Methods of controlling Anti-methanogenic halomethane production

Abstract

The present invention relates to methods of controlling the anti-methanogenic content of ruminant animal feed and feed supplements, and producing stabilised halomethylcarbonyl compounds for ruminant animal feed and feed supplements.

Description

METHODS OF CONTROLLING ANTI-METHANOGENIC HALOMETHANE PRODUCTIONTechnical Field

[0001] The field of invention relates to methods of controlling the anti-methanogenic content of ruminant animal feed and feed supplements, and producing stabilised halomethylcarbonyl compounds for ruminant animal feed and feed supplements.Background of Invention

[0002] Methane (CH4) is a greenhouse gas (GHG) produced primarily by methanogenic microbes that are found in natural ecosystems (e.g. wetlands, oceans and lakes) and the gastrointestinal tract of invertebrates and vertebrates, such as termites and ruminants. Every year "'429-507 Tg of CH4 are removed from the atmosphere and ~40 Tg from the stratosphere through reactions with hydroxyl (OH) radicals; and ~30Tg by CH4-oxidizing bacteria in soil.

[0003] Nevertheless, anthropogenic GHG emissions have been increasing rapidly, with the CH4 concentration in the atmosphere now more than twofold higher than in the early 1800s. Methane is very effective in absorbing solar infrared radiation and has a global warming potential 28 times greater than CO2. Consequently, its accumulation in the atmosphere contributes considerably to climate change. One of the main sources of anthropogenic CH4 can be attributed to agricultural activities, including ruminant livestock.

[0004] According to a recent UN report, cattle-rearing generates more global warming greenhouse gases, as measured in CO2 equivalent, than transportation. In Australia, ruminants are estimated to contribute ~10% of the total GHG emissions. Ruminants produce CH4 as a by-product of the anaerobic microbial fermentation of feeds in the rumen and, to a lesser extent, in the large intestine. The ruminal microbial community is highly diverse and composed of bacteria, protozoa, fungi, and bacteriophages that act collectively to ferment ingested organic matter (OM), resulting in CO2, H2, volatile fatty acids (VFAs), and formates. Methanogenic archaea present in the rumen use these end-products and produce CH4. Although the production of CH4 reduces the partial pressure of H2, which could otherwise inhibit rumen fermentation, it also reduces the amount of energy and carbon available for formation of VFAs essential for ruminant nutrition. Most of the CH4 produced in ruminants is exhaled and belched by the animal and represents a loss of up to 12% of gross energy intake.

[0005] Previous work has focused on the use of Asparagopsis species as feed supplements for reducing total gas production and / or methane production in ruminant animals. This work has led to the use of Asparagopsis biomass and Asparagopsis derived products for reducing methane, but there remains a need for improved mitigation strategies that reduce enteric CP formation.Summary of Invention

[0006] In one aspect, the present invention provides a method of producing an anti-methanogenic halomethane composition comprising one or more anti-methanogenic halomethane compounds, the method comprising contacting one or more halomethylcarbonyl compounds with one or more protic solvent.

[0007] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is contacted with the one or more protic solvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form the one or more anti-methanogenic halomethane compounds.

[0008] In another embodiment, the present invention provides a method as described herein, wherein the one or more -halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0009] In a further embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0010] In a further embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0011] In a further embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

[0012] In a further embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl,14, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, 1, 1,1, 3, 5,5,5-heptabromopentan-2, 4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, 1, 1,3, 3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

[0013] In a further embodiment, the present invention provides a method as described herein, wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

[0014] In a further embodiment, the present invention provides a method as described herein, wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

[0015] In a further embodiment, the present invention provides a method as described herein, further comprising contacting the one or more halomethylcarbonyl compounds with one or more protic solvent in the presence of a base.

[0016] In a further embodiment, the present invention provides a method as described herein, wherein the base is selected from the group consisting of hydroxide, bicarbonate, carbonate, ammonia, aqueous ammonia, methanolic ammonia, and ethanolamine.

[0017] In another aspect, the present invention provides a method as described herein, method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with a one or more protic solvent.

[0018] In a further aspect, the present invention provides a method of increasing the levels of at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent.

[0019] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis comprises one or more halomethylcarbonyl compounds.

[0020] In another embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof is contacted with the one or more protic solvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form the one or more anti-methanogenic halomethane compounds.

[0021] In another embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0022] In another embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0023] In another embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0024] In another embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound

[0025] In another embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, 1, 1,1, 3, 5,5,5-heptabromopentan-2, 4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, 1, 1,3, 3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

[0026] In another embodiment, the present invention provides a method as described herein, wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, ethylene glycol monomethyl ether.

[0027] In another embodiment, the present invention provides a method as described herein, wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

[0028] In another embodiment, the present invention provides a method as described herein, wherein the Asparagopsis is A. armata or A taxiformis

[0029] In another embodiment, the present invention provides a method as described herein, further comprising a step of separating the at least one protic solvent from the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

[0030] In another aspect, the present invention provides a method of producing bromoform, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with rumen fluid within the rumen of a ruminant animal.

[0031] In another aspect, the present invention provides a method as described herein, wherein the level of the at least one anti-methanogenic halomethane compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, contacted with the at least one protic solvent is increased relative to the level of the at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, not contacted with a protic solvent.

[0032] In another aspect, the present invention provides a method of preparing a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, having an increased levels of at least one or more halomethylcarbonyl compounds, said method comprising a step of decreasing the hydrolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

[0033] In another embodiment, the present invention provides a method as described herein, wherein the method comprises a step of increasing the integrity of intact gland cells in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

[0034] In another embodiment, the present invention provides a method as described herein, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with an aprotic solvent or solvent.

[0035] In another embodiment, the present invention provides a method as described herein, wherein the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, / V-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0036] In another embodiment, the present invention provides a method as described herein, wherein the method comprises a step of reducing the levels of protic solvent in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or a mixture thereof.

[0037] In another embodiment, the present invention provides a method as described herein, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with at least one acid.

[0038] In another embodiment, the present invention provides a method as described herein, further comprising a step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with at least one acid and at least one further solvent.

[0039] In another embodiment, the present invention provides a method as described herein, wherein the at least one acid is selected from the group consisting of formic acid, acetic acid,propanoic acid, pyruvic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, malonic acid, malic acid, phosphoric acid, tartaric acid, trichloroacetic acid, and tribromoacetic acid.

[0040] In another embodiment, the present invention provides a method as described herein, wherein the at least one further solvent is a protic solvent.

[0041] In another embodiment, the present invention provides a method as described herein, wherein the protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

[0042] In another embodiment, the present invention provides a method as described herein, wherein the step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with the at least one acid reduces the pH of the mixture to 6 or less, 5 or less, 4 or less, 3 or less, 2 or less or 1 or less.

[0043] In another aspect, the present invention provides a method of preparing an anti-methanogenic halomethane composition, said method comprising: contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with an extraction liquid comprising at least one aprotic solvent or an acid to decrease the hydrolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, and to extract the one or more halomethylcarbonyl compounds or the one or more halomethylcarbonyl compounds and the one or more anti-methanogenic halomethane compounds into the extraction liquid.

[0044] In one embodiment, the present invention provides a method as described herein, further comprising separating the extraction liquid from the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, to obtain an anti-methanogenic halomethane composition comprising one or more halomethylcarbonyl compounds, or one or more halomethylcarbonyl compounds and one or more anti-methanogenic halomethane compounds.

[0045] In another embodiment, the present invention provides a method as described herein, wherein the at least one aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butylacetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, / V-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tertamyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0046] In another embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of separating the at least one aprotic solvent from the extraction liquid.

[0047] In another embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of removing the aprotic solvent from the composition.

[0048] In another embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of removing the aprotic solvent from the composition under vacuum.

[0049] In another embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of contacting the composition with a further solvent.

[0050] In another embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of adsorbing the composition into a solid support, or encapsulating the composition.

[0051] In another embodiment, the present invention provides a method as described herein, wherein the level of the at least one halomethylcarbonyl compound in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof contacted with an aprotic solvent is increased relative to the level of at least one halomethylcarbonyl compound not contacted with an aprotic solvent.

[0052] In another embodiment, the present invention provides a composition produced by a method described herein.

[0053] In another aspect, the present invention provides an anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and at least one aprotic solvent.

[0054] In another aspect, the present invention provides an anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and one or more halomethylcarbonyl compound stabilising excipient.

[0055] In one embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0056] In another embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0057] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0058] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

[0059] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1, 1,1, 7,7,7-hexabromoheptan-2, 6-dione, l,l,l,3,5,5,5-heptabromopentan-2,4-dione, 1, 1,1, 3, 3, 5,5,5-octabromopentan-2, 4-dione, l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

[0060] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more synthetic halomethylcarbonyl compounds.

[0061] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0062] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compound stabilising excipient comprises an edible wax, grease, oil, cyclodextrins, molasses and a saturated fat.

[0063] In another aspect, the present invention provides a feed supplement for reducing total gas production and / or methane production in a ruminant animal, said supplement comprising an effective amount of composition produced by a method as described herein, or an effective amount of an anti-methanogenic composition of as described herein

[0064] In another embodiment, the present invention provides a feed for a ruminant animal, wherein said feed is supplemented with a feed supplement described herein.

[0065] In another embodiment, the present invention provides a method for reducing total gas production and / or methane production in a ruminant animal comprising administering to said ruminant animal an effective amount of a composition, a feed supplement, or a feed as described herein.Brief Description of Drawings

[0066] Figure 1: Representative isotope splitting pattern for the bromoform (CHBra) molecular ion. This sample was generated by extracting lyophilised sample A10 with methanol (CH3OH), where a negligible amount of deuterium incorporation is possible. Boxed labels show the isotopic composition of each ion.

[0067] Figure 2: Detail of the molecular ion of the 70 eV El mass spectrum of the bromoform peak for the sample A10 dried by lyophilisation, that was extracted with methanol-c / 4 (CD3OD). Boxed labels indicate a pair of ions with identical isotopic composition apart from the substitution of deuterium for hydrogen; the relative peak areas of these ions indicate the amount of deuterium substitution.

[0068] Figure 3: Detail of the molecular ion of the 70 eV El mass spectrum of the bromoform peak for the microwave-dried sample A9, that was extracted with methanol-c / 4 (CD3OD).

[0069] Figure 4: Mass spectra (70eV El) of synthetic bromoform standard under different H / D exchange conditions. Upper trace: Bromoform in natural-abundance methanol solution (CH3OH). Middle trace: Bromoform (1 mg / mL) at 11% deuteration after H / D exchange for 22 h with deuterated methanol (CD3OD, 99.8%). Lower trace: Complete deuteration was observed after equilibration of bromoform (1 mg) with methanol-c / 4 (CD3OD, 1 mL) + ammonium deuteroxide (ND3 in D2O, 20 pL) for 22 h.

[0070] Figure 5: Partial 70 eV El mass spectrum of chlorodibromomethane from the sample A10 dried by lyophilisation, that was extracted with methanol-c / 4, CD3OD. This ion cluster is assigned the formula CHClBr, corresponding to loss of bromine from the molecular ion. Boxed labels show the isotopic composition of each ion; the ion in brackets (12C79Br35CI) is a further fragment that does not contain hydrogen and was therefore not used for quantitation.

[0071] Figure 6: Partial 70 eV El mass spectrum of chlorodibromomethane from the microwave-dried sample, that was extracted with methanol-c / 4, CD3OD. This ion is assigned the formula CHClBr, corresponding to loss of bromine from the molecular ion.

[0072] Figure 7: GC-MS Total Ion Chromatogram (70 eV El) of the mixture obtained by dissolution of 1,1,1,3,3,3-hexabromoacetone in methanol. Additional small peaks due to siloxanes (base peak m / z = 73) are not integrated.

[0073] Figure 8: GC-MS Total Ion Chromatogram (70 eV El) of the mixture obtained by dissolution of 1,1,1,3,3,3-hexabromoacetone in methanolic ammonia (7N).

[0074] Figure 9: GC-MS chromatograms showing detail of minor components produced during extractions of lyophilised sample A10 (50 mg) with DCM (1 mL) for 16 h. Lower trace: GC-MS chromatogram of sample extracted with DCM only, showing few peaks apart from halomethanes and haloacetones. Upper trace: GC-MS chromatogram of sample extracted with the addition of aqueous ammonia (20 pL) and ultrasonication at 50 kHz, showing the presence of amide and nitrile sideproducts. For peak assignments see Table 13.

[0075] Figure 10: Selected GC-MS chromatograms for sample 018-02 in Table 19. Upper trace: Extraction of freeze-dried seaweed into DCM (1 mL) in the presence of aqueous ammonia (20 pL). Lower trace: Extraction into MeOH (1 mL) in the presence of aqueous ammonia (20 pL).

[0076] Figure 11: Partial1H-NMR spectra of extracts obtained by sonicating 200 mg of freeze-dried Asparagopsis with 1 mL of DCM-ct- Lower trace: sonication was performed for 1 h. Upper trace: an identical sample was sonicated for 16 h. Note the significant increase in the methylene resonances marked with arrows. The starred peaks are the13C satellites of the solvent peak at 8 = 5.32 ppm; as these are at the same concentration in the two samples they can be used visually as an internal standard for relative quantitation.

[0077] Figure 12: Partial HSQC spectrum of DCM extract (200 mg, 16 h). Chemical shift coordinates for selected cross-peaks are marked.

[0078] Figure 13: Stacked1H-NMR spectra showing chemical changes occurring in the 200 mg-scale DCM extract after addition of MeOD (0.1 mL). Traces 1-7 were recorded on days 1-7 after the addition, and trace 8 was recorded on day 11. The bromoform proton resonates at 8 = 6.94 ppm in this solvent mixture, referenced to CDHCL at 8 = 5.32 ppm.

[0079] Figure 14: GC-MS chromatograms comparing extraction of 200 mg of freeze-dried sample A10 with two different solvents for 1 hour with sonication, followed by storage of the extracts in glass for 1 month before analysis. Upper trace: Aging of DCM-ct extracts yields a mixture of halomethanes and halogenated acetones. Lower trace: Aging of methanol-c / 4 extracts yields no detectable haloacetones, instead halomethanes and methyl esters dominate. For peak assignments see Table 25.

[0080] Figure 15: Non-Haloform reactivity of haloform precursors can proceed through enolization.

[0081] Figure 16: Scheme 2 Reasonable chemical transformations of candidate bromoform precursor molecules. Structures coloured gray are theoretical only; structures coloured black are experimentally supported as Asparagopsis metabolites.

[0082] Figure 17: A. GC-MS analysis of HBA (hexabromoacetone) contacted with methanol. B. GC-MS analysis methanol extract of lyophilized seaweed sample 018-02. C. GC-MS analysis of DCM extract of lyophilized seaweed sample 018-02. D. GC-MS analysis HBA contacted with methanolic ammonium and water. E. GC-MS analysis of MeOH / NH4OH extracts of lyophilized seaweed sample 018-02 . F. GC-MS analysis of DCM / NH4OH extracts of lyophilized seaweed sample 018-02. G. Compounds identified by GC-MS and the reactions that generate them from haloacetones.

[0083] Figure 18: Live Asparagopsis is subjected to osmotic shock; distinctive pink colour is released into the water.

[0084] Figure 19: Example cleavage pathways for precursor candidate molecules

[0085] Figure 20: APCI-mass spectra of various Asparagopsis extracts compared against the HBA standard.Detailed Description

[0087] The present invention is based in part on establishing that the production of anti-methanogenic halomethane compounds such as bromoform, from halomethylcarbonyl precursors in gland cells of Asparagopsis species, requires a nucleophile for attack of the carbonyl, and a proton source to protonate the nascent halomethyl carbanion. As a result, it is possible to produce anti-methanogenic halomethane compounds by contacting one or more halomethylcarbonyl compounds with one or more protic solvent, including increasing the levels of anti-methanogenic halomethane compounds in Asparagopsis and / or compositions derived from Asparagopsis. As will also be discussed in more detail below, because halomethylcarbonyl precursors are demonstrated herein to be hydrolysed to produce actives upon damage to the Asparagopsis gland cells, it is possible to treat Asparagopsis biomass and parts thereof and compositions comprising halomethylcarbonyl precursors to reduce the production of anti-methanogenic halomethane compounds, for example, for storage with reduced loss of volatile actives, or for the production of volatile anti-methanogenic halomethane compounds in vivo.

[0088] Importantly, the present inventors have demonstrated herein that halomethylcarbonyl precursor compounds can be used to form bromoform, including that synthetic halomethylcarbonyl precursor compounds can be used to form bromoform.

[0089] For example, Example 1 demonstrates that when the content of bromoform in Asparagopsis is examined, bromoform is generated from solvolysis of unhydrolysed tribromocarbonyl precursors during processing, and the conditions used can determine the amount of bromoform produced. In Example 2, the present inventors have demonstrated that bromoform can be produced by solvolysis from a precursor compound, either synthetic or Asporogop / s-derived. In Examples 3 and 4, the present inventors have demonstrated that the amounts of unhydrolysed tribromocarbonyl precursors and the amounts of anti-methanogenic halomethanes in Asparagopsis samples and extracts can be controlled and increased.

[0090] Importantly, Asparagopsis contains a large number of halogenated small molecules varying in molecular weight thought to be stored in gland cells and then the stored components released to the environment as chemical defence against herbivores and antagonistic bacteria. The present inventors have demonstrated herein that halomethane compounds capable of inhibiting methanogenesis - when administered to ruminants - can be produced by hydrolysis of precursor molecules stored in gland cells when the gland cells are damaged / release their contents to an environment that allows for solvolysis.

[0091] Asparagopsis species are able to store large quantities of haloforms in specialised gland cells.5Previous workers have assumed the precise nature of the chemical species stored in the gland cells to be 'free' halomethanes; that is, all the chemical steps in Scheme 1 (below) are complete before the metabolites are stored.

[0092] Scheme 1: Mechanism of the haloform reaction, illustrated for bromoform. Nucleophilic attack of a protic solvent such as water on a halomethylcarbonyl precursor leads to expulsion of a carbanion leaving group, in this case tribromomethyl carbanion. Protonation of this carbanion yields bromoform.

[0093] However, the present inventors have surprisingly demonstrated that the bulk of the anti-methanogenic halomethane compounds (including bromoform) are instead stored as halomethylcarbonyl precursors. The present inventors propose that the electron-translucent (i.e. low-water) inclusions reported to be present in the gland cells of Asparagopsis910do not primarily store free halomethanes such as bromoform, but rather the halomethane precursors are safely and stably accumulated in a low-aqueous environment.

[0094] The demonstration that the bulk of the compounds are stored as precursors allows for the preparation of compositions of known characteristics with regard to anti-methanogenic halomethane compounds and / or halomethylcarbonyl precursor compounds. For example, under suitable conditions halomethylcarbonyl precursors can be retained using the methods described herein, until the release of haloforms is required. Reinterpreting confusing and contradictory results from the literature in the light of this finding, the present inventors demonstrate improved methods for controlling the potency and stability of Asparagopsis products and extracts, enabled by an understanding of the role of precursor chemistry.

[0095] The present inventors propose that it is therefore useful to consider the anti-methanogenic halomethane content of a composition, seaweed product or seaweed extract etc. as the sum of two fractions; the "free" anti-methanogenic halomethane compound fraction that is present as such, and the "potential" halomethane compounds (precursors) that will be produced on complete hydrolysis of all available halomethylcarbonyl precursor compounds. The "total" halomethane content that is available for reducing methane production of a ruminant animal is therefore the sum of both "free" and "potential" fractions.

[0096] In Examples 5 and 7, the present inventors have demonstrated that halomethylcarbonyl precursors can be extracted into oil, and bromoform produced by solvolysis using methanol. In Example 5 the present inventors have demonstrated that the amounts of unhydrolysed tribromocarbonyl precursors can be increased in Asparagopsis and extracts.

[0097] Accordingly, in one aspect, the present invention provides a method of producing an anti-methanogenic halomethane composition comprising one or more anti-methanogenic halomethane compound, the method comprising contacting the one or more halomethylcarbonyl compounds with one or more protic solvent.

[0098] In one embodiment, the composition comprising one or more anti-methanogenic halomethane compound comprises one or more synthetic anti-methanogenic halomethane compound, and / or one or more Asparagopsis derived anti-methanogenic halomethane compound.

[0099] Asparagopsis, for example A. taxiformis, has been determined to contain an abundance of anti-methanogenic compounds including: bromoform, dibromochloromethane, bromochloroacetic acid, dibromoacetic acid, dichloromethane, bromochloromethane, 2-bromoethanesulfonate and 2-chloroethanesulfonate.

[0100] Bromoform is the most abundant anti-methanogenic compound found in A. taxiformis, and has been shown to inhibit enzymatic activities of CoM methyltransferase and methyl-CoM reductase, which are needed for methanogenesis.

[0101] As used herein the term "anti-methanogenic halomethane composition" refers to a composition comprising one or more halomethane compounds that reduce the production of methane from enteric fermentation in the rumen of ruminant animals. Such anti-methanogenic halomethane compounds include bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane,dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

[0102] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein the one or more anti-methanogenic halomethane compounds produced are selected from the group consisting of dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichloromethane, bromodichloromethane, bromochloromethane, diiodomethane, and carbon tetrabromide.

[0103] In a preferred embodiment, the one or more anti-methanogenic halomethane compounds comprises bromoform.

[0104] As used herein, the term "anti-methanogenic" refers to the ability of a halomethane compound, a composition, an extract, or a biomass etc to - when fed to ruminant animals - reduce the amount of methane produced by enteric fermentation in the gastrointestinal tract of ruminant animals. The term includes the specific volume of methane generated as a result of anaerobic fermentation. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The term therefore also includes halomethylcarbonyl compounds and a composition, an extract, or a biomass etc comprising halomethylcarbonyl compounds which, when fed to ruminant animals - reduce the amount of methane produced by enteric fermentation in the gastrointestinal tract of ruminant animals.

[0105] For context, the process of rechewing the cud to further break down plant matter and stimulate digestion is called "ruminating". Ruminants have a digestive tract with four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, the rumen and the reticulum, the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud, or bolus. The cud is then regurgitated, chewed slowly to completely mix it with saliva, which further breaks down fibers. Fiber, especially cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa and fungi. The broken-down fiber, which is now in the liquid part of the contents, then passes through the rumen and the reticulum into the next stomach chamber, the omasum. The food in the abomasum is digested much like it would be in the monogastric stomach. Digested gut contents are finally sent to the small intestine, where the absorption of the nutrients occurs. Almost all the glucose produced by the breaking down of cellulose is used by the symbiotic bacteria. Ruminants get their energy from the volatile short chain fatty acids (VFAs) produced by the bacteria, namely acetate, propionate, butyrate, valerate, andisovalerate. In vitro studies using rumen fluid have demonstrated that the production of CH4 is significantly affected by synthetic halomethanes, such as bromoform, when these synthetic compounds are added to rumen fluid.

[0106] Bromoform - one of the secondary metabolites contained in Asparagopsis biomass - is volatile and has physical properties (including its volatility) which are considered to make impractical its use in vivo or in vitro. Initial work demonstrated that manufactured bromoform in DMSO is unable to significantly inhibit methane production at low doses (e.g. doses of less than 5uM) (Machado et al. (2016) J Appl Phycol 28:3117-3126). Subsequent work by Machado et al. (2018) (Microbial Ecology 75(D1)) demonstrated that the inclusion of Asparagopsis biomass at 2% OM inhibits methane production more effectively than an equivalent dose of manufactured bromoform in DMSO, indicating that the other components of Asparagopsis contribute to its anti-methanogenic activity.

[0107] However, previous work has demonstrated that the effects of Asparagopsis biomass containing secondary metabolites - or Asparagopsis derived products such as extracts - both in rumen fluid and in vivo are variable and contradictory due to the differences in extracts / compositions comprising such compounds, doses of compounds, and the influence type and quality of basal diet. The present inventors propose that previous work produced inconsistent results because the way in which the compositions (including extracts and / or biomass-based compositions) were made did not include processing to control the levels of anti-methanogenic halomethane compounds. Importantly, the present inventors demonstrate herein that Asparagopsis biomass comprises stored halomethylcarbonyl compounds (that can be considered 'precursors' of the anti-methanogenic halomethane compounds referred to herein) and which can be solvolysed to form anti-methanogenic halomethane compounds, including in a controlled manner. Some example reaction pathways for precursor solvolysis are shown in Figure 19.

[0108] Without wishing to be bound by theory, using the demonstration herein that Asparagopsis biomass comprises stored halomethylcarbonyl compound precursors, the present inventors propose a range of halomethylcarbonyl precursors described previously can be utilised in the methods described herein. For example, numerous halogenated acetones and butenones have been identified in seaweed extracts by GC-MS,2along with the acrylic acid derivatives likely formed from them by Favorskii rearrangement.11Additionally, significant quantities of halogenated acetic acids11and acetamides12have been identified; the present inventors propose that these are formed as the by-products of haloform-type cleavage of haloacetones by water (Scheme 2) or by ammonia (Scheme 3). Although hexabromoacetone has not been previously isolated from Asparagopsis, theisolation by Sugano et al. of an enol ester likely formed from this molecule (Scheme 4) the present inventors propose this provides indirect evidence of its existence. The central role that haloacetones likely play in the formation of a range of known Asparagopsis metabolites is summarised in Figure 15. Finally, Thapa et al.3identified a series of halogenated pentanediones including 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione by LC-MS analysis of Asparagopsis extracts. Those workers proposed a biosynthetic pathway producing bromoform via polyketide ketosynthase enzymes, and noted that such a pathway also allows for halogenated acetones as "intermediates or shunt metabolites". However, the laboratory-reconstructed biosynthetic pathway of Thapa et al. did not include a reconstruction of the Asparagopsis gland-cells. With the understanding by the present inventors that these structures could impart stability to the halomethylcarbonyl precursors in the absence of water, such metabolites can now be viewed not as short-lived intermediates but as useful products to be controlled and manipulated. Some possible biosynthetic pathways that could produce bromoform from polyketide intermediates are set out in Figure 16, including the pathways reconstructed by Thapa et al. in the absence of gland cells. The intermediates shaded in black in Figure 16 are those that are known to be present in Asparagopsis. The intermediates that have been isolated and identified are necessarily the more stable intermediates with lower levels of bromination. Highly brominated intermediates such as those shaded in grey in Figure 16 are less likely to survive the extraction process and be detected. However, if the intermediates were protected against hydrolysis, for example by synthesising them in the absence of water under laboratory conditions or within the Asparagopsis gland cell, then some of the more highly brominated molecules of Figure 16 could be stored and used as bromoform precursors.

[0109] Scheme 2: Hydrolysis reaction of an example haloacetone (hexabromoacetone) to yield bromoform and a haloacetic acid.+ CHBr3

[0110] Scheme 3: Haloform-type ammonolysis of an example haloacetone (hexabromoacetone) to yield bromoform and a haloacetamide.

[0111] Scheme 4: A reasonable mechanism for the formation of the known13Asparagopsis metabolite pentabromopropen-2-yl tribromoacetate, by condensation of pentabromoacetone with hexabromoacetone.

[0112] As a result, the levels of bromoform in Asparagopsis can be increased by hydrolysing halomethylcarbonyl compounds stored in biomass. Similarly, extracts of Asparagopsis can be treated to convert halomethylcarbonyl compounds to anti-methanogenic halomethane compounds. Additionally, compositions comprising synthetic halomethylcarbonyl compounds can be treated to convert the halomethylcarbonyl compounds to anti-methanogenic halomethane compounds.

[0113] Once it is known that Asparagopsis stores halomethylcarbonyl compounds which can be converted to anti-methanogenic halomethane compounds by solvolysis, it is possible to reduce the production of volatile anti-methanogenic halomethane compounds by preventing solvolysis. As a result, precursors could be extracted in their precursor state, for later solvolysis under desired circumstances. As a result, some of the problems associated with the "free" halomethane fraction -which are mostly due to the higher volatility of the halomethanes compared to their higher-molecular weight precursors - can be addressed.

[0114] Without wishing to be bound by theory, the present inventors propose that controlling potency loss (loss of anti-methanogenic capacity) at the precursor cleavage stage using the methods claimed herein, and which involves preventing solvolysis, may be more convenient than attempting to control volatilisation of free halomethanes present. Additionally, the toxic nature of the volatile halomethanes when no rumen bacteria are present to metabolise them gives reason for controlling and reducing the "free" fraction as much as practicable to protect workers and users alike.

[0115] Conversely, when conversion of the halomethylcarbonyl compounds to anti-methanogenic halomethane compounds is desired, such as upon administration into the rumen of a ruminant animal, the halomethylcarbonyl compounds may be brought into contact with water andother nucleophiles, whereby solvolysis to the active anti-methanogenic halomethane compounds can occur.

[0116] As used herein, the term "halomethane" refers to a compound that is structurally similar to methane (CH4) with one or more of the hydrogen atoms replaced with halogen atoms (e.g. F, Cl, Br, or I).

[0117] It has also been shown that A. taxiformis reduces CH4 production during enteric fermentation more effectively than highly concentrated halogenated methane analogs, and it has been suggested that the increased efficiency of A. taxiformis may be due to multiple anti-methanogenic bioactives working synergistically.

[0118] Accordingly, in one embodiment, the present invention provides a method of producing an anti-methanogenic halomethane composition comprising two or more anti-methanogenic halomethane compounds, the method comprising contacting two or more halomethylcarbonyl compounds with one or more protic solvent.

[0119] As used herein the term a "halomethylcarbonyl compound" refers to a compound which is capable of being solvolysed to form a halomethane compound. A halomethyl group is an alkyl derived from methane, containing one carbon atom bonded to one or more halogen atoms and zero or more hydrogen atoms. In a halomethylcarbonyl compound, the carbon atom of a halomethyl group is bonded to the carbon atom of a carbonyl group with the formula C=O. in one embodiment, halomethylcarbonyl compounds refers to a compound which is capable of being solvolysed to form an anti-methanogenic halomethane compound.

[0120] As used herein, the term "solvolysis" means nucleophilic cleavage by a solvent, a type of substitution or elimination reaction in which the solvent acts as a nucleophile. The term includes hydrolysis, which refers to a type of substitution or elimination reaction in which water acts as a nucleophile, in which case the term "hydrolysis" is synonymous with "solvolysis".

[0121] As used herein a "solvent" includes a substance, often in liquid form, that is capable of dissolving a solute. The solvent may be present in stoichiometric excess. As defined herein, the term "solvent" includes mixtures of solvents, and substances in solution in a solvent or a mixture of solvents.

[0122] As used herein, the term "protic solvent" includes a solvent comprising available acidic protons, capable of cleaving a halomethylcarbonyl compound that it contacts, whereby said protonsare incorporated into the produced halomethane compound. The term includes a solvent that has a hydrogen atom bound to an oxygen (e.g. a hydroxyl group -OH), a nitrogen (e.g. an amine group -NH2 or -NH-), or fluoride (e.g. hydrogen fluoride). The term includes any solvent that contains a labile H+. In the context of the present invention, the molecules of such protic solvents readily donate protons (e.g. H+) to solutes, often via acid-base chemistry. In contrast, aprotic solvents cannot donate protons. The term protic solvent also includes mixtures of one or more protic solvents.

[0123] As used herein the term "at least one protic solvent" includes a single type of protic solvent, or compositions comprising a single type of protic solvent, or a mixture of two or more protic solvents, or a composition comprising a mixture of two or more protic solvents. As used herein, the term includes protic substances, or protic substances dissolved in or mixed with other solvents, whether protic or aprotic. The term protic solvent also includes mixtures of one or more protic solvents. Based on the data presented herein, the present inventors propose that any sufficiently nucleophilic molecule could conceivably take the place of water in the haloform reaction of Scheme 1, as long as a proton source is available to protonate the halomethyl carbanion. For example, haloform-type solvolysis reactions occur in alcohol solution as illustrated in Scheme 5.

[0124] As used herein, the term "aprotic solvent" includes a solvent that does not comprise protons of sufficient acidity to be incorporated into the produced halomethane compound resulting from cleavage of a halomethylcarbonyl compound. The term aprotic solvent also includes mixtures of one or more aprotic solvents.

[0125] As used herein the term "at least one aprotic solvent" includes a single type of aprotic solvent, or compositions comprising a single type of aprotic solvent, or a mixture of two or more aprotic solvents, or a composition comprising a mixture of two or more aprotic solvents. As used herein, the term includes aprotic substances, or aprotic substances dissolved in or mixed with other aprotic solvents. The term aprotic solvent also includes mixtures of one or more aprotic solvents.

[0126] As used herein the term "contacting" includes mixing the one or more halomethylcarbonyl compounds with the one or more protic solvent, and retaining the one or more halomethylcarbonyl compounds with the one or more protic solvent under conditions suitable to form the anti-methanogenic halomethane composition, e.g. by solvolysis of the one or more halomethylcarbonyl compounds to from one or more halomethane compounds. The term also includes mixing the one or more halomethylcarbonyl compounds with the one or more aprotic solvent, and retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent under conditions suitable to reduce or prevent the solvolysis of the one or morehalomethylcarbonyl compounds to form one or more halomethane compounds. As will be discussed in more detail below, the step of contacting can be performed under any suitable conditions, with a number of parameters that can be varied to control the formation of one or more anti-methanogenic halomethane compounds.

[0127] In one embodiment, the halomethylcarbonyl compounds to be contacted with a solvent are present in a composition comprising Asparagopsis biomass or a part thereof (e.g. biomass in oil), or are present in a composition comprising halomethylcarbonyl compounds extracted from Asparagopsis biomass or a part thereof.

[0128] In one embodiment, the halomethylcarbonyl compounds to be contacted with a solvent are present in Asparagopsis biomass or a part thereof.

[0129] In another embodiment, the halomethylcarbonyl compounds to be contacted with a solvent are wholly or partially derived from a source other than Asparagopsis biomass or a part thereof, for example, wholly or partially derived by chemical synthesis or semi-synthesis. For example, in one embodiment, the halomethylcarbonyl compounds comprise Asparagopsis derived halomethylcarbonyl compounds and / or synthetically produced halomethylcarbonyl compounds.

[0130] In preferred embodiment, the halomethylcarbonyl compounds to be contacted with a solvent comprise synthetic halomethylcarbonyl compounds.

[0131] When the one or more halomethylcarbonyl compounds are present in Asparagopsis, the term contacting includes mixing a biomass of Asparagopsis with the one or more protic solvent and retaining the biomass comprising the one or more halomethylcarbonyl compounds with the one or more protic solvent under conditions to form the anti-methanogenic halomethane composition, e.g. by solvolysis of the one or more halomethylcarbonyl compounds to from one or more anti-methanogenic compounds.

[0132] When the one or more halomethylcarbonyl compounds are not present in Asparagopsis, the term contacting includes mixing a composition comprising the one or more halomethylcarbonyl compound with the one or more protic solvent and retaining the composition comprising the one or more halomethylcarbonyl compounds with the one or more protic solvent under conditions to form the anti-methanogenic halomethane composition, e.g. by solvolysis of the one or more halomethylcarbonyl compounds to form one or more anti-methanogenic compounds.

[0133] The term contacting also includes mixing an extract or composition with the one or more protic solvent and retaining the extract or composition comprising the one or more halomethylcarbonyl compounds with the one or more protic solvent under conditions to form the anti-methanogenic halomethane composition, e.g. by solvolysis of the one or more halomethylcarbonyl compounds to form one or more anti-methanogenic compounds.

[0134] Importantly, the present inventors have demonstrated that the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds can be controlled. For example, in one embodiment, the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds can be reduced by controlling one or more parameters. In another embodiment, the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds can be increased by controlling one or more parameters.

[0135] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more protic solvent at a suitable temperature. In one embodiment the suitable temperature allows for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0136] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent at a suitable temperature. In one embodiment the suitable temperature allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0137] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more protic solvent at a suitable pH. In one embodiment the suitable pH allows for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0138] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent at a suitable pH. In one embodiment the suitable pH allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0139] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more protic solvent at a suitable pressure. In one embodiment the suitable pressure allows for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0140] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent at a suitable pressure. In one embodiment the suitable pressure allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0141] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds in biomass with the one or more protic solvent under suitable biomass gland cell integrity, where the biomass gland cell integrity allows for protic solvent access. In one embodiment the suitable biomass gland cell integrity allows for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0142] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds in biomass with the one or more protic solvent under suitable biomass gland cell integrity, where the biomass gland cell integrity does not allow for protic solvent access. In one embodiment the suitable biomass gland cell integrity allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0143] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent under suitable biomass gland cell integrity, where the biomass gland cell integrity allows for aprotic solvent access. In one embodiment the suitable biomass gland cell integrity allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0144] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds in biomass with the one or more protic solvent under suitable solvent accessibility conditions, where one or more halomethylcarbonyl compounds are accessible by the protic solvent. In one embodiment the suitable solvent accessibility conditions allow for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0145] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent under suitable solvent accessibility conditions, where one or more halomethylcarbonyl compounds are accessible by the aprotic solvent. In one embodiment the suitable solvent accessibility conditions allow for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0146] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more protic solvent for a suitable time. In one embodiment the suitable time allows for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0147] The term "contacting" includes retaining the one or more halomethylcarbonyl compounds with the one or more aprotic solvent for a suitable time. In one embodiment the suitable time allows for reducing or preventing solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0148] Suitable conditions are discussed in more detail below.

[0149] As used herein, the term "reducing" includes the reduction of amount of substance in comparison with a reference. For example, the term includes the reduction of the production of the amount of an anti-methanogenic halomethane compounds from an amount of a halomethylcarbonyl compound.

[0150] In the context of the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds, reducing includes reducing the amount of anti-methanogenic halomethane compounds produced from halomethylcarbonyl compounds relative to the theoretical maximum amount of anti-methanogenic halomethane compounds that could be produced if all available halomethylcarbonyl compounds are solvolysed.

[0151] As used herein, the term "preventing" includes the reduction of the production of an anti-methanogenic halomethane compounds from a halomethylcarbonyl compound.

[0152] As used herein, the term "increasing" includes the increase of amount of substance in comparison with a reference.

[0153] In the context of the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds, increasing includes increasing the amount of anti-methanogenic halomethane compounds produced from halomethylcarbonyl compounds relative to the theoretical maximum amount of anti-methanogenic halomethane compounds that could be produced if all available halomethylcarbonyl compounds are hydrolysed.

[0154] In the context of the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds, in one embodiment, the one or more parameters is selected fromthe group consisting of Asparagopsis gland cell integrity, solvent availability, temperature, time, pH and volatilisation.Asparagopsis gland cell integrity

[0155] The importance of the gland-cell structures in storage and deployment of anti-methanogenic halomethane compounds by Asparagopsis has been suggested previously. Gland cells are known to be activated by the organism in response to numerous triggers, causing the release of metabolites such as halomethanes including bromoform into the environment. Importantly, the present inventors have demonstrated herein that Asparagopsis biomass stores halomethylcarbonyl compounds that are converted to anti-methanogenic halomethane compounds by hydrolysis when Gland cells are activated. As a result, if Gland cell integrity is increased, halomethylcarbonyl compounds and any anti-methanogenic halomethane compounds stored can be retained in that state, whereas if gland cell integrity is reduced or gland cells activated, anti-methanogenic halomethane compounds (including volatile anti-methanogenic halomethane compounds such as bromoform) will be produced from the stored halomethylcarbonyl compounds.

[0156] The present inventors proposed that during manufacture of anti-methanogenic compositions based on whole seaweed biomass or seaweed biomass meal (e.g. milled biomass), losing the (active) anti-methanogenic halomethane compounds by reducing gland cell integrity is undesirable, as it can reduce the anti-methanogenic potency of the product by loss of volatile anti-methanogenic halomethane compounds during processing.

[0157] However, if extraction of free anti-methanogenic halomethane compounds from Asparagopsis is desired, the present inventors propose that activation / disruption of gland cells is beneficial in the production of the (active) anti-methanogenic halomethane compounds if volatilisation and / or other parameters described herein are considered.

[0158] For example, the experiments in Example 5 were performed on cultured tetrasporophytes of A. taxiformis that were grown under neutral to acidic pH conditions (cf. normal seawater pH = 8.0-8.2). Sample 018-1 was derived by lyophilisation on small scale of a snap-frozen, very high-water content (>95%) sample from this batch, leading to a porous product that was extremely hygroscopic. The difficulty in keeping this sample protected from atmospheric moisture during processing and storage lead to relatively high haloform losses through halomethylcarbonyl precursor compound hydrolysis to form anti-methanogenic halomethane compounds and volatilisation of those formed compounds (see for example Table 14 and Table 15).

[0159] In contrast, a sample from the same culture that was harvested and prepared under different conditions yet lyophilised together in the same run as sample 018-01, resulted in a product with lowered hygroscopicity and increased stability. In particular, sample 018-02 was harvested on 10-fold larger scale, centrifuged to reduce water content to ~80%, and frozen more slowly at -20°C before lyophilisation. Previous workers taught (16) that only the low temperature sublimation conditions employed during freeze drying prevented the volatilisation and loss of bromoform from the cells, which would have been extensively damaged during freezing (18).

[0160] However, the present inventors have demonstrated herein that lyophilisation - rather than preventing volatilisation - provides cold-stabilisation of halomethylcarbonyl compounds (precursors) until the removal of water is largely complete, at which point the rate of precursor hydrolysis is greatly slowed / reduced. On this basis, the present inventors propose that the lyophilised material 018-02 contained mostly intact gland cells and very little free bromoform as only 0.80 mg / g could be extracted into DCM solution (Table 18). When the extraction was repeated with the addition of a protic solvent - aqueous ammonia (20 pL per mL of DCM) - to allow precursor hydrolysis the recovery of bromoform from sample 018-02 was 11.58 mg / g.

[0161] The maximum bromoform that could be recovered from the very hygroscopic sample 018-01 using these extraction techniques was 4.60 mg / g on dried basis. Without being bound by theory, the present inventors suggest that the hygroscopicity of this sample meant it remained at higher water content throughout its lyophilisation, even as its lower thermal mass may have allowed it to thaw earlier next to the larger sample 018-02. Higher water content means greater disruption of gland cells and increased precursor hydrolysis, followed by loss of bromoform through volatilisation.

[0162] Accordingly in one embodiment, the present invention provides a method as described herein, wherein when halomethylcarbonyl compounds (precursors) are to be hydrolysed to form anti-methanogenic halomethane compounds, gland cell integrity is reduced.

[0163] Accordingly, in one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis or mixture thereof has been treated to reduce Asparagopsis gland cell integrity.

[0164] Gland cell integrity is reduced by ultrasonication, osmotic shock, light stress, and physical pressure and / or partial desiccation, which have all been shown to trigger gland cell activation.

[0165] For example, in one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with a one or more protic solvent, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis or mixture thereof has been treated to reduce Asparagopsis gland cell integrity by a treatment selected from the group consisting of freezing, freeze / thaw cycling, ultrasonication, osmotic shock, light stress, physical pressure and partial desiccation.

[0166] In one embodiment, gland cell integrity is reduced by milling the biomass of Asparagopsis or part thereof.

[0167] In another embodiment, gland cell integrity is reduced by increasing disruption of gland cells by friction and physical pressure during milling.

[0168] In a preferred embodiment, gland cell integrity is reduced by freezing the biomass of Asparagopsis or part thereof.

[0169] In another preferred embodiment, gland cell integrity is reduced by allowing frozen biomass of Asparagopsis or part thereof to thaw.

[0170] In one embodiment, gland cell integrity is reduced by increasing disruption of gland cells by freezing to expand intracellular and / or extracellular ice.

[0171] In another embodiment, gland cell integrity is reduced by increasing osmotic disruption of gland cells by contact with concentrated electrolyte solutions excluded from crystallising ice during freezing.

[0172] In another embodiment, gland cell integrity is reduced by increasing osmotic disruption of gland cells by contact with altered electrolyte concentrations in melt waters during thawing.

[0173] In another embodiment, the present invention provides a method as described herein, wherein when halomethylcarbonyl compounds (precursors) are to be hydrolysed to form anti-methanogenic halomethane compounds, gland cell integrity is reduced by freezing wet Asparagopsis.

[0174] As used herein, 'wet Asparagopsis' refers to Asparagopsis biomass that has not had its internal moisture content (completely) dried or substantially dried. For example, 'wet Asparagopsis' may comprise Asparagopsis biomass that is fresh or has been freshly or recently cultivated, collected and / or removed from its growing environment, for example, a seawater or salt water environment; itmay also comprise Asparagopsis biomass that has been cultivated, collected and / or removed from its growing environment and then stored, cooled, and / or transported for a period of time prior to use in the methods described herein.

[0175] The Asparagopsis biomass is preferably cooled between collection and the step of contacting with at least one protic solvent or at least one aprotic solvent, for example, to reduce the premature or uncontrolled hydrolysis of halomethylcarbonyl compounds leading to loss of volatile anti-methanogenic halomethane compounds. Cooling preferably comprises storing the collected or collected and washed macroalgae on ice or refrigerated container during storage and / or transport prior to the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent.

[0176] In another embodiment, gland cell integrity is reduced by contacting the Asparagopsis biomass or part thereof prior to the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent, and / or during the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent under conditions substantially dissimilar to seawater.

[0177] In another embodiment, gland cell integrity is reduced by contacting the Asparagopsis biomass or part thereof prior to the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent, and / or during the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent under pH conditions substantially dissimilar to seawater.

[0178] In another embodiment, gland cell integrity is reduced by contacting the Asparagopsis biomass or part thereof prior to the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent, and / or during the step of contacting the Asparagopsis biomass or part thereof with the at least one protic solvent in freshwater to induce osmotic shock.

[0179] In another embodiment, gland cell integrity is reduced by breaking down the Asparagopsis into smaller portions in the presence of at least one protic solvent or at least one aprotic solvent.

[0180] The step of contacting the biomass with the at least one oil may also include homogenising the biomass or part thereof. As used herein, the term "homogenising" means to break up the biomass to facilitate release of one or more halomethylcarbonyl compounds and / or one or more anti-methanogenic halomethane compounds from the biomass, for example, into a solvent. Thehomogenising can take place by any means known in the art, such as crushing, grinding, milling, blending, cutting, slicing, or dicing.

[0181] After the death of the alga, the gland cell is known to continue to act as a physical barrier against release of halogenated metabolites. Thus, previous research taught that losses of volatile compounds from seaweed could result from rupture of gland cells by osmotic lysis or expansion of intracellular ice on freezing. Using the data described herein, the present inventors now know that non-enzymatic halomethane precursor hydrolysis can occur even in dead algae. As a result, rather than breaches of gland cell integrity inevitably leading to halomethane release, disruption of gland cells is only one step in the release of free halomethanes.

[0182] Accordingly, in one aspect, the present invention provides a method as described herein, wherein when solvolysis of halomethylcarbonyl compounds (precursors) to form anti-methanogenic halomethane compounds is to be reduced, gland cell integrity is increased.

[0183] In one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more aprotic solvent, wherein the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or mixture thereof has been treated to increase Asparagopsis gland cell integrity.

[0184] Gland cell integrity can be increased by avoiding the use of freezing, freeze / thaw cycling, ultrasonication, osmotic shock, light stress, and physical pressure and / or partial desiccation, which have all been shown to trigger gland cell disruption.

[0185] In one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more aprotic solvent, wherein the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or mixture thereof has been treated to increase Asparagopsis gland cell integrity by avoiding a treatment selected from the group consisting of freezing, freeze / thaw cycling, ultrasonication, osmotic shock, light stress, physical pressure and partial desiccation.

[0186] In one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent, wherein the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or mixture thereof has beentreated to increase Asparagopsis gland cell integrity by avoiding a treatment selected from the group consisting of freezing, freeze / thaw cycling, ultrasonication, osmotic shock, light stress, physical pressure and partial desiccation.

[0187] In a preferred embodiment, gland cell integrity is increased by avoiding freezing the biomass of Asparagopsis or part thereof.

[0188] In another preferred embodiment, gland cell integrity is increased by avoiding thawing the frozen biomass of Asparagopsis or part thereof.

[0189] In one embodiment, gland cell integrity is increased by reducing disruption of gland cells by freezing to expand intracellular and / or extracellular ice.

[0190] In another embodiment, gland cell integrity is increased by reducing osmotic disruption of gland cells by contact with concentrated electrolyte solutions excluded from crystallising ice during freezing.

[0191] In another embodiment, gland cell integrity is increased by reducing osmotic disruption of gland cells by contact with altered electrolyte concentrations in melt waters during thawing.

[0192] In another embodiment, gland cell integrity is increased by contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent under osmotic conditions substantially similar to seawater. In another embodiment, gland cell integrity is increased by maintaining the Asparagopsis biomass or part thereof at osmotic conditions substantially similar to seawater prior to contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent.

[0193] In another embodiment, gland cell integrity is increased by contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent under pH conditions substantially similar to seawater. In another embodiment, gland cell integrity is increased by maintaining the Asparagopsis biomass or part thereof at pH conditions substantially similar to seawater prior to contacting the Asparagopsis biomass or part thereof with the at least one protic solvent or at least one aprotic solvent.

[0194] As used herein with reference to gland cell integrity, "increased" refers to maintaining gland cell integrity so that the gland cells to not rupture or degrade etc. thereby releasing their contents. After the death of the alga, the gland cell is known to continue to act as a physical barrieragainst release of halogenated metabolites. Thus, previous research taught that losses of volatile compounds from seaweed could result from rupture of gland cells by osmotic lysis or expansion of intracellular ice on freezing. Using the data described herein, the present inventors now know that non-enzymatic halomethane precursor hydrolysis can occur even in dead algae. As a result, rather than breaches of gland cell integrity inevitably leading to halomethane release, disruption of gland cells is only one step in the release of free halomethanes.

[0195] Accordingly, in another embodiment, the present invention provides a method as described herein, wherein when solvolysis of halomethylcarbonyl compounds (precursors) to form anti-methanogenic halomethane compounds is to be reduced, gland cell integrity is increased.

[0196] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or mixture thereof is snap frozen.

[0197] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or mixture thereof is slowly frozen.

[0198] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or mixture thereof is lyophilised.

[0199] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or mixture thereof is slowly frozen before lyophilisation.

[0200] In one embodiment, the present invention provides a method as described herein, wherein the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or mixture thereof is centrifuged to reduce water content.Solvent availability

[0201] As discussed above, the present inventors have demonstrated that a number of known methods of treating Asparagopsis biomass - such as lyophilisation - rather than preventing volatilisation, provide cold-stabilisation of halomethylcarbonyl compounds (precursors) until the removal of water is largely complete, at which point the rate of precursor hydrolysis is greatlyslowed / reduced. As a result, and as will be discussed in more detail below, altering the availability of protic solvent for halomethylcarbonyl compound (precursor) solvolysis (or altering the availability of aprotic solvent for preventing precursor solvolysis) can control the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds.

[0202] For example, the treatment to reduce gland cell integrity discussed above allows for increased accessibility of protic solvent to produce by solvolysis anti-methanogenic halomethane compounds from halomethylcarbonyl compounds, or of aprotic solvent to reduce solvolysis of halomethylcarbonyl compounds. The treatment to increase gland cell integrity discussed above allows for reduced accessibility of protic solvent to produce by hydrolysis anti-methanogenic halomethane compounds from halomethylcarbonyl compounds.

[0203] As will be discussed in more detail below, the accessibility of halomethylcarbonyl compounds to protic solvent can also be controlled by using an aprotic solvent, such as oil, to solubilise halomethylcarbonyl compounds and thereby limit access of protic solvent to the halomethylcarbonyl compounds.

[0204] The present inventors have demonstrated herein that production of halomethanes from halomethylcarbonyl precursors involves a nucleophile for attack of the carbonyl, and a proton source to protonate the nascent halomethyl carbanion. The reagent (solvent) acting as a proton source may conveniently be water (Scheme 1), or an alcohol such as methanol (Scheme 5), which the present inventors have determined can be used to solvolyse halomethylcarbonyl compounds to form anti-methanogenic halomethane compounds. This previously unappreciated non-enzymatic production of anti-methanogenic halomethane compounds such has bromoform indicates that previous data in the field as to bromoform levels in Asparagopsis derived compositions does not reflect the available bromoform (and / or other available anti-methanogenic halomethane compounds) content of those compositions, because the bromoform (and / or other anti-methanogenic halomethane compound) are formed during the step of quantifying bromoform levels. Put another way, because bromoform (an anti-methanogenic halomethane compound) is formed by solvolysis with methanol when determining levels of bromoform in a sample of a biomass or composition, the levels detected by the assay are higher than the true levels of free bromoform (and / or anti-methanogenic halomethane compound) in the biomass or composition assayed.

[0205] Scheme 5: A simplified version of Scheme 1 showing the products of the haloform reaction when performed in methanol solution instead of water. In this case, methyl esters are formed instead of carboxylic acids.

[0206] The present inventors propose that the production of halomethanes upon methanolysis of stored precursors explains why methanol has become the standard extraction solvent for bromoform assay of Asparagopsis; it can both extract free bromoform and also solvolyse bromoform precursors, thus giving a measure of total bromoform content. As indicated above, the latter has not been appreciated by previous workers attempting to maximise the anti-methanogenic potency of Asparagopsis products and extracts, who have followed the teaching of the early Asparagopsis literature and ruled out the contribution of halomethane precursors.

[0207] For example, in an influential early paper on extraction of fresh Asparagopsis, McConnell and Fenical14noted the presence of methyl esters in their specific methanol extracts, and ethyl esters in their specific ethanol extracts; correctly concluding that ester formation was an artefact of the extraction process. However, they excluded the haloform reaction as a mechanism for ester formation, instead proposing simple esterification of pre-existing acids:"One possible method of producing esters not involving esterification reactions is the solvolysis of 1,1,1-trihaloketones in alcohol, which could produce esters and haloforms. To discount this possibility, we treated 1,1,3,3,3-pentabromoacetone with ethanol and traces of HBrfor 12 hr. GC-MC analysis showed that ethyl esters were not produced."

[0208] In contrast, as is shown in Example 2, treating 1,1,1,3,3,3-hexabromoacetone with methanol does yield a mixture of bromoform and the di- and tribrominated methyl esters. Similarly, treating 1,1,1,3,3,3-hexabromoacetone with methanolic ammonia yields a mixture of bromoform and the di- and tribrominated acetamides. This confirms that halomethylketones such as 1,1,1,3,3,3-hexabromoacetone have the required reactivity to act as anti-methanogenic halomethane compound precursors.

[0209] The Applicant proposes that the general involvement of nucleophilicity and a proton source can be satisfied by a range of other protic solvents or solvent mixtures providing for controlled formation of anti-methanogenic halomethane compounds and / or increased levels of anti-methanogenic halomethane compounds. Similarly, aprotic solvents can be used to reduce the formation of anti-methanogenic halomethane compounds from precursors.

[0210] In one embodiment, the protic solvent is selected from the group consisting of group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

[0211] Conversely, the methods of excluding nucleophilic protic molecules and / or replacing them with solvents (including mixtures of solvents) that are unreactive towards the halomethylcarbonyl compounds described herein provides for the preparation of Asparagopsis products or extracts comprising increased levels of halomethane precursors. The ability to control the levels of halomethylcarbonyl compounds, and thereby reduce the production of volatile anti-methanogenic halomethane compounds, allows for the formation of compositions with increased anti-methanogenic compound forming capacity, and / or increased stability (e.g. reduced loss of anti-methanogenic halomethane compounds).

[0212] Accordingly, in one aspect, the present invention provides herein methods of increasing the stability of an anti-methanogenic composition by excluding nucleophilic and protic solvents that solvolyse or otherwise cleave the halomethane precursors. For example, removing the water from the seaweed under conditions where precursor hydrolysis is inhibited, such as by lyophilisation as is demonstrated in Example 3, and which increases the levels of halomethylcarbonyl compounds (precursors).

[0213] In another embodiment, the present invention provides a method described herein, wherein the use of a protic solvent is avoided, and is replaced with another non-nucleophilic / non-protic solvent, such as an aprotic organic solvent, aprotic solvent mixture, or an oil.

[0214] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein an aprotic solvent is used to solubilise one or more halomethylcarbonyl compounds. In another embodiment, the method comprises one or more halomethylcarbonyl compound with one or more aprotic solvent under conditions to solubilise the one or more halomethylcarbonyl compound. In a further embodiment, the method comprises contacting comprising one or more halomethylcarbonyl compound with an aprotic solvent under conditions to solubilise any one or more anti-methanogenic halomethane compound present. In another embodiment, the method comprisesone or more halomethylcarbonyl compound with one or more aprotic solvent under conditions to reduce solvolysis of the one or more halomethylcarbonyl compound by one or more protic solvent.

[0215] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein one or more protic solvent is used to solubilise and / or solvolyse one or more halomethylcarbonyl compounds. In another embodiment, the method comprises contacting one or more halomethylcarbonyl compounds with a protic solvent under conditions to solubilise and / or solvolyse the one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds. In a further embodiment, the method comprises contacting one or more anti-methanogenic halomethane compounds formed with a protic solvent under conditions to solubilise the one or more anti-methanogenic halomethane compounds present.

[0216] For example, in one embodiment, the present invention provides a method as described herein, wherein the method comprises mixing the seaweed directly with an aprotic solvent, aprotic solvent mixture or an oil. For example, in Example 4, a sample of Asparagopsis was steeped intact in oil in order to prepare an oil-stabilised bromoform product similar to those described previously.19-20After decanting the oil-based product, the residue comprised of extracted seaweed biomass and residual canola oil was retained at 2-8°C for 2 years. As described herein, a further quantity of oil was decanted from the residue, extracted with methanol and with methanol-c / 4 and analysed by GC-MS. The oil was found to contain 3.85 mg / g of bromoform, of which 35.5% was in the form of unhydrolysed bromoform precursors.

[0217] That such a quantity of halomethylcarbonyl precursors remained intact after 2 years of storage demonstrates the advantages of maintaining the precursors in a dry state (e.g. in oil without access to protic solvent). The oil phase contains "'0.5% water, which is demonstrated herein to be insufficient for complete precursor hydrolysis over the refrigerated storage period.

[0218] Accordingly, in one aspect the present invention provides compositions as described herein, wherein the level of halomethylcarbonyl compounds in the composition is maintained following at least one, two, three, or four weeks in storage.

[0219] In another aspect the present invention provides compositions as described herein, wherein the level of halomethylcarbonyl compounds in the composition is maintained following at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months in storage. In another aspect the present invention provides compositions as described herein, wherein the level ofhalomethylcarbonyl compounds in the composition is maintained following at least one or two years in storage.

[0220] From the work described herein, the present inventors note that previous observations on the stability of oil-based Asparagopsis extracts made by published methods is likely due to the unknown presence of halomethane precursors in these extracts. As summarised by previous workers,21counter-intuitive increases in bromoform assay are observed on reanalysis of stored oil extracts:"The study by Magnusson et al. (2020) reported an increase of bromoform content in Asp-Oil after 12 weeks of storage at 4 °C which is in agreement with the findings of this study. However, the increment observed in this study was only 8.4%, compared to the 26.7% reported by Magnusson et al. (2020) at the same storage temperature. The increase of bromoform over time is counter intuitive. However, it may be due to the particulate algal biomass (< 100 pm) remaining in the oil after filtering Asp-Oil to remove biomass, that continues to release bromoform into the oil during storage."

[0221] The present inventors propose that instead of residual particulate algal biomass, slow hydrolysis of oil-soluble bromoform precursors occurred in the compositions referred to above, to increase bromoform over time. The large difference in magnitude between the two reported increases demonstrates that, unlike the methods described herein, it is only once it is known that the production of anti-methanogenic halomethane compounds from halomethylcarbonyl compounds can be controlled, that the retention of halomethylcarbonyl compounds, or the production of anti-methanogenic halomethane compounds, can be increased or decreased, including to desired levels.

[0222] For example, the method of Example 4 demonstrates some enrichment (35.5%) and stabilisation of bromoform precursors, but in the context of other methods described herein, the quantity of precursors stabilised is relatively small, and the 2-year preparation time is impractical. However, using the methods described herein, such as excluding protic solvents, provides for improved, and controlled, production of anti-methanogenic halomethane compounds, or retention of halomethylcarbonyl compounds.

[0223] For example, in Example 5, neutral-acidic seaweed samples lyophilised using appropriate methods can be extracted into aprotic solvents, such as DCM, yielding solutions that are high in bromoform precursors and low in water and free bromoform. The present inventors propose that the further removal of water (a protic solvent) from these extracts using techniques known in the art, suchas drying of solutions using anhydrous solid desiccants, allows for a further increase bromoform precursors.

[0224] In Example 2, the present inventors have demonstrated that methanol is a relatively inefficient solvent for extraction of the total halomethane fraction, possibly because the solubility of halomethane precursors in this solvent is limited. Importantly, the present inventors have demonstrated that aprotic solvents such as DCM, reported by some workers to be a "surprisingly poor" solvent for extraction of seaweed metabolites,15are in fact excellent choices for extraction of total halomethanes once the (previously unappreciated) principle of precursor hydrolysis is accounted for. As DCM is aprotic and non-nucleophilic toward halomethylcarbonyl compounds, it does not participate in precursor solvolysis. The rate of bromoform formation in DCM solution is therefore much slower, even with the addition of small amounts of methanol (see Example 7).

[0225] The present inventors propose that without an understanding of the contribution of the stored halomethane precursor fraction, literature in the field has led to seemingly contradictory recommendations for preparing compositions. For example, Paul et al. extracted freeze-dried seaweed with methanol and with dichloromethane (DCM), stating that "[...] methanol extraction of freeze-dried material consistently yielded the highest amounts of the major halogenated metabolites."-. However, in direct contrast to this, Machado et al. who also compared extraction solvents for freeze-dried seaweed, stated that “The DCM extract had the highest yield of bromoform [...] corresponding to 1723.2 gg^DW of biomass. This is 5.7 times higher than the methanol (301.2 pgg^DW) and hexane (297.0 pgg^DW) extracts."

[0226] Using the principles discussed herein, the present inventors propose that the contradiction in the literature as to conflicting approaches to form effective anti-methanogenic compositions can be resolved. The present inventors propose that extraction with an aprotic solvent such as DCM can recover both the "free" and "potential" halogenated metabolites from a seaweed sample, but only the "free" halomethanes are detected as such in the GC-MS assay. Therefore, unless the DCM extract is solvolysed in contact with a nucleophilic, protic molecule such as water or methanol, the measured halomethane assay result will be erroneously low. Extraction of seaweed samples with a protic solvent such as methanol converts non-volatile "potential" halomethanes to volatile "free" halomethanes, allowing the "total" halomethanes to be detected by GC-MS analysis. The difference between the extraction procedures of Machado et al. and Paul et al. is that the former added extra processing steps including evaporation to dryness and resolvation. The present inventors propose that without the principles described herein, the levels of haloform precursors are notcontrolled during processing, and give widely varying results in relation to actives, including results in which the actives are generated as part of the measurement process.

[0227] Without wishing to be bound by theory, two complementary effects of this additional processing by Machado et al. could have acted to increase the apparent bromoform content of the DCM extract and reduce that of the methanol extract. Evaporation to dryness lowers the volatile free halomethane fraction, therefore the methanol extract would suffer relatively higher potency loss. At the same time these extra processes, and the time taken to perform them, could have enabled slow hydrolysis of halomethane precursors, increasing the free halomethane fraction in the DCM extract and thereby also increasing its apparent bromoform assay.

[0228] Since the work of Paul et al. who taught that methanol was preferred for extraction of bromoform from seaweed samples for GC-MS analysis, methanol has been the standard solvent used by subsequent workers for this purpose. A validated bromoform assay using methanol for extraction was reported by Romanazzi et al.™ and this method has been influential in the field. However, as is demonstrated herein, aprotic solvents are also usable for extraction of total bromoform, as long as the requirement for solvolysis is taken into account before GC-MS quantitation.

[0229] Accordingly, in one embodiment, the present invention provides a method of solubilising one or more halomethylcarbonyl compounds with an aprotic solvent capable of solubilising the halomethylcarbonyl compounds, wherein the aprotic solvent is dichloromethane (DCM).

[0230] Aprotic solvents can also be used to stabilise halomethane precursors (halomethylcarbonyl compounds) by excluding water and other substances that hydrolyse halomethylcarbonyl compounds.

[0231] In some aspects, the aprotic solvent or mixture is contacted with the Asparagopsis biomass, part thereof, extract thereof or a mixture thereof, for a period of time to stabilise halomethane precursors therein. Time of contacting is discussed in more detail below.

[0232] In one embodiment, the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone,acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0233] In a preferred embodiment, the aprotic solvent is selected from the group consisting of chlorinated solvents such as dichloromethane or chloroform, ester solvents such as ethyl acetate, ketone solvents such as acetone, hydrocarbon solvents such as heptane and an oil.

[0234] As used herein, the term "oil" means any non-polar, hydrophobic substance which is typically a liquid at ambient temperature and pressure. Oils may be derived from animals, vegetables, or petrochemicals, and typically have a high carbon and hydrogen content. The oil is preferably an edible oil, and preferably digestible by a ruminant animal. Typically, an oil of vegetable origin is extracted from the seeds or fruits of plants, and is typically comprised primarily of triglycerides. The term "vegetable oil" is a generic term to indicate that the oil is of primarily or exclusively vegetable origin, and may comprise a mixture of one or more oils of vegetable origin or from differing origins.

[0235] As used herein the term "at least one oil" includes a single type of oil, or compositions comprising a single type of oil, or a mixture of two or more oils, or a composition comprising a mixture of two or more oils. The at least one oil includes an oil suitable for application to, administration to, or feeding to, an animal.

[0236] In one embodiment, the at least one oil comprises an edible oil.

[0237] In one embodiment, the edible oil is selected from the group consisting of almond oil, apricot oil, argan oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, colza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, and vegetable oil or any combination thereof.

[0238] In one aspect, the aprotic solvent is an animal- or vegetable-derived fat product.

[0239] In one aspect, the aprotic solvent comprises a medium chain triglyceride.

[0240] In another aspect, the present invention provides a method as described herein, wherein the method further comprises removing the aprotic solvent from the composition formed by contacting one or more halomethylcarbonyl compounds with one or more aprotic solvent.

[0241] Methods of removing the (aprotic) solvent from the composition formed are known in the art, and include filtering or decanting the composition to remove unwanted solids such as biomass, removing the aprotic solvent or mixture by concentrating or evaporating under vacuum to yield an enriched sample of halomethylcarbonyl compounds, or exchanging the aprotic solvent for a different solvent, for example, by evaporation and resolvation, evaporation of a low-boiling solvent or mixture with simultaneous replacement with a higher-boiling solvent or mixture (e.g. solvent swapping), liquid-liquid extraction, or adsorption onto a stabilising excipient, or encapsulating the composition with a stabilising excipient.

[0242] Stabilising excipients are discussed in more detail below.

[0243] In another embodiment, the present invention provides replacing the one or more aprotic solvent in the composition formed by contacting the one or more halomethylcarbonyl compounds with the one more aprotic solvent with one or more protic solvents.

[0244] In a further embodiment, the present invention provides replacing the one or more aprotic solvent in the composition formed by contacting the one or more halomethylcarbonyl compounds with the one or more protic solvents capable of producing one or more anti-methanogenic halomethane compounds from one or more halomethylcarbonyl compounds.

[0245] For example, the present inventors have demonstrated herein that mixtures of strong aprotic solvents such as DCM with substances that promote producing one or more anti-methanogenic halomethane compounds from one or more halomethylcarbonyl compounds (such as ammonia) can increase extraction efficiency of the total halomethane fraction from Asparagopsis biomass compared to poor solvents such as methanol. One advantage of this approach is reduced solvent use; the prior art teaches that preferred extraction volumes with methanol are 10 mL of solvent per 100 mg of lyophilised seaweed.15Our data shows that extraction of 50 mg of lyophilised seaweed with a mixture of aqueous ammonia (20 pL) with either of DCM (1 mL) or methanol (1 mL) gives a greater yield of free bromoform than methanol alone, at approximately one fifth of the relative volume of extraction solvent (Table 16 and Table 18). A further advantage of the DCM / aqueous ammonia solvent system is that a more complete recovery of minor halomethane components is achieved, in particular the iodinated metabolites bromodiiodomethane and bromoiodoacetamide(Table 19). Although these minor components may be components of Asparagopsis or may be produced by extraction chemistry, their presence and quantity is proposed to give an indication of the iodine levels of the Asparagopsis biomass sample. This is important due to the possible toxic effects from feeding seaweed containing high levels of iodine to animals.22

[0246] Although the high precursor-content seaweed samples and extracts are useful in themselves for preparation of anti-methanogenic Asparagopsis products such as feed supplements and feed, the present inventors propose they can also be further improved through chemical transformation. For example, the Haloform reaction of Scheme 1 yields a carboxylic acid by-product. This is the likely source of the haloacetic acids known to be present in Asparagopsis products and extracts, and which could result from hydrolysis of haloacetones as in Scheme 2.

[0247] Importantly, haloacetic acids are inactive as methanogenesis inhibitors.23 4However, Haloform-type cleavage of haloacetones with ammonia as in Scheme 3 is known to give haloacetamides in good yield.25Haloacetamides such as trichloroacetamide are known to be methanogenesis inhibitors.24

[0248] Therefore, one advantage of the methods described herein is that when levels of halomethane precursors are controlled, their breakdown into useful anti-methanogenic compounds can likewise be controlled, or altered, so that desired compounds are produced and / or effective amounts of desired compounds can be produced. In Example 3 and Example 5, seaweed samples were prepared containing increased levels of halomethane precursors. When these samples were extracted into DCM, precursors such as haloacetones were extracted intact, and levels of free bromoform in the extract were low. When a sample was extracted into DCM in the presence of aqueous ammonia, the precursors were cleaved to yield increased levels of free bromoform in the extract, along with haloacetamides formed via chemistry similar to Scheme 3. As haloacetamides are more active methanogenesis inhibitors than the inactive haloacetic acids formed by hydrolysis in the absence of ammonia, this method can yield Asparagopsis products with increased potency.

[0249] Accordingly, in one aspect, the present invention provides a method as described herein, wherein the method comprises contacting a composition comprising one or more halomethylcarbonyl compounds with ammonia or a composition comprising ammonia to form one or more anti-methanogenic haloacetamides in the composition.

[0250] In one embodiment, the composition comprising ammonia is selected from the group consisting of aqueous ammonia, methanolic ammonia, and a formate, an acetate, or a bicarbonate salt of ammonia.

[0251] In another embodiment, the present invention provides a method as described herein, wherein when the composition described herein comprises increased amounts halomethylcarbonyl compounds, the composition is contacted with ammonia that is present in the rumen fluid of ruminant animals, to form haloacetamides in the ruminant animal.

[0252] In another embodiment, levels of ammonia that are naturally present in the rumen fluid of ruminant animals may be supplemented by the controlled addition of suitable sources of ammonia, such as urea, to feed compositions comprising halomethylcarbonyl compounds.

[0253] When preparing an anti-methanogenic halomethane compositions it is desirable to have a reproducible metabolite profile and / or a standardised anti-methanogenic activity.

[0254] Based on the data set out in the Examples, the present inventors propose that the methods described herein can be used to control the levels of halomethane precursors and / or anti-methanogenic halomethane compounds in the compositions described herein, the levels of which are uncontrolled using the methods of the prior art. An advantage of this control is the ability to control the levels and composition of anti-methanogenic halomethane compounds administered to, and / or produced upon, feeding the compositions described herein to ruminants.Temperature

[0255] Low temperatures are known to be beneficial during extraction or processing of samples containing free halomethanes. This effect is through slowing the rate of loss of halomethanes by volatilisation and by chemical degradation. In their validated assay for bromoform content of Asparagopsis samples, previous workers15teach that "The temperature of the samples was kept as low as was practically possible throughout the initial sample handling, transport, and storage to minimize loss of bromoform through evaporation", and the extraction with methanol was performed at 0°C in an ice slurry.

[0256] Similarly, recognised methods for analysis of Volatile Organic Compounds (VOCs) including bromoform by GC-MS such as US EPA method 524.4 recommend low-temperature controls such as refrigerated autosamplers (10°C).

[0257] However, based on the data and principles described herein, the present inventors have demonstrated that low temperatures slow the rate of solvolysis reactions that generate free halomethanes. As a result of the principles described herein, compositions comprising significant quantities of halomethane precursor compounds will require sufficient time for the solvolysis to go to completion, or the measured anti-methanogenic halomethane compounds content will be erroneously low. Without an appreciation of the contribution of precursor chemistry described herein, certain samples may confusingly give poorly reproducible analysis despite rigorous attention to maintaining cold temperatures.

[0258] Accordingly, in one aspect the present invention provides a method as described herein wherein contacting is performed at a temperature suitable for solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds. In another embodiment, contacting is also performed at a temperature suitable for reducing evaporation of one or more anti-methanogenic halomethane compounds.

[0259] In one aspect the present invention provides a method as described herein wherein contacting is performed at a temperature suitable for complete solvolysis of one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds. In another embodiment, contacting is also performed at a temperature suitable for reducing evaporation of one or more anti-methanogenic halomethane compounds.

[0260] The present inventors propose that the particular temperatures required may also depend on the other parameters described herein, such as the water content of the Asparagopsis sample or extract. As Example 3 shows, a sample of seaweed (initial water content "'80%) that was subjected to temperatures of <50°C for 100 minutes during microwave vacuum drying lost half of its extractable bromoform content through destruction and / or hydrolysis of precursors and volatilisation of free bromoform. However, an identical sample held at low temperature while the bulk of its water was removed by lyophilisation was then able to tolerate an extended secondary drying phase at elevated temperature (20°C for 6 h) without loss of bromoform (recovery 107%).

[0261] The temperature at which the biomass can be contacted with the at least one protic solvent is not limited, insofar as the one or more halomethylcarbonyl compounds and the anti-methanogenic halomethane compounds do not evaporate / sublime appreciably from the solvent or composition, and / or does not degrade appreciably at the temperature at which the contacting takes place. The temperature may be selected from the group consisting of -78°C to -50°C, -50°C to -20°C, -20°C to -5°C, -5°C to 0°C, -5°C to 4°C, 0°C to 4°C, 4°C to 10°C, 10°C to 20°C, 20°C to 25°C, 25°C to30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C. In some embodiments, the temperature may be selected from the group consisting of -78°C, -20°C, -5°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C and 30°C. In one embodiment, the temperature is preferably about 4°C. In another embodiment, the temperature is preferably about 25°C. Suitable temperatures can be achieved using freezers, chillers, fridges, air-conditioners and the like.

[0262] As used herein, the term "room temperature" is used to indicate normal ambient temperature. As one of skill in the art will appreciate, ambient conditions will differ depending on the geographical location and time of year, such that room temperatures may vary from below zero Celsius to above 40°C. Typically, room temperature is considered to be between about 20°C and about 25°C.Time

[0263] A further parameter that affects the rate of hydrolysis of halomethane precursors is the reaction time. Higher temperatures do not necessarily lead to hydrolysis of precursors, if the seaweed sample or seaweed extract containing them is exposed to high temperatures for a relatively short period of time.

[0264] In the unoptimized microwave vacuum drying procedure of Example 3, a sample suffered destruction and / or hydrolysis of the majority of its contained halomethane precursors during drying at <50°C for 100 minutes.

[0265] Other drying technologies are available with a much shorter residence time. For example the Vortair cyclonic drying system operates at a similar temperature of <50°C, but the residence time of the drying material is only a few seconds.

[0266] The time for which the one or more halomethylcarbonyl compounds are contacted with the protic solvent to produce the one or more anti-methanogenic halomethane compounds is not limited, insofar as the one or more halomethylcarbonyl compounds and the anti-methanogenic halomethane compounds do not evaporate / sublime appreciably from the mixture, and / or does not degrade appreciably during heating. The time period may be selected from the group consisting of 1 second to 1 minute, 1 minute to 2 minutes, 2 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 60 minutes, 60 minutes to 70 minutes, 70 minutes to 80 minutes, 80 minutes to 90 minutes, 1.5 hours to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, and 4 hours to 5 hours. In someembodiments, the time period may be selected from the group consisting of 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, and 3 hours.£H

[0267] The Haloform reaction (Scheme 1) is known to be catalysed by base. In extreme cases such as trichloroacetophenone, no reaction at all is observed at acidic pH; as reviewed by Fuson & Bull:4"[...] trichloroacetophenone, although unchanged by seven hours' heating with water at 170°C., is immediately decomposed by the addition of one drop of potassium hydroxide solution to a methyl alcohol solution of the ketone."

[0268] As documented in the same review, other structural types undergo Haloform-type cleavage under much milder conditions. For example, the weak base sodium acetate is sufficient to catalyse the hydrolysis of hexabromodiacetyl.

[0269] As is shown in Example 5, the present inventors have demonstrated that Asparagopsis halomethane precursor compounds undergo significant solvolysis even at acidic pH. Seaweed samples cultured at neutral-acidic pH and freeze-dried from dilute acetic or citric acid solution yield bromoform upon solvolysis with methanol (Table 17). The reaction is not inhibited by acid, but simply proceeds at its minimum rate as the concentration of catalytic hydroxide ions is very low at acidic pH.

[0270] Previous workers claim26that control of pH is important for extraction of bromoform from Asparagopsis seaweed into oil. Although they provide no examples where pH was controlled, the pH ranges that they claim are neutral on average (5-9, 6-8, or about 7).

[0271] In surprising contrast, the present inventors have demonstrated that control of pH gives control over the rate of conversion of halomethylcarbonyl compounds to free halomethanes (anti-methanogenic halomethane compounds). The present inventors propose that maintaining an acidic pH prevents base-catalysed enhancement of precursor hydrolysis, thereby minimising levels of free bromoform. This provides advantages, for example during processing and storage of Asparagopsis where loss of bromoform by volatilisation is undesirable.

[0272] In Example 6, live seaweed placed into tap water for two hours lost bromoform equivalent to 3.5 mg / g of seaweed on dried basis; up to a third of what the present inventors havedemonstrated are its available stores. This is the well-known consequence of the activation of gland cells under conditions of osmotic shock.16

[0273] However, live seaweed placed into 3% aqueous acetic or citric acid solutions lost only 0.7 mg / g of bromoform (Table 17), and when these samples were freeze-dried, the standard extraction with methanol gave an erroneously low bromoform assay. Addition of base to the methanol extraction solvent gave respective 2.0- and 3.7-fold increases in the recovery of bromoform (Table 16); higher in the case of citric acid which is both a stronger acid and much less able to be removed by volatilisation under freeze-drying. These results indicate that minimisation of the rate of precursor hydrolysis occurs under acidic conditions.

[0274] Conversely, basic pH increases the rate of precursor hydrolysis, which is usefully employed when high levels of free bromoform are desired, or to maximise the yield of bromoform that is extracted for some other purpose such as the preparation of a stabilised bromoform product. Complete conversion of precursors to bromoform is necessary in order to correctly assay total bromoform in a sample, including an Asparagopsis sample, and the present inventors show this may be accelerated by the addition of base if preferred.

[0275] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein the step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at an acidic pH. In another embodiment, the present invention provides a method as described herein, wherein the step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a basic pH.

[0276] In a preferred embodiment, the base used to accelerate hydrolysis solvolysis of halomethane precursors is a weak base, preferably ammonium hydroxide. This may be added in the form of an aqueous solution of ammonia. Alternatively, some other form, salt, mixture or solution containing ammonia may be added, such that ammonia mixes with the water present in the seaweed sample or product or extract to form ammonium hydroxide. Ammonia in the absence of water, such as methanolic ammonia solution, may be less effective as a base for catalysing precursor hydrolysis than mixtures that can form hydroxide ions (see examples in Table 18), however note previous discussion of ammonia's effectiveness as a nucleophilic reagent. Other weak bases that may be usefully employed include salts of weak acids such as acetate or bicarbonate salts.

[0277] In the corresponding experiment in Example 5, live seaweed that was cultured under neutral-acid conditions and then placed into 1.3% aqueous sodium bicarbonate lost an intermediate amount of its stored bromoform into the solution (1.46 mg / g on dried basis; Table 17). This solution was chosen to be more similar in buffer composition and pH to natural seawater than either of the tap water or acid solutions used in the previous experiments, and therefore likely to cause less activation of gland cells. On decanting and freeze-drying of this seaweed sample, the standard methanol extraction gave a high recovery of contained bromoform without added base, due to the presence of residual sodium bicarbonate (Table 16) which was available to catalyse the solvolysis of precursors.

[0278] Generally, the seaweed samples that were cultured and / or treated under acid conditions (Table 17) tended to show a relatively wide variability in the bromoform assay results obtained through the standard methanol extraction, and a larger increase in the assay value when base was added to catalyse the solvolysis of halomethane precursors (Table 16). Seaweed samples grown under slightly basic conditions such as ocean-harvested samples showed a closer agreement between the base-catalysed and uncatalyzed methanol extractions (Table 2). Note that hydrolysis of halomethylcarbonyl compounds in the presence of water produces carboxylic acids according to the reaction of Scheme 1, and therefore a fall in the pH of stored samples may be at least partly indicative of ongoing precursor hydrolysis.

[0279] In one embodiment, wherein the step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at an acidic pH, the pH is 6 or less, 5.5 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.5 or less.

[0280] In one embodiment, wherein the step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a basic pH, the pH is 8 or more, 8.5 or more, 9 or more, 9.5 or more, 10 or more, 10.5 or more, 11 or more, 11.5 or more, 12 or more, 12.5 or more, 13 or more, or 13.5 or more.Volatilisation

[0281] Previous workers16taught that lyophilisation conditions must be carefully chosen to avoid the loss of bromoform by volatilisation. Those skilled in the art know that volatilisation occurs when the pressure is lower than the vapour pressure of bromoform at the particular temperature used.

[0282] The saturated vapour pressure of pure bromoform at 20°C is 6.70 hPa.27The vapour pressure at a different temperature may be calculated using the Clausius-Clapyron equation, from the data at 20°C and the molar enthalpy of vaporisation (46.05 kJ / mol).28

[0283] Sample A9 (Example 3) was dried by microwave vacuum drying at a temperature of <50°C and a pressure of 20-30 hPa. As detailed above, during the manufacture of this sample the conversion of precursors into free haloforms was almost complete with only 5.8% remaining as measured for the bromoform peak using the deuterated methanol assay. The vapour pressure of bromoform at 50°C can be calculated with the Clausius-Clapyron equation yielding a value of 38.71 hPa. In this case, since the pressure used for microwave vacuum-drying was lower than the vapour pressure of bromoform, then loss of bromoform by volatilisation would be expected. Indeed, this was observed in the sample A9 which was microwave vacuum-dried, which contained only 13.4 mg / g of bromoform corresponding to a recovery of 56%.

[0284] The lyophilised sample A10 (Example 3) demonstrates the benefits of drying an Asparagopsis sample where the bulk of the halomethanes remain in halomethylcarbonyl precursor form (48-75.8% at the time of analysis). In this case the lyophilisation program included an extended secondary drying step at 20°C and 2 hPa for 6 h. As the vapour pressure of bromoform (6.70 hPa) is higher than the pressure during lyophilisation, free bromoform is expected to be lost under these conditions, but the total bromoform content is preserved using the methods described, with a recovery of 107%.

[0285] Using the methods claimed herein, a wider range of drying conditions can be employed to allow finer control over other properties of the product in addition to potency, such as final water activity (control of microbial growth), porosity (susceptibility to oxidation; hygroscopicity), bulk density and particle form (dust formation; rate of rehydration).

[0286] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a pressure above the vapour pressure of the one or more anti-methanogenic halomethane compounds produced. In another embodiment, the present invention provides a method as described herein, wherein step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a pressure below the vapour pressure of the one or more anti-methanogenic halomethane compounds produced.

[0287] In another embodiment, the present invention provides a method as described herein, wherein step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a pressure above the vapour pressure of the one or more halomethylcarbonyl compounds contacted with the one or more protic solvent or one or more aprotic solvent.

[0288] In a further embodiment, the present invention provides a method as described herein, wherein step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a pressure above the vapour pressure of the one or more halomethylcarbonyl compounds contacted with the one or more protic solvent or one or more aprotic solvent and the vapour pressure of the one or more halomethylcarbonyl compounds contacted with the one or more protic solvent or one or more aprotic solvent.

[0289] In a preferred embodiment, the present invention provides a method as described herein, wherein step of contacting one or more halomethylcarbonyl compounds with one or more protic solvent or one or more aprotic solvent is performed at a pressure above an equivalent to 6.7 hPa at 20°C.

[0290] In the context of the present invention, the term "precursors" as used herein refers to halomethylcarbonyl compounds that can be used to produce anti-methanogenic halomethane compounds, for example, by hydrolysis / solvolysis. For example, in the context of Asparagopsis gland cells, the term precursors or halomethylcarbonyl compounds refers to naturally occurring halomethylcarbonyl compounds of Asparagopsis, such as those stored in gland cells, and which are used to produce halomethane compounds also present in - and released from - Asparagopsis.

[0291] In one embodiment, the protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

[0292] As used herein, and as indicated above, the term "aprotic solvent" refers to a solvent that cannot readily donate protons (H+) to solutes. The term aprotic solvent also includes mixtures of one or more aprotic solvents.

[0293] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is contacted with the one or more proticsolvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0294] Importantly, the present inventors have demonstrated that not only can halomethylcarbonyl compounds be solvolysed to form anti-methanogenic halomethane compounds, extracts of Asparagopsis biomass comprising one or more halomethylcarbonyl compounds can be used for solvolysis to form anti-methanogenic halomethane compounds

[0295] Accordingly, the one or more halomethylcarbonyl compounds can be present in a composition derived from Asparagopsis biomass or a part thereof. For example, the one or more halomethylcarbonyl compounds could be present in a composition formed by extracting biomass or a part thereof into an extraction liquid.

[0296] For example, in one embodiment, the present invention provides a method as described herein, wherein the extract of Asparagopsis is formed by providing a biomass of Asparagopsis; providing an extraction liquid; and contacting the biomass with an extraction liquid under conditions to extract at least one bioactive agent from the biomass into the extraction liquid to form an Asparagopsis extract.

[0297] In one embodiment, the extraction liquid comprises an oil.

[0298] In another embodiment, the extraction liquid is a heterogeneous liquid.

[0299] As used herein, the term a "heterogeneous liquid" refers to a liquid comprises two or more phases. A "phase" refers to any part of a sample that has a uniform composition and properties. For example, when an aqueous solution and at least one oil is mixed they form a heterogeneous liquid comprising an oil phase and an aqueous solution phase.

[0300] In one embodiment, the extraction liquid comprises an oil and a protic solvent, such as water.

[0301] In another embodiment, the present invention provides a methods as described herein, wherein the extract of Asparagopsis is formed by providing a biomass of Asparagopsis or part thereof; providing at least one oil; and contacting the biomass with the at least one oil under conditions to extract at least one bioactive agent from the biomass into the at least one oil to form the Asparagopsis extract.

[0302] As used herein, the term a "heterogeneous liquid" refers to a liquid comprises two or more phases. A "phase" refers to any part of a sample that has a uniform composition and properties. For example, when an aqueous solution and at least one oil is mixed they form a heterogeneous liquid comprising an oil phase and an aqueous solution phase.

[0303] In one embodiment, following contacting the Asparagopsis biomass with the extraction liquid, the extraction liquid is separated from the Asparagopsis biomass or part thereof, to obtain the Asparagopsis extract.

[0304] Techniques for recovery of the extract that are known in the art include separating the extraction liquid from the biomass by decanting or filtration

[0305] In another embodiment, the Asparagopsis extract is formed following contacting the Asparagopsis biomass or part thereof with the extraction liquid under conditions to extract the one or more anti-methanogenic halomethane compounds and / or the one or more halomethylcarbonyl compounds into the extraction liquid.

[0306] In another embodiment, an Asparagopsis extract comprising one or more anti-methanogenic halomethane compounds and / or the one or more halomethylcarbonyl compounds is contacted with a protic solvent to solvolyse the one or more halomethylcarbonyl compounds, according to a method as described herein.

[0307] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0308] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0309] In one embodiment, the present invention provides a method as described herein, wherein the halomethylcarbonyl compound is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0310] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

[0311] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, 1, 1,1, 3, 5,5,5-heptabromopentan-2, 4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, 1, 1,3, 3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one..

[0312] In one embodiment, the present invention provides a method as described herein, wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

[0313] In one embodiment, the present invention provides a method as described herein, wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

[0314] As indicated above, the present inventors have demonstrated herein that parameters including pH, solvent accessibility, temperature and time can be used to control the rate of solvolysis, and pressure can be used to control detectable yield. Accordingly, suitable parameters can be selected to control the solvolysis of extracts of biomass, as is discussed above.

[0315] As a simple example also discussed above, the present inventors have demonstrated that basic pH increases the rate of precursor solvolysis.

[0316] Accordingly, in one embodiment, the present invention provides a method as described herein, further comprising contacting the one or more halomethylcarbonyl compounds with one or more protic solvent in the presence of a base.

[0317] In one embodiment, the present invention provides a method as described herein, wherein the base is selected from the group consisting of hydroxide, bicarbonate, carbonate, ammonia, aqueous ammonia, methanolic ammonia, and ethanolamine.

[0318] Conversely, acidic pH does not increase the rate of precursor solvolysis. Accordingly, in one embodiment, the present invention provides a method as described herein, further comprising contacting the one or more halomethylcarbonyl compounds with one or more protic solvent in the presence of an acid.

[0319] Importantly, the present inventors have demonstrated that not only can halomethylcarbonyl compounds be solvolysed to form anti-methanogenic halomethane compounds, and extracts comprising halomethylcarbonyl compounds be solvolysed to form anti-methanogenic halomethane compounds, but Asparagopsis compositions can be treated to solvolyse halomethylcarbonyl compounds to form anti-methanogenic halomethane compounds, or treated to prevent the solvolysis of halomethylcarbonyl compounds to reduce formation of anti-methanogenic halomethane compounds.

[0320] Importantly, as indicated above, the present inventors have demonstrated herein that parameters including pH, solvent accessibility, temperature and time can be used to control the rate of solvolysis, and pressure can be used to control perceived yield. Additionally, for Asparagopsis biomass, gland cell integrity can be used to control solvolysis.

[0321] Therefore, suitable parameters can be selected to control the solvolysis of Asparagopsis compositions, as is discussed above.

[0322] In one embodiment, the present invention provides a method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent.

[0323] In one embodiment, following contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with the one or more protic solvent, the protic solvent is separated from the Asparagopsis biomass or part thereof.

[0324] In another embodiment, following contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with the one or more aprotic solvent, the aprotic solvent is separated from the Asparagopsis biomass or part thereof.

[0325] In one embodiment, the present invention provides a method of increasing the levels of at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent.

[0326] Asparagopsis has a heteromorphic life history with two free-living life history stages - a gametophyte (large foliose form) and a sporophyte (or tetrasporophyte - smaller, filamentous form). Historically, the tetrasporophyte was recognised as a separate genus (Falkenbergia). Therefore, the term "Asparagopsis" as used herein refers to the genus Asparagopsis, and other taxonomic classifications now known to belong to the genus Asparagopsis.

[0327] There are at least two recognised species of Asparagopsis, one tropical / sub-tropical (Asparagopsis taxiformis) and one temperate (Asparagopsis armata) which are present throughout the world.

[0328] In one embodiment, the species of Asparagopsis is selected from Asparagopsis taxiform is or Asparagopsis armata.

[0329] In another aspect, a biomass of at least one species of red marine macroalgae selected from a species of belonging to the other genera of red seaweed in the family Bonnemaisoniaceae to which Asparagopsis belongs (for example, Bonnemaisonia, Delisea, Ptilonia, Leptophyllis and Pleuroblepharidella) is used in place of a biomass of Asparagopsis in the methods and compositions provided herein. Without wishing to be bound by theory, the six genera of red seaweed in the family Bonnemaisoniaceae (for example Asparagopsis, Bonnemaisonia, Delisea, Ptilonia, Leptophyllis and Pleuroblepharidella), produce and store bioactive halogenated secondary metabolites with bioactive properties, including the anti-methanogenic compounds described herein.

[0330] As used herein the term "providing a biomass" includes the provision or use of a biomass of Asparagopsis or a part thereof removed from water immediately prior to contacting the biomass with the at least one solvent. In one embodiment the biomass of Asparagopsis or part thereof is collected from its environment and placed directly into at least solvent. In another embodiment the biomass of Asparagopsis is contacted with the at least one aprotic solvent or the at least one proticsolvent within 5, 4, 3, 2, or 1 hours from the removal of biomass or part thereof from its environment. In another embodiment the biomass of Asparagopsis is contacted with the at least one aprotic solvent or the at least one protic solvent following treating the biomass or part thereof using the parameters referred to herein.

[0331] As use herein "a part thereof" with reference to Asparagopsis includes parts of Asparagopsis, such as parts of a particular lifecycle stage (e.g. plumose branches and extruding barbs of gametophytes, or gland cells of tetrasporophytes etc).

[0332] As used herein "an extract" of Asparagopsis refers to a composition formed by contacting a biomass of Asparagopsis or a part thereof, or cells of Asparagopsis with one or more solvents to extract one or more anti-methanogenic halomethane compounds and / or one or more one or more halomethylcarbonyl compounds into the one or more solvents.

[0333] As used herein the term "cells" refer to cells of Asparagopsis, including Asparagopsis gland cells which comprise one or more anti-methanogenic halomethane compounds and / or one or more one or more halomethylcarbonyl compounds.

[0334] In one embodiment, the biomass of Asparagopsis comprises one or more halomethylcarbonyl compounds.

[0335] In another embodiment, the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof is contacted with the one or more protic solvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form one or more anti-methanogenic halomethane compounds.

[0336] In one embodiment, the present invention provides a method as described herein, wherein the halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0337] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0338] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of ahaloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0339] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound

[0340] In one embodiment, the present invention provides a method as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, 1, 1,1, 3, 5,5,5-heptabromopentan-2, 4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, 1, 1,3, 3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

[0341] In one embodiment, the present invention provides a method as described herein, wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, ethylene glycol monomethyl ether.

[0342] In one embodiment, the present invention provides a method as described herein, wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, carbon tetrabromide or any combination thereof.

[0343] In one embodiment, the present invention provides a method as described herein, wherein the Asparagopsis is A. armata or A. taxiformis.

[0344] In a preferred embodiment, the present invention provides a method as described herein, further comprising a step of separating the at least one protic solvent from the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof.

[0345] The present inventors propose that using the principles described herein, a composition of the invention can be contacted with rumen fluid, with rumen fluid acting as a protic solvent to solvolyse the one or more halomethylcarbonyl compounds to from one or more anti-methanogenic halomethane compounds. Furthermore, as described herein, ammonia present in the rumen fluid can form anti methanogenic haloacetamides by solvolysis of the one or more halomethylcarbonyl compounds.

[0346] Accordingly, in one embodiment, the present invention provides a comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with rumen fluid within the rumen of a ruminant animal.

[0347] In one embodiment, the present invention provides a method as described herein, wherein the level of the at least one anti-methanogenic halomethane compounds in the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, contacted with the at least one protic solvent is increased relative to the level of the at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, not contacted with a protic solvent.

[0348] Importantly, the present inventors have demonstrated herein that Asparagopsis biomass or parts thereof can be treated using the principles described herein to control the solvolysis of one or more halomethylcarbonyl compounds. As a result, Asparagopsis biomass or parts thereof can be treated to increase the levels of one or more halomethylcarbonyl compounds relative to untreated Asparagopsis biomass or parts thereof, or treated to increase the levels of one or more anti-methanogenic halomethane compounds relative to untreated Asparagopsis biomass or parts thereof.

[0349] For example, gland cell integrity, solvent accessibility, pH, time, temperature and pressure can be used to control solvolysis, as is discussed above.

[0350] Accordingly, in one embodiment, the present invention provides a method of preparing a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, having an increased levels of at least one or more halomethylcarbonyl compounds, said method comprising a step of decreasing the solvolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof.

[0351] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein the method comprises a step of increasing the integrity of intact gland cells in the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof.

[0352] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein the method comprises a step of decreasing the rupture of intact gland cells in the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof.

[0353] In another embodiment, the present invention provides a method as described herein, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with one or more aprotic solvent or mixture of aprotic solvents.

[0354] In a further embodiment, the present invention provides a method as described herein, wherein the one or more aprotic solvent or mixture of aprotic solvents is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, / V-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0355] In a further embodiment, the present invention provides a method as described herein, wherein the method comprises a step of reducing the levels of protic solvent or protic substance in the biomass of Asparagopsis or a part thereof, cells, or extract of Asparagopsis.

[0356] In a further embodiment, the present invention provides a method as described herein, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with at least one acid.

[0357] In a further embodiment, the present invention provides a method as described herein, further comprising a step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with at least one acid and at least one further solvent.

[0358] In a further embodiment, the present invention provides a method as described herein, wherein the at least one acid is selected from the group consisting of formic acid, acetic acid, propanoic acid, pyruvic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, malonic acid, malic acid, phosphoric acid, tartaric acid, trichloroacetic acid, tribromoacetic acid.

[0359] In a further embodiment, the present invention provides a method as described herein, wherein the at least one further solvent is a protic solvent.

[0360] In a further embodiment, the present invention provides a method as described herein, wherein the protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, ethylene glycol monomethyl ether

[0361] In a further embodiment, the present invention provides a method as described herein, wherein the step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with the at least one acid reduces the pH of the mixture to 6 or less, 5 or less, 4 or less, 3 or less, 2 or less or 1 or less.

[0362] In another aspect, the present invention provides a method of preparing an anti-methanogenic halomethane composition, said method comprising: contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with an extraction liquid comprising at least one aprotic solvent or an acid to decrease the hydrolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, and to extract the one or more halomethylcarbonyl compounds or the one or more halomethylcarbonyl compounds and the one or more anti-methanogenic halomethane compounds into the extraction liquid.

[0363] In a further embodiment, the present invention provides a method as described herein, further comprising separating the extraction liquid from the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, to obtain an anti-methanogenic halomethane composition comprising one or more halomethylcarbonyl compounds, or one or more halomethylcarbonyl compounds and one or more anti-methanogenic halomethane compounds.

[0364] Techniques for the removal or biomass are known and include decanting or filtration.

[0365] In one embodiment, the at least one aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0366] In a further embodiment, the present invention provides a method as described herein, further comprising the step of separating the at least one aprotic solvent from the extraction liquid.

[0367] In a further embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of removing the aprotic solvent from the composition.

[0368] Methods of removing solvents are known in the art.

[0369] In a further embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of removing the aprotic solvent from the composition under vacuum.

[0370] In a further embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of contacting the composition with a further solvent.

[0371] In a further embodiment, the present invention provides a method as described herein, wherein the method further comprises a step of adsorbing the composition or a fraction thereof into a stabilising excipient, or encapsulating the composition or a fraction thereof with a stabilising excipient.

[0372] In one embodiment, stabilising excipient is selected from the group consisting of an edible protein, an edible wax, a carbohydrate, a grease, an oil, a cyclodextrin, a molasses, a saturated fat and mixtures thereof.

[0373] Carbohydrates include molasses, cyclodextrins, lactose, dextrose, sucrose, glucose, fructose, galactose, xylose, arabinose, beta-glucans, galactans, pectins, and the like.

[0374] In another embodiment, the stabilising excipient is a cyclodextrin.

[0375] Cyclodextrins (sometimes called cycloamyloses) are cyclic oligosaccharides that contain glucose units, i.e., (a-l,4)-linked a-D-glucopyranose units, bound together in a ring. Cyclodextrins are typically produced from starch by means of enzymatic conversion. Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone or hollow stopper shape; for example, a (alpha)-cyclodextrin comprises a 6-membered sugar ring molecule, p (beta)-cyclodextrin comprises a 7-membered sugar ring molecule; and y (gamma)-cyclodextrin comprises an 8-membered sugar ring molecule. The cyclodextrins suitable for the compositions of the present invention can, if desired, be modified by the addition of substituents. As used herein, "cyclodextrins" include both modified and unmodified cyclodextrins. Substituents generally replace either the entire hydroxyl group or the hydrogen atom on one or more of the hydroxyl groups of the cyclodextrin ring.

[0376] The cyclodextrin may be selected from a-cyclodextrin, p-cyclodextrin or y- cyclodextrin or derivatives thereof which may be naturally and / or synthetically produced.

[0377] As used herein, "molasses" includes a syrup produced as a by-product of processing sugar cane or other vegetable products. A few examples include sugarcane waste (by-product of sugar production from sugarcane); high test (cane) molasses (primary product squeezed from sugarcane); sugarcane molasses (Byproduct of the process of refining unrefined brown sugar into white sugar); Sugar radish molasses (byproduct when sugar is produced from sugar beet); Citrus molasses (fruit juice squeezed in the production of dried citrus pulp); amongst others. In one embodiment, the bromoform stabilising excipient is an edible non-polar substance.

[0378] Edible non-polar substances include edible oils, which are discussed herein.

[0379] In a further embodiment, the present invention provides a method as described herein, wherein the level of the at least one halomethylcarbonyl compound in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof contacted with an aprotic solvent is increased relative to the level of at least one halomethylcarbonyl compound not contacted with an aprotic solvent.

[0380] In another aspect, the present invention provides a composition produced by a method as described herein.

[0381] As discussed above, in one embodiment, the present invention provides methods and compositions as described herein wherein the composition comprises one or more synthetic halomethylcarbonyl compound and / or one or more synthetic anti-methanogenic halomethane compound. This can also include compositions comprising both a) one or more Asparagopsis derived halomethylcarbonyl compound and / or one or more Asparagopsis derived anti-methanogenic halomethane compound, or b) one or more synthetic halomethylcarbonyl compound and / or one or more synthetic anti-methanogenic halomethane compound in combination with one or more Asparagopsis derived halomethylcarbonyl compound and / or one or more Asparagopsis derived anti-methanogenic halomethane compound.

[0382] In one embodiment, the present invention provides methods and compositions as described herein wherein the one or more halomethylcarbonyl compound consists of one or more synthetic halomethylcarbonyl compound and / or the one or more anti-methanogenic halomethane compound consists of one or more synthetic anti-methanogenic halomethane compound.

[0383] In one embodiment, the present invention provides methods and compositions as described herein wherein the one or more halomethylcarbonyl compound consists of one or more Asparagopsis-demed halomethylcarbonyl compound and / or more synthetic halomethylcarbonyl compound, and / or the one or more anti-methanogenic halomethane compound consists of one or more Asparagopsis-demed anti-methanogenic halomethane compound and / or one or more synthetic anti-methanogenic halomethane compound.

[0384] The present inventors have demonstrated herein that halomethylcarbonyl precursor compounds can be used to form bromoform, including that synthetic halomethylcarbonyl precursor compounds can be used to form bromoform.

[0385] Accordingly, in one embodiment, the present invention provides an anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and at least one aprotic solvent.

[0386] Suitable aprotic solvents are discussed herein.

[0387] In another embodiment, the present invention provides an anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and one or more halomethylcarbonyl compound stabilising excipient.

[0388] As used herein the term "halomethylcarbonyl compound stabilising excipient" refers to an excipient which prevents loss of halomethylcarbonyl compound from the composition. In one embodiment, the halomethylcarbonyl compound stabilising excipient allows the halomethylcarbonyl compound to be bioavailable in a ruminant animal. Halomethylcarbonyl compound stabilising excipients include those described herein, including edible solids, liquids and semi-solids that are able to stabilise halomethylcarbonyl compounds, and also include medium-chain triglyceride derived compositions.

[0389] Suitable halomethylcarbonyl compound stabilising excipients are known to those skilled in the art and include acidic buffering / binding agents such as stearic acid, moisture-absorbing compounds such as colloidal silica, or materials that form a barrier including waxes such as carnauba wax, modified celluloses such as cellulose acetate, and polymers such as polyvinyl acetate or methacrylate copolymers. Preferably, the stabilising excipient is safe for consumption by animals at final concentration in the dose of the composition to be provided to animals.

[0390] In one embodiment, the anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and one or more halomethylcarbonyl compound stabilising excipient is provided in a form whereby the one or more halomethylcarbonyl compounds are prevented from contacting a protic solvent by the halomethylcarbonyl compound stabilising excipient. For example, a solid composition of one or more halomethylcarbonyl compound is coated with one or more halomethylcarbonyl compound stabilising excipient.

[0391] In one aspect the present invention provides a method of making an anti-methanogenic composition comprising contacting one or more halomethylcarbonyl compounds with one or more halomethylcarbonyl compound stabilising excipient, wherein the halomethylcarbonyl compound stabilising excipient is selected from the group consisting of an edible oil or edible carbohydrate.

[0392] In one aspect the present invention provides a process for preparing a composition comprising one or more synthetic halomethylcarbonyl compounds and a halomethylcarbonyl compound stabilising excipient, said process comprising the steps of:

[0393] (a) providing one or more synthetic halomethylcarbonyl compounds;

[0394] (b) providing a halomethylcarbonyl compound stabilising excipient; and

[0395] (c) contacting the one or more synthetic halomethylcarbonyl compounds with the halomethylcarbonyl compound stabilising excipient under conditions to form the composition.

[0396] In one embodiment, the halomethylcarbonyl compound stabilising excipient is selected from the group consisting of an edible wax, grease, oil, cyclodextrins, molasses and a saturated fat.

[0397] Stabilising excipients are discussed herein, wherein halomethylcarbonyl compound stabilising excipients are stabilising excipients which prevent solvolysis of one or more halomethylcarbonyl compounds they are contacted with.

[0398] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

[0399] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

[0400] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

[0401] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

[0402] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1, 1,1, 7,7,7-hexabromoheptan-2, 6-dione, l,l,l,3,5,5,5-heptabromopentan-2,4-dione, 1, 1,1, 3, 3, 5,5,5-octabromopentan-2, 4-dione, l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

[0403] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compounds comprises one or more synthetic halomethylcarbonyl compounds.

[0404] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein,, wherein the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

[0405] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compound stabilising excipient comprises an edible wax, grease, oil, cyclodextrins, molasses and a saturated fat.

[0406] In a further embodiment, the present invention provides an anti-methanogenic composition as described herein, wherein the one or more halomethylcarbonyl compound stabilising excipient comprises a medium chain triglyceride.

[0407] In another embodiment, the present invention provides a feed supplement for reducing total gas production and / or methane production in a ruminant animal, said supplement comprising an effective amount of composition produced by a method as described herein.

[0408] In one embodiment, the present invention provides a feed or a feed supplement as described herein wherein the feed or feed supplement comprises one or more synthetic halomethylcarbonyl compound and / or one or more synthetic anti-methanogenic halomethane compound.

[0409] In another embodiment, the present invention provides a feed or a feed supplement as described herein wherein the feed or feed supplement comprises a) one or more Asparagopsis derived halomethylcarbonyl compound and / or one or more Asparagopsis derived anti-methanogenic halomethane compound, or b) one or more synthetic halomethylcarbonyl compound and / or one or more synthetic anti-methanogenic halomethane compound in combination with one or more Asparagopsis derived halomethylcarbonyl compound and / or one or more Asparagopsis derived anti-methanogenic halomethane compound.

[0410] In one embodiment, the present invention provides a feed or a feed supplement as described herein wherein the one or more halomethylcarbonyl compound consists of one or more synthetic halomethylcarbonyl compound and / or the one or more anti-methanogenic halomethane compound consists of one or more synthetic anti-methanogenic halomethane compound.

[0411] In another embodiment, the present invention provides a feed or a feed supplement as described herein wherein the one or more halomethylcarbonyl compound consists of one or more Asparagopsis derived halomethylcarbonyl compound and / or the one or more anti-methanogenic halomethane compound consists of one or more Asparagopsis-demed anti-methanogenic halomethane compound.

[0412] In one embodiment the present invention provides a feed supplement when used as described herein, wherein the feed supplement is formulated to provide a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of organic matter provided to the ruminant animal.

[0413] In another embodiment the present invention provides a feed supplement when used as described herein, wherein the supplement further comprises one or more edible excipients.

[0414] In another aspect, the present invention provides a feed for a ruminant animal, wherein said feed is supplemented with a feed supplement described herein.

[0415] In one embodiment the present invention provides a feed for a ruminant animal as described herein, wherein the animal feed comprises a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of organic matter of the ruminant animal feed.

[0416] In one embodiment, the present invention provides an animal feed when used as described herein, wherein the composition is formulated to provide a dose of at least 0.005, 0.01,0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of the organic matter of the ruminant animal feed.

[0417] In another aspect, the present invention provides a method for reducing total gas production and / or methane production in a ruminant animal comprising administering to said ruminant animal an effective amount of a composition described herein, a feed supplement described herein or a feed described herein.

[0418] As used herein, the term 'reducing total gas production' refers to the reduction of the total amount of gas produced, for example the amount of total gas produced in the gastro-intestinal tract. The term includes the collective volume of all gasses generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of gas including methane. The present invention aims to reduce this process, such as to reduce the total amount of gas produced in the gastro-intestinal tract. It is within the knowledge and skill of those trained in the art to assess total gas production by a ruminant animal.

[0419] As used herein, the term 'reducing methane production' refers to the reduction of methane produced in the gastro-intestinal tract. The term includes the specific volume of methane generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The present invention aims to reduce this process, such as to reduce the total amount of methane produced in the gastro-intestinal tract. It is within the knowledge and skill of those trained in the art to assess methane production by a ruminant animal.

[0420] In preferred embodiments of the invention, the amount of total gas produced is reduced by at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to a reference. In one embodiment the reference is the amount of total gas produced when animals are not administered an effective amount of a composition described herein. In another embodiment, the reference is the amount of total gas produced when animals are administered a control feed. In another embodiment, the reference is the amount of total gas produced when a control feed is subjected to in vitro anaerobic fermentation.

[0421] In preferred embodiments of the invention, the amount of methane produced is reduced by at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to a reference. In one embodiment the reference is the amount of methane produced when animals are not administeredan effective amount of a composition described herein. In another embodiment, the reference is the amount of methane produced when animals are administered a control feed. In another embodiment, the reference is the amount of methane produced when a control feed is subjected to in vitro anaerobic fermentation.

[0422] By "effective amount", is meant a quantity of a composition as described herein sufficient to allow improvement, e.g. reduction in the amount of methane production in comparison with a reference or control, reduction in the amount of total gas produced in comparison with a reference or control, maintenance of effective levels of desirable volatile fatty acids in comparison with a reference or control, reduction in the acetate to propionate ratio in comparison with a reference or control, maintenance of liveweight, dry matter intake and / or organic matter intake in comparison with a reference or control, or improvement in growth performance relative in comparison with a reference or control. Within the meaning of the present invention, the methane reductive effect can be measured in the rumen with an artificial rumen system, such as that described in T. Hano., J. Gen. Appl. Microbiol., 39, 35-45,1993 or by in vivo oral administration to ruminants.

[0423] An effective amount of a composition described herein may be determined by the methods described herein, including the in vitro and in vivo studies and in vivo dose-response studies described in Kinley et al. (2020) Journal of Cleaner Production 259:120836 and WO2015109362, incorporated herein by reference. For example, the Applicant has demonstrated that ruminal fermentation in vitro can be used to examine the effect of amounts of a composition on total gas, hydrogen and methane production, and can be used to examine levels of volatile fatty acids, including acetate and propionate. Measures of growth performance can be measured using the in vivo studies described herein. Therefore, ruminal fermentation in vitro can be used to characterize doses of the composition that may be an effective amount sufficient to allow improvement, e.g. reduction in the amount of methane production in comparison with a reference or control, reduction in the amount of total gas produced in comparison with a reference or control, maintenance of effective levels of desirable volatile fatty acids in comparison with a reference or control, or reduction in the acetate to propionate ratio in comparison with a reference or control.

[0424] As used herein, the term "providing" includes the provision of a composition as described herein as a feed additive in feed provided to an animal or to an animal system, (e.g. animals in a feedlot, or animals in a farming system, etc). Providing also includes the administration of a composition as described herein.

[0425] In one embodiment, the farming system is a pastoral, feedlot, regularly supplemented system, or a combination thereof.

[0426] As used herein, "administer" and "administered", includes the action of introducing a composition as described herein into the animal's gastro-intestinal tract. More particularly, this administration is an administration by oral route. This administration can in particular be carried out by supplementing the feed intended for the animal with the composition, the thus supplemented feed then being ingested by the animal. This administration can also include providing the composition in a form that an animal consumes (e.g. a lick block, supplement, etc). The administration can also be carried out using a stomach tube or any other means making it possible to directly introduce said composition (e.g. a dosage form (e.g. a bolus) of composition) into the animal's gastro-intestinal tract.

[0427] Accordingly, the present invention provides a method as described herein wherein the step of providing said ruminant animal an effective amount of a composition comprises administering said ruminant animal with the effective amount of a composition described herein

[0428] The composition may be provided to the ruminant in one of many ways. A composition can be provided in a solid form as a veterinary pharmaceutical, may be distributed in an excipient, and directly fed to the animal, may be physically mixed with feed material in any suitable form (e.g. dry form, in solution, or in suspension etc.) or the composition may be formed into a solution and thereafter sprayed onto feed material. For example, a dry mixture can be used that is prepared by adsorption or deposition of a solution onto / into a dry excipient.

[0429] The method of administration of the composition to the animal is considered to be within the skill of the artisan.

[0430] When used in combination with a feed material, the feed material is preferably grain / hay / silage / grass-based. Included amongst such feed materials are improved and / or grass or legume-based forages either grazed directly or prepared as a conserved forage hay, any feed ingredients and food or feed industry by-products as well as bio-fuel industry by-products and corn meal and mixtures thereof, or feed lot and dairy rations, such as those high in grain content.

[0431] The composition may be provided for consumption in an animal system (e.g. a feedlot, a native grass land / pasture farming system), in an animal feed supplement as an inclusion in any suitable form, including, a mineral loose lick, wet lick, pellets, water suspension, or lick block. As is known to those skilled in the art such loose licks, wet licks, pellets, water suspensions, or lick blocks are particularly convenient for feeding mineral supplements (as well as proteins and carbohydrates)to ruminants grazing pastures. Such loose licks, wet licks, pellets, water suspensions, or lick blocks etc. may comprise, in addition to the composition of the invention, various types of binders, e.g. cements, gypsum, lime, calcium phosphate, carbonate, and / or gelatin; and optionally further additives such as vitamins, trace elements, mineral salts, sensory additives, etc.

[0432] The time of administration is not crucial so long as the reductive effect on total gas production, methane production and / or growth performance is shown. As long as the feed is retained in the rumen, administration is possible at any time. However, since the composition is preferably present in the rumen at about the time methane is produced, the composition is preferably administered with or immediately before feed.

[0433] In a particular embodiment of the invention, said effective amount of the composition is administered to a ruminant animal by supplementing a feed intended for said animal with the composition. By "supplementing", within the meaning of the invention, is meant the action of incorporating the effective amount of the composition according to the invention directly into the feed intended for the animal. Thus, the animal, when feeding, ingests the composition according to the invention which can then act to maintain the digestibility of the fibres and / or cereals contained in the animal's feed. Alternatively, supplements, such as loose lick, wet lick, pellets, water suspension, or lick blocks and other feed supplements, can be provided in an animal system without incorporating directly into animal feed.

[0434] Thus, another subject of the invention relates to a feed supplement for a ruminant animal comprising a composition described herein.

[0435] In another aspect the present invention also provides a feed supplement for reducing total gas production and / or methane production in a ruminant animal, said supplement comprising an effective amount of a composition described herein.

[0436] In one embodiment, the effective amount of a composition described herein is administered to said ruminant animal by supplementing food intended for said animal with said composition described herein.

[0437] In another embodiment the present invention provides methods for improving the growth performance of a ruminant animal comprising the step of providing said ruminant animal with an effective amount of a composition described herein.

[0438] In a preferred embodiment, the growth performance is increased average daily weight gain and / or increased feed utilisation efficiency.

[0439] In one embodiment the present invention provides a method of increasing average daily weight gain of a ruminant animal comprising the step of providing said ruminant animal with an effective amount of a composition described herein.

[0440] In one embodiment the present invention provides a method of increasing feed utilisation efficiency of a ruminant animal comprising the step of providing said ruminant animal with an effective amount of a composition described herein.

[0441] In one embodiment, the present invention provides a method as described herein wherein the effective amount of a composition described herein is provided in the farming system, thereby reducing total gas production and / or methane production and / or improving the growth performance of the livestock animal in the farming system.

[0442] In one embodiment, the present invention provides a method as described herein wherein the effective amount of a composition described herein is provided to a Stocker operation to enable consumption of the composition described herein by a livestock animal in the Stocker operation, thereby reducing total gas production and / or methane production and / or improving the growth performance of the livestock animal in Stocker operation.

[0443] In one embodiment, the present invention provides a method as described herein wherein the effective amount of a composition described herein is provided to a feedlot system to enable consumption of the composition described herein by a livestock animal in the feedlot system, thereby reducing total gas production and / or methane production and / or improving the growth performance of the livestock animal in the feedlot system.

[0444] In one embodiment, the present invention provides a method as described herein wherein the effective amount of a composition described herein is provided in the pasture system to enable consumption of the composition described herein by a livestock animal in the pasture system thereby reducing total gas production and / or methane production and / or improving the growth performance of the livestock animal in the pasture system.

[0445] A ruminant is a mammal of the order Artiodactyla that digests plant-based food by initially softening and partially fermenting it within the animal's first stomach chambers, then regurgitating the semi-digested mass, now known as cud, and chewing it again.

[0446] Examples of ruminants are listed below. However, preferably compositions described herein are used as an additive for foodstuffs for domesticated livestock such as cattle, goats, sheep and llamas. The present invention is particularly useful in cattle and sheep. Therefore, in one embodiment, said ruminant animal is selected from the members of the Ruminantia and Tylopoda suborders. In another embodiment, said ruminant animal is cattle or sheep. In a further embodiment, said ruminant animal is a cattle.ExamplesGeneral Methods

[0447] Seaweed assay data are reported in units of mg of extractable bromoform per g of seaweed (mg / g), either on wet-mass basis (WMB) or dried solids basis (DSB) as indicated. Bromoform quantitation and GC-MS analysis was performed in-house and at two commercial analytical labs, further described as follows:

[0448] AST methods (Analytical Services Tasmania, New Town TAS): Bromoform was quantified using NATA-accredited method 3429-Biota: "Bromoform in oil and seaweed by GC-MS". This method is performed according to Australian Standard AS 5404:2025 using a 16-hour methanol extraction for solid samples and a 30-minute extraction for oil samples.

[0449] Qualitative GC-MS analysis of seaweed volatiles was performed using method 2429-Soil: "VOC in solids by GC-MS" using a 16-hour methanol extraction identical to the above. No NATA accreditation covers this test.

[0450] Extraction with deuterated methanol (CD3OD) was performed according to method 2429-Soil: "VOC in solids by GC-MS" with substitution of deuterated methanol which was purchased from Merck or from Cambridge Isotopes. Raw data files were transferred from AST to FutureFeed for analysis using MestReNova software vl5.0.1-35756 (Mestrelab Research S.L.).

[0451] ChemCentre method (ChemCentre, Bentley WA): Bromoform was quantified in aqueous samples using NATA-accredited method QRG002W "VOC in water by purge and trap GC-MS".

[0452] FutureFeed methods (FutureFeed, Brisbane QLD): Three FF methods were developed and used as indicated. The column and GC parameters remain unchanged, and retention times remain comparable across all FF methods. Chromatographic separation was performed with a general-uselow-bleed 5% phenyl arylene column (Zebron ZB-5plus, 30m x 0.25mm x 1 pm; Phenomenex Inc., USA) and ultrapure helium 5.5 was used as a mobile phase with a constant flow of 0.98 mL / min. The injector temperature was set to 250°C. The injection was performed using a split mode in the proportion of 1:50. Gradient: 60°C for 1 min., rising to 300°C at 25° / min., final hold 3 min., total run-time 13.6 min. The mass spectrometer was operated in electron ionization mode at 70eV. The inlet pressure was 57.7 kPa, and the ion source and interface temperatures were 230°C and 250°C respectively.

[0453] SIM was not used; rather the detector was used in Acquisition mode so that metabolites other than bromoform could be analysed. Single-point quantitation against a 1 mg / mL bromoform standard was used during initial development. Bromoform was found to elute at a retention time of 5.34 minutes, and was identified by the ions at m / z 171, 175, 91, and 252. Bromoform was quantified using the ion at m / z 173 using Shimadzu software.

[0454] Halogenated analytes other than bromoform were assigned structures by comparison with literature data where available, including the NIST standard reference database. Compounds with no available literature data were tentatively identified based on mass spectral fragmentation. MestReNova software vl5.0.1-35756 (Mestrelab Research S.L.) was used for all data processing other than bromoform quantitation.GC-MS analysis (FF Method 1)

[0455] Seaweed sample (50-200 mg on dried basis) was chopped with scissors if necessary, then accurately weighed into an appropriately sized glass vial. Extraction solvent (1-5 mL) was added and the vial was sealed using a PTFE-lined lid. Samples were vortexed briefly, then shaken for 16h using an end-over-end orbital mixer (Joanlab RMO-80). Finally, an aliquot of the supernatant was decanted into a GC-MS vial and analysed directly.

[0456] Sample extracts were directly analysed by gas chromatography mass spectrometry (GC-MS) using instrument model QP2020 NX (Shimadzu Australasia).

[0457] Table 1: Summary of analytical data collected for various prepared and dried seaweed samples‘likely an artefact of H / D exchangeGC-MS analysis (FF Method 2)

[0458] The general extraction conditions used for FF Method 1 were optimised and standardised in preparation for method validation, and an internal standard (either 1,2- or 1,4-dichlorobenzene) was introduced for calibration against a 6-point calibration curve. Bromoform quantitation (excluding seaweed extraction) using this method was successfully validated under Eurachem and ICH guidelines.

[0459] Seaweed sample (100-200 mg) was chopped with scissors if necessary, then accurately weighed into an 8 mL amber glass vial with a Teflon-sealed cap. To this was added DCM (2.5 mL), MeOH (2.4 mL), NH4OH (0.1 mL), followed by the internal standard as a solution in methanol (10 mg / mL; 50 pL). Samples were vortexed briefly, then shaken for 16h using an end-over-end orbital mixer (Joanlab RMO-80). Finally, an aliquot of the supernatant was decanted into a GC-MS vial and analysed directly.

[0460] Calibration standards were prepared by dilution into the same solvent mixture as used for extraction of samples.GC-MS analysis (FF Method 3)

[0461] FF Method 3 was developed for more accurate quantitation in solvent mixtures other than the standard DCM / MeOH / NH4OH used for validation of FF Method 2. A mass-based calibration was introduced where any volumetric units were either not used, or cancelled out, in any calculations.This avoids any errors from density and volume changes due to non-ideal mixing of solvents. The present inventors reverted to methanol solvent for calibration standards for convenience.

[0462] Seaweed sample (100-200 mg) was chopped with scissors if necessary, then accurately weighed into an 8 mL amber glass vial with a Teflon-sealed cap. To this was added the internal standard and the extraction solvents; for example, an aliquot of the internal standard stock solution in MeOH (2.5 mL), followed by DCM (2.5 mL) and aqueous ammonia (0.1 mL). Samples were vortexed briefly, then shaken for 16h using an end-over-end orbital mixer (Joanlab RMO-80). Finally, an aliquot of the supernatant was decanted into a GC-MS vial and analysed directly.

[0463] Table 2: Bromoform assay results obtained on dried samples using the final analytical method (FF Method 3). Samples were freshly dispensed from frozen storage before analysis.Table 2:Development and validation of general extraction solvent

[0464] Most of the previously reported methods for bromoform analysis in Asparagopsis samples rely on methanol as extraction solvent. As demonstrated herein, methanol can support precursor hydrolysis, however once this is complete, other solvents could be used for extraction of the produced bromoform. The present inventors tested various solvents and mixtures for extraction and analysis of fresh-frozen algae ("'80% water content) as well as dried samples, comparing the results in the first instance with 16-hour methanol extraction. Averaged bromoform assay results (FF Method 1) are summarised in Table 3

[0465] Effectiveness of different solvents was similar between dried and non-dried batches, in that chlorinated solvents tended to perform well, and other solvents less so unless MeOH was also included. However, the combination of a chlorinated solvent in the presence of NH4OH, which was ineffective in non-dried samples, yielded some of the highest recoveries from dried samples as described below. This effect was variable, such that in one example, two samples of dried product AlOb extracted side-by-side gave bromoform assays of 20.9 and 30.2 mg / g (Table 23).

[0466] The present inventors propose that the greater variability in extractable bromoform in non-milled, dried samples is due to non-homogeneity of residual moisture throughout the samples. The importance of small quantities of water in the Haloform-type cleavage of precursors is demonstrated in Example 2.

[0467] Table 3: Comparison of bromoform assay data measured between two different laboratories (AST method and FutureFeed method 1), using methanol or DCM as extraction solvents, and examining the effect of added base in the form of aqueous ammonia. Samples A9 & A10 were obtained by drying of ocean-grown gametophyte sample A2. Sample 018-02 is a neutral-acidic cultured tetrasporophyte sample.Table 3:>>Preparation and drying of seaweed samplesAST Moisture ais method

[0468] Moisture analysis was performed gravimetrically using method 3001-Biota: "Moisture in Biota dried at 104±3°C".FF Moisture analysis method

[0469] Residual moisture analysis was performed by loss on drying at 120°C using a Halogen Rapid Moisture Tester (Ningbo Scientz Biotechnology Co.).

[0470] Gametophytes of A. taxiformis were harvested as described previously. (Vucko et al. 2016) Seaweed was centrifuged to ~80% water content and stored frozen at -20°C in mylar pouches until required. Frozen samples were transported where necessary maintaining frozen state using validated -20°C shippers (CryoPDP Australia). Methanol-extractable bromoform assay (AST method) of the fresh-frozen sample was 4.21 mg / g (WMB).

[0471] Seaweed sample A2 (2.4 kg) was dried using a 10 kW microwave-energy vacuum drier (Enwave Corporation). Frozen seaweed was transferred to ambient-temperature carousel trays for drying, with no attempt made to maintain frozen state during this process. Drying was carried out at a vacuum of 20-30 hPa (mbar), and the product temperature was maintained under 50°C at all times. Power Level: 2.6kw for 1401seconds, 1.3kw for 1681 seconds, 0.75kw for 972 seconds, 0.35kw for 2082 seconds. Yield 470g (19.6%); residual moisture 10.9% (AST method).

[0472] Seaweed sample A2 (1.2 kg) was dried by lyophilisation as follows. The product frozen at - 18°C was transferred directly to the lyophiliser shelves at -30°C, and was not allowed to thaw during this process. Drying was carried out at a vacuum of 2 hPa (mbar). Temperature program: -30°C (Freeze), -10°C for 4 hours, 0°C for 4 hours, 10°C for 4 hours, 20°C for 6 hours. Yield 225 g (18.8%); residual moisture 6.5% (AST method).Analysis:

[0473] The dried products A9 & A10 were heat-sealed into food-grade Mylar bags and transported at ambient temperature to Analytical Services Tasmania (AST; New Town, TAS) for analysis in duplicate.Sample AlOb (Lyophilized)

[0474] Preparation of sample A10 was repeated using the Autodry function of the CryoDry CD8 freeze-drier (CryoDry Australia). This program uses automated temperature control to maintain a target vacuum of 0.5 mbar throughout the lyophilisation. Final drying temperature 30°C; total drying time 24 h. Residual moisture 1.87% (Ohaus MB25); water activity 0.10 (Meter Pawkit).Neutral-acidic tetrasporophyte samples

[0475] Samples 018-01 to 019-10 were prepared from cultured Asparagopsis taxiformis tetrasporophytes that were grown at neutral to acidic pH. This was achieved using an automated system that sparged the culture with carbon dioxide gas whenever the pH of the culture medium rose to 7.

[0476] Samples were harvested on the same day and were separately treated. The treated samples were lyophilized together in the same run, using a CryoDry CD-8 lyophiliser (CryoDry Australia). Lyophilisation program used is reported in Table 4. Solution pH and salinity was measured using a calibrated probe (YSI ProQuatro). Water content was measured by loss on drying at 120°C (FF moisture method). Methanol-extractable bromoform content of the dried samples was measured by GC-MS (AST method).

[0477] Table 4: Typical lyophilisation program used with the CryoDro CD8 freeze-drier.Sample 018-01(liquid nitrogen frozen and lyophilized

[0478] This sample was handled and processed to the minimum possible level during harvest. The sample ("'15g) was harvested from the culture using a mesh bag and immediately snap-frozen in liquid nitrogen. The water content of a similar sample was measured as 95.35%. With such a high water content, the lyophilised product was extremely porous and hygroscopic. After recovery from the freezedrier, via 11 ing and sampling, the water content of the sample was ~64% and the sample was visibly damp.

[0479] This sample was processed using standard harvest procedures. After harvest, the sample was dried to ~80% water content by centrifugation. The sample ("'120 g) was spread evenly on a stainless-steel tray and frozen at -20°C overnight. After lyophilisation, 27.2 g of product was obtained, at a residual water content of 13.97%.

[0480] The sample ("'10g) was harvested using a mesh bag, and immediately suspended in commercial white distilled vinegar (4.2%, 50 mL). The pH of the resulting suspension was 2.31. After aging for 2 h, the solid was decanted and immediately snap-frozen in liquid nitrogen. The frozen solid was stored at -20°C before lyophilisation. The decanted solution was assayed for bromoform content by GC-MS (ChemCentre method).

[0481] The sample ("'10g) was harvested using a mesh bag, and immediately suspended in 3% aqueous citric acid (50 mL). The pH of the resulting suspension was 1.85. After aging for 2 h, the solid was decanted and immediately snap-frozen in liquid nitrogen. The frozen solid was stored at -20°C before lyophilisation. The decanted solution was assayed for bromoform content by GC-MS (ChemCentre method).

[0482] The sample ("'10g) was harvested using a mesh bag, and immediately suspended in 1.3% aqueous sodium bicarbonate (50 mL). The pH of the resulting suspension was 7.87. After aging for 2 h, the solid was decanted and immediately snap-frozen in liquid nitrogen. The frozen solid was stored at-20°C before lyophilisation. The decanted solution was assayed for bromoform content by GC-MS (ChemCentre method).

[0483] The sample ("'10g) was harvested using a mesh bag, and immediately suspended in tap water (50 mL). The pH of the resulting suspension was 6.03 and the salinity was 3.4 ppt. After aging for 2 h, the solid was decanted and immediately snap-frozen in liquid nitrogen. The frozen solid was stored at -20°C before lyophilisation. The decanted solution was assayed for bromoform content by GC-MS (ChemCentre method).Example 1: Quantifying free and potential halomethanes using deuterium incorporation

[0484] The potential halomethane fraction, or halomethane precursor compounds (referred to herein as halomethylcarbonyl compounds), can be quantified by mass spectrometry after deuterium incorporation. The chemistry behind this test is set out in Scheme 6. Solvolysis of trihalomethylcarbonyl compounds such as the tribromocarbonyl derivative depicted requires a proton source, and these protons are incorporated into the product (in this case, tribromomethane). Replacement of the methanol solvent with its deuterated analogue yields the equivalent deuterium-substituted product, which can be distinguished by mass spectrometric analysis. Since the natural abundance of deuterium (0.0156%)29is negligible compared to the added deuterated solvent (99.8% D), any deuteriumsubstituted product detected can only have been formed after addition of the deuterated solvent, and therefore confirms the presence of trihalomethylcarbonyl precursor-compounds prior to that point.

[0485] Scheme 6: Methanolysis of a tribromomethylcarbonyl compound yields a bromoform product that incorporates a hydrogen atom derived from the methanol. Similarly, replacement of methanol (CH3OH) with deuterated methanol (CD3OD, at bottom) yields a bromoform product with deuterium incorporation.

[0486] Deuterium incorporation was measured by examination of the isotope splitting patterns in the bromoform molecular ions. As an example, the mass spectra and data used for calculation of residual precursor for samples A9 and A10 (Example 3) are provided.

[0487] Figure 1 shows the negligible level of deuterium incorporation expected when extracting sample A10 with natural-abundance methanol. The theoretical isotope splitting pattern30for an ion containing three bromine atoms is four peaks in the ratio of 100:293:286:93.4, which is close to the observed ratio of 100:260:252:90.2.

[0488] Table 5: Abundance data for the ions shown in Figure 1 (bromoform molecular ion; lyophilised sample A10; CH3OH extraction). The "Relative Abundance" values are normalised to the base peak from the full mass spectrum (m / z = 170.946), whereas the "Normalised Abundance" values are normalised to the lightest molecular ion (visible here at m / z = 249.802).m / ,ztIn 4t.ensi 4t,y R .e. lativ .e N . o. rma .lised' Abundance Abundance1 249.802 9426459.625 22.48 1002 251.804 24484357.750 58.38 2603 253.802 23773777.250 56.68 2524 254.810 63178.891 0.155 255.788 8500729.375 20.27 90.26 256.821 100765.749 0.24

[0489] Figure 2 shows the isotope splitting pattern for the same sample extracted with deuterated methanol. Instead of four peaks, eight are seen; consisting of the four peaks due to proteo-bromoform identical to those in Figure 1, and four peaks due to deutero-bromoform that are each one mass-unit heavier than their proteo-counterparts.

[0490] The ratio of proteo- to deutero-bromoform can be calculated from relative peak areas of pairs of corresponding ions; when averaged over all four pairs this ratio equals "'52:48. Therefore, of the 24 mg of total bromoform in each gram of lyophilised sample A10 (as-is basis), approximately 12.5 mg is present as free bromoform, and 11.5 mg is present as unhydrolysed tribromocarbonyl precursors.

[0491] Table 6: Abundance data for the ions shown in Figure 2 (bromoform molecular ion; lyophilised sample A10; CD3OD extraction). The "Relative Abundance" values are normalised to the base peak from the full mass spectrum (m / z = 170.946; 100%).m / z Intensity Rel. Abundance (%)dRat° P' ' ' deutero-brroomte°ofotrom1 249.805 7174312.969 16.02 “|2 249.900 131570.688 0.29 I 53.9:46.13 250.807 6143210.625 13.72 -14 250.870 661353.500 1.485 251.810 18975539.625 42.37 16 251.870 399398.594 0.89 | 51.4:48.67 252.813 17962046.875 40.10J8 253.808 18926261.813 42.261 51.2:48.89 254.807 18041522.406 40.28 J10 255.797 6595858.625 14.73 “|11 255.860 250632.469 0.56 I 51.7:48.312 256.796 6171682.164 13.78 -113 257.827 63130.531 0.1414 257.909 3667.971 0.01Average = 52.0:48.0

[0492] A protiur deuterium ratio of ~94:6 for the bromoform peak can be similarly calculated for the case of microwave vacuum-dried sample A9 using the mass spectral data in Figure 3 and Table 3. Compared to the lyophilised product, a much lower incorporation of deuterium has occurred.

[0493] Table 7: Abundance data for the ions shown in Figure 7 (bromoform molecular ion; microwave-dried sample A9, CD3OD extraction). The "Relative Abundance" values are normalised to the base peak from the full mass spectrum (m / z = 170.946; 100%).m / z In 4t.ens -i4t.y r R>e 1. Abundance (%) . Rat .io of . prroteo ,- to'7 v' deutero-bromoform1 249.788 5923576.750 17.65 j 94.5:5.52 250.790 342585.344 1.02 J3 251.793 15882667.625 47.34 j 94.7:5.34 252.783 885520.250 2.64 J5 252.900 247587.453 0.746 253.788 15451806.125 46.05 j 93.8:6.27 254.787 1026853.734 3.06 J8 254.860 109758.469 0.339 255.780 5327571.094 15.88 j 93.7:6.310256.783 356813.523 1.06 JAverage = 94.2:5.8H / D exchange as a potential confounding variable

[0494] One limitation of the early unoptimized version of the precursor (e.g. halomethylcarbonyl compounds) assay is the possibility for protium-deuterium (H / D) exchange reactions under high-pH conditions. The mechanism of the H / D-exchange (Scheme 7) proceeds through the same trihalomethyl carbanion as the Haloform reaction (Scheme 1) but formed by deprotonation instead of leaving-group elimination. The deprotonation reaction is the rate-determining step and is not subject to general base catalysis in aqueous solution.31-32This means that adding another base to the reaction in Scheme 7 would only increase the reaction rate to the extent that the additional base increases the concentration of hydroxide ions. The rate of H / D exchange therefore depends on the pH of the aqueous extraction solvent and not on the specific base used. Haloforms are extremely stable in the absence of base, with the environmental half-life of chloroform at pH 7 and 25°C estimated as 1850 years.

[0495] Scheme 7: Mechanism of H / D exchange reaction of halomethanes, illustrated for bromoform in alkaline D2O>"

[0496] H / D exchange of a synthetic bromoform standard in methanol-c / 4 was slow in the absence of base catalysis (Figure 4). After 22 hours equilibration at 20°C the extent of H / D exchange between CHBra and CD3OD was only 11% as measured by GC-MS. This was sufficiently slow that precursor hydrolysis could be monitored by deuterium incorporation during the standard 16-hour extraction.

[0497] However, when hydroxide ion was present as a basic catalyst, the rate of H / D exchange was accelerated, and full deuteration of the bromoform standard (1 mg / mL) was observed within 22 h when treated with methanol-c / 4 containing 2% ammonium-c / 4 deuteroxide (Figure 4). For this reason, base catalysis was not employed in the deuterium incorporation experiments to monitor precursor hydrolysis.

[0498] At the normal pH of dried Asparagopsis samples the rate of H / D exchange is acceptably low during a standard 16h extraction period. For example, the microwave vacuum dried sample A9 (Example 3) showed 5.4% deuterium incorporation into the bromoform peak after a 16h extraction with deuterated methanol. As this is roughly equal to the rate of H / D exchange in the bromoform standard, the contribution from deuterolysis of residual bromoform precursors in this sample was therefore very low.

[0499] However, measurement of pH in low-aqueous solvent mixtures such as aqueous methanol is more difficult and less meaningful, and measuring the pH within compartments such as gland cells in seaweed samples is not practicable. In cases such as these, the dependence of the H / D exchange rate on the acidity of the halomethane may be used to confirm a low rate of H / D exchange under the experimental conditions.

[0500] This was achieved through GC-MS examination of the isotope ratio for dibromochloromethane, which is the second-most abundant halomethane in the Asparagopsis samples. The conjugate base of this compound, the dibromochloromethyl carbanion, is less stable than the anion derived from bromoform (Scheme 7) due to the lesser stabilising effect of chlorine compared to bromine. Both the Haloform reaction and the H / D exchange reaction pass through this intermediate, and both reactions are slower in the case of dibromochloromethane.

[0501] A slower haloform reaction means that more dibromochloromethane remains in precursor (e.g. halomethylcarbonyl compound) form in incompletely hydrolysed seaweed samples. Therefore, the ratio of CDBr2CI / CHBr2CI will tend to be higher than the ratio of CDBr3 / CHBr3 in extracts derived by complete solvolysis of haloform precursors with deuterated methanol.

[0502] On the other hand, a slower H / D exchange means that the ratio of CDBr2CI / CHBr2CI will tend to be lower than the ratio of CDBr3 / CHBr3 in extracts derived largely by H / D exchange of free halocarbons.

[0503] Therefore, if the measured degree of deuterium incorporation is greater in the more acidic haloform, then H / D exchange has dominated. Conversely, if the less acidic haloform is observed to bemore highly deuterated, then precursor solvolysis was the dominant mechanism of deuterium incorporation.

[0504] The relative quantities of bromoform and dibromochloromethane in dried seaweed samples A9 and A10 as measured by relative peak areas is reported in Table 4. Although the two samples had similar compositions within the error of the experiment, there was a trend toward the microwave-dried sample A9 being relatively depleted in CHBr2CI.

[0505] Table 8: Estimation of dibromochloromethane (CHBr2CI) in the dried seaweed samples by peak area percentage. The total ion chromatograms for both methanol (CH3OH) and deutero-methanol (CD3OD) extracts were integrated using MestreNova V14.3.3 automatic integration, and average values for the two different drying methods were calculated.„ . Percent Average, Extraction Peak area , ,Sample abundance CHBr3: CHBr2CIS0 VenCHBr3CHBr2CI CHBr3CHBr2CI (a / a) A9 CH3OH 357966815 20862939 94.5% 5.5% 194.5:5.5A9 CD3OD 355711328 20394995 94.6% 5.4% JA10 CH3OH 447900446 28778778 94.0% 6.0% 193.4:6.6A10 CD3OD 691713025 53299010 92.8% 7.2% J

[0506] Due to the lower abundance and greater fragmentation of the molecular ion of dibromochloromethane compared to bromoform, deuterium incorporation was instead measured on the major fragment ion with atomic composition CHBrCI, resulting from loss of one bromine atom from CHBr2CI. Mass spectrometric data for the freeze-dried sample A10 are presented in Figure 5, while data for the microwave-dried sample A9 are presented in Figure 6. In both samples, the ratio of CDBr2CI: CHBr2CI is higher than the ratio of CDB^CHBrs, i.e. the level of deuterium incorporation is higher for dibromochloromethane than for bromoform, irrespective of drying process employed. Without being bound by theory, there are two ways that this difference could arise.

[0507] Firstly, dibromochloromethane is more volatile than bromoform, and could have been preferentially lost by evaporation under either of the two drying processes. The trihalomethylcarbonyl precursors for both compounds much less volatile due to their higher molecular mass, and it is these precursors that yield deuterium-labeled product. However, since approximately half of the total bromoform was lost under microwave-drying conditions with only a slight non-significant depletion in relative amount of dibromochloromethane, then the two compounds must be being lost at approximately similar rates proportional to their abundance.

[0508] Secondly, the rate of solvolysis of a dibromochloromethylcarbonyl compound is known to be slower than for a tribromomethylcarbonyl compound. This is because bromine lends greater stability than chlorine to the trihalomethyl carbanion leaving-group introduced in Scheme 1; as demonstrated by Hine et al., a-halogen substituents facilitate carbanion formation in the order I ~ Br > Cl > F33. The slower hydrolysis means that the quantity of dibromochloromethylcarbonyl precursor remains closer to its initial maximum value than does the tribromomethylcarbonyl precursor. That this maximum value for "potential" halomethanes could be higher than 75.8% as measured for lyophilised sample A10 shows the room for improvement over these unoptimized lyophilisation and microwave-drying conditions.

[0509] The data for all samples analysed by deuterium incorporation are reported in Table 11, and the results are consistent with the above. All samples show a higher level of deuteration in the less acidic halomethane (i.e. the ratio CDBr2CI%:CDBr3% is >1) confirming that the dominant deuterium source is precursor-hydrolysis, except for sample 019-09 which was treated with sodium bicarbonate. This sample serves as a negative control, confirming that CDBr2CI%:CDBr3% ratios less than 1 arise under high-pH conditions.

[0510] The highest ratios of CDBr2CI%:CDBr3% are associated with samples that underwent significant precursor hydrolysis and volatilisation of CHBr2CI before the deuterated methanol was added. These are sample A9 (CDBr2CI%:CDBr3% = 4.62) which suffered almost complete precursor hydrolysis during the microwave-drying process, and sample 019-10 which suffered the well-known activation of gland cells under conditions of osmotic shock upon exposure to tap water.16

[0511] Table 9: Abundance data for ions shown in Figure 5 (chlorodibromomethane fragment ion; lyophilised sample A10; CD3OD extraction)m / z In 4t.ens -i4t.y r R>e 1. Abundance ( ,n%, .) R .ati .o of proteo- to .'1' deutero-compound1 125.884 91544.166 0.902 126.880 2458749.813 24.151 23.5:76.53 127.889 8025386.813 78.83 J4 128.880 3221772.094 31.64 24.0:76.05 129.890 10181256.406 100.00 J6 130.880 847715.465 8.331 25.0:75.07 131.884 2548366.961 25.03 JAverage = 24.2:75.8

[0512] Table 10: Abundance data for the ions shown in Figure 6 (chlorodibromomethane fragment; microwave-dried sample A9; CD3OD extraction)m / z In 4t.ensi 4t,ynRe 1. Abundance ™ (% 4) R .ati .o of proteo- to .'1' deutero-compound1 125.836 47908.488 1.302 126.880 2859349.234 77.401 72.5:27.53 127.886 1085661.336 29.39 J4 128.875 3694277.438 100.00 73.7:26.35 129.883 1321233.211 35.76 J6 130.873 893593.504 24.191 73.6:26.47 131.883 320592.111 8.68 JAverage = 73.2:26.8

[0513] Table 11: Incorporation of deuterium into halomethanes derived by precursor hydrolysis. Samples were extracted into methanol-d4and analysed by GC-MS (non-validated AST method).Example 2: Solvolysis of hexabromoacetone to yield bromoform

[0514] The present inventors sought to repeat the experiment of McConnell and Fenical14which had ruled out the Haloform-type alcoholysis of haloacetones during extraction of seaweed. Since pentabromoacetone (PBA) 10 was not available, a sample of 1,1,1,3,3,3-hexabromoacetone was purchased from Epichem (Bentley, WA), and its reactivity with methanol was investigated. The sample was a cream-coloured powder melting at 106-109°C (lit. 109°C)25The13C-NMR spectrum was consistent with product,34and no significant resonances were present in the1H-NMR spectrum.

[0515] Hexabromoacetone (1.0 mg, 1.9 pmol) was separately dissolved in DCM (1 mL), methanol (1 mL) and in methanolic ammonia (7N) for analysis by GC-MS. No significant ions were detected in the chromatogram for the DCM solution under electron ionisation at 70 eV, and it should be noted that GC-MS data for hexabromoacetone has not been published previously to our knowledge. However, the methanol solution (Figure 7) contained bromoform at 0.87 mg / mL (3.4 pmol, 1.8 eq.) as quantifiedagainst an authentic sample (FutureFeed method). Three additional halogenated compounds were identified by their mass spectra as reported in Table 12. In the presence of methanolic ammonia and small quantities of water, the product esters were replaced with the corresponding amides (Figure 8). Identical ester and amide products in comparable quantities were obtained by extraction of seaweed under similar conditions (Figure 17 B,E,F). This demonstrates that haloacetones can in fact have the necessary reactivity to constitute the stored bromoform precursors, contradicting the earlier14conclusion.

[0516] Table 12: Peak table for the chromatograms of Figure 7 and Figure 8

[0517] The analytes reported in Table 12 are consistent with the reactions set out in Scheme 8 and Scheme 9. However, the precise mixture of products obtained by methanolysis of HBA 1 was unexpected, and this may be another reason why concluded thatHaloform-type chemistry was not involved. Based on the data presented herein, it was expected that HBA 1 would react via a single Haloform cleavage as depicted in Scheme 8, yielding a straightforward 1:1 molar ratio of the bromoform and acid products. Instead, the observed yield of 1.8 equivalents of bromoform as quantified by GC-MS suggests that reactions similar to Scheme 8 account for a maximum of 20% of the product, with the bulk of the hexabromoacetone undergoing a double haloform cleavage formally similar to Scheme 9.

[0518] The present inventors propose that the production of unexpectedly minor quantities of esters by ethanolysis of their impure sample of pentabromoacetone could have led the previous workers (McConnell and Fenical 1977)14to conclude that no esters were produced, and hence to incorrectly dismiss the haloacetones as transient intermediates.

[0519] Scheme 8: Haloform-type methanolysis of hexabromoacetone is expected to yield 1 molar equivalent of bromoform

[0520] Scheme 9: One possible mechanism to explain the experimentally observed stoichiometry of hexabromoacetone cleavage

[0521] Scheme 9 depicts the loss of the carbonyl group as carbon dioxide, but this was not experimentally confirmed. Scheme 9 would formally require ~34 pg of adventitious water for complete reaction of 1 mg of hexabromoacetone, but nucleophiles other than water could also contribute and the product esters in Scheme 8 could react further. Traces of other possible carbonyl elimination products such as dimethyl carbonate and methyl carbamate were tentatively identified by GC-MS in some samples but were not quantified or compared against authentic samples. It should be noted that decarboxyation of trihaloacetates is generally expected to require elevated temperature,35and could therefore even occur in the GC injection port; however complete decarboxylation of trichloroacetate has also been reported at room temperature.36

[0522] Dibromomethane and methyl dibromoacetate are likely to be formed by solvolysis of traces of pentabromoacetone, which may have been present as an impurity in the hexabromoacetone sample. However, pentabromoacetone is also known to be generated from hexabromoacetone by bromonium abstraction.34The products observed on reaction of hexabromoacetone with methanolic ammonia solution (Figure 8 and Table 12) provide further evidence for such a side-reaction; the trace of carbon tetrabromide is probably formed through abstraction of a bromonium ion by the tribromomethyl carbanion in a process similar to that reported for carbon tetrachloride.35Bromonium abstraction becomes more favourable under low-water conditions such as methanolic ammonia in place of aqueous ammonia, when the easily abstractable water protons are unavailable.

[0523] However, it appears that some minimum amount of water is required for good conversion of precursors by ammonolysis. When lyophilized seaweed was extracted with anhydrous methanolic ammonia, few volatile products were produced (Table 18), and addition of aqueous ammonia was necessary for good recovery as in Figure 17E. In the case of pure HBA 1 reacting with methanolic ammonia, the present inventors propose that rigorously anhydrous conditions were not used and therefore adventitious water appears sufficient. However, lyophilized seaweed is hygroscopic and may need to be partially rehydrated before sufficient unbound water is available for reaction.

[0524] To determine whether precursors extracted into DCM could be detected by converting them to bromoform, a model precursor - HBA (1 mg) - was dissolved in DCM (1 mL), and the solution showed no peaks upon GC-MS analysis apart from a trace of bromoform (0.02 mg / mL). The sample was then diluted with MeOH (0.1 mL) and immediately analysed by GC-MS. Approximately 8% of the precursor was consumed within 1 minute of addition of the methanol based on bromoform produced, and the final yield of bromoform was 0.71 mg / mL after 7.5 hours reaction. This data demonstrates that synthetic HBA can be solvolysed to produce bromoform.

[0525] This data demonstrates that aprotic solvents such as DCM cannot support precursor hydrolysis, and therefore appear to be poor at what was previously characterised as 'extracting' bromoform from Asparagopsis. The addition of a small amount of methanol to the precursor solution in DCM gave very rapid production of bromoform by solvolysis.

[0526] Importantly, the data allows for the production of anti-methanogenic compositions using synthetic compounds and / or extracted compounds that can be provided to produce bromoform upon solvolysis under desired conditions (e.g. in vivo in ruminant animals).

[0527] A similar experiment was performed with precursors extracted from Asparagopsis to confirm they behaved similarly to the model precursor HBA. High-precursor sample A10 was extracted with DCM without milling. The bromoform assay of the DCM extract (FF Method 1) was 1.64 mg / g(DSB), which was 5.5% of the amount recovered in the control sample [29.60 mg / g(DSB)] obtained by 16-hour sonication of batch A10 with DCM / NH4OH. A series of less-reactive haloacetones were identified in trace amounts in the chromatogram of the DCM extract (Figure 9 and Table 13).

[0528] To test whether these trace haloacetones behaved as Haloform-cleavable precursors, NH4OH (10 pL) was added to the DCM extract in the GC-MS vial (1 mL), and after vortexing briefly and standing for 45 min., the sample was reanalysed. The bromoform content increased slightly to 2.67 mg / g(DSB) or 9.0% of the control sample. After standing for 3 hours, the bromoform assay [2.30mg / g(DSB)] did not further change significantly. However, the haloacetones were completely destroyed upon addition of NH4OH; most within 45 minutes apart from a trace of 1,1,3,3-tetrabromoacetone, but this too was lost after standing 3 hours (Table 13). Loss of tetrabromoacetone was accompanied by formation of dibromoacetamide as a likely cleavage product, but the corresponding formation of dibromomethane was not observed; instead dibromoiodomethane was formed. This confirms that the concentration of water transferred from the added NH4OH into the DCM extract was low as expected, and that ammonia is not sufficiently acidic to act as a proton source for quenching the reactive dibromomethyl anion; instead, this species has scavenged an iodonium ion from an undetected iodine-containing compound. Therefore, similar to the production of carbon tetrabromide above, the chemically reactive precursor species can occasionally react via alternative non-standard Haloform chemistry, abstracting a convenient halonium ion if the required protic solvent is not available to act as a proton source.

[0529] No haloacetones were detected in the control sample where NH4OH was included in the extract from the beginning; here the same haloacetone cleavage products were detected (Table 13), but it much greater quantities. The present inventors propose that less-reactive haloacetones are quickly consumed by Haloform-type cleavage in the presence of base, accounting for their absence from chromatograms of such extracts.

[0530] Table 13: Haloacetones as Haloform-cleavable precursors. Haloacetones extracted into DCM were rapidly cleaved to yield haloforms upon vortexing the DCM extract with NH4OHExample 3: Effect of sample processing on residual precursor content

[0531] The present inventors have tested seaweed batches prepared under various conditions using a deuterated methanolysis assay described herein, demonstrating that existing methods to retain bromoform tend to yield samples with increased residual precursor content (Table 11). Conversely, treatments that promote the Haloform reaction are associated with production and loss of volatile free bromoform from seaweed samples.

[0532] For example, the present inventors identified two closely related seaweed batches with significantly different precursor content, coded as samples A9 and A10. Both batches were derived from the same collection of ocean-grown A. taxiformis (gametophyte phase) and differed only in the manner of their drying. Under microwave vacuum drying conditions, sample A9 was maintained at a temperature of 50°C for nearly two hours, resulting in almost complete hydrolysis and / or destruction of methanol-extractable bromoform precursors (Table 11) along with loss of up to half of the produced bromoform by volatilization (Table 14). In contrast, sample A10 was lyophilized to dryness without ever allowing it to thaw, which - prior to the present invention - resulted in the highest bromoform assays. Indeed, these conditions preserved around half of the methanol-extractable bromoform in precursor form (Table 11), in turn maintaining a high bromoform assay even through a secondary drying stage at 20°C / 2 mmHg - at this pressure pure bromoform would be expected to boil at 2°C. This result suggests that precursors are quite stable if the water activity of the sample is low enough, and the present inventors proposed that secondary drying could be safely extended even further once the bulk of the water had been removed. This was confirmed via a repeat batch (AlOb) that was lyophilized from the same frozen stock using a final secondary drying temperature of 30°C, yielding a product with a lower water content (1.87% vs 6.5%) at the cost of a slight reduction in bromoform (Table 2) and precursor content.

[0533] Table 14: Recovery of bromoform through drying of microwave vacuum-dried sample A9 and lyophilised sample A10Example 4: Precursors in Asparagopsis oil extract

[0534] Wild-harvested Asparagopsis taxiformis gametophytes were mixed with canola oil (1:1 by mass) and the mixture was aged for 5 weeks. The oil was decanted to yield an anti-methanogenic product as per published procedures.

[0535] The residue from the oil extraction which included solid seaweed and residual oil was retained at 2-8°C for 2 years, at which time a further small oil sample was decanted for analysis. Analiquot was extracted with methanol for 30 min., and the extract was analysed by GC-MS. The major halogenated compounds detected are reported in Table 15. Bromoform content was 3.85 mg / g as quantified against an authentic sample (FutureFeed method). Other detected components not reported in Table 15 include fatty acid methyl esters.

[0536] The extraction and GC-MS analysis was repeated using tetradeuterated methanol CD3OD. By this method, the fraction of bromoform remaining in precursor form was 35.5%.

[0537] Table 15: Halogenated metabolites detected upon GC-MS analysis of an aged Asparagopsis oil extraction.Example 5: Control of precursor hydrolysis via control of pH

[0538] Having demonstrated in Example 3 that heat treatment could destroy precursors in the presence of water, alternative methods of retaining precursors were examined. Conditions such as osmotic shock, light stress, partial desiccation and physical pressure have been reported to result in bromoform release from live algae, and so the present inventors resolved to freeze a seaweed sample as quickly as possible after harvest with the absolute minimum of handling. A fast rate of freezing should also benefit gland cell integrity through the formation of smaller ice crystals throughout the sample, as disruption by expanding ice crystals during freezing was thought to result in bromoform loss from thawed samples. As the Haloform reaction is catalysed by base, we further sought to preserve precursors through maintaining seaweed samples at neutral to acidic pH.

[0539] The seaweed samples analysed in Example 3 were wild-harvested, and therefore grew under normal ocean pH conditions (pH ~8). However, seaweed grown in culture can be produced under a range of pH conditions. A batch of cultured Asparagopsis taxiformis tetrasporophytes was grown at neutral to acidic pH. This was achieved using an automated system that sparged the culture with carbon dioxide gas whenever the pH of the culture medium rose to 7. Sample 018-02 which was harvested from this culture, frozen and lyophilized with minimal handling or delay showed only a slight increase in precursor content over the ocean-grown samples (Table 11). In contrast, sample 019-10 which wassubjected to osmotic shock by exposure to tapwater after harvest, contained only 23% residual precursors.

[0540] Based on the data described herein, the present inventors proposed that increased preservation of precursors is achieved by harvesting a sample of this culture directly into liquid nitrogen, as such rapid freezing would allow no time for gland cell activation or bromoform release while also forming smaller ice crystals. Unfortunately, sample 018-01 did not dry completely and was difficult to work with; having skipped the standard harvest procedure (drying to ~80% water content by centrifugation) and the live tetrasporophytes as decanted through a nylon mesh were greater than 95% seawater, resulting in a highly porous, salty, hygroscopic product upon lyophilization. The precursor content of this sample was lowered (Table 11), presumably by reaction with water during drying and processing.

[0541] Harvesting the cultured seaweed directly into acidic solutions to prevent any basecatalyzed enhancement of precursor hydrolysis led to increased precursor content. After "pickling" in this way for 2 hours, the samples were gently squeezed to remove excess liquid and immediately snap-frozen in liquid nitrogen before lyophilization. The highest residual precursor levels (76%) were measured after pickling in 3% aqueous citric acid solution at pH 1.85 (Table 11: 019-08). Although the increased protection given by citric acid compared to acetic acid may be because the former is a stronger acid, acetic acid is also more volatile and could be partially lost during lyophilisation.

[0542] However, the low pH of the samples resulted in poor bromoform recoveries when the samples were extracted with the standard methanol solvent. Increased recoveries were obtained when a small quantity of aqueous ammonia was included in the extraction solvent. The present inventors propose that this increased recovery of extractable bromoform is due to aqueous ammonia acting as a basic catalyst to promote the Haloform-type cleavage of residual bromoform precursors. Furthermore, promoting the Haloform reaction leaves a lesser opportunity for consumption of residual precursors in non-bromoform productive side-reactions.

[0543] Thus, a DCM extract of lyophilized seaweed sample 018-02 showed very few peaks on GC-MS analysis (Figure 17C) and only a trace of bromoform (0.80 mg / g); as DCM is aprotic and non-nucleophilic it cannot support precursor hydrolysis. Adding a small amount of aqueous ammonia (20 pL per mL of DCM) to the extraction allowed precursor hydrolysis to proceed under base catalysis and gave a striking 14-fold increase (Figure 17 F) in the measured bromoform assay, to 11.82 mg / g. This result, constituting the highest bromoform recovery of the four seaweed extractions in Figure 17, resolves the contradictions of in the field regarding DCM and MeOH extraction; the present inventorshave demonstrated that both solvents are suitable for extraction of bromoform from Asparagopsis as long as the requirement for precursor hydrolysis is considered.

[0544] On addition of aqueous ammonia to MeOH extraction of sample 018-02, the amount of bromoform extracted increased from 5.97 to 9.22 mg / g. This increase is smaller than that observed for DCM with NH4OH, as MeOH itself can support precursor hydrolysis. However, the chromatograms for extracts with MeOH / NH4OH were very similar to those for DCM / NH4OH; both conditions yielded good recovery of bromoform with similar component profiles, reflecting similar precursor reactivity (Figure 10, Figure 17 E,F, Table 19).

[0545] This data also demonstrates that the conditions and methods for processing of Aspragopsis will impact bromoform content and precursor content of compositions formed from Asparagopsis.

[0546] Without wishing to be bound by theory, this data may explain why Asparagopsis products were more efficacious than their measured bromoform content would suggest. If precursor hydrolysis were more efficient in the rumen (reaction temperature >39°C in cattle; possible base catalysis by saliva) than in the laboratory (MeOH extraction at room temperature or below), then compounds that are somewhat resistant to methanolysis could be hydrolysed to bromoform in vivo. On this basis, laboratory bromoform assays could be systematically underestimating the anti-methanogenic potency of the Asparagopsis products. Alternatively, if precursor hydrolysis in the rumen is not instantaneous, then Asparagopsis products may constitute a "slow release" formulation of bromoform. Unlike a bolus dose of synthetic bromoform, non-volatile precursors may be resistant to clearance in exhaled breath.

[0547] Table 16: Comparison of bromoform assay data after extraction with methanol (AST method) versus extraction with methanol plus aqueous ammonia (FutureFeed method 1; data from Table 17).

[0548] Table 17: Bromoform assay results for Asparagopsis samples treated with various solutions before drying by lyophilisation. Solutions were analysed directly by headspace gas chromatography (ChemCentre method). Solids were assayed using the standard methanol extraction (AST method), giving erroneously low results for acid-treated samples.

[0549] Table 18: Extractable bromoform content of sample 018-02 as measured using a range of extraction conditions with FF Method 1.

[0550] Table 19: Halogenated compounds detected in the chromatograms of Figure 10Example 6: Disruption of gland cells in non-dried samplesExtraction using osmotic shock.

[0551] A sample of live sporophytes of A. armata were placed in tap water to cause osmotic shock.

[0552] In brief, cultured tetrasporophytes of A. armata were dried by centrifuging to a water content of 75.1%. A sample (10g) was placed into tap water (400 mL) in an open vessel at 20°C. The supernatant developed a pale pink colour over 5.5 hours, which further developed overnight. Samples of the supernatant (40 mL) were decanted into an EPA GC-MS vial, and these were stored at 2-8°C until they were assayed for bromoform content by purge & trap GC-MS (ChemCentre method).

[0553] The water developed a pale pink colour over 5.5 hours (Figure 18), consistent with release of photosynthetic accessory pigments from disrupted chloroplasts. This was accompanied by the release of large quantities of bromoform, to a maximum concentration of at least 6.2 mg / L. This corresponds to 990 pg / g(DSB) which is 890 times the natural hourly bromoform release for tetrasporophytes of this species which was given (Paul et al. 2006)5as 1110 ng.g(DSB)1.h1, confirming a degree of gland cell activation and / or disruption. While the pink colour further strengthened overnight, the bromoform assay of the water fell; this suggests that bromoform release had already peaked. Assuming exponential decay, the natural logarithm of the bromoform assay in pg / g(DSB) was plotted against sampling time. The curve was roughly linear with R2= 0.969 and a slope of -0.4745 d1, corresponding to a half-life for bromoform loss from the solution of 1.42 days. Possible reasons for bromoform loss include volatilisation from the open container, or metabolism by various organisms.Cell disruption by freeze-thaw treatment

[0554] A frozen sample of A. taxiformis tetrasporophytes was allowed to thaw at room temperature. The defrosted sample was squeezed by hand, separating it into an expressed aqueous fraction and a damp solid residue. The pink colour of the aqueous fraction was the same as that obtained through osmotic shock (Figure 18). Again, this is consistent with disruption of chloroplasts, however, a high level of bromoform release [380 mg / L; 1520 pg / g(DSB) at 80% water content] confirmed that some amount of gland cell disruption had also occurred. The remaining solid residue was assayed at 3.95 mg / g(WMB) of bromoform; ignoring water content of this sample (which was not measured) about % to 1 / 3 of the total bromoform has been expressed into the aqueous sample.

[0555] The major compounds that were quantified in the expressed water are reported in Table 20. Bromoform and dibromoacetic acid were detected in a relative mass ratio of 2.375:1. Similar results were reported by Paul et al.5who measured release rates of 1110 ng.g(DW)-1.h-1(± 393 SE) for bromoform, and 539 ng.g(DW)-1.h-1(±166 SE) for DBA, equivalent to a relative mass ratio of 2.05:1. They also report ratios of 1.88:1 within the algae, and 1.93:1 released into the medium, as averages of highly variable data. (Paul et al. 2006)5The similar proportions observed here suggest that the precursors released by freeze-thaw cell disruption are hydrolysing in a manner close to the naturalrelease mechanism in live algae.

[0556] Since the sample had been held at -20°C for ~3 months prior to thawing, bromoform production on thawing by intact haloperoxidase enzymes in the sample was ruled out; haloperoxidase enzymes are reported to decay rapidly over a period of days upon frozen storage.

[0557] Table 20: Analysis of aqueous liquid expressed from freeze-thaw-treated tetrasporophytes. Raw results in pg / L are converted to ng / g(DSB) of seaweed using a water content of 80%.ResultResult Result (ng / gAnalyte (pg / L) (pM) DSB)Dibromoacetic acid 160000 734 40000 Monobromoacetic acid 17000 122 4250 Monochloroacetic acid 13000 138 3250 Bromochloroacetic acid 6700 39 1675Dichloroacetic acid 770 6.0 192.5Trichloroacetic acid 440 2.7 110Bromoform 380000 1504 95000 Dibromochloromethane 1800 8.6 450Chloroform 120 1.0 30 Bromodichloromethane 81 0.49 20.25 Dibromomethane 48 0.28 12

[0558] The analytical method used by Paul et al.5for quantification of metabolite release into seawater (Paul et al. 2006)5relied on extraction of metabolites into TBME after acidification with H2SO4, followed by esterification of the haloacetic acids with MeOH, and finally a wash to remove H2SO4 before analysis by GC-MS. In contrast, the present inventors directly analysed the aqueous samples by LC-MS and purge & trap GC-MS, avoiding any losses inherent in the multistep extraction and derivatisation process. However, since the measured bromoform / dibromoacetic acid ratios are similar between the two methods, the losses through the method of Paul et al.5are proposed to be small.Complete disruption of gland cells at 50°C

[0559] Experiments on disruption of gland cells in non-dried samples were performed using a Ratek circulating water bath containing ~16L of water. This was set up in a fume hood and the water was heated to 50°C.

[0560] A control sample (023-01) of cultivated sporophytes of A. taxiformis was lyophilised without allowing it to thaw, to preserve gland cell integrity as much as possible. One bag of frozen tetrasporophytes ("'150 g at ~80% water content) was cut open and spread on a stainless-steel tray, which was placed directly in the pre-chilled lyophilizer. The dried sample contained 2.78 mg / g of methanol-extractable bromoform.

[0561] A second sample (023-02) of A. taxiformis was allowed to thaw before hydrolysis at 50°C. This sample (~300 g at ~80% water content) was placed directly in a heated water bath, still vacuum-packed in the food-grade polyethylene in which it had been frozen. The sample was kept fully submerged by the weight of the Perspex sample rack. As polyethylene is permeable to lipophilic molecules such as bromoform, but relatively impermeable to polar molecules such as water, this packaging acted as a semi-permeable dialysis membrane. Under dialysis against a large volume of water at 50°C, which was open to the fumehood for further loss of bromoform by volatilisation, almost complete depletion of the bromoform from the sample was observed. After one hour the water bath was drained (Caution! Contains bromoform). The bag was recovered and cut open, and the seaweed was frozen at -20°C and lyophilized to dryness. Yield 64.7 g at a moisture content of 5.675%. Methanol-extractable bromoform content of the dried sample was 0.10 mg / g(DSB) which is 3.6% of the control sample.

[0562] Importantly, the observation of essentially complete bromoform depletion confirms that gland cell disruption was complete. Since bromoform depletion represents complete transfer of bromoform from one equilibrium phase (the seaweed) to another (the surrounding water and fume hood exhaust), this shows that no barrier remained to complete hydrolysis of bromoform precursors, and free diffusion of the produced bromoform.

[0563] In the following closed-system experiment, the displacement of the equilibrium position is comparatively minor, making it reasonable to assume that the system reached complete equilibration within the time allowed.

[0564] A third sample (023-04) of A. taxiformis was allowed to thaw at room temperature. In brief, the thawed sample was diluted in a small volume of water in an enclosed system then heat-treated as for 023-02, using the following procedure. The sample (99.2 g at ~80% water content) was allowed to thaw at ambient temperature for two hours, then the bag was cut open and the contents transferred into a IL Schott bottle containing tapwater (300 mL). Thus, the mixture contained approximately 19.8 g of solids on dried basis, and 379 mL of water. The bottle was placed on the sample rack in the water bath with sample 023-02 and heat-treated for the same period. The seaweed was decanted through a stainless-steel colander, frozen at -20°C and lyophilized. The decanted solution was assayed for bromoform content by GC-MS (ChemCentre method).

[0565] The decanted aqueous fraction contained bromoform at 0.015 mg / mLmg / g). A seaweed-water partition coefficient Kswcan be formulated as the ratio of seaweed-bound to aqueous bromoform:

[0566] Since the system is sealed and the aqueous bromoform concentration [CHBr3]aqueous= 0.015 mg / g is small, then [CHBr3]seaweedis approximately equal to the total bromoform concentration of the lyophilised control sample, giving Ksw= 185, or log / fsvl / = 2.27. This value of [CHBr3]seaweed assumes that all the bromoform within the control sample was methanol-extractable, and that no bromoform was lost during lyophilisation of the control sample, and therefore the "true" value of Kswwill be higher. For example, Error! Reference source not found.Table 16 shows a 1.9-fold increase in methanol-extractable bromoform for a different batch of this same culture, measured at a later date upon the addition of NH4OH to the extraction solvent. Hence, we derive an upper estimate of Kswby doubling the measured value to give Ksw= 370, or log / fsw= 2.57.

[0567] The n-octanol-water partition coefficient P is commonly used to model membrane permeability of lipophilic molecules and has been measured for bromoform as log P = 2.4.(Atlanta (GA): Agency for Toxic Substances and Disease Registry (US) 2005) Although obvious differences exist between the octanol-water and seaweed-water systems, to a first approximation log / ^ = log? is what we expect if the majority of the methanol-extractable bromoform in the seaweed is in equilibrium with the aqueous phase, i.e. the gland cells have been completely disrupted and any methanol-labile bromoform precursors fully hydrolysed to free bromoform.

[0568] Therefore, in fully disrupted and equilibrated samples, the seaweed-bound bromoform concentration is around two log units higher than the aqueous bromoform concentration. This result confirms that even when gland cells are disrupted, water remains a poor solvent for bromoform extraction due to the lipophilicity of bromoform.

[0569] Recovery of methanol-extractable bromoform from the dried product was 64.6% of the control sample using the measured value of [CHBra aweed, and the aqueous phase accounted for another 8.9% of the total bromoform. The remaining 26.5% represents an additional quantity of bromoform lost due to heat treatment in tap water. Using instead the upper estimate of [CHBra aweed, the recoveries are halved, and the loss doubled. Therefore, in a fully disrupted and equilibrated sample, roughly 30-60% of the bromoform content is lost during drying and processing compared to a minimally disrupted lyophilised control. This loss may be primarily due to volatilisation during lyophilisation, with a contribution from diversion of precursors into non-productive side-reactions.The bromoform-depleted sample 023-02

[0570] The data for sample 023-02 demonstrates that it is possible to prepare bromoform from fresh-frozen Asparagopsis samples whereby >95% of bromoform was removed compared to a lyophilised control. As detailed above, this was achieved by dialysis of a thawed seaweed sample at ~80% water content, vacuum-packed in polyethylene heat-seal tubing, against a large volume of water. After lyophilisation, the bromoform-depleted sample 023-02 contained bromoform at 0.10 mg / g according to the standard commercially available assay with MeOH extraction (Table 21), increasing only slightly (to 0.13-0.16 mg / g; Table 22) when ammonia was added to catalyse precursor hydrolysis. Therefore sample 023-02 is essentially depleted of both bromoform and bromoform precursors, showing that no barrier remained to hydrolysis of bromoform precursors with water or free diffusion of the resulting bromoform. Therefore, the present inventors have demonstrated essentially complete disruption of gland cells in a thawed seaweed sample at 80% water content that was heated at 50°C for an hour.

[0571] This data also demonstrates that the conditions and methods for processing of Aspragopsis, including gland cell integrity, impact bromoform content and precursor content of compositions formed from Asparagopsis.

[0572] Importantly, this data allows for the assessment of the anti-methanogenic capacity of Asparagopsis compositions.

[0573] Table 21: Extractable bromoform content of bromoform-depleted sample 023-02 as determined using various methods and extraction solvents

[0574] Table 22: Extractable bromoform content of bromoform-depleted sample 023-02 as determined by sequential re-extraction with different solvents and GC-MS analysis (FF Method 3)Effect of acid and base treatment on gland cell integrity

[0575] In Example 5 the control sample 018-02 was dried to ~80% water by centrifugation then frozen at -20°C before lyophilisation, retaining high levels of bromoform during this process. However, the highest bromoform levels were retained in the sample treated with bicarbonate buffer at pH 7.87, which is close to normal seawater pH (~8). As any bromoform precursors released into basic solution will be rapidly hydrolysed, but aqueous bromoform levels were lower than the tap water-treated sample, then gland cell disruption is proposed to have been minimal under these conditions. The retention of high bromoform levels through the lyophilisation process suggests that losses of free bromoform by volatilisation were minimal, consistent with the precursors remaining largely intact until the sample was dry. This requires that the gland cells remained intact at least until the sample was snap-frozen, successfully forming a barrier between precursors and the surrounding aqueous base until the rate of precursor hydrolysis could be slowed in the cold. Precursor hydrolysis may have even been delayed until re-addition of protic solvent for bromoform analysis, where the extra bicarbonate base was well positioned to promote the Haloform reaction, minimising non-bromoform-producing sidereactions of the precursors and yielding a higher bromoform recovery than the control.

[0576] This level of bromoform retention was unexpected, as the low salt concentration in the bicarbonate experiment was expected to cause a higher level of osmotic shock in the algae. 1.3% aqueous bicarbonate is isotonic with human blood ("'300 mOsm / L). Another 100 mOsm / L should be added due to residual culture medium; the same dilution of culture medium in the tap water treatment yielded a salinity of 3.4 ppt, which is ~10% of the value normally given for seawater. Therefore, the bicarbonate treatment involved a sudden drop in osmolarity from culture medium at ~1000 mOsm / L to an estimated 400 mOsm / L, but bromoform release was significantly less than the tap water experiment where osmolarity fell to ~100 mOsm / L.

[0577] The present inventors propose that harvesting seaweed into 1.3% aqueous bicarbonate may have the advantages of preserving high extractable bromoform levels compared to a tap-water rinse, while still removing some salts by dilution of seawater or culture medium. Residual salts yield a product that is hygroscopic when dried and may have a reduced bromoform content as illustrated by sample 018-01, which was too hygroscopic for effective lyophilisation as discussed previously.Example 7: Disruption of gland cells in dried samplesComplete extraction of free CHBrg from fully disrupted samples

[0578] Bromoform-depleted sample 023-02 is assessing the recovery of free CHBra by a particular extraction solvent. As the sample has been shown to be fully disrupted, bromoform added as a solution in a solvent will freely partition between sample and solvent according to its affinity for both.

[0579] A solution of bromoform in the DCM / MeOH / NH4OH extraction solvent preferred for FF Method 2 was allowed to equilibrate with an aliquot of sample 023-02 over 16 h. At this time, GC-MS analysis (FF Method 2) of the solution measured a bromoform recovery of 112%. This shows that partition of bromoform out of DCM / MeOH / NH4OH solvent into the seaweed sample was minimal. In other words, extraction of free CHBra from fully disrupted samples is complete when DCM / MeOH / NH4OH extraction solvent is employed.

[0580] This data demonstrates the utility of the DCM / MeOH / NH4OH solvent mixture for efficient extraction of bromoform.Sequential re-extraction of minimally disrupted seaweed samples

[0581] The ability of various solvent mixtures to penetrate gland cells was assessed using sequential re-extraction. In this procedure, a sample that has been extracted with one solvent or mixture is re-extracted with another solvent or mixture. Detection of significant quantities of bromoform in the second extract indicates that the bromoform recovery of the first extraction was incomplete.

[0582] Extraction and analysis was performed on non-milled samples according to FF Method 3 with dichlorobenzene internal standard and 5-point mass-based calibration curve. Sonication was not used.

[0583] Samples ("'100 mg) were weighed into tared vials and extracted by shaking with the first set of extraction solvents (including internal standard) according to Table 23. The extracts weredecanted by pipette for GC-MS analysis, and the mass of bromoform in the extract is reported in Table 23 as mg of bromoform per g of seaweed.

[0584] The sample vials were re-weighed to determine the mass of extract removed and the mass of extract remaining with the sample. The masses of residual internal standard and residual extracted bromoform remaining with the sample were then calculated.

[0585] Complete decantation was difficult for the bulky freeze-dried samples, with ~25% of the extract remaining after decantation of sample 018-02, and ~8% remaining for sample A10. Decantation was almost complete for microwave-dried sample A9, with ~2% residual extract.

[0586] The sample in the same vial was re-extracted with the appropriate solvent (including internal standard) according to Table 23. Bromoform was quantified in the decanted extract by GC-MS, subtracting the mass of residual internal standard carried over from the first extraction. The mass of newly extracted bromoform was then determined by subtracting the mass of residual extracted bromoform from the GC-MS assay result, and the difference is reported in Table 23.

[0587] Table 23: Bromoform assay data from repeat extractions of the same sample with various solvents / mixtures are reported on mass basis.

[0588] Results for high-precursor sample AlOb are as expected based on the data described herein. Extracted totals have a wide variance as discussed above, with as much as 30.2 mg / g(DSB) recoverable in a single extraction in favourable cases. A first extraction with DCM / MeOH / NH4OH solvent recovered 99-100% of the extractable bromoform, compared to 97% for a first extraction with methanol. This confirms that the variance is within sample AlOb rather than due to incomplete extraction. A first extraction with DCM behaved as expected, with only 8% of the total bromoform recovered; successful re-extraction of the remainder with DCM / MeOH / NH4OH confirms that very few bromoform precursors are extractable from this sample using DCM.

[0589] The present inventors propose that DCM alone is unable to penetrate intact gland cells to extract free bromoform or bromoform precursors. Only when a suitable aprotic solvent such as methanol, or a suitable base such as aqueous ammonia is added to the DCM extraction solvent can the gland cell barrier be penetrated, allowing solvent access to the bromoform and / or bromoform precursors within.

[0590] The results for low-precursor sample A9 are also straightforward. First extractions with DCM / MeOH / NH4OH or MeOH are again 99% and 97% effective respectively. Total extractable by any method was around 20 mg / g(DSB), as these measurements were performed when analysis of batch A9 had stabilised at this higher value. Apart from the lower variance, the major difference between this sample and the high-precursor sample A10 is that a first extraction of sample A9 with DCM recovered as much as a third of the extractable bromoform, with the remainder recoverable using DCM / MeOH / NH4OH.

[0591] The present inventors propose that since more bromoform is extractable using DCM alone, this implies a greater level of gland cell disruption in the microwave vacuum-dried sample A9.

[0592] An initial extraction of neutral-acidic cultured tetrasporophyte sample 018-02 with either of DCM or methanol alone only recovers a part of the bromoform recovered by extraction with DCM / MeOH / NH4OH. Unlike in the case of samples A9 and A10 , the missing bromoform cannot be recovered by re-extraction with DCM / MeOH / NH4OH.

[0593] The present inventors propose that the missing bromoform from re-extraction of sample 018-02 is the result of consumption of precursors in non-productive side-reactions. Therefore, extraction in the first instance with solvents such as DCM / MeOH / NH4OH, which reduce losses through side-reactions by base-catalysis of the Haloform reaction, is preferred for neutral-acidic samples.Extraction of intact precursors from milled samples

[0594] The extraction of bromoform and haloform precursors from lyophilised and milled Asparagopsis was examined by1H- and13C-NMR spectroscopy, followed by GC-MS.

[0595] Freeze-dried seaweed sample A10 (2 g) was milled in a Qiagen planetary ball-mill at 30 Hz for 1 min. Portions of the milled algae (50 mg or 200 mg) were weighed into centrifuge tubes along with 1 m Lof deuterated solvent (methanol-^ or dichloromethane-ck). The sealed tubes were extracted with ultrasonication at 50 kHz for periods of lh or 16 h; the 200 mg DCM extraction suffered a partial loss of solvent through this period of "'170 pLand this volume was replaced with fresh DCM-ck- Samples were centrifuged at 10000 rpm for 1 min., and the clarified supernatants were examined by NMR spectroscopy (Bruker BioSpin GmbH). The following experiments were performed for each sample:1H-NMR (600.13 MHz): 16 scans with 1 s delay and a spectral width of 20.02 ppm13C NMR (150.92 MHz): Proton decoupled acquisition using 1,024 scans, 2 s delay, and a spectral width of 240.07 ppmHSQC (sensitivity improved + multiplicity editing): 16 scans, 1.5 s delay, and a spectral width of 13.018 ppm.

[0596] Significant differences in both chemical shift and relative peak area were observed between the samples extracted for 1 h and 16 h, confirming the ongoing chemical changes associated with precursor solvolysis during the extraction. Of most interest was the appearance of a distinctive methylene resonance in the HSQC spectrum of the DCM extracts, with an unusually low-field shift 8 = 4.68 ppm (1H) / 111.23 ppm (13C). This resonance can be assigned to the terminal methylene group of an enol ester; formation of such compounds via a haloform reaction (Scheme 10) could account for some of the production of bromoform in low-water environments such as in DCM solution, particularly the 50 mg DCM extract which showed full conversion of precursors to bromoform on extended storage without the addition of methanol.

[0597] Scheme 10: A reasonable Haloform-type mechanism for the formation of an enol ester, allowing the formation of bromoform in Asparagopsis extracts under low-water conditions.

[0598] The observed changes slowed greatly when ultrasonication was ceased and the extract was decanted from the biomass. The 200 mg DCM extract was monitored by1H-NMR spectroscopy over 30 hours after decanting, and no significant chemical changes were observed. The sample was then diluted with MeOD (0.1 mL) and significant slow chemical changes were monitored over a period of 11 days (Figure 13).

[0599] On completion of the NMR experiments, the samples were stored in glass for 1 month and then recovered for GC-MS analysis. Bromoform assay results are given in Table 24. Selected chromatograms are provided in Figure 14 and Table 25, which demonstrate the difference in extraction chemistry between DCM and methanol. In contrast to freshly prepared DCM extracts of non-milled samples in which few components are detected by GC-MS (lower trace in Figure 9), the chromatograms of aged DCM extracts of milled samples are dominated by the presence of haloacetones and halomethanes.

[0600] Ball-milling of biological samples is known to result in significant disruption of cellular structure. This data confirms that disruption of gland cell barriers, for example through milling of dried Asparagopsis samples, is required before stored haloacetones are accessible for extraction by DCM solvent alone.

[0601] Note that the GC-MS results support the earlier NMR analysis (Figure 13) which showed significant changes in the region where haloacetone protons are expected to resonate based on literature values [8 (ppm; CDCh): 1,1-dibromoacetone 2.58 (s, 3H), 5.76 (s, 1H); 1,1,3-tribromoacetone 4.54 (s, 2H), 6.24 (s, 1H); 1,1,3,3-tetrabromoacetone 6.48; 1-bromo-l-chloroacetone 2.50 (s, 3 H), 5.86 (s, 1 H)]37

[0602] Table 24: Final bromoform content (by GC-MS) of Asparagopsis extracts from the NMR spectroscopy study, after 1 month extended storage / reaction time. Proteo-bromoform (CHBr3) assay of the extracts was measured using FF Method 1, and is expressed as mg / g of the original lyophilised sample A10, on dried basis. Total bromoform (CHBr3 + CDBr3) was measured by integration of the total ion count for the bromoform peak using MestreNova software, and comparing with the 1 mg / mL bromoform standard.

[0603] Table 25: Components identified by their mass spectra in the chromatograms of Figure 14.The effect of milling and sonication on extractable bromoform in high-precursor sample 018-02

[0604] Example 5 demonstrated the reactivity of the bromoform precursors under acidic and basic conditions. Further experiments were carried out with the addition of disruption / homogenisation of the sample by grinding in a planetary ball-mill (Table 26), and / or the addition of sonication during extraction (Table 27). These preliminary experiments were analysed by GC-MS using FF Method 1 (single-point calibration).

[0605] Table 26: Effect of sample milling before extraction on bromoform recovery from high-precursor sample 018-02 using a range of extraction conditions. Data for non-milled, non-sonicated extractions have been reported previously in Table 18.Average 6.4 7.31.1 13%

[0606] Table 27: Effect of sonication during extraction on bromoform recovery from high-precursor sample 018-02 using a range of extraction conditions. Data for non-milled, non-sonicated extractions have been reported previously in Table 18.Average 7.7 6.0 -1.6 -21%

[0607] Ball-milling of high-precursor sample 018-02 generally caused a reduction in bromoform recovery. For extractions with protic solvents, i.e. those featuring MeOH and / or NH4OH, milling caused a decrease in extractable bromoform of 19-34% (average 24%).

[0608] Extractions that included MeOH saw a positive effect on bromoform recovery from sonication of 8-19%.

[0609] It is clear that ball-milling of the sample under non-cryogenic conditions has a significant negative effect on bromoform recovery. The fact that milling and sonication give similar and nonadditive increases in bromoform recovery through DCM extraction suggests they might work via the same mechanism of gland cell disruption.Effect of milling and sonication on repeat extractions of high-precursor samples AlOb and 018-02

[0610] The effects of milling and sonication on neutral-acidic cultured tetrasporophyte sample 018-02 were re-examined with the further comparison of ocean-grown gametophyte-phase sample AlOb. Repeat extractions of the same sample were analysed using FF Method 3 (full calibration curve).

[0611] Table 28 compares non-milled samples extracted by sonication for 1 hour or 16 hours, reporting the bromoform recovered in each extraction, and the total amount recovered.

[0612] Table 27 showed a positive effect of sonication on initial extraction of bromoform into DCM from sample 018-02. However, Table 28 shows that this effect masks a large negative effect on extractability of the remaining bromoform in the sample. Sonication of sample 018-02 in DCM for 16 hours has reduced total bromoform recovery by >95%, with only 0.58 mg / g recovered over two extractions compared to 13.5 mg / g recovered in a comparable extraction in the presence of NH4OH (Table 27). Sonication for only 1 hour has a lesser negative effect (33% reduction in total recovery) for this sample.

[0613] The present inventors propose that ball-milling of a dried seaweed sample results in disruption of gland cells and release of stored precursors. As no protic solvent is available under these conditions to support the Haloform reaction apart from residual moisture content of the sample, the released precursors may instead be consumed in non-Haloform side-reactions, reducing the extractable bromoform content of the sample.

[0614] Given that full recovery of extractable bromoform is achievable from non-disrupted samples, it may be preferable to avoid non-cryogenic disruption of dried samples before bromoform extraction is attempted.

[0615] The positive effect of sonication on initial extraction of bromoform into DCM is again apparent for sample A10. Re-extraction of sample AlOb after 16-hour sonication in DCM yielded a total of 19.41 mg / g of bromoform, which is at the low end of the variable range normally observed for this sample. It is likely that sonication in DCM is having a small negative effect on overall bromoform recovery for this sample. However, the negative effect is more pronounced for the neutral-acidic cultured tetrasporophyte sample 018-02.

[0616] Table 28: Effect of sonication on bromoform recovery through repeat extractions of the same sample with various solvents / mixtures. Results are reported on mass basis. Vial tares were unavailable for most samples, and thus results marked with an asterisk include an estimate of residual internal standard and bromoform based on the decantation behaviour noted in the earlier experiment.

[0617] A sample of batch AlOb was milled with a planetary ball-mill before sonication for 1 hour with DCM, MeOH, or DCM / MeOH / NH4OH. Bromoform quantitation of the resulting extracts is reported in Error! Reference source not found.Table 29.

[0618] Milling the sample has further increased the initial recovery of bromoform into DCM. A milled sample sonicated in DCM for 1 hour yields more bromoform (8.63 mg / g; Table 29) than a nonmilled sample sonicated for 16 hours (6.39 mg / g; Table 28)

[0619] Table 29: Bromoform recovered on extraction of a milled sample of ocean-grown high-precursor lyophilised gametophyte batch AlOb with various solvents under sonication for 1 hour

[0620] Extraction of samples that were both milled and sonicated resulted in the detection of significant quantities of extracted haloacetones. Without milling and sonication, a 16-hour extraction of sample A10 into DCM yielded only traces of haloacetones (Table 13). A DCM extract of a milled sample of batch AlOb obtained by sonication for just one hour featured numerous haloacetones on GC-MS analysis (Table 31). The most abundant of these was 1,1,3,3-tetrachloroacetone, at 38% of the area of the bromoform peak. A MeOH extract obtained under the same conditions featured a similar amount of 1,1,3,3-tetrachloroacetone by peak area (Table 30), along with the expected methyl esters of dibromoacetic and dibromoacrylic acids as reported herein.

[0621] Further in line with expectations based on the data presented herein, a DCM / MeOH / NH4OH extract obtained by sonication for 1 hour contained haloacetones in reduced quantity compared to the other extracts, along with their ester and amide cleavage products (Table 32). This confirms that a 1-hour sonication is insufficient for full precursor extraction and hydrolysis from milled samples.

[0622] Table 30: Components detected in GC-MS chromatogram for sample of high-precursor batch AlOb that was extracted into MeOH with sonication for 1 hourdibromochloromethane1,1 -DibromopropaneDichloroiodomethaneBromoformNOT 1-Bromo-3-chloroacetone1 , 1 ,3-trichloroacetoneAlkaneZ-olMethyl dibromoacetateIS (dichlorobenzene)dibromoiodomethane1,1,3,3-tetrachloroacetone1-bromo-1,3-dichloroacetoneCarbon tetrabromideBromochloroacetamideMethyl dibromoacrylate1-Bromo-1,3,3-trichloroacetone1,1-Dibromo-3-chloroacetoneHeptadecaneAlkenol?Fatty acid (ester?)Glycerol-1 -palmitate?

[0623] Table 31: Components detected in GC-MS chromatogram for sample of high-precursor batch AlOb that was extracted into DCM with sonication for 1 hourChloroiodomethaneToluene (laboratory contaminant)DibromochloromethaneAlkaneAlkaneBromoform1,1,3-trichloroacetoneIS (dichlorobenzene)dibromoiodomethane1,1,3,3-tetrachloroacetone1-bromo-1,3-dichloroacetoneCarbon tetrabromide1 -Bromo-1 , 3, 3-trich loroacetone1 , 1 -dibromo-3-chloroacetone1.1.3-Tribromoacetone1.3-Dibromo-1 ,3-dichloroacetonebromo compoundTetrabromopropeneAlkaneAlkenolHexadecanoic acid

[0624] Table 32: Components detected in GC-MS chromatogram for sample of high-precursor batch AlOb that was extracted into DCM with sonication for 1 hourBromodichloromethaneMethyl chloroacetateToluene (laboratory contaminant)Dibromochloromethane1,1 -DibromopropaneDichloroiodomethaneMethyl chloroacrylateBromoformMethyl bromochloroacetateBromochloroiodomethaneMethyl dichloroacrylate1,1,3-trichloroacetoneMethyl dibromoacetateDichloroacetamideIS (dichlorobenzene)Dibromoiodomethane1,1,3,3-TetrachloroacetoneDichloro sulfur compoundCarbon tetrabromideChlorobromoacetamideMethyl dibromoacrylateDichloroacrylamideDibromoacetamideDichloroacrylate derivativeTribromoacetonitrileHeptadecane?Tetradecanoic acidAlkenol?11.118 1.52 Alkenol?11.467 5.52 Hexadecanoic acid 13.338 2.3 Glycerol-1 -palmitateReferences1. Glasson, C. R. K. et al. Benefits and risks of including the bromoform containing seaweed Asparagopsis in feed for the reduction of methane production from ruminants. Algal Research 64, 102673 (2022).2. Burreson, B. J., Moore, R. E. & Roller, P. Haloforms in the essential oil of the alga Asparagopsis taxiformis (rhodophyta). Tetrahedron Lett. 16, 473-476 (1975).3. Thapa, H. R. et al. Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends Insights into Seaweed Reactive Oxygen Species Enzymology. ACS Chem. Biol. 15, 1662-1670 (2020).4. Fuson, R. C. & Bull, B. A. The Haloform Reaction. Chem. Rev. 15, 275-309 (1934).5. Paul, N. A., de Nys, R. & Steinberg, P. D. Chemical defence against bacteria in the red alga Asparagopsis armata: linking structure with function. Mar. Ecol. Prog. Ser. 306, 87-101 (2006).6. Kladi, M., Vagias, C. & Roussis, V. Volatile halogenated metabolites from marine red algae.Phytochem. Rev. 3, 337-366 (2004).7. Hutchings, J. L., Grebneva, Y., Dilmetz, S. J., Pincher, D. W. M. & Hoffmann, P. Analytical methods for the analysis of bromoform in red seaweed Asparagopsis armata and Asparagopsis taxiformis - A review. Algal Research 79, 103478 (2024).8. Hargrave, M. S., Islam, S., Agarwal, V. & Smith, J. E. The influence of light stress on bromoform synthesis and concentration in the red seaweed Asparagopsis taxiformis. J. Appl. Phycol. 36, 321-329 (2024).9. Fenical, W. HALOGENATION IN THE rhodophyta2A REVIEW. J. Phycol. 11, 245-259 (1975). 10. Paul, N. A., Cole, L., de Nys, R. & Steinberg, P. D. Ultrastructure of the gland cells of the red alga Asparagopsis armata (bonnemaisoniaceae)l. J. Phycol. 42, 637-645 (2006).11. Woolard, F. X., Moore, R. E. & Roller, P. P. Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis. Phytochemistry 18, 617-620 (1979).12. Woolard, F. X., Moore, R. E. & Roller, P. P. Halogenated acetamides, but-3-en-2-ols, and isopropanols from Asparagopsis taxiformis (delile) trev. Tetrahedron 32, 2843-2846 (1976).13. Sugano, M. et al. Aldose reductase inhibitors from the red alga, Asparagopsis taxiformis.Tetrahedron Lett. 31, 7015-7016 (1990).14. McConnell, O. & Fenical, W. Halogen chemistry of the red alga Asparagopsis. Phytochemistry 16, 367-374 (1977).15. Romanazzi, D. et al. Rapid Analytical Method for the Quantification of Bromoform in the Red Seaweeds Asparagopsis armata and Asparagopsis taxiformis Using Gas Chromatography- Mass Spectrometry. ACSAgric. Sci. Technol. 1, 436-442 (2021).16. Vucko, M. J., Magnusson, M., Kinley, R. D., Villart, C. & de Nys, R. The effects of processing on the in vitro antimethanogenic capacity and concentration of secondary metabolites of Asparagopsis taxiformis. J. Appl. Phycol. 29, 1577-1586 (2016).17. Packer, M., Romanazzi, D. & Svenson, J. Animal Feed Additive and Methods for its Preparation. (2022).18. Muldrew, K. & McGann, L. E. The osmotic rupture hypothesis of intracellular freezing injury.Biophys. J. 66, 532-541 (1994).19. WQ2020113279A1.20. Magnusson, M., Vucko, M. J., Neoh, T. L. & de Nys, R. Using oil immersion to deliver a naturally-derived, stable bromoform product from the red seaweed Asparagopsis taxiformis. Algal Research 51, 102065 (2020).21. Tan, S., Harris, J., Roque, B. M., Askew, S. & Kinley, R. D. Shelf-life stability of Asparagopsis bromoform in oil and freeze-dried powder. J. Appl. Phycol. 35, 291-299 (2023).22. Council, N. R., Division on Earth and Life Studies, Board on Agriculture and Natural Resources & Committee on Minerals and Toxic Substances in Diets and Water for Animals. Mineral Tolerance of Animals: Second Revised Edition, 2005. 510 (National Academies Press, 2006).23. Machado, L. et al. Identification of bioactives from the red seaweed Asparagopsis taxiformis that promote antimethanogenic activity in vitro. J. Appl. Phycol. 28, 3117-3126 (2016). 24. Clapperton, J. L. The effect of trichloroacetamide, chloroform and linseed oil given into the rumen of sheep on some of the end-products of rumen digestion. Br. J. Nutr. 32, 155-161 (1974).25. Gilbert, E. E. Perhaloketones— XVII. Tetrahedron 25, 1801-1806 (1969).26. Cassidy, P. Process for preparing anti-methanogenic composition. (2023).27. National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 5558, Bromoform. https: / / pubchem.ncbi.nlm.nih.gov / compound / Bromoform.28. CRC Handbook of Chemistry and Physics, 99th Edition. 1550 (CRC Press, 2018).29. Hagemann, R., Nief, G. & Roth, E. Absolute isotopic scale for deuterium analysis of natural waters. Absolute D / H ratio for SMOW. Tellus 22, 712-715 (1970).30. Silverstein, R. M., Webster, F. X., Kiemle, D. J. & Bryce, D. L. Spectrometric Identification of Organic Compounds. 464 (Wiley, 2014).31. Margolin, Zafra. & Long, F. A. Acidic behavior of chloroform. J. Am. Chem. Soc. 95, 2757-2762 (1973).32. Strait, D. Kinetic and thermodynamic acidities of selected weakly acidic hydrocarbons.(Missouri S&T, 1974).33. Parham, W. E. & Schweizer, E. E. Halocyclopropanes from Halocarbenes. in Organic Reactions (ed. Denmark, S. E.) 55-90 (Wiley, 2004). doi:10.1002 / 0471264180.or013.02.34. Menezes, F. G., Kolling, R., Bortoluzzi, A. J., Gallardo, H. & Zucco, C. Hexabromoacetone as tribromoacetylating agent of alcohols and amines and as mediator in the conversion of carboxylic acids into amides in the presence of triphenylphosphine. Tetrahedron Lett. 50, 2559-2561 (2009).35. Wagner, W. M., Kloosterziel, H. & Bickel, A. F. The thermal decarboxylation of alkali trichloroacetates in aprotic solvents IV decarboxylation in the presence of hexa- and pentachloroacetone. Reel. Trav. Chim. Pays-Bas 81, 933-946 (1962).36. Thomas, A., Gasch, B., Olivieri, E. & Quintard, A. Trichloroacetic acid fueled practical amine purifications. BeilsteinJ. Org. Chem. 18, 225-231 (2022).37. Burreson, B. J., Moore, R. E. & Roller, P. P. Volatile halogen compounds in the alga Asparagopsis taxiformis (Rhodophyta). J. Agric. Food Chem. 24, 856-861 (1976).

Claims

1. The claims defining the invention are as follows:

1. A method of producing an anti-methanogenic halomethane composition comprising one or more anti-methanogenic halomethane compounds, the method comprising contacting one or more halomethylcarbonyl compounds with one or more protic solvent.

2. A method of claim 1, wherein the one or more halomethylcarbonyl compounds is contacted with the one or more protic solvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form the one or more anti-methanogenic halomethane compounds.

3. A method of claim 1 or claim 1, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

4. A method of any one of claims 1 to 3, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

5. A method of any one of claims 1 to 4, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

6. A method of any one of claims 1 to 5, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

7. A method according to any one of claims 1 to 5, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1, 1,1, 3,3,3- hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, l,l,l,3,5,5,5-heptabromopentan-2,4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2,2- tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo- 3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts oresters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

8. A method according to any one of claims 1 to 7 , wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

9. A method according to any one of claims 1 to 8, wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

10. A method according to any one of claims 1 to 9 further comprising contacting the one or more halomethylcarbonyl compounds with one or more protic solvent in the presence of a base.

11. A method according to claim 10 wherein the base is selected from the group consisting of bicarbonate, carbonate, ammonia, aqueous ammonia, methanolic ammonia, and ethanolamine.

12. A method of producing an anti-methanogenic composition, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with a one or more protic solvent.

13. A method of increasing the levels of at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with one or more protic solvent.

14. A method according to claim 12 or 13, wherein the biomass of Asparagopsis comprises one or more halomethylcarbonyl compounds.

15. A method according to any one of claims 12 to 14, wherein the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof is contacted with the one or more protic solvent under conditions to solvolyse the one or more halomethylcarbonyl compounds to form the one or more anti-methanogenic halomethane compounds.

16. A method of according to any one of claims 12 to 15, wherein the one or more halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

17. A method according to claim 12 or 14, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

18. A method according to any one of claims 12 to 17, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

19. A method according to any one of claims 12 to 18, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

20. A method according to any one of claims 12 to 18, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1,1,3,3-pentabromoacetone, 1, 1,1, 3,3,3- hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1,1, 1,5, 5, 5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, l,l,l,3,5,5,5-heptabromopentan-2,4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2,2- tribromoacetate and l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2-dibromoacetate, 4,4,4- tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6-tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2- tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

21. A method according to any one of claims 12 to 18 wherein the one or more protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane,1,2-dihydroxypropane, 1,3-dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

22. A method according to any one of claims 12 to 21 wherein the one or more anti-methanogenic halomethane compounds is selected from the group consisting of bromoform, dibromochloromethane, dibromoiodomethane, bromoiodomethane, bromodiiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, bromodichloromethane, bromochloromethane, dichloromethane, diiodomethane, and carbon tetrabromide.

23. A method according to any one of claims 12 to 22, wherein the Asparagopsis is A. armata or A.24.taxiformis.

24. A method according to any one of claims 12 to 23, further comprising a step of separating the at least one protic solvent from the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

25. A method of producing bromoform, the method comprising contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with rumen fluid within the rumen of a ruminant animal.

26. A method according to any one of claims 12 to 24 wherein the level of the at least one anti- methanogenic halomethane compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, contacted with the at least one protic solvent is increased relative to the level of the at least one anti-methanogenic halomethane compounds in a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, not contacted with a protic solvent.

27. A method of preparing a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, having an increased levels of at least one or more halomethylcarbonyl compounds, said method comprising a step of decreasing the hydrolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

28. A method according to claim 27, wherein the method comprises a step of increasing the integrity of intact gland cells in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof.

29. A method according to claim 26 or claim 27, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with an aprotic solvent or solvent.

30. A method according to claim 29, wherein the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1- trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3-trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y-valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, / V-methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p- cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetrahydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

31. A method according to claim 27, wherein the method comprises a step of reducing the levels of protic solvent in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or a mixture thereof.

32. A method according to any one of claims 27, 30, or 31, wherein the method comprises a step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with at least one acid.

33. A method according to claim 32, further comprising a step of contacting the biomass of Asparagopsis or a part thereof, cells, an extract of Asparagopsis, or a mixture thereof with at least one acid and at least one further solvent.

34. A method according to claim 32 or 33, wherein the at least one acid is selected from the group consisting of formic acid, acetic acid, propanoic acid, pyruvic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, malonic acid, malic acid, phosphoric acid, tartaric acid, trichloroacetic acid, and tribromoacetic acid.

35. A method according to claim 33, wherein the at least one further solvent is a protic solvent.

36. A method according to claim 35, wherein the protic solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, ethyl lactate, ammonia, n-butanol, isobutanol, t-butanol, 1,2-dihydroxyethane, 1,2-dihydroxypropane, 1,3- dihydroxypropane, glycerol, ethanolamine, and ethylene glycol monomethyl ether.

37. A method according to any one of claims 32 to 36, wherein the step of contacting the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof with the at least one acid reduces the pH of the mixture to 6 or less, 5 or less, 4 or less, 3 or less, 2 or less or 1 or less.

38. A method of preparing an anti-methanogenic halomethane composition, said method comprising:38.contacting a biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, with an extraction liquid comprising at least one aprotic solvent or an acid to decrease the hydrolysis of one or more halomethylcarbonyl compounds in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, and to extract the one or more halomethylcarbonyl compounds or the one or more halomethylcarbonyl compounds and the one or more anti-methanogenic halomethane compounds into the extraction liquid.

39. A method according to claim 38, further comprising separating the extraction liquid from the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis, or a mixture thereof, to obtain an anti-methanogenic halomethane composition comprising one or more halomethylcarbonyl compounds, or one or more halomethylcarbonyl compounds and one or more anti-methanogenic halomethane compounds.

40. A method according to claim 38 or claim 39 wherein the at least one aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3- trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y- valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, N- methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetra hydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

41. A method according to any one of claims 38 to 40, wherein the method further comprises a step of separating the at least one aprotic solvent from the extraction liquid.

42. A method according to any one of claims 38 to 40, wherein the method further comprises a step of removing the aprotic solvent from the composition.

43. A method according to any one of claims 38 to 40, wherein the method further comprises a step of removing the aprotic solvent from the composition under vacuum.

44. A method according to claim 42 or claim 43, wherein the method further comprises a step of contacting the composition with a further solvent.

45. A method according to any one of claims 42 to 44, wherein the method further comprises a step of adsorbing the composition into a solid support, or encapsulating the composition.

46. A method according to any one of claims 38 to 41, wherein the level of the at least one halomethylcarbonyl compound in the biomass of Asparagopsis or a part thereof, an extract of Asparagopsis or a mixture thereof contacted with an aprotic solvent is increased relative to the level of at least one halomethylcarbonyl compound not contacted with an aprotic solvent.

47. A composition produced by the method according to any one of claims 1 to 46.

48. An anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and at least one aprotic solvent.

49. An anti-methanogenic composition comprising one or more halomethylcarbonyl compounds and one or more halomethylcarbonyl compound stabilising excipient.

50. An anti-methanogenic composition of claim 48 or claim 49, wherein the one or more - halomethylcarbonyl compounds comprise one or more halomethyl group selected from the group consisting of monohalomethyl, dihalomethyl, and trihalomethyl.

51. An anti-methanogenic composition of any one of claims 48 to 50, wherein the one or more halomethylcarbonyl compounds comprises one or more halogen selected from the group consisting of chlorine, bromine and iodine.

52. An anti-methanogenic composition of any one of claims 48 to 51, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of a haloacetic acid or salt or ester or amide, a halomethyl ketone or ketone derivative, and a haloacetaldehyde or acetaldehyde derivative.

53. An anti-methanogenic composition of any one of claims 48 to 52, wherein the one or more halomethylcarbonyl compounds is a a,a,a-tribromomethylcarbonyl compound.

54. An anti-methanogenic composition of any one of claims 48 to 53, wherein the one or more halomethylcarbonyl compounds is selected from the group consisting of 1,1, 1,3,3- pentabromoacetone, 1,1,1,3,3,3-hexabromoacetone, 1,1,1,4,4,4-hexabromodiacetyl, 1.1.1.5.5.5-hexabromopentan-2, 4-dione, 1,1, 1,7, 7, 7-hexabromoheptan-2, 6-dione, 1.1.1.3.5.5.5-heptabromopentan-2,4-dione, l,l,l,3,3,5,5,5-octabromopentan-2,4-dione, l,l,3,3,3-pentabromoprop-l-en-2-yl 2,2,2-tribromoacetate and 1,1,3,3,3-pentabromoprop-l- en-2-yl 2,2-dibromoacetate, 4,4,4-tribromo-3-ketobutanoic acid or its salts or esters, 6,6,6- tribromo-3,5-diketohexanoic acid or its salts or esters, 2,2,2-tribromoacetic acid or its salts or esters or amides; 2,2,2-tribromoacetaldehyde and l,l,l-tribromobut-3-en-2-one.

55. An anti-methanogenic composition of any one of claims 48 to 54 wherein the one or more halomethylcarbonyl compounds comprises one or more synthetic halomethylcarbonyl compounds.

56. An anti-methanogenic composition of any one of claims 48 to 55 , wherein the aprotic solvent is selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, 1,2,3- trichloropropane, benzene, toluene, xylene, methyl formate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, isoamyl acetate, methyl propionate, glycol diacetate, 2-ethoxyethyl acetate, y- valerolactone, diethylsuccinate, dimethylformamide, dimethylacetamide, / V- methylpyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, acetonitrile, propionitrile, butane, pentane, hexane, cyclohexane, methylcyclohexane, heptane, isooctane, dodecane, undecane, limonene, p-cymene, t-butyl methyl ether, t-butyl ethyl ether, diethyl ether, diisopropyl ether, tert-amyl methyl ether, dimethoxyethane, tetra hydrofuran, methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, dimethyl carbonate, ethylene carbonate, propylene carbonate, trimethylene carbonate, animal or vegetable oil or oil-derived products such as fatty acid esters, mineral oil, and super-critical CO2.

57. An anti-methanogenic composition of any one of claims 49 to 56 wherein the one or more halomethylcarbonyl compound stabilising excipient comprises an edible wax, grease, oil, cyclodextrins, molasses and a saturated fat.

58. A feed supplement for reducing total gas production and / or methane production in a ruminant animal, said supplement comprising an effective amount of composition produced by a method according to any one of claims 1 to 47, or an effective amount of an anti-methanogenic composition of any one of claims 48 to 57.

59. A feed for a ruminant animal, wherein said feed is supplemented with a feed supplement according to claim 58.

60. A method for reducing total gas production and / or methane production in a ruminant animal comprising administering to said ruminant animal an effective amount of a composition according to claim 47, a feed supplement according to claim 58 or a feed according to claim 59.

1. A method for improving the growth performance of a ruminant animal comprising the step of providing said ruminant animal an effective amount of a composition according to claim 47, a feed supplement according to claim 58 or a feed according to claim 59.

Images