CN116669564A - Composition for reducing methane emissions, method for improving the metabolic efficiency of ruminants and method for administration of methanogenesis inhibitors - Google Patents

Abstract

Compositions for reducing methane emissions and/or inhibiting one or more methanogens are provided. The composition comprises an organohalogen compound and an organosulfur compound, preferably bromoform and allicin. The composition may further comprise a polyphenol compound. Animal feeds comprising the composition are also described.

Description

Composition for reducing methane emissions, for improving metabolic efficiency of ruminants Methods and methods for administration of methanogenesis inhibitors

field of invention

The present invention relates to compositions for reducing methane emissions and/or for inhibiting one or more methanogenic organisms (ie methanogens). The present invention also relates to methods for improving the metabolic efficiency of ruminants and methods of administering methanogenesis inhibitors to ruminants, particularly agricultural ruminants.

Background of the invention

Ruminants consume feedstuffs containing carbon and nitrogen, which are converted into a variety of substances, including carbon-rich lipids, fats, fatty acids and carbohydrates as well as nitrogen-rich proteins, nucleic acids, amino acids and nucleotides.

Feed that is converted to these substances aids animal growth and is a major component of a variety of valuable animal products.

However, ruminants, such as cattle, sheep and goats, are inefficient users of carbon and nitrogen. Ruminant methane production from carbon represents an energy loss ranging from 2% to 12% of total energy intake from feed.

In addition, ruminants can excrete more than 50% of their nitrogen from feed, mainly in the form of urinary and fecal nitrogen.

Loss of carbon and nitrogen from ruminants acts both as a loss of valuable animal growth and has significant environmental impacts, including release of the potent greenhouse gases methane and nitrous oxide into the atmosphere and nitrogen leaching from soils. Feed supplements administered to ruminants to improve animal growth and reduce carbon and nitrogen emissions into the environment are expensive and difficult to administer optimally and economically.

One of the known ways to reduce methane production in ruminants is through the use of seaweed. However, feeding large amounts of seaweed to animals may have potential risk factors. One of the risk factors is reduced feed intake and performance. Feeding seaweed at higher levels in the diet has resulted in reduced dry matter intake in beef cattle (Roque et al., 2021) and dairy cattle (Roque et al., 2019, Stefenoni et al., 2021, Muizelaar et al., 2021). It has been observed that cows often reject seaweed or choose against it when mixed with fresh feed for cows, indicating poor palatability of seaweed (Muizelaar et al., 2021). Lower feed intake can also lead to lower performance when cows are fed high dosage levels of seaweed, as shown by reduced milk production (Roque et al., 2019, Stefenoni et al., 2021, Muizelaar et al., 2021). Seaweed is also known to contain high levels of iodine (Makkar et al., 2016), and its transfer to livestock products has been studied. Seaweed (Asparagopsis taxiformis) was fed to beef cattle at levels of 0.25% and 0.5% in the diet, resulting in a daily iodine intake of 106 to 225 mg/day of iodine (Roque et al., 2021) . This exceeds the recommended daily iodine intake level of about 5 mg/day based on 0.5 mg/kg DMI (NRC, 2006) and 10 kg DM intake in this study. Transfer of iodine into milk is a bigger problem. According to Lean et al. (2021), feeding Asparagopsistaxiformis at 0.5% in the diet increased iodine levels 5-fold to 3 mg/L.

There is clearly a need to develop improved compositions for reducing methane emissions, in particular wherein the aforementioned risk factors are reduced and/or wherein the compositions have improved palatability compared to seaweed.

There is also a clear need to improve the metabolic efficiency of ruminants in order to reduce methane emissions and nitrogen excretion to the environment, and to improve animal growth levels and increase the amount of valuable animal products.

The present invention addresses this need by providing a feed supplement that improves metabolic efficiency in ruminants. The feed supplement reduces methane emissions and nitrogen excretion into the environment and converts energy that would otherwise be converted into emitted methane and otherwise excreted nitrogen into valuable animal products and thus improves rumination Metabolic efficiency of animals.

There is a clear need for improved administration of feed supplements to ruminants to improve animal growth and reduce carbon and nitrogen losses to the environment.

The present invention addresses this need by providing compositions and methanogenesis inhibitor feed supplement administration that optimally and economically reduce methane emissions and nitrogen excretion, and will The energy otherwise converted to emitted methane and otherwise excreted nitrogen is converted into valuable animal products.

statement of invention

According to the present invention, in a first aspect, there is provided a composition for reducing methane emission, which comprises an organohalogen compound and an organosulfur compound.

In some embodiments, the organohalogen compound is an organobromine compound. In a preferred embodiment, the organohalogen compound is bromoform ( _CHBr3 ).

In some embodiments, the organosulfur compound is from a plant of the Allium species. In some embodiments, the organosulfur compound is selected from allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); diallyl disulfide (C ₆ H ₁₀ S ₂ ); and allyl mercaptan (C ₃ H ₆ S). In a preferred embodiment, the organosulfur compound is allicin.

In some embodiments, the ratio of organohalogen compound to organosulfur compound is 1:10 to 1:3500, more preferably 1:1000 to 1:2500.

In some embodiments, the composition further comprises a polyphenolic compound. In some embodiments, the polyphenolic compound includes or is a bioflavonoid. In some embodiments, the polyphenolic compound includes naringin, neohesperidin, or combinations thereof.

In some embodiments, the compositions are used to inhibit one or more methanogens. In some embodiments, the compositions are used to improve the metabolic efficiency of an animal, more particularly a ruminant such as a cow, goat or sheep.

The present inventors have found that when an organohalogen compound such as bromoform is combined with an organosulfur compound such as allicin, the composition of the present invention exhibits a high % inhibition of methanogens.

In particular, it was shown that organohalogen compounds and organosulfur compounds can act synergistically to reduce methane production. Synergistic combinations provide an effect greater than the sum of the individual components. The inventors have additionally found that compositions comprising organohalogen compounds such as bromoform and powder mixtures comprising organosulfur compounds and polyphenolic compounds such as NXRH214 powder also lead to effective inhibition of methanogens.

Also according to the invention, in a second aspect, is an animal feed comprising a composition of the invention or as otherwise disclosed herein.

Also according to the present invention, in a third aspect, is the use of a composition of the invention or an animal feed of the invention for reducing methane emissions and/or for inhibiting methanogens and/or improving the metabolic efficiency of an animal.

Also according to the present invention, in a fourth aspect, is a method of reducing methane emissions, the method comprising administering to an animal, more particularly a ruminant, a composition or an animal feed according to the invention. In some examples, the ruminant is a cow, goat or sheep.

Also according to the present invention, in a fifth aspect, is a method of inhibiting one or more methanogens, the method comprising administering to an animal, more particularly a ruminant, a composition or an animal feed according to the invention. In some examples, the ruminant is a cow, goat or sheep.

Also according to the present invention, in a sixth aspect, is a method of improving the metabolic efficiency of an animal, the method comprising administering to an animal, more particularly a ruminant, a composition or an animal feed according to the invention. In some examples, the ruminant is a cow, goat or sheep.

Also disclosed herein are compositions for inhibiting one or more methanogens comprising an organohalogen compound and an organosulfur compound. Organohalogen compounds and organosulfur compounds are as otherwise described herein.

Also disclosed herein is a composition for improving metabolic efficiency in an animal, the composition comprising an organohalogen compound and an organosulfur compound. Organohalogen compounds and organosulfur compounds are as otherwise described herein.

Also disclosed herein is a method of reducing nitrogen excreted by a ruminant and/or reducing methane emitted by a ruminant and/or increasing nitrogen- and carbon-rich substances in a ruminant, said method comprising administering to said ruminant an effective amount of at least A type of methanogenesis inhibitor step. In one embodiment, the method comprises administering to said ruminant any composition disclosed herein.

In one embodiment, the methanogenesis inhibitor is selected from the group consisting of: organohalogen compounds; organohalogen-enriched marine macroalgae; organosulfur compounds; organosulfur-enriched plants; polyphenolic compounds;

_CH3Br ; CH3I _{; CH2Cl2} ; _CH2Br2 ; _{CH2I2 ; CHCl3 ; CHBr3 ; CHI3 ; CCl4 CH2ClBr ; CH2ClI ; CH2BrI ; CHBr2Cl ; CHBrI2 ; CHBrClI ; CHBr2I ; _} I; _CH3 ( _CH2 ) _3I ; _CH3 ( _CH2 ) _{4Br ; CH3} ( _CH2 ) _4I ; (CH3) _2CHI ; _CH3CH2CH ( _CH3 )I; ( _CH3 ) _{2CHCH2I ; BrCH2CH2Br ; ClCH} = _CCl2 ; _{and CH3CH2CH2CH2I .}

In one embodiment, the organohalogen-rich marine macroalgae are selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; Oedogonium species ; Ulva species; and Cladophora patentiramea.

In one embodiment, the organosulfur compound is selected from the group consisting of: organosulfur secondary metabolites; allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); diallyl di Sulfides (C ₆ H ₁₀ S ₂ ); and Allyl Mercaptans (C ₃ H ₆ S).

In one embodiment, the organosulfur-rich plant is an Allium species selected from the group consisting of: Allium sativum; Allium ampeloprasum; and Allium cepa.

In one embodiment, the polyphenolic compound is selected from: flavonoids; bioflavonoids; non-bioflavonoids. The at least one polyphenolic compound may for example comprise at least one bioflavonoid; anthocyanins; flavones; flavonols; flavanones; dihydroflavonols; Isoflavones; proanthocyanidins; phenolic acids; hydroxycinnamic acids; coumarins; stilbenes; anthraquinones; lignans; lignin; tannins; polyphenolic proteins; catechins; rutin; Flavonoids; Kaempferol; Gallocatechin; Catechin Gallate; Epigallocatechin; Epigallocatechin; Epigallocatechin Gallate; Quercetin; Allocatechin; Gallate Theophyl gallate; epicatechin; epigallocatechin; epicatechin gallate; epigallocatechin gallate; kaempferol; quercetin; naringin; neohesperidin; eriocitrin; isonaringin; naringenin; hesperidin; roifolin; diosmin; melissain; hesperetin; citrusin; epicatechin; gallatechin epigallocatechin; coumaric acid; cinnamic acid; gallic acid; ellagic acid; protocatechuic acid; chlorogenic acid; caffeic acid; ferulic acid; punicalagin; and garnetin.

In one embodiment, the polyphenol-rich plant is selected from the group consisting of: Allium species; Brassica species; Camelia species; Capsicum species; Citrus species ; Citrusaurantium; Cucumis species; Malus species; Musa species; Phaseolus species; Prunus species; Punica species; pear species of the genus Pyrus; species of the genus Solanum; and species of the genus Vaccinium.

In another aspect, the present invention provides a method for reducing excreted nitrogen and emitted carbon and increasing valuable nitrogen- and carbon-rich materials in a ruminant, said method comprising administering to said animal a Feed supplements or feeds as described herein.

Also disclosed herein is a method of reducing nitrogen excreted by a ruminant and/or reducing methane emitted by a ruminant and/or increasing nitrogen- and carbon-rich substances in a ruminant, said method comprising gradually administering to said ruminant an effective amount of At least one type of methanogenesis inhibitor. In one embodiment, the method comprises administering to said ruminant a composition as disclosed herein.

In one embodiment, the methanogenesis inhibitor is administered stepwise with at least one dose, as a percentage of the weight of said ruminant selected from: 0.1%; 0.2%; 0.3%; 0.4%; 0.5%; 0.6%; 0.7% ; 0.8%; 0.9%; 1.0%; 1.1%; 1.2%; 1.3%; 1.4%; 1.5%; 2.0%; 2.5%; 3.0%;

In one embodiment, the methanogenesis inhibitor is administered stepwise with at least one dose, as a percentage by weight of feed of said ruminant selected from the group consisting of: 0.1%; 0.2%; 0.3%; 0.4%; 0.5%; 0.6%; 0.7%; 0.8%; 0.9%; 1.0%; 1.1%; 1.2%; 1.3%; 1.4%; 1.5%; 2.0%; 2.5%; 3.0%; 3.5%; 4.0%; 4.5%; 5.0%; %.

In one embodiment, the stepwise administration has at least one interval between consecutive doses selected from the group consisting of: 1 minute; 1 hour; 1 day; 2 days; 3 days; 4 days; 5 days; 6 days; 7 days ; 10 days; 2 weeks; 3 weeks; 4 weeks; 6 weeks; 2 months; 3 months; 4 months; 6 months; 9 months; and 12 months.

Brief description of the drawings

Figures 1 and 2 show the % methane inhibition of various compositions of the invention after incubation with Methanococcus maripaludis cultures.

Detailed description of the invention

According to the present invention, there is provided a composition for reducing methane emission, which comprises an organohalogen compound and an organosulfur compound. The compositions are also useful for inhibiting one or more methanogens. Any "composition" referred to herein may also be referred to as an "animal feed supplement".

In some embodiments, the one or more methanogens have the genera Methanobacterium, Methanosarcina, Methanobrevibacter, Methanosarcina, Methanosarcina Methanoculleus, Methanosphaera, Methanocorpusculum, Methanofollis, Methanogenium, Methanomicrobium, Methanofollis Methanopyrus, Methanoregula, Methanasaeta, Methanthermobacter or Methanococcus. In some embodiments, the one or more methanogens are selected from the group consisting of Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacterium millerae , Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, Methanosarcina barkeri, Methanosarcina barkeri (Methanobrevibacter boviskoreani), Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methanosarcina mazei, Methanosarcina mazei ( Methanobrevibactergottschalkii), Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter woesei, Methanobrevibacterwolinii ). In some examples, the one or more methanogens are Methanococcus maripaludis.

Hereinafter, the present invention will be described according to preferred embodiments of the invention with reference to the accompanying description. It is to be understood, however, that the limitation of the description to the preferred embodiments of the invention is only for the purpose of facilitating the discussion of the invention, and that it is envisaged that various modifications may be devised by those skilled in the art without departing from the scope of the appended claims.

-Organohalogen compounds

The compositions of the present invention comprise an organohalogen compound (ie, at least one organohalogen compound).

Organohalogen compounds are organic compounds that contain halogens. In some embodiments, the organohalogen compound is a C ₁ -C ₆ alkylhalogen compound. In some embodiments, the organohalogen compound comprises chlorine, bromine, iodine, or combinations thereof. _{CH3Br; CH3I} ; CH2Cl2 _{; CH2Br2} ; _CH2I2 ; _{CHCl3 ; CHBr3 ; CHI3 ; CCl4 ;} CBr _{4 ; CH2ClBr ; CH2ClI ; CH2BrI ; CHBr2Cl ; CHBrI2 ; _ _ _ CH3} ( _CH2 ) _3I ; _CH3 ( _CH2 ) _{4Br ; CH3} ( _CH2 ) _4I ; (CH3) _2CHI ; _CH3CH2CH ( _CH3 )I; ( _CH3 ) _{2CHCH 2} I; BrCH ₂ CH ₂ Br; ClCH=CCl ₂ ; and CH ₃ CH ₂ CH ₂ CH ₂ I. In some embodiments, the organohalogen compound is a trihalomethane. In some embodiments, the organohalogen compound is an organobromine compound, more preferably, wherein the organohalogen compound is bromoform ( _CHBr3 ).

The biological sources of organohalogen compounds are marine macroalgae rich in organohalogens. For example, the organohalogen-rich marine kelp comprises at least one species of marine kelp selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; Oedogonium species; Ulva species; and Cladophora patentiramea. Thus, in some embodiments, the organohalogen compound is derived from organohalogen-rich marine microalgae, for example selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; Oedogonium species; Ulva species; and Cladophorapatentiramea.

In other embodiments, organohalogen compounds are produced by bacteria, fungi, and cyanobacteria. For example, the bacterium includes a bacterium selected from the group consisting of Streptomyces sp. and Zobellia galactanivorans. For example, fungi include a fungus selected from the group consisting of Pyricularia oryzae, Curvularia inaequalis, Pyrenophoratritici-repentis, and Embellisia didymospora. For example, cyanobacteria include one selected from the group consisting of Trichodesmium erythraeum, Synechococcus sp., and Acaryochloris marina.

In other embodiments, the organohalogen is synthetic, ie, the organohalogen is chemically synthesized. In other embodiments, the organohalogen is produced by recombinant yeast.

In some embodiments, the concentration of the organohalogen compound in the composition is greater than 100 nM, or greater than 110 nM, or greater than 120 nM, or greater than 130 nM, or greater than 140 nM, or greater than 150 nM. In some embodiments, the concentration of the organohalogen compound in the composition is less than 10000 nM, or less than 1000 nM, or less than 500 nM, or less than 200 nM, or less than 175 nM, or less than 160 nM, or less than 150 nM, or less than 140 nM, or less than 130 nM. In some embodiments, the concentration of the organohalogen compound in the composition is from 100 nM to 10000 nM, from 100 nM to 1000 nM, from 100 nM to 500 nM, from 100 nM to 300 nM, from 100 nM to 200 nM, or from 110 nM to 175 nM. In some examples, the organohalogen compound is an organobromine compound, preferably bromoform.

-Organosulfur compounds

The compositions of the present invention comprise an organosulfur compound (ie at least one organosulfur compound).

Organosulfur compounds are organic compounds that contain sulfur. In certain embodiments, each organosulfur compound may be independently selected from the group consisting of thioethers, thioesters, thioacetals, thiols, disulfides, polysulfides, sulfoxides, sulfones, thiosulfinates, Sulfonimide, sulfinimide, sulfodiimide, thioketone, thial, sulfonide, sulfone, thiocarboxylic acid (including dithiocarboxylic acid), sulfonic acid, sulfinic acid, sulfenyl Acid, sulfonate, sulfinate, sulfenate, sulfonamide, sulfenamide, sulfenamide, sulfonium compound, sulfoxonium compound, sulfonium ylide, sulfoxonylide, thiocarbonyl ylide Salt, sulfurane and persulfurane. In certain embodiments, each organosulfur compound is independently selected from the group consisting of thioesters, sulfoxides, thioethers, disulfides, polysulfides (including trisulfides), and thiols. In certain embodiments, each organosulfur compound is independently selected from the group consisting of thioesters, sulfoxides, thioethers, disulfides, and polysulfides (including trisulfides). In certain embodiments, each organosulfur compound is independently selected from disulfides and polysulfides (including trisulfides). In certain embodiments, each organosulfur compound is a disulfide.

In some embodiments, the organosulfur compound is from a plant of the Allium species. In some embodiments, the organosulfur compound is a disulfide compound, and more specifically a diallyl disulfide compound.

In certain embodiments, each organosulfur compound is independently selected from the group consisting of allicin, allicin, allyl propyl disulfide, diallyl trisulfide, s-allyl cysteine, ethylene Vinyldithiines (vinyldithiines) (3-vinyl-4H-1,2-dithiine (3-vinyl-4H-1,2-dithiin) and 2-vinyl-4H-1,3-dithiine (2-vinyl-4H-1,3-dithiin)) and diallyl disulfide.

In some embodiments, the organosulfur compound is selected from allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); diallyl disulfide (C ₆ H ₁₀ S ₂ ); and allyl mercaptan (C ₃ H ₆ S). In certain embodiments, at least one organosulfur compound is or includes allicin.

Allicin is an organosulfur compound having the chemical formula C ₆ H ₁₀ OS ₂ and the structure shown below.

Organosulfur compounds such as allicin can be obtained, for example, from garlic or another Allium species. For example, organosulfur compounds such as allicin can be obtained from extracts of Allium species such as Allium sativum. The term extract includes aqueous extracts, non-aqueous extracts, alcoholic extracts, concentrates, oils, infusions, powders, granules and combinations of two or more thereof. For example, organosulfur compounds such as allicin can be obtained from raw garlic, dried garlic, or a combination thereof. Organosulfur compounds such as allicin may, for example, be derived from any Allium subspecies and varieties currently known or later discovered, such as Allium sativum, Allium ursinum, Allium fistulosum, Allium onion cepa) and shallots (Allium tricoccum). For example, organosulfur compounds such as allicin can be independently derived from garlic of the subspecies ophioscorodon (Garlicum hardneck) and garlic of the subspecies sativum (Garlicum softneck). For example, organosulfur compounds such as allicin can be independently derived from porcelain garlic, rocambole garlic, purple stripe garlic, marbled purple stripe garlic, glaze Glazed purple stripe garlic, artichoke garlic, silverskin garlic, asiatic garlic, turban garlic, and creole garlic . In particular, organosulfur compounds such as allicin are obtainable from garlic (Allium sativum).

For example, Allium plants from which organosulfur compounds such as allicin can be derived may have been treated or processed. For example, Allium plants can be "aged" or "black" (such as aged or black garlic) by storing Allium plants under controlled conditions at a specific temperature, humidity Heating with a solvent, for example over days or weeks, darkens the garlic cloves after undergoing a Maillard or browning reaction. For example, Allium plants (Allium) can be "dried" or "dehydrated" by freshly Fresh or unaged garlic is obtained by heating to a temperature of 30° C. to 120° C. and to a moisture content of about 3% to 10%. Allium, for example, may be "fresh" or "unaged" (as in fresh or unaged garlic), obtained without knowingly undergoing special handling or processing that transforms or transforms its constituents into different compounds . Fresh or unaged Allium (Allium) may eg have been treated or processed to remove odor (deodorized) (eg deodorized garlic extract). Often, encapsulation or coating methods can be used to mask or reduce odors. Alternatively or additionally, taste-masking ingredients such as green tea, parsley, basil, spinach, etc. may be added to mask or reduce odors in the composition.

The organosulfur compound (eg, allicin) may or may not be isolated and/or purified prior to incorporation into the compositions described herein. As such, in certain embodiments, the compositions described herein may comprise raw garlic, dried garlic, and/or garlic extract. In other embodiments, the organosulfur compound (eg, allicin) is chemically synthesized. In certain embodiments, alliin can be enzymatically converted to Allicin and optionally obtained by extracting allicin. Suitable methods are further described, for example, in WO03/004668, the contents of which are incorporated herein by reference.

In other embodiments, the organosulfur compound, such as allicin, may be synthetic, ie, chemically synthesized.

In some embodiments, the concentration of the organosulfur compound in the composition is greater than 10 μM, or greater than 100 μM, or greater than 150 μM, or greater than 175 μM, or greater than 200 μM, or greater than 225 μM, or greater than 250 μM, or greater than 275 μM. In some embodiments, the concentration of the organosulfur compound in the composition is less than 350 μM, or less than 325 μM, or less than 300 μM, or less than 275 μM, or less than 250 μM, or less than 225 μM. In some embodiments, the concentration of the organosulfur compound in the composition is 10 μM to 350 μM, 100 μM to 350 μM, 150 μM to 350 μM, and in some instances, 200 to 300 μM.

- Ratio of organohalogen compounds to organosulfur compounds

In some embodiments, the ratio of organohalogen compound to organosulfur compound in the composition is preferably from 1:10 to 1:3500. In some embodiments, the ratio of organohalogen compound to organosulfur compound in the composition is 1:50 to 1:3500, or 1:100 to 1:3500, or 1:500 to 1:3500, or 1:750 to 1:3500, or 1:1000 to 1:3500, or 1:1500 to 1:3500, or 1:2000 to 1:3500. In some embodiments, the ratio of organohalogen compound to organosulfur compound in the composition is 1:500 to 1:3000, or 1:500 to 1:2750, or 1:500 to 1:2500, or 1:500 to 1:2000, or 1:500 to 1:1500. In a preferred embodiment, the ratio of organohalogen compound to organosulfur compound in the composition is from 1:750 to 1:3000, or from 1:1000 to 1:2500.

In some examples, the organohalogen is an organobromine compound, such as bromoform, and the organosulfur compound is a disulfide, such as allicin.

- polyphenolic compounds

The compositions of the present invention may further comprise a polyphenolic compound (ie, one or more polyphenolic compounds).

The term phenol refers to a chemical compound comprising a hydroxyl group (-OH) bonded directly to an aromatic hydrocarbon group. The term polyphenolic compound refers to a compound comprising more than one phenolic group. The polyphenolic compounds described herein can include bioflavonoids, non-bioflavonoid polyphenolic compounds, or combinations thereof. The at least one polyphenolic compound may for example comprise at least one bioflavonoid.

The term bioflavonoids refers to a class of plant and fungal secondary metabolites and has a general structure with a 15-carbon skeleton consisting of two benzene rings (A and B) and a heterocycle (C), sometimes abbreviated as _C6 -C ₃ -C ₆ . Thus, bioflavonoids are polyphenols. The term bioflavonoids includes anthoxanthins (including flavones and flavonols), flavanones, flavanols, flavans and anthocyanins. The term bioflavonoids also includes compounds with a flavonoid skeleton (2-phenyl-1,4-benzopyrone), an isoflavane skeleton (3-phenylchromen-4-one) or a neoflavan skeleton (4-benzene base coumarin) compounds. The term non-bioflavonoid polyphenolic compounds refers to other classes of polyphenolic compounds known in the art which do not fall within the definition of the term bioflavonoids set forth herein. The term non-bioflavonoid polyphenolic compounds includes compounds containing 6 or more carbons, 7 or more carbons, 8 or more carbons, 9 or more carbons, 10 or more carbons, Polyphenolic compounds of 13 or more carbons, 14 or more carbons, 16 or more carbons, 18 or more carbons, or 30 or more carbons. The term non-bioflavonoid polyphenolic compounds includes, but is not limited to, polyphenolic acids (C ₆ -C ₁ structure), stilbenes (C ₆ -C ₂ -C ₆ structure), anthraquinones (C ₆ -C ₂ -C ₆ structure) and lignans ((C ₆ -C ₃ ) ₂ structure). In some embodiments, non-bioflavonoid polyphenolic compounds are plant polymers including, but not limited to, lignin, catechol melanin, flavolans, polyphenolic proteins, and polyphenols. In certain embodiments, the one or more bioflavonoids are each independently selected from the group consisting of anthocyanins (including flavones and flavonols), flavanones (including flavanone glycosides), dihydroflavonols, flavans, Isoflavones, anthocyanins and proanthocyanidins. In certain embodiments, each of the one or more bioflavonoids is independently selected from anthocyanins and flavanones (including flavanone glycosides). In certain embodiments, all bioflavonoids are anthocyanins and/or flavanones. In certain embodiments, the one or more bioflavonoids are independently flavones or flavanones. In certain embodiments, all bioflavonoids are flavones and/or flavanones. Flavones and flavanones may, for example, be independently flavone glycosides and flavanone glycosides, respectively. In certain embodiments, the one or more bioflavonoids are flavanones. In certain embodiments, all bioflavonoids are flavanones. In certain embodiments, the one or more bioflavonoids are flavanone glycosides. In certain embodiments, all bioflavonoids are flavanone glycosides. The one or more bioflavonoids may for example be selected from the group consisting of naringin, neohesperidin, eriocitrin, isonaringin, naringenin, hesperidin, rhodorin, diosmin, lemon balm Glycosides, hesperetin, citrusin, catechin, rutin, acacia, genistein, kaempferol, quercetin, epicatechin, gallocatechin, epigallocatechin, catechin Gallate, epicatechin gallate, epigallocatechin gallate, and gallocatechin gallate. In certain embodiments, the one or more bioflavonoids include naringin and neohesperidin. In certain embodiments, the one or more bioflavonoids are a combination of naringin and neohesperidin. In certain embodiments, the one or more bioflavonoids include catechin, rutin, acacia, genistein, kaempferol, gallocatechin, catechin gallate, epicatechin, One or more of epigallocatechin, epicatechin gallate, and quercetin. In certain embodiments, the one or more bioflavonoids include one or more of catechin, rutin, acacinin, genistein, and kaempferol. In certain embodiments, the one or more bioflavonoids are a combination of catechin, rutin, acacinin, genistein, and kaempferol. In certain embodiments, the one or more bioflavonoids include gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate, gallocatechin One or more of vegetarian gallate, epigallocatechin gallate, kaempferol and quercetin. In certain embodiments, the one or more bioflavonoids include gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate, kaempferol, and One or more of quercetin. In certain embodiments, the one or more bioflavonoids are gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate, gallate Combination of catechin gallate, gallocatechin gallate, kaempferol and quercetin. In certain embodiments, the one or more bioflavonoids are gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate, kaempferol Combination of phenol and quercetin.

In some embodiments, the polyphenols include one or more non-bioflavonoid polyphenolic compounds. In some embodiments, the one or more non-bioflavonoid phenolic compounds are each independently selected from the group consisting of phenolic acids, stilbenes, anthraquinones, lignans, lignins, tannins, polyphenolic proteins, and polyphenols. In certain embodiments, each of the one or more non-bioflavonoid polyphenolic compounds is independently selected from tannins and polyphenols. In certain embodiments, all non-bioflavonoid polyphenolic compounds are tannins and/or polyphenols.

The compositions described herein comprise one or more polyphenolic compounds. For example, the composition may comprise two or more polyphenolic compounds or three or more polyphenolic compounds or four or more polyphenolic compounds or five or more or six or more or Seven or more or eight or more or nine or more or ten or more polyphenolic compounds. For example, the composition may contain one, two, three, four or five polyphenolic compounds. The compositions described herein comprise one or more bioflavonoids. For example, the composition may comprise two or more bioflavonoids, or three or more bioflavonoids, or four or more bioflavonoids, or five or more or six or more Multiple or seven or more or eight or more or nine or more or ten or more bioflavonoids. For example, the composition may contain one, two, three, four or five bioflavonoids. For example, the composition may contain two bioflavonoids, which may be naringin and neohesperidin. In another example, the composition may comprise five bioflavonoids, which may be catechin, rutin, acacinin, genistein, and kaempferol. In an alternative embodiment, the composition may comprise seven bioflavonoids which may be gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate Esters, Kaempferol and Quercetin. In an alternative embodiment, the composition may comprise nine bioflavonoids which may be gallocatechin, catechin gallate, epicatechin, epigallocatechin, epicatechin gallate Ester, Gallocatechin Gallate, Epigallocatechin Gallate, Kaempferol and Quercetin.

One or more polyphenolic compounds, such as one or more bioflavonoids, can be obtained, for example, from a part of a plant such as a fruit or vegetable. For example, flavonols can be obtained from tomatoes, beans, almonds and/or radishes. For example, flavan-3-ols can be obtained from peaches, plums, strawberries and/or green tea. For example, flavonoids can be obtained from watermelon and/or peppers. For example, flavanones can be obtained from the fruit of Citrus species. For example, anthocyanins can be obtained from blueberries, bananas, strawberries, cranberries and/or plums. One or more polyphenolic compounds, eg one or more bioflavonoids, may eg be obtained from Citrus species fruits such as oranges, lemons, grapefruit, pomelo or limes. In particular, one or more polyphenolic compounds, such as one or more bioflavonoids, may be obtained from oranges. One or more polyphenolic compounds, eg one or more bioflavonoids, may eg be obtained from a fruit of a species of the genus Punica, such as Punica granatum or Punica protopunica. In particular, one or more polyphenolic compounds, such as one or more bioflavonoids, may be obtained from pomegranate (Punica granatum).

One or more polyphenolic compounds, such as one or more bioflavonoids, can be obtained, for example, from parts (such as leaves) of plants of the genus Camellia, such as Camellia spp. Camellia sinensis), Camellia taliensis, Camellia oleifera, Camellia assimilis, Camellia azalea, Camellia brevistyla, Camellia caudata, Camellia chekiangoleosa, Camellia chrysantha, Camellia chrysanthoides, Camellia connata, Camellia crapnelliana, Camellia cuspidata, Camellia euphlebia, Camellia euryoides, Camellia flava, Camellia fleuryi, Camellia forrestii, Camellia fraterna, Rough fruit tea ( Camellia furfuracea), Camellia gilbertii, Camellia granthamiana, Camellis grijsii, Camelliahengchunensis, Camellia hiemalis, Hong Kong Camellia hongkongensis, Camellia irrawadiensis, Camellia japonica, Camellia oleifera (Camelliakissii), Camellia lutchuensis, Camellia miyagii, Camellia nitidissima ), Camellia nokoensis, Camellia parviflora, Camellia pitardii, Camellia pleurocarpa, Camellia polyodonta, Camellia pubupetala ), Camellia (Camelliareticulata), Camellia rosiflora, Camellia rusticana, Camellia salicifolia, Camellia saluenensis, Camellia sasanqua, Camellia semiserrata, Alpine Camellia trasnokoensis, Camellia tunghinensis, Camellia tunghinensis, Camellia vietnamensis, Camellia x williamsii and Camellia yunnanensis. In particular, one or more bioflavonoids may be obtained from Camellis sinensis (tea plant). Any subspecies or variety of Camellia sinensis can be used. Parts (eg leaves) of Camellia sinensis may be untreated or may be treated eg by steaming, withering, rolling, oxidation, fermentation and/or drying. One or more polyphenolic compounds, such as one or more bioflavonoids, can be obtained, for example, from green tea (Camellia sinensis) leaves.

For example, one or more polyphenolic compounds, such as one or more bioflavonoids, can be extracted from Citrus species fruit, Punica species fruit, or parts of Camellia species plants things get. The term extract includes aqueous extracts, non-aqueous extracts, alcoholic extracts, concentrates, oils, infusions, powders, granules and combinations of two or more thereof. For example, one or more polyphenolic compounds, such as one or more bioflavonoids, can be obtained from dried Citrus fruits, dried Punica fruits, or dried Camellia plant parts (like a leaf) to obtain. For example, one or more polyphenolic compounds, such as one or more bioflavonoids, can be obtained from raw Citrus fruits, raw Punica fruits, or raw Camellia plant parts (such as leaves )get.

One or more polyphenolic compounds, such as one or more bioflavonoids, may or may not be isolated and/or purified prior to incorporation into the compositions described herein Compounds such as one or more bioflavonoids. Thus, in certain embodiments, the compositions described herein may comprise raw Citrus fruit, dried Citrus fruit and/or Citrus fruit extract, or raw Pomegranate ( Punica fruit, dried Punica fruit and/or Punica fruit extract, or raw Camellia plant, dried Camellia plant and/or Camellia plant Extract.

In other embodiments, one or more polyphenolic compounds, such as one or more bioflavonoids, can each be independently chemically synthesized.

In certain embodiments, the compositions described herein comprise two polyphenolic compounds, eg, two bioflavonoids. The ratio of the first polyphenolic compound to the second polyphenolic compound, such as the first bioflavonoid to the second bioflavonoid may, for example, be in the range of about 0.5:5 to about 3:1. For example, the ratio of the first polyphenolic compound to the second polyphenolic compound, e.g., the first bioflavonoid to the second bioflavonoid, can be from about 0.5:5 to about 2.5:1, or from about 0.5:5 to about 2:1 , or about 0.5:5 to about 1.5:1, or about 0.5:5 to about 1:1 range. For example, the ratio of the first polyphenolic compound to the second polyphenolic compound, e.g., the first bioflavonoid to the second bioflavonoid can be from about 1:5 to about 3:1, or from about 1.5:5 to about 3:1 , or about 2:5 to about 3:1, or about 2.5:5 to about 3:1, or about 3:5 to about 3:1, or about 3.5:5 to about 3:1, or about 4:5 to about 3:1, or about 4.5:5 to about 3:1, or about 5:5 to about 3:1 range. The ratio is preferably 2:1.

In certain embodiments, the compositions described herein comprise naringin and neohesperidin. In certain embodiments, the at least one polyphenol comprises a major portion of naringin, neohesperidin, or a combination thereof, wherein the major portion means at least 50% by weight, or at least 60% by weight of the total weight of the polyphenolic compound, Or at least 70% by weight, or at least 80% by weight, or at least 90% by weight. The ratio of naringin to neohesperidin may, for example, range from about 0.5:5 to about 3:1. For example, the ratio of naringin to neohesperidin can be from about 0.5:5 to about 2.5:1, or from about 0.5:5 to about 2:1, or from about 0.5:5 to about 1.5:1, or about 0.5:5: 5 to 1:1 range. For example, the ratio of naringin to neohesperidin can be from about 1:5 to about 3:1, or from about 1.5:5 to about 3:1, or from about 2:5 to about 3:1, or from about About 2.5:5 to about 3:1, or about 3:5 to about 3:1, or about 3.5:5 to about 3:1, or about 4:5 to about 3:1, or about 4.5:5 to about 3:1, or about 5:5 to about 3:1 range. The ratio is preferably 2:1.

In certain embodiments, the ratio of total organosulfur compounds to total polyphenolic compounds (eg, the ratio of total organosulfur compounds to total bioflavonoids) may range from about 16:1 to about 1:30. For example, the ratio of total organosulfur compounds to total polyphenolic compounds (e.g., the ratio of total organosulfur compounds to total bioflavonoids) can be from about 15:1 to about 1:30, or from about 14:1 to about 1:30, Or about 13:1 to about 1:30, or about 12:1 to about 1:30, or about 10:1 to about 1:30, or about 16:1 to about 1:16 range. For example, the ratio of total organosulfur compounds to total polyphenolic compounds (e.g., the ratio of total organosulfur compounds to total bioflavonoids) can be from about 9:1 to about 1:25, or from about 8:1 to about 1:20, Or about 7:1 to about 1:15, or about 6:1 to about 1:10, or about 5:1 to about 1:8, or about 4:1 to about 1:7, or about 3:1 to A range of about 1:6, or about 2:1 to about 1:5, or about 1:1 to about 1:4. For example, the ratio of total organosulfur compounds to total polyphenolic compounds (e.g., the ratio of total organosulfur compounds to total bioflavonoids) can range from about 1:1 to about 1:3, or from about 2:1 to about 1:4. scope. For example, the ratio of total organosulfur compounds to total polyphenolic compounds (eg, the ratio of total organosulfur compounds to total bioflavonoids) can be about 1:3.

In certain embodiments, the ratio of organosulfur compounds to total polyphenolic compounds (eg, the ratio of total organosulfur compounds to total bioflavonoids) ranges from about 16:1 to about 1:30. For example, the ratio of organosulfur compounds to total polyphenolic compounds (e.g., the ratio of total organosulfur compounds to total bioflavonoids) can be from about 15:1 to about 1:30, or from about 14:1 to about 1:30, or A range of about 13:1 to about 1:30, or about 12:1 to about 1:30, or about 10:1 to about 1:30, or about 16:1 to about 1:16. For example, the ratio of organosulfur compounds to total polyphenolic compounds (e.g., the ratio of organosulfur compounds to total bioflavonoids) can be from about 9:1 to about 1:25, or from about 8:1 to about 1:20, or about 7:1 to about 1:15, or about 6:1 to about 1:10, or about 5:1 to about 1:8, or about 4:1 to about 1:7, or about 3:1 to about 1 :6, or from about 2:1 to about 1:5, or from about 1:1 to about 1:4. For example, the ratio of organosulfur compounds to total polyphenolic compounds (e.g., the ratio of total organosulfur compounds to total bioflavonoids) can be from about 1:4 to about 1:8, or from about 1:1 to about 1:3, or About 2:1 to about 1:4 range. For example, the ratio of organosulfur compounds to total polyphenolic compounds (eg, the ratio of total organosulfur compounds to total bioflavonoids) can be about 1:6 or can be about 1:3. In a preferred embodiment, the organosulfur compound is a disulfide compound. In a preferred embodiment, the organosulfur compound is allicin and/or the polyphenolic compound is a bioflavonoid including naringin and neohesperidin.

In some embodiments, the organosulfur compound and at least one polyphenolic compound may be provided as a mixture. The mixture may be one as described in WO 2018/220340 A1, which is incorporated herein by reference. In the examples disclosed herein, the ratio of organosulfur compounds to total polyphenolic compounds in the mixture is 1:3, and the ratio of garlic powder to citrus extract is 93:7, which is referred to in the examples disclosed herein as "NXRH214 ". In some examples disclosed herein, the ratio of organohalogen compound (such as bromoform) to powder mixture (such as NXRH214 powder) comprising organosulfur compound and polyphenol compound is 1:100 to 1:100000, more preferably 1:30000 to 1:100000, or 1:35000 to 1:83000.

-Other additives

The composition may, for example, further comprise other animal feed supplements including, for example, vitamins, minerals, antibiotics, growth stimulants, and combinations thereof. For example, the composition may comprise other biologically active animal feed supplements, such as animal feed supplements suitable for reducing methane production/emissions and/or increasing nutrient availability to animals. Vitamins can be vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, cyanocobalamin, carotenoids (including beta-carotene, zeaxanthin, lutein, and lycopene Any one or more of niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol and their salts and derivatives. The mineral may be any one or more of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, cobalt, boron, iodine, sodium, potassium, molybdenum, selenium, chromium, fluorine and chlorine. The animal feed composition may, for example, comprise from about 0.001% to about 5% by weight of each additional animal feed supplement, or from about 0.01% to about 5% by weight, or from about 0.1% to about 5% by weight of each Additional animal feed supplement.

In addition to organosulfur compounds, organohalogen compounds and optionally at least one polyphenolic compound, the composition may, for example, also comprise other components such as, for example, flavorings, colorants, stabilizers, antioxidants, buffers, emulsifying additives, dispersants, thickeners, solubilizers, micronutrients (e.g. selenium), vitamins, other feed substances (e.g. carbohydrates such as sugars and starches), soluble and insoluble fibers, cellulose, lignocellulose, grains , bran, grain middlings, chaff, fruit and vegetable seeds, skins, peels, etc.

-Animal food

Also disclosed herein are animal feeds comprising the compositions described herein. Animal feed can be solid (eg powder, granules, pellets), semi-solid (eg gel, ointment, cream, paste) or liquid (eg solution, suspension, emulsion). Animal feed may independently be solid, semi-solid (eg gel, ointment, cream, paste) or liquid (eg solution, suspension, emulsion). For example, animal feed can be all liquid, or all semi-solid, or all solid. Alternatively, the animal feed and composition may each be in a different physical state. For example, the animal feed can be solid or semi-solid, while the composition can be liquid. The composition may, for example, be used in a "top-dress" (added on top) ruminant feedlot ration, or may be used for blending into an overall mixed ration. For example, the composition can be added to an animal's drinking water. In certain embodiments, the composition can be added to the animal's brain immediately before ingestion, for example, up to 1 hour before ingestion, or up to 30 minutes before ingestion, or up to 15 minutes before ingestion, or up to 5 minutes before ingestion. drinking water. The three main types of animal feed include roughage, concentrate and mix. Generally, roughage contains a higher percentage of crude fiber and a lower percentage of digestible nutrients than concentrate feed. For example, a roughage may be defined as comprising equal to or greater than 20% by weight crude fiber and equal to or less than 60% by weight total digestible nutrients. Roughage can include, for example, dry roughage (such as hay, straw, artificially dehydrated forages containing at least 90% dry matter by weight), silage (formed from green forages such as grass, alfalfa, sorghum and corn, and mixed with 20% to 50% dry matter content kept in silos) and pastures (such as green growing pastures that provide forage with a high moisture content and typically less than 30% dry matter). The two basic types of roughage include grasses and legumes. Grasses are generally higher in fiber and dry matter than legumes. Beans are generally higher in protein, metabolizable energy, vitamins and minerals. Concentrated feed contains a relatively lower percentage of crude fiber and a higher percentage of digestible nutrients than roughage. For example, a concentrated feed can be defined as comprising less than 20% by weight crude fiber and greater than 60% by weight total digestible nutrients. Concentrates may include, for example, energy-rich grains and molasses. Corn, wheat, oats, barley and milo (sorghum grains) are energy-dense grains containing about 70 to 80% by weight of total digestible nutrients.

Mixed feed is usually a mixture of roughage and concentrate to provide a "complete" balanced ration and can be high or low in energy, protein or fiber. At least one organosulfur compound and at least one polyphenolic compound (such as at least one bioflavonoid) can be combined with the animal feed, for example, in various amounts, depending on the organohalogen compound(s) intended to be administered to the animal, ( Total amount of organosulfur compound(s) and optionally polyphenolic compound(s) such as bioflavonoid(s).

The animal feed may, for example, comprise from about 0.0001% to about 10% by weight of an organosulfur compound such as allicin, based on the total dry weight of the animal feed. The animal feed may, for example, comprise from about 0.3% to about 10% by weight of an organosulfur compound such as allicin, based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.001% to about 9.5%, or from about 0.005% to about 9%, or from about 0.01% to about 8.5%, or from about 0.05% to about 8% by weight, or from about 0.1% to about 7.5% by weight, or from about 0.9% to about 7% by weight, or from about 1% to about 6% by weight, or from about 1.5% to about 5.5% by weight, Or about 2% to about 5% by weight, or about 2.5% to about 4.5% by weight, or about 3% to about 4% by weight of organosulfur compounds (such as allicin). For example, the animal feed may comprise about 0.4% to about 9.5% by weight, or about 0.5% to about 9% by weight, or about 0.6% to about 8.5% by weight, or about 0.7% by weight based on the total dry weight of the animal feed to about 8 wt%, or about 0.8 wt% to about 7.5 wt%, or about 0.9 wt% to about 7 wt%, or about 1 wt% to about 6 wt%, or about 1.5 wt% to about 5.5 wt%, Or about 2% to about 5% by weight, or about 2.5% to about 4.5% by weight, or about 3% to about 4% by weight of organosulfur compounds (such as allicin). The concentration of total organosulfur compounds (eg, allicin) present in the animal feed supplements or animal feed compositions described herein generally exceeds the concentration of each organohalogen compound. As indicated above, the ratio of organohalogen compound to organosulfur compound in the animal feed may be from about 1:10 to 1:3500, or from 1:100 to 1:3500, or more preferably from 1:1000 to 1:2500. Thus, in some embodiments, the animal feed may comprise from about 0.00015% to about 0.01% by weight of an organosulfur compound (eg, allicin) based on the total dry weight of the animal feed.

If present, the animal feed can, for example, comprise from about 0.0001% to about 10% by weight total polyphenolic compounds (eg, total bioflavonoids) based on the total dry weight of the animal feed. The animal feed may, for example, comprise from about 0.1% to about 10% by weight total polyphenolic compounds (eg total bioflavonoids) based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.001% to about 10% by weight, or from about 0.005% to about 10% by weight, or from about 0.01% to about 9.5% by weight, or about 0.05% by weight based on the total dry weight of to about 9 wt%, or about 0.1 wt% to about 8.5 wt%, or about 0.7 wt% to about 8 wt%, or about 0.8 wt% to about 7.5 wt%, or about 0.9 wt% to about 7 wt%, Or about 1% by weight to about 6% by weight, or about 1.5% by weight to about 5.5% by weight, or about 2% by weight to about 5% by weight, or about 2.5% by weight to about 4.5% by weight, or about 3% by weight to About 4% by weight total polyphenolic compounds (eg total bioflavonoids). For example, the animal feed may comprise from about 0.2% to about 10% by weight, or from about 0.3% to about 10% by weight, or from about 0.4% to about 9.5% by weight, or about 0.5% by weight based on the total dry weight of the animal feed to about 9 wt%, or about 0.6 wt% to about 8.5 wt%, or about 0.7 wt% to about 8 wt%, or about 0.8 wt% to about 7.5 wt%, or about 0.9 wt% to about 7 wt%, Or about 1% by weight to about 6% by weight, or about 1.5% by weight to about 5.5% by weight, or about 2% by weight to about 5% by weight, or about 2.5% by weight to about 4.5% by weight, or about 3% by weight to About 4% by weight total polyphenolic compounds (eg total bioflavonoids). The concentration of total organosulfur compounds (eg, allicin) present in the animal feed supplements or animal feed compositions described herein may exceed the concentration of total polyphenolic compound(s).

- Methods of the present disclosure

Disclosed herein are methods of reducing methane, eg, reducing methane production by an animal, comprising administering to an animal, more particularly a ruminant, a composition or animal feed described herein.

The compositions and methods described herein can, for example, reduce methane production and/or emissions by at least about 10% (compared to methane production and/or emissions if the animal feed supplement was not consumed). For example, the animal feed supplement can reduce methane production and/or emissions by at least about 10%, or at least about 15%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, Or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%. The animal feed supplements described herein can, for example, reduce methane production and/or emissions by up to 100%. For example, the animal feed supplement can reduce methane production and/or emissions by up to about 99%, or up to about 98%, or up to about 97%, or up to about 96%, or up to about 95%, or up to about 90%, Or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%. This can be measured eg by a Hohenheim gas test or by using a manometer.

Also disclosed herein is a method of inhibiting one or more methanogens comprising administering to an animal, more particularly a ruminant, a composition or animal feed described herein. In some embodiments, the compositions and combinations disclosed herein are useful for reducing one or more methanogens selected from the group consisting of: Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacterium rumen (Methanobrevibacterruminantium), Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, Pasteur's methane Methanosarcinabarkeri, Methanobrevibacter boviskoreani, Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methanobacillus Methanosarcinamazei, Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter thaueri Methanobrevibacter woesei, Methanobrevibacter wolinii.

Also disclosed herein is a method of improving the metabolic efficiency of an animal comprising administering to the animal a composition or animal feed of the invention. Improvements in metabolic efficiency can result in increased production of animal products, such as one or more of meat, fat, wool (ie, fiber), and milk. Thus, the compositions or methods of the invention may improve the production of meat and/or fat and/or wool and/or milk of an animal.

The compositions, animal feeds and methods described herein can, for example, increase milk and/or meat and/or wool production by at least about 20% (compared to milk and/or meat and/or fat and / or wool production). For example, the composition or animal feed can increase milk and/or meat and/or fat and/or gross production by at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%. A composition or animal feed as described herein may eg increase milk and/or meat and/or fat and/or wool production by up to 100%. For example, the composition or animal feed can increase milk and/or meat and/or fat and/or gross production by up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%. This can eg be measured by the volume of milk produced per day or by the weight of the animal or by the weight of wool and/or fat and/or meat produced.

For example, the compositions and animal feeds described herein can increase the efficiency of milk and/or meat and/or wool production by at least about 20% (compared to the milk and/or meat and/or fat produced if the compositions or animal feed were not consumed). and/or gross production efficiency). For example, the composition or animal feed described herein can increase the efficiency of milk and/or meat and/or fat and/or gross production by at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%. A composition or animal feed as described herein may eg increase the efficiency of milk and/or meat and/or fat and/or wool production by up to 100%. For example, the composition or animal feed described herein can increase the efficiency of milk and/or meat and/or fat and/or gross production by up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%. Efficiency is related to the degree to which a particular biological process (eg milk, meat, fat, wool production) occurs per unit of nutrient consumed. For example, this can be measured by the volume of milk produced or the change in animal weight or weight of gross or fat divided by the total nutrients consumed by the animal per day. For example, a composition or animal feed described herein can increase nutrient availability by at least about 20% (compared to milk and/or meat and/or fat and/or gross production if the composition or animal feed is not consumed) . For example, a composition or animal feed described herein can increase nutrient availability by at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%. A composition or animal feed described herein may, for example, increase nutrient availability by up to 100%. For example, the compositions or animal feeds described herein can increase nutrient availability by up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70%. Nutrient availability refers to the amount of a nutrient available to an animal for biological/metabolic function.

In some embodiments, the ruminant is a cow, goat, sheep, yak, deer, or antelope. In some embodiments, the ruminant is a cow, goat or sheep.

Compositions or animal feeds can be administered orally to animals. In some embodiments, the composition or animal feed can be administered to the animal on a daily basis.

Improved metabolic efficiency in ruminants

The present disclosure provides feed supplement formulations incorporating biologically or synthetically derived organohalogen, organosulfur and polyphenolic compounds suitable for oral administration to ruminants to improve their metabolic efficiency for reducing emitted methane and reducing excreted nitrogen , and for increasing valuable animal products such as meat, fat, fiber and milk.

The present invention is based on the unexpected discovery that when certain organohalogen compounds, organosulfur compounds and polyphenolic compounds are administered to ruminants to reduce methane emissions in said ruminants, these organohalogen compounds, organosulfur compounds and polyphenolic compounds Phenolic compounds also improve the metabolic efficiency of the ruminant and also reduce urinary nitrogen excretion and increase the production of valuable animal products. Furthermore, when the organohalogen compounds, organosulfur compounds, and polyphenol compounds were given in certain combinations, there was a surprising enhancement in the reduction of both methane emitted and nitrogen excreted, and in valuable animal product There is a surprising enhancement in the increase in production of .

The present inventors have recognized the unexpected and surprising effect of feed supplements comprising organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants on metabolic efficiency improvement.

The combination of organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants of the present invention, when administered to a ruminant, reduces methanogenesis and reduces ruminant methane production. The reduction in methanogenesis occurs through different modes including: reduction of methanogenic organisms by limiting their growth or killing them; reduction of methanogenesis by limiting or terminating the enzymes involved in methanogenesis.

Methanogens identified in cattle, sheep, and goats include Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobiummobile, Methanoculleus olentangyi, Methanosarcina barkeri, Methanobrevibacter boviskoreani , Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methanosarcina mazei, Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter woesei and Methanobrevibacter wolinii coexist. Accordingly, in some embodiments, the compositions and combinations disclosed herein are useful for reducing one or more methanogens selected from the group consisting of: Methanobacterium formicicum, Methanobacterium bryantii, Rumen M. Methanobrevibacter ruminantium, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleusolentangyi, Pasteur Methanosarcina barkeri, Methanobrevibacter boviskoreani, Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methane Methanosarcina mazei, Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaerastadtmanae, Worthian methane Methanobrevibacter woesei and Methanobrevibacter wolinii.

The inventive combination of organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants has unexpected properties in reducing methane emissions and excreted urinary nitrogen and in increasing valuable animal products and surprising enhancement, which may be due to a synergistic effect between different modes of inhibition of methanogenesis including, for example:

Organohalogen compounds from the marine macroalgae Asparagopsis species include organobromine, especially bromoform (CHBr ₃ , tribromomethane), which reacts with the reduced vitamin The B12 cofactor reacts to inhibit the efficiency of methyltransferases and also competitively inhibits methane production by acting as a terminal electron acceptor.

Organosulfur compounds from plants of Allium species, including allicin and diallyl disulfide, are resistant to oxidative stress due to oxidative interactions with important thiol-containing enzymes and through inhibition of the enzyme HMG-CoA reductase. Methanogen activity.

Polyphenolic compounds from plants of the Citrus species include the flavonoids neohesperidin and naringin, which have anti-methanogenic activity.

The present inventors have realized that the unexpected and surprising improvement in metabolic efficiency due to the supplements of the present invention may also be due to additional health benefits including, for example: an anthelmintic effect leading to a reduction in gastrointestinal parasites; leading to an antimicrobial effect in reduction of bacterial infections including e.g. mastitis; and, when administered to ruminants, present in the combination of organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants of the present invention Supplement of trace minerals and vitamins.

Combinations of organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants of the present invention may be administered as feed supplements in certain combinations and in certain ratios.

The combination of organohalogen-rich marine macroalgae and organosulfur-rich plants and polyphenol-rich plants of the present invention may be administered as a separate feed supplement or combined into a mixed composition feed supplement.

use

The compositions or animal feed supplements described herein (including all embodiments and combinations of embodiments) can be used to reduce methane production and/or emissions by animals, reduce nitrogen excretion by animals, increase nutrient availability by animals, and/or Or increase valuable nitrogen-rich and carbon-rich animal products from animals.

In certain embodiments, the animal is a ruminant. Ruminants include animals selected from members of the suborders Ruminantia and Tylopoda, and include domesticated ruminants: eg, cattle (eg, cows), goats, sheep, buffaloes, yaks, deer, or antelope.

In particular, the compositions or feed supplements of the present invention, when administered to ruminants in an effective amount, result in a reduction in ruminant methane production that would otherwise be emitted to the atmosphere primarily through exhaled breath through the mouth and nostrils and represent energy losses accounting for from 2% to 12% of the total energy intake of the feed.

Methane is a greenhouse gas with 28 times the global warming potential of carbon dioxide. Intestinal methane is a byproduct of ruminant digestion and is produced by a complex microbial community that includes ciliate protozoa, bacteria, archaea, and anaerobic fungi through a process known as methanogenesis. Cattle produce about 7 and 9 times as much methane as sheep and goats, respectively. Enteric methane is mainly produced in the rumen (87%-90%) and to a lesser extent (13%-10%) in the large intestine.

The compositions and feed supplements of the invention cause the diversion of metabolic energy from methane production and direct it to anabolic growth processes. Thus, feed supplementation resulted in an increase in ruminant live weight, which was determined by: direct weighing of animal mass; computed tomography (CT) scans to measure empty body mass (gross mass minus intestinal contents); carcass mass and composition Analysis (lean, fat, and major fat distribution; and changes in organs including liver).

Examples of valuable nitrogen- and carbon-rich animal products are tissue-based commodities and include, for example: meat; offal; and hides.

Another example of valuable nitrogen- and carbon-rich animal products are excrement-based commodities and products of those commodities, including, for example: milk; whole milk; milk powder; cream; ice cream; cheese; and yogurt.

Another example of a valuable nitrogen- and carbon-rich animal product is a fiber-based commodity, and includes, for example: wool; horn; and antler.

The compositions and feed supplements of the present invention lead to an unexpected and surprising improvement in metabolic efficiency, which may lead to a reduction in the excretion of urinary nitrogen, which is deposited in urine plaques on pastures after urination. When excess nitrogen excreted in urine patches is greater than that required for optimal pasture plant efficiency, the excess nitrogen is leached via nitrate (NO ₃ ⁻ ) as well as ammonia (NH ₃ ), nitrous oxide (N ₂ O) and Nitrogen (N ₂ ) is lost by volatilization. Nitrous oxide is particularly harmful to the atmosphere as a greenhouse gas with a global warming potential 298 times that of carbon dioxide. Nitrogen loss to groundwater can lead to uncontrolled growth of aquatic biota, which can damage ecosystems, lead to toxic algal blooms and eutrophication of water bodies.

Manufacturing method

The compositions or animal feed supplements described herein can be prepared by combining one or more organohalogen compounds with one or more organosulfur compounds and one or more polyphenol compounds.

Organohalogen compounds can be synthesized or extracted from suitable biological sources and used in raw or processed form. CH3Br _{; CH3I} ; _{CH2Cl2 ; CH2Br2} ; CH2I2 _{; CHCl3 ; CHBr3 ; CHI3} ; _{CCl4 ; CBr4} ; CH _{CH2ClBr ; CH2ClI ; CH2BrI ; CHBr2Cl ; CHBrI2 ; CHBrClI ; CHBr2I ; _ CH2} ) _3I ; _CH3 ( _{CH2 ) 4Br} ; _CH3 ( _CH2 ) _4I ; (CH3) _2CHI ; CH3CH2CH( _CH3 )I _{; ( CH3} ) _2CHCH2I ; _{BrCH2CH2Br ; ClCH = CCl2} ; and _{CH3CH2CH2CH2I .}

The biological sources of organohalogen compounds are marine macroalgae rich in organohalogens. For example, the organohalogen-rich marine macroalgae includes at least one marine macroalgae selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; species of the genus Oedagonium; species of the genus Ulva; and Cladophora patentiramea.

Organosulfur compounds can be synthesized or extracted from suitable biological sources and used in raw or processed form. Organosulfur compounds include: organosulfur secondary metabolites; allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); diallyl disulfide (C ₆ H ₁₀ S ₂ ); and allyl mercaptan (C ₃ H ₆ S).

Biological sources of organosulfur compounds are organosulfur-rich plants. For example, organic sulfur-rich plants include: Allium species; A. sativum (garlic); A. ampeloprasum (leek); A. cepa (onions and shallots). The one or more organosulfur compounds can be obtained from one or more parts of a plant including, for example: leaves; stems; bark; roots; bulbs; flowers; fruits; and seeds.

Polyphenolic compounds can be synthesized or extracted from suitable biological sources and used in raw or processed form. For example, polyphenolic compounds include bioflavonoids and phenolic compounds. The term phenolic compound refers to a class of chemical compounds that contain a hydroxyl group (-OH) bonded directly to an aromatic hydrocarbon group. The phenolic compounds described herein may include bioflavonoids, non-bioflavonoid phenolic compounds, or combinations thereof. The at least one polyphenolic compound may for example comprise at least one bioflavonoid. The term bioflavonoids refers to a class of plant and fungal secondary metabolites and has a general structure with a 15-carbon skeleton consisting of two benzene rings (A and B) and a heterocycle (C), sometimes abbreviated as _C6- C ₃ -C ₆ . The term bioflavonoids includes anthocyanins (including flavones and flavonols), flavanones, flavanols, flavans and anthocyanins. The term bioflavonoids also includes compounds with a flavonoid skeleton (2-phenyl-1,4-benzopyrone), an isoflavane skeleton (3-phenylchromen-4-one) or a neoflavan skeleton (4-benzene base coumarin) compounds. Thus, the term polyphenolic compounds includes, but is not limited to: anthocyanins; flavanones (including flavanone glycosides); flavonols; dihydroflavonols; flavans; isoflavones; anthocyanins; proanthocyanidins; phenolic acids; hydroxyl Cinnamic acid; coumarin; stilbenes; anthraquinone; lignan; lignin; tannin; polyphenol protein; catechin; rutin; locustin; genistein; kaempferol; gallocatechin; catechin Theophyl gallate; epicatechin; epigallocatechin; epicatechin gallate; quercetin; allocatechin; gallocatechin gallate; epicatechin; epigallocatechin Theophylline; epicatechin gallate; epigallocatechin gallate; kaempferol; quercetin; naringin; neohesperidin; eriocitrin; isonaringin; naringenin; Hesperidin; Rhodorin; Dianotin; Melitin; Hesperetin; Citrusin; Epigallocatechin; Gallocatechin; Epigallocatechin; Coumaric Acid; Cinnamic Acid; Gallic Acid ; ellagic acid; protocatechuic acid; chlorogenic acid; caffeic acid; ferulic acid; punicalagin; pomegranate tannin.

Biological sources of polyphenolic compounds are polyphenol-rich plants. For example, polyphenol-rich plants include: Allium species; Brassica species; Camelia species; Capsicum species; Citrus species; ) species; Malus species; Musa species; Phaseolus species; Prunus species; Punica species; Pyrus species; Solanum species Species; Vaccinium species. One or more polyphenolic compounds can be obtained from one or more parts of a plant including, for example: leaves; stems; bark; roots; bulbs; flowers; fruits; and seeds.

The compositions or animal feed supplements described herein may be obtained by combining one or more organohalogen-rich marine macroalgae with one or more organosulfur-rich plant compounds and one or more polyphenol-rich A combination of plants to prepare.

The components are combined in suitable amounts to obtain a composition having the desired amount of each component. Each component may be combined with one or more other components in any order and combination suitable to obtain the desired product. For example, each component can be combined by mixing or blending. For example, one or more organohalogen compounds and one or more organosulfur compounds and one or more polyphenolic A halogen compound and one or more organosulfur compounds and one or more polyphenol compounds are combined with an animal feed.

Compositions may be prepared in dry solid form, eg powder form, and subjected to further processing steps depending on the type of formulation in which the end product is desired. The method may further comprise a forming step, wherein the mixture is molded, pressed, spray-dried or otherwise formed into a shape (e.g. bars, pellets, pellets, clusters, tablets), preferably of the type described herein. The size and/or texture of the animal's food. The method may include containing the animal feed or animal feed supplement in a specific delivery device such as a syringe. The method may comprise bolusing the animal feed supplement or animal feed, which is intended to remain in the animal's stomach, such as the rumen of a ruminant.

Administration of methanogenesis inhibitors

Also disclosed herein is a method of gradually administering a methanogenesis inhibitor feed supplement incorporating biologically derived organohalogen compounds and/or organosulfur compounds and/or polyphenol compounds, the method being suitable for It is given orally to ruminants to improve their metabolic efficiency, to reduce emitted methane and reduce excreted nitrogen, and to increase valuable animal products such as meat, fat, fiber and milk.

The term "gradually" as used herein refers to the administration of at least one dose of an effective amount of at least one methanogenesis inhibitor, and optionally at least one consecutive dose of an effective amount of at least one methanogenesis inhibitor after some effective time interval Inhibitors.

The present disclosure is based on the unexpected discovery that the feed supplement, when gradually administered to the animal in an effective dosage amount and at an effective time interval, improves metabolic efficiency, reduces methane emissions, and reduces excreted methane. Nitrogen, as well as increasing valuable animal products such as meat, fat, fiber and milk, provides surprising economic benefits.

Inhibition of methanogenesis occurs through different modes of action including, for example: reducing the methanogenic process; by limiting or terminating the enzymes involved in methanogenesis; or reducing methanogenic organisms by limiting their growth or killing them .

Methanogenic inhibitors include for example: _{organic halogen} compounds; _{CH3Cl ;} CH3Br _{; CH3I} ; _CH2Cl2 ; _CH2Br2 ; _CH2I2 ; _CHCl3 ; _CHBr3 ; _CHI3 ; _CCl4 ; _{CH2ClBr ; CH2ClI ; CH2BrI ; CHBr2Cl ; CHBrI2 ; CHBrClI ; _ _ _ CH3} ( _CH2 ) _3I ; _CH3 ( _CH2 ) _4Br ; _{CH3 ( CH2} ) _4I ; (CH3) _2CHI ; _CH3CH2CH ( _CH3 )I; ( _CH3 ) ₂ CHCH ₂ I; BrCH ₂ CH ₂ Br; ClCH=CCl ₂ ; CH ₃ CH ₂ CH ₂ CH ₂ I; Organosulfur compounds; Organosulfur secondary metabolites; Allicin (C ₆ H ₁₀ S ₂ O); Diene Propyl sulfide (C ₆ H ₁₀ S); Diallyl disulfide (C ₆ H ₁₀ S ₂ ); Allyl mercaptan (C ₃ H ₆ S); Polyphenolic compounds; Flavonoids; Biological Flavonoids; Abiotic flavonoids; Anthoxanthins; Flavones; Flavonols; Flavanones; Dihydroflavonols; Flavans; Anthocyanins; Isoflavanes; ; hydroxycinnamic acid; coumarin; stilbenes; anthraquinone; lignan; lignin; tannin; polyphenol protein; catechin; rutin; locustin; genistein; kaempferol; gallocatechin ; Catechin gallate; Epigallocatechin; Epigallocatechin; Epigallocatechin gallate; Quercetin; Gallocatechin; epicatechin gallate; epigallocatechin gallate; kaempferol; quercetin; naringin; neohesperidin; eriocitrin; isonaringin; naringin Hesperidin; Rhodorin; Dianotin; Melissain; Hesperetin; Citrusin; Epigallocatechin; Gallocatechin; Epigallocatechin; Coumaric Acid; Cinnamic Acid; Gallic acid; ellagic acid; protocatechuic acid; chlorogenic acid; caffeic acid; ferulic acid; punicalagin; and garnetin.

The biological sources of organohalogen compounds are marine macroalgae rich in organohalogens. For example, the organohalogen-rich marine macroalgae includes at least one marine macroalgae selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; species of the genus Oedagonium; species of the genus Ulva; and Cladophora patentiramea. The organohalogen compounds, organobromine compounds and bromoforms disclosed herein may also be synthetic.

Biological sources of organosulfur compounds are organosulfur-rich plants. For example, organic sulfur-rich plants include: Allium species; A. sativum (garlic); A. ampeloprasum (leek); A. cepa (onions and shallots). The one or more organosulfur compounds can be obtained from one or more parts of a plant including, for example: leaves; stems; bark; roots; bulbs; flowers; fruits; and seeds. The organosulfur compounds disclosed herein may also be synthetic.

Biological sources of polyphenolic compounds are polyphenol-rich plants. For example, polyphenol-rich plants include: Allium species; Brassica species; Camelia species; Capsicum species; Citrus species; ) species; Malus species; Musa species; Phaseolus species; Prunus species; Punica species; Pyrus species; Solanum species Species; Vaccinium species. One or more polyphenolic compounds can be obtained from one or more parts of a plant including, for example: leaves; stems; bark; roots; bulbs; flowers; fruits; and seeds. The polyphenolic compounds disclosed herein may also be synthetic.

The inventors optimized stepwise administration by optimizing both the amount of effective dose and the effective time interval between successive doses, recognizing that:

1. To maximize the increase of valuable animal products in said ruminants, contributing to economic benefits; and

2. Prevents overfeeding or other overfeeding, which reduces the cost of feed supplements and contributes to economics; and

3. Prevention of overfeeding or other overfeeding, which prevents counter-productivity caused by the feed supplement in the ruminant, contributing to economic efficiency.

The present inventors have recognized the unexpected and surprising economic benefit of carefully controlled gradual administration of a methanogenesis inhibitor feed supplement comprising organohalogen-rich marine macroalgae and and/or certain combinations of organosulfur-rich plants and/or polyphenol-rich plants.

A combination of organohalogen-rich marine macroalgae and/or organosulfur-rich plants and/or polyphenol-rich plants, when administered to a ruminant, reduces methanogenesis and reduces ruminant methane production. The reduction in methanogenesis occurs through different modes including: reduction of methanogenic organisms by limiting their growth or killing them; reduction of methanogenesis by limiting or terminating the enzymes involved in methanogenesis.

Gradual administration of certain combinations of organohalogen-enriched marine macroalgae and organosulfur-enriched plants and polyphenol-enriched plants to ruminants was effective in reducing methane emissions and excreted urinary nitrogen as well as in increasing valuable The unexpected and surprising enhancement in animal products may be due to a synergy between different modes of inhibition of methanogenesis including, for example:

1. Organohalogen compounds from the marine macroalgae Asparagopsis species include organobromine, especially bromoform (CHBr ₃ , tribromomethane), which undergo reduction with the second to final steps required for methanogenesis The vitamin B12 cofactor reacts to inhibit the efficiency of methyltransferases and also competitively inhibits methane production by acting as a terminal electron acceptor.

2. Organosulfur compounds from plants of the Allium species include allicin and diallyl disulfide, which are released due to oxidative interactions with important thiol-containing enzymes and through inhibition of the enzyme HMG-CoA reductase Possesses activity against methanogens.

3. Polyphenolic compounds from plants of the genus Citrus, including the flavonoids neohesperidin and naringin, which have anti-methanogenic activity.

The present inventors have realized that the unexpected and surprising improvement in metabolic efficiency due to gradual administration of the methanogenesis inhibitor feed supplement or composition of the present invention may also be due to additional health benefits including, for example: causing Anthelmintic effect with reduced gastrointestinal parasites; antibacterial effect resulting in reduced bacterial infection including e.g. mastitis; and when administered to ruminants, providing the organohalogen-rich marine macroalgae and organosulfur-rich plants present in the present invention As well as supplementary trace minerals and vitamins in a combination of polyphenol-rich plants.

The gradual administration of organohalogen-rich marine macroalgae and/or organosulfur-rich plants and/or polyphenol-rich plants of the present invention may be administered as a feed supplement in certain combinations and in certain ratios.

The stepwise administration of the combination of organohalogen-rich marine macroalgae and/or organosulfur-rich plants and polyphenol-rich plants of the present invention may be administered as a separate feed supplement or combined into a mixed composition feed supplement (eg as a composition described herein).

use

Gradual administration of the methanogenesis inhibitor animal feed supplements described herein (including all embodiments and combinations of embodiments) can be used to optimize feed supplement administration to reduce methane production and/or emissions by animals, reduce nitrogen excretion by animals, Increase the availability of nutrients to animals and/or increase the animal's valuable nitrogen- and carbon-rich animal products.

In certain embodiments, the animal is a ruminant. Ruminants include animals selected from members of the suborders Ruminantia and Tylopoda, and include domesticated ruminants: eg, cattle (eg, cows), goats, sheep, buffaloes, yaks, deer, or antelope.

In particular, when administered to a ruminant, stepwise administration of a methanogenesis inhibitor feed supplement or composition of the invention results in a reduction in the ruminant's production of methane that would otherwise be emitted to the atmosphere primarily through the mouth and nostrils of exhaled air, and Represents energy loss, ranging from 2% to 12% of total energy intake from feed.

Methane is a greenhouse gas with 28 times the global warming potential of carbon dioxide. Intestinal methane is a byproduct of ruminant digestion and is produced by a complex microbial community that includes ciliate protozoa, bacteria, archaea, and anaerobic fungi through a process known as methanogenesis. Cattle produce about 7 and 9 times as much methane as sheep and goats, respectively. Enteric methane is mainly produced in the rumen (87%-90%) and to a lesser extent (13%-10%) in the large intestine.

Gradual administration of the methanogenesis inhibitor feed supplement of the present invention results in a diversion of metabolic energy from methane production and directs it towards anabolic growth processes. Thus, the composition or feed supplement results in an increase in ruminant live weight, which is determined by: direct weighing of animal mass; computed tomography (CT) scans to measure empty body mass (gross mass minus intestinal contents); carcass Mass and composition analysis (lean, fat, and major fat distribution; and changes in organs including liver).

Examples of valuable nitrogen- and carbon-rich animal products are tissue-based commodities and include, for example: meat; offal; and hides.

Another example of valuable nitrogen- and carbon-rich animal products are excrement-based commodities and products of those commodities, including, for example: milk; whole milk; milk powder; cream; ice cream; cheese; and yogurt.

Another example of a valuable nitrogen- and carbon-rich animal product is a fiber-based commodity, and includes, for example: wool; horn; and antlers.

Gradual administration of the methanogenesis inhibitor feed supplement of the present invention results in an unexpected and surprising improvement in metabolic efficiency, which may lead to a reduction in the excretion of urinary nitrogen, which is deposited in urine plaques on pastures after urination middle. When excess nitrogen excreted in urine patches is greater than that required for optimal pasture plant efficiency, the excess nitrogen is leached via nitrate (NO ₃ ⁻ ) as well as ammonia (NH ₃ ), nitrous oxide (N ₂ O) and Nitrogen (N ₂ ) is lost by volatilization. Nitrous oxide is particularly harmful to the atmosphere as a greenhouse gas with a global warming potential 298 times that of carbon dioxide. Nitrogen loss to groundwater can lead to uncontrolled growth of aquatic biota, which can damage ecosystems, lead to toxic algal blooms and eutrophication of water bodies.

step-by-step approach

Gradual administration of the compositions or methanogenesis inhibitor feed supplements described herein may be by administering at least one dose of an effective amount of at least one methanogenesis inhibitor and optionally at least one consecutive dose after some effective time interval. an effective amount of at least one methanogenic inhibitor.

It will be understood that successive doses of the methanogenesis inhibitor feed supplement at certain time intervals constitute gradual administration. Furthermore, the amount of successive doses, the time interval between doses, and the methanogenesis inhibitor can be the same from dose to dose, from time interval to time interval, and from methanogenesis inhibitor to methanogenesis inhibitor , or they may be different dosage amounts and/or different time intervals, and/or different methanogenic inhibitors.

The animal feed supplements or compositions described herein can be obtained by combining one or more organohalogen-rich marine macroalgae with one or more organosulfur-rich plant compounds and one or more polyphenol-rich A combination of plants to prepare.

The components are combined in suitable amounts to obtain a composition having the desired amount of each component. Each component may be combined with one or more other components in any order and combination suitable to obtain the desired product. For example, each component can be combined by mixing or blending. For example, one or more organohalogen compounds and one or more organosulfur compounds and one or more polyphenolic A halogen compound and one or more organosulfur compounds and one or more polyphenol compounds are combined with an animal feed.

Methanogenic inhibitor feed supplements may be prepared to facilitate gradual administration in forms including dry solid form, eg powder form, and subject to further processing steps depending on the type of formulation the final product is intended for. The method may further comprise a forming step, wherein the mixture is molded, pressed, spray-dried or otherwise formed into a shape (e.g. bars, pellets, pellets, clusters, tablets), preferably of the type described herein. The size and/or texture of the animal's food. The method may comprise containing the animal feed or the animal methanogenesis inhibitor feed supplement in a specific delivery device such as a syringe. The method may comprise forming a bolus of the composition or animal feed, which is expected to remain in the animal's stomach, such as the rumen of a ruminant.

The methanogenesis inhibitor feed supplement can be administered stepwise, for example, in dosage amounts based on the weight percentage of ruminants selected from the group consisting of: 0.1%; 0.2%; 0.3%; 0.4%; 0.5% ;0.6%;0.7%;0.8%;0.9%;1.0%;1.1%;1.2%;1.3%;1.4%;1.5%;2.0%;2.5%;3.0%;3.5%;4.0%;4.5%;5.0 %; and 10%.

The methanogenesis inhibitor feed supplement may be dosed stepwise, for example, in dosage amounts based on the percentage of feed weight consumed by the ruminant selected from the group consisting of: 0.1%; 0.2%; 0.3% ;0.4%;0.5%;0.6%;0.7%;0.8%;0.9%;1.0%;1.1%;1.2%;1.3%;1.4%;1.5%;2.0%;2.5%;3.0%;3.5%;4.0 %; 4.5%; 5.0%; and 10%.

The stepwise administration of the methanogenesis inhibitor feed supplement may have, for example, at least one time interval between consecutive doses selected from the group consisting of: 1 minute; 1 hour; 1 day; 2 days; 3 days; 4 days; 5 days ; 6 days; 7 days; 10 days; 2 weeks; 3 weeks; 4 weeks; 6 weeks; 2 months; 3 months; 4 months; 6 months; 9 months; and 12 months.

All uses and methods described herein are considered purely non-therapeutic.

The invention will now be described by reference only to the following non-limiting examples.

Example 1 - Inhibition of methane production by the methanogenic archaea Methanococcus maripaludis

i) Bromoform and allicin

The purpose of this experiment was to determine whether bromoform and allicin act synergistically in their ability to inhibit methane production by the methanogenic archaea Methanococcus maripaludis.

For this experiment, 100 mM bromoform was prepared by adding 8.75 μl bromoform to 991 μl DMSO. It was diluted 10-fold to yield a 10 mM solution and further diluted to yield 0.12 mM and 0.156 mM stock solutions. Allicin stock solutions were prepared by adding 48.6 μl allicin to 951.4 μl DMSO to make a 300 mM stock solution, and 32.5 μl allicin to 967.5 μl DMSO to make a 200 mM stock solution. By adding 5 ml of M141 medium (https://www.dsmz.de/microorganisms/medium/pdf/D SMZ_Medium141.pdf) to a screw cap Hungate tube followed by 5 μl of allicin and/or 5 μl of bromoform or 10 μl of DMSO for experiments:

Tube 1: DMSO (10 μl)

Tube 2: Bromoform (5 μl 0.120 mM) (final 120 nM) + 5 μl DMSO

Tube 3: Bromoform (5 μl 0.156 mM) (final 156 nM) + 5 μl DMSO

Tube 4: Allicin (5 μl 200 mM) (final 200 μM) + 5 μl DMSO

Tube 5: Allicin (5 μl 300 mM) (final 300 μM) + 5 μl DMSO

Tube 6: Bromoform (5 μl 0.120 mM) (final 120 nM) + allicin (5 μl 200 mM) (final 200 μM)

Tube 7: Bromoform (5 μl 0.120 mM) (final 120 nM) + allicin (5 μl 300 mM) (final 300 μM)

Tube 8: Bromoform (5 μl 0.156 mM) (final 156 nM) + allicin (5 μl 200 mM) (final 200 μM)

Tube 9: Bromoform (5 μl 0.156 mM) (final 156 nM) + allicin (5 μl 300 mM) (final 300 μM)

After the addition of the test substances, 500 [mu]l of an overnight M. maripaludis culture was added to each reaction tube. Each tube was gassed to 240 kPa with 80% _H2 /20% _CO2 and incubated at 37 °C for 24 h.

After the 24 h incubation, measure the pressure inside the tube using a manometer. Assuming the consumption of 5 moles of _H2 / _CO2 to produce 1 mole of _CH4 , the observed pressure drop was used to calculate the amount of methane for the control and test reactions, from which percent inhibition was calculated.

ii) Bromoform and powder containing organic sulfur and polyphenols (NXRH214 powder)

The purpose of this experiment was to determine whether bromoform and powders comprising organosulfur and polyphenols were synergistic in their ability to inhibit methane production by the methanogenic archaea Methanococcus maripaludis.

For this experiment, 100 mM bromoform was prepared by adding 8.75 μl bromoform to 991 μl DMSO. It was diluted 10-fold to yield a 10 mM solution and further diluted to yield 0.10 mM and 0.156 mM stock solutions. Samples were prepared by adding 245mg NXRH214 powder to 35ml M141 medium (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium141.pdf) to create a 7mg/ml stock solution. N XRH214 powder is garlic powder (allicin) and citrus extract (polyphenolic flavonoid mixture) in a ratio of 93:7, where the flavonoid mixture mainly includes naringin and neohesperidin.

The reaction tube was set up as follows:

Tube 1: 0ml NXRH214 (No NXRH214) + 5ml M141 + 5μl DMSO

Tube 2: 1ml NXRH214 (1.4μg/ml NXRH214) + 5ml M141 + 5μl DMSO Tube 3: 1.5ml NXRH214 (2.8μg/ml NXRH214) + 3.5ml M141 + 5μl DMSO Tube 4: 0ml NXRH214 (without NXRH214) + 5ml M141 +5μl 0.1mM bromoform (100nM bromoform)

Tube 5: 1ml NXRH214 (1.4μg/ml NXRH214)+5ml M141+5μl 0.1mM bromoform (100nM bromoform)

Tube 6: 1.5ml NXRH214 (2.8μg/ml NXRH214) + 3.5ml M141 + 5μl 0.1mM bromoform (100nM bromoform)

Tube 7: 0ml NXRH214 (No NXRH214) + 5ml M141 + 5μl 0.156mM Bromoform (156nM Bromoform)

Tube 8: 1ml NXRH214 (1.4μg/ml NXRH214) + 5ml M141 + 5μl 0.156mM bromoform (156nM bromoform)

Tube 9: 1.5ml NXRH214 (2.8μg/ml NXRH214) + 3.5ml M141 + 5μl 0.156mM bromoform (156nM bromoform)

After the addition of the test substances, 500 [mu]l of an overnight M. maripaludis culture was added to each reaction tube. Each tube was gassed to 240 kPa with 80% _H2 /20% _CO2 and incubated at 37 °C for 24 h.

After the 24 h incubation, measure the pressure inside the tube using a manometer. Assuming the consumption of 5 moles of _H2 / _CO2 to produce 1 mole of _CH4 , the observed pressure drop was used to calculate the amount of control and test reactive methane, from which percent inhibition was calculated.

Experimental results

The experimental results of i) bromoform and allicin and ii) bromoform and powder containing organic sulfur and polyphenol (ie, NXRH214 powder) are shown in Figures 1 and 2, respectively, and shown in Tables 1 and 2 below.

Table 1: Methane inhibition with various compositions, including compositions comprising bromoform and allicin.

Inhibited gas% DMSO 0 DMSO/bromoform 120nM 0 DMSO/bromoform 156nM 0 DMSO/Allicin 200μM 0 DMSO/Allicin 300μM 19 Bromoform 120nM/Allicin 300μM 75 Bromoform 156nM/Allicin 200μM 39 Bromoform 156nM/Allicin 300μM 99

The results show a clear synergy of the composition comprising bromoform and allicin, which significantly exceeds the % gas inhibition exhibited by either component alone.

Table 2: Methane inhibition with various compositions, including compositions comprising bromoform and a powder comprising organosulfur and polyphenols (ie, NXRH214 powder), which is a mixture of allicin and bioflavonoid compounds.

sample Inhibited gas% DMSO 0 Bromoform 100nM 0 Bromoform 156nM 0.5 NXRH214 powder 1.4mg/ml 2.2 Bromoform 100nM+NXRH214 powder 1.4mg/ml 44.8 Bromoform 156nM+NXRH214 powder 1.4mg/ml 100 NXRH214 powder 2.1mg/ml 88.5 Bromoform 100nM+NXRH214 powder 2.1mg/ml 96.2 Bromoform 156nM+NXRH214 powder 2.1mg/ml 100

The results also show a clear synergy of compositions comprising bromoform and a powder comprising organic sulfur and polyphenols (ie NXRH214 powder), which significantly exceeds the % gas inhibition exhibited by either component in the presence of DMSO. NXRH214 powder contains allicin and polyphenolic compounds, more specifically, the bioflavonoid compounds naringin and neohesperidin. Therefore, compositions comprising bromoform, organosulfur compounds and polyphenols can also be used to effectively inhibit methane production.

in conclusion

The above data clearly show high % inhibition of methanogens when organohalogen (ie bromoform) is combined with organosulfur (ie allicin) alone or in combination with polyphenols (ie bioflavonoids from citrus extracts).

Example 2

Preparations of the feed supplement Mootral ^TM and the marine macroalgae Asparagopsis armata, developed and marketed by Mootral ^TM SA of Switzerland, were combined in various ratios and given progressively on a completely grass-fed diet in various dosage amounts and at various time intervals sheep. Methane emissions, blood, body and feces were measured and compared to control animals that did not receive the supplement.

This disclosure can also be described by the following paragraphs

A. A method of reducing nitrogen excreted by ruminants and/or reducing methane emitted by ruminants and/or increasing nitrogen-rich and carbon-rich substances in ruminants, said method comprising administering to said ruminants an effective amount of at least one Types of methanogenesis inhibitor steps.

B. The method according to paragraph A, wherein the methanogenesis inhibitor is selected from the group consisting of: organohalogen compounds; organohalogen-rich marine macroalgae; organosulfur compounds; organosulfur-rich plants; polyphenolic compounds; plant.

C. The method according to claim paragraph _B , wherein the organohalogen compound _is selected from _the group consisting of _{: CH3Cl} ; _CH3Br ; CH3I; _CH2Cl2 ; _CH2Br2 ; _CH2I2 ; _CHCl3 ; _CHBr3 ; _{CHI3 ; CCl4 ; CBr4 ; CH2ClBr ; CH2ClI ; CH2BrI ;} CHBr2Cl; _{CHBrI2 ; 3} CH ₂ CH ₂ I; CH ₃ (CH ₂ ) ₃ I; CH _{3 (} CH _{2 ) 4} Br; _{CH 3} (CH ₂ ) ₄ I; (CH3) ₂ CHI; _{; ( CH3} ) _{2CHCH2I ; BrCH2CH2Br} ; ClCH= _CCl2 ; _{and CH3CH2CH2CH2I .}

D. The method according to claim paragraph B, wherein the organohalogen-rich marine macroalgae is selected from the group consisting of: Asparagopsis armata; Asparagopsis taxiformis; Dictyota species; Oedogonium species; Ulva species; and Cladophorapatentiramea.

E. The method according to claim paragraph B, wherein the organosulfur compound is selected from the group consisting of: organosulfur secondary metabolites; allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); diallyl disulfide (C ₆ H ₁₀ S ₂ ); and allyl mercaptan (C ₃ H ₆ S).

F. The method according to paragraph B, wherein the organosulfur-rich plant is an Allium species selected from the group consisting of: garlic (Allium sativum); scallions (Allium ampeloprasum); and onions (Allium cepa).

G. The method according to paragraph B, wherein the polyphenolic compound is selected from the group consisting of: flavonoids; bioflavonoids; non-bioflavonoids. The at least one polyphenolic compound may for example comprise at least one bioflavonoid; anthocyanins; flavones; flavonols; flavanones; dihydroflavonols; Isoflavones; proanthocyanidins; phenolic acids; hydroxycinnamic acids; coumarins; stilbenes; anthraquinones; lignans; lignin; tannins; polyphenolic proteins; catechins; rutin; Flavone; Kaempferol; Gallocatechin; Catechin Gallate; Epigallocatechin; Epigallocatechin; Epigallocatechin Gallate; Quercetin; Allocatechin; Gallocatechin Gallate Epicatechin; Epigallocatechin; Epigallocatechin Gallate; Epigallocatechin Gallate; Kaempferol; Quercetin; Naringin; Neohesperidin; Eriophonium Glycoside; Isonaringin; Naringenin; Hesperidin; Rhonocardin; Dianoside; Melitrin; Hesperetin; Citrusin; Epicatechin; Gallocatechin; Epigallocatechin coumaric acid; cinnamic acid; gallic acid; ellagic acid; protocatechuic acid; chlorogenic acid; caffeic acid; ferulic acid; punicalagin; and garnetin.

H. The method according to paragraph B, wherein the polyphenol-rich plant is selected from the group consisting of: Allium species; Brassica species; Camelia species; Capsicum species; Citrus ( Citrus species; Citrus aurantium; Cucumis species; Malus species; Musa species; Phaseolus species; Prunus species; Punica species ) species; Pyrus species; Solanum species and Vaccinium species.

I. A method for reducing nitrogen excreted by ruminants and/or reducing methane emitted by ruminants and/or increasing nitrogen-rich and carbon-rich substances in ruminants, said method comprising gradually administering to said ruminants an effective amount of at least one types of methanogenesis inhibitors.

J. The method according to paragraph I, wherein the methanogenesis inhibitor is administered stepwise with at least one dose, as a percentage by weight of said ruminant selected from the group consisting of: 0.1%; 0.2%; 0.3%; 0.4%; 0.5%; 0.6% ; 0.7%; 0.8%; 0.9%; 1.0%; 1.1%; 1.2%; 1.3%; 1.4%; 1.5%; 2.0%; 2.5%; 3.0%; 10%.

K. The method according to paragraph I, wherein the methanogenesis inhibitor is administered stepwise with at least one dose, as a percentage by weight of feed of said ruminant selected from the group consisting of: 0.01%, 0.03%, 0.05%, 0.075%, 0.1%; 0.2%; 0.3%; 0.4%; 0.5%; 0.6%; 0.7%; 0.8%; 0.9%; 1.0%; 1.1%; 1.2%; 1.3%; 1.4%; 1.5%; 2.0%; 2.5%; 3.0% ; 3.5%; 4.0%; 4.5%; 5.0%; and 10%.

L. The method according to any one of paragraphs I to K, wherein the stepwise administration has at least one interval between consecutive doses selected from the group consisting of: 1 minute; 1 hour; 1 day; 2 days; 3 days; 4 days ; 5 days; 6 days; 7 days; 10 days; 2 weeks; 3 weeks; 4 weeks; 6 weeks; 2 months; 3 months; 4 months; 6 months; 9 months; and 12 months.

Claims (19)

CLAIMS 1. A composition for reducing methane emissions, comprising an organohalogen compound and an organosulfur compound. 2. The composition of any preceding claim, wherein the organohalogen compound is an organobromine compound. 3. The composition according to any one of the preceding claims, wherein the organohalogen is bromoform. 4. The composition according to any one of the preceding claims, wherein the organosulfur compound is from a plant of the Allium species. 5. The composition according to any one of the preceding claims, wherein the organosulfur compound is allicin (C ₆ H ₁₀ S ₂ O); diallyl sulfide (C ₆ H ₁₀ S); allyl disulfide (C ₆ H ₁₀ S ₂ ); and allyl mercaptan (C ₃ H ₆ S). 6. _The composition according to any one of the preceding claims, wherein the organosulfur compound is _allicin ( _C6H10S2O ). 7. Composition according to any one of the preceding claims, wherein the ratio of organohalogen to organosulfur is from 1:10 to 1:3500. 8. Composition according to any one of the preceding claims, wherein the ratio of organohalogen to organosulfur is from 1:1000 to 1:2500. 9. A composition according to any preceding claim, further comprising at least one polyphenolic compound. 10. The composition of claim 9, wherein the at least one polyphenolic compound comprises at least one bioflavonoid. 11. The composition of claim 9, wherein the at least one polyphenolic compound comprises naringin, neohesperidin, or a combination thereof. 12. A composition as claimed in any preceding claim for use in inhibiting one or more methanogens. 13. A composition as claimed in any preceding claim for use in improving the metabolic efficiency of an animal. 14. An animal feed comprising the composition of any one of claims 1-13. 15. Use of the composition according to claims 1-13 or the animal feed according to claim 14 for reducing methane emissions and/or inhibiting one or more methanogens and/or improving the metabolic efficiency of animals . 16. A method of reducing methane emissions, said method comprising administering to an animal a composition according to any one of claims 1-13 or an animal feed according to claim 14. 17. A method of inhibiting one or more methanogens, said method comprising administering to an animal a composition according to any one of claims 1-13 or an animal feed according to claim 14. 18. A method of improving the metabolic efficiency of an animal, said method comprising administering to the animal a composition according to any one of claims 1-13 or an animal feed according to claim 14. 19. The method according to claims 16-18, wherein the animal is a ruminant, preferably wherein the ruminant is a cow, goat or sheep.