The benzene ring of the above quinoline group may be substituted with R3 at one or more of the 5, 6, 7, or 8 positions simultaneously or independently, and The above pyridine group may be substituted with one or more R4s simultaneously or independently, and The above R2 is selected from the group consisting of H, C1 to C6 alkyl groups, and The above R3 is selected from the group consisting of H, C1 to C6 alkoxy groups, and The above R4 is selected from the group consisting of H and C1 to C6 alkyl groups).

2. A composition for inhibiting methane production according to claim 1, comprising a compound wherein R1 in the chemical structure formula I is a quinoline group, the quinoline group is bonded to the chemical structure formula I at position 5, the pyridine ring of the quinoline group at position 2 is substituted with R2, and the benzene ring of the quinoline group at position 8 is substituted with R3.

3. A composition for inhibiting methane production according to paragraph 2, comprising a compound of the following chemical structure formula II. Chemical Structure Formula II 4. A composition for inhibiting methane production according to claim 1, wherein R1 in chemical structural formula I is a pyridine group, and the compound is bonded to chemical structural formula I at the 4th position of the pyridine group.

5. A composition for inhibiting methane production according to claim 4, comprising a compound of the following chemical structure formula III. Chemical Structure Formula III 6. A composition for inhibiting methane production according to claim 1, wherein the compound binds to the protein structure of methyl-coenzyme M reductase (MCR) of methane-producing bacteria in the rumen of a ruminant animal and inhibits the activity of said methane-producing bacteria.

7. In paragraph 6, the said methane-producing bacteria are rumen methane-producing bacteria, namely Methanobrevibacter smithii, Methanobrevibacter millerae, Methanobrevibacter thaurei, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanobrevibacter gottschalkii, Methanosarcina barkeri, and Methanobacterium A composition for inhibiting methane production, comprising one or more selected from Methanobacterium bryantii, Methanobacterium formicicum, and Methanocaldococcus jannaschii.

8. A composition for inhibiting methane production that reduces the relative abundance of methane-producing bacteria in the rumen, in accordance with claim 1.

9. A composition for inhibiting methane production according to claim 1, wherein the compound is contained at a concentration of 0.1 mg / mL or higher.

10. A composition for inhibiting methane production according to claim 1, wherein the compound is contained at a concentration of 0.4 mg / mL or higher.

11. A methane-inhibiting composition according to claim 1, wherein the pH of the methane-inhibiting composition is in the range of 6.0 to 7.

0.

12. A methane-inhibiting composition according to claim 1, wherein the methane-inhibiting composition further comprises one or more selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline solution, injectable solution, glycerol, methanol, and ethanol.

13. A methane-inhibiting composition according to claim 1, wherein the methane-inhibiting composition further comprises one or more selected from the group consisting of antioxidants, buffers, bacteriostatic agents, excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, flavoring agents, diluents, dispersants, pH adjusters, and surfactants.

14. A methane-inhibiting composition according to claim 1, wherein the methane-inhibiting composition is an aqueous solution, suspension, emulsion, injection, rumen formulation, powder, granule, tablet, capsule, pellet, block, or sustained-release block formulation.

15. A methane production inhibiting composition according to claim 1, wherein the methane production inhibiting composition can be administered via one of the following routes: oral, rectal, intravenous, arterial, intraperitoneal, intramuscular, transdermal, nasal, inhalation, topical, ocular, or intradermal.

16. A composition for inhibiting methane production according to claim 1, wherein the ruminant animal is one or more selected from cattle, goats, sheep, deer, reindeer, giraffes, camels, alpacas, llamas, and okapis.

17. A feed additive for ruminants comprising the methane production inhibiting composition of claim 1.

18. A feed additive for ruminants characterized by reducing the methane production rate by 10% or more when added to the feed compared to when not added, in accordance with claim 17.

19. A feed additive for ruminants according to claim 17, further comprising a protein-degrading enzyme or a cellulose-degrading enzyme for the digestive efficiency of ruminants.

20. Feed for ruminants comprising the methane production inhibiting composition of claim 1.

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