WO2023212773A1

WO2023212773A1 - Process for preparing anti-methanogenic composition

Info

Publication number: WO2023212773A1
Application number: PCT/AU2023/050366
Authority: WO
Inventors: Philip Cassidy; Rocky De Nys
Original assignee: Sea Forest Limited
Priority date: 2022-05-06
Filing date: 2023-05-02
Publication date: 2023-11-09

Abstract

The present disclosure is directed to the preparation of an anti-methanogenic oil composition from a biomass of red marine algae using a heterogeneous extraction liquid comprising an aqueous solution and at least one oil. The present disclosure is also directed to the anti- methanogenic oil composition, and methods and use thereof as an anti-methanogenic agent for administration to ruminants.

Description

PROCESS FOR PREPARING ANTI-METHANOGENIC COMPOSITION

Field

The present disclosure generally relates to a process for preparing an anti-methanogenic composition comprising one or more anti-methanogenic agents from a biomass of red marine algae.

Background

Ruminant animals produce a large amount of methane which has a significant role in global warming. Methods of reducing methane production in ruminant animals have been developed. One of the methods of reducing methane production is feeding the ruminant animals with seaweed such as red marine algae including Asparagopsis species which contain anti-methanogenic agents such as bromoform.

Feeding red marine algae directly to the ruminant animals has certain disadvantages. For example, a large amount of red marine algae need to be produced, processed and transported to the end users, thus, the cost and timing of such process would be significant.

Extracting anti-methanogenic agents red marine algae and feeding the extract to the ruminant animals may be more efficient than feeding red marine algae directly to the ruminant animals to reduce methane production in ruminant animals.

Certain methods of extracting anti-methanogenic agents from red marine algae have been developed. An existing method for extracting anti-methanogenic agents from red marine algae is based on the extraction of biomass directly into an edible oil. The volume of oil limits the concentration of anti-methanogenic agents in the oil and consequently the amount of anti-methanogenic agents that can be carried to the ruminant animals. This limits the use of the oil product due to the large volume of oil required to transfer the amount of anti- methanogenic agents into the ruminant animal.

The disadvantage of existing methods is that the concentration of anti-methanogenic agents is relatively low in the oil and requires a significantly increased volume of oil to achieve the intended anti-methanogenic agent quantity to result in the intended reduction in methane emissions. Additionally, the existing method does not allow for batches of lower quality biomass (lower concentrations of anti-methanogenic agents per gram of red marine algae material) to be processed into a viable oil product achieving a minimum target concentration of anti-methanogenic agents. Therefore, any lower quality (reduced anti-methanogenic agent concentration) biomass would inherently deliver a lower quality oil product (low concentration anti- methanogenic agents).

Accordingly, there is a need for developing more effective methods of extracting anti- methanogenic agents from a biomass of red marine algae.

Any reference to methods, apparatus or documents of the prior art are not to be taken as consulting any evidence or admission that they formed, or form part of the common general knowledge.

Summary

The subject matter of the present disclosure is predicated in part on the surprising discovery of a process for preparing an anti-methanogenic oil composition from a biomass of red marine algae using a heterogeneous extraction liquid comprising an aqueous solution and at least one oil.

According to one aspect, there is provided a process for preparing an anti-methanogenic oil composition, wherein the process comprises: contacting a biomass of red marine algae with a heterogeneous extraction liquid comprising an aqueous solution and at least one oil, and/or with at least one of the aqueous solution and the at least one oil prior to forming the heterogeneous extraction liquid, under conditions to extract one or more anti-methanogenic agents from the biomass into the heterogeneous extraction liquid; and separating the oil from the heterogeneous extraction liquid to obtain an anti- methanogenic oil composition comprising one or more anti-methanogenic agents.

According to another aspect, there is provided an anti-methanogenic oil composition comprising one or more oils and one or more anti-methanogenic agents selected from bromoform, dibromochloromethane, bromoiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichlorobromomethane, bromoiodomethane, diiodomethane, carbon tetrabromide or combination thereof, wherein the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (w/v) is between about 1 to 100 mg/ml, about 0.5 to 70 mg/ml, about 1 to 50 mg/ml, about 2 to 30 mg/ml, 3 to 20 mg/ml, 0.1 to 10 mg/ml, about 0.5 to 9 mg/ml, about 1 to 8 mg/ml, or about 2 to 5 mg/ml.

According to another aspect, there is provided an anti-methanogenic oil composition prepared from the process according to any embodiments or examples thereof as described herein.

According to another aspect, there is provided a ruminant animal feed or supplement comprising the anti-methanogenic oil composition according to any embodiments or examples thereof as described herein.

According to another aspect, there is provided a method of reducing methane production in a ruminant animal by administration to the ruminant animal of a ruminant animal feed or supplement, or anti-methanogenic oil composition, according to any embodiments or examples thereof as described herein.

According to another aspect, there is provided use of the ruminant animal feed or supplement, or anti-methanogenic oil composition, according to any embodiments or examples thereof as described herein for reducing methane production in a ruminant animal.

The present disclosure according to at least some embodiments or examples as described herein allows for improved use of oil by significantly enhancing the concentration of anti- methanogenic agents, such as bromoform, in the oil, which can significantly improve its various commercial applications.

The present disclosure according to at least some embodiments or examples as described herein can also deliver a low volume oil product with lower effective levels of anti- methanogenic agents, such as bromoform, concentration, to produce high efficacy anti- methanogenic effects in ruminant animals.

The oil can be concentrated with anti-methanogenic agents, such as bromoform, to levels comparable to freeze-dried red marine algae material without the inherent risks or costs of a freeze-dried product. In comparison to the freeze-dried red marine algae material comprising anti-methanogenic agents, such as bromoform, the loss over time in the oil composition according to at least some embodiments or examples thereof as described herein can be lower and financial/energy cost associated with commercial scale freeze drying can be mitigated.

The method utilises the addition of an aqueous solution such as water to biomass of red marine algae while at least in some embodiments or examples using a smaller volume of oil. This can act to significantly increase the biomass loading per unit of oil.

In at least some embodiments or examples as described herein, concentrations or ratios of the source biomass, water and oil, can each be manipulated to deliver a consistent, standardised anti-methanogenic agents, such as bromoform, concentration in the oil product. It is understood that the existing oil processing methods cannot deliver this.

Detailed Description

General definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, natural product chemistry, analytical chemistry, animal feed science and technology, and the like).

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, the terms “a”, “an” and “the” include both singular and plural aspects, unless the context clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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.

As used herein, the term a “polar solvent” refers to a type of solvent that has large partial charges or dipole moments. It will be appreciated that polar solvent can dissolve ions and other polar compounds.

As used herein, the term "reducing" includes the reduction of amount of substance in comparison with a reference. For example, the reduction of methane production in a ruminant a ruminant animal or animals administered an anti-methanogenic oil composition according to the present disclosure, is relative to an animal or animals not administered an anti- methanogenic oil composition of the present disclosure. The reduction can be measured in vitro with an artificial rumen system that simulates anaerobic fermentation, or in vivo with animals confined in respiration chambers. It is within the knowledge and skill of those trained in the art to assess enteric methanogenesis by a ruminant animal.

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 administration of the anti-methanogenic oil composition of the present disclosure reduces 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.

As used herein, the term "animal feed supplement" refers to a concentrated additive premix comprising the active ingredients, which premix or supplement may be added to an animal's feed or ration to form a supplemented feed in accordance with the present disclosure. The terms "animal feed premix," "animal feed supplement," and "animal feed additive" are generally considered to have similar or identical meanings and are generally considered interchangeable.

Heterogeneous liquid extraction process

In one embodiment, there is provided a process for preparing an anti-methanogenic oil composition, wherein the process comprises: contacting a biomass of red marine algae with a heterogeneous extraction liquid comprising an aqueous solution and at least one oil under conditions to extract one or more anti-methanogenic agents from the biomass into the heterogeneous extraction liquid; and separating the oil from the heterogeneous extraction liquid to obtain an anti- methanogenic oil composition comprising one or more anti-methanogenic agents.

In one embodiment, there is provided a process for preparing an anti-methanogenic oil composition, wherein the process comprises: contacting a biomass of red marine algae with at least one of the aqueous solution and at least one oil, forming a heterogeneous extraction liquid comprising the aqueous solution and the at least one oil, wherein the contacting of the biomass and forming of the heterogeneous extraction liquid is under conditions to extract one or more anti-methanogenic agents from the biomass into the heterogeneous extraction liquid; and separating the oil from the heterogeneous extraction liquid to obtain an anti- methanogenic oil composition comprising one or more anti-methanogenic agents.

In one embodiment, the anti-methanogenic oil composition comprises one or more oils and one or more anti-methanogenic agents. In one example, the anti-methanogenic oil composition consists essentially of one or more oils and one or more anti-methanogenic agents.

In one embodiment, the biomass of red marine algae is selected from a species belonging to any one or more of the five genera of red seaweed in the family Bonnemaisoniaceae Asparagopsis, Bonnemaisonia, Delisea, Ptilonia, Leptophyllis. In one embodiment, the biomass of red marine algae is Asparagopsis. In another embodiment the biomass of Asparagopsis is Asparagopsis taxiformis. In another embodiment, the biomass of Asparagopsis is Asparagopsis armata. In another embodiment, the biomass of Asparagopsis is Asparagopsis taxiformis and Asparagopsis armata.

In one embodiment, the ratio of the biomass of red marine algae to the heterogeneous extraction liquid is in a range of between about 1:20 to 20:1, 1:19 to 19:1, 1:18 to 18:1, 1:17 to 17:1, 1:16 to 16:1, 1:15 to 15:1, 1:14 to 14:1, 1 :13 to 13:1, 1:12 to 12:1, 1:11 to 11:1, 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.5 to 1.5:1. In other examples, the ratio of the biomass of red marine algae to the heterogeneous extraction liquid is about 1:2, about 1:1.5, or about 1:1.

In one embodiment the ratio of aqueous solution to oil in the heterogeneous extraction liquid is in a range of between about 100:1 to 1:1, 95:1 to 1:1, 90:1 to 1:1; 85:1 to 1:1, 80:1 to 1:1, 75:1 to 1:1, 70:1 to 1:1, 65:1 to 1:1, 60:1 to 1 :1, 55:1 to 1:1, 50:1 to 2:1, 45:1 to 2:1, 40:1 to 2:1, 35:1 to 2:1, 30:1 to 2:1, 25:1 to 3:1, 20:1 to 3:1, 15:1 to 3:1, 14:1 to 3:1, 13:1 to 3:1, 12:1 to 3:1, 11:1 to 3:1, 10:1 to 4:1, or 9:1 to 4:1. In other examples the ratio of aqueous solution to oil in the heterogeneous extraction liquid is about 8:1, about 7:1, about 6:1, about 5:1, or about 4:1. Heterogeneous extraction liquid

In one embodiment, a heterogeneous extraction liquid comprises an aqueous solution and at least one oil. In one example, the heterogeneous extraction liquid is a heterogeneous liquid mixture comprising or consisting of water and one or more oils.

In one embodiment, the aqueous solution comprises or consists of water. In one embodiment, the water is fresh water, tap water, purified water, brine, salt water, or seawater.

In one embodiment, the aqueous solution further comprises a polar organic solvent. In one embodiment, the polar organic solvent is selected from one or more of acetic acid, formic acid, acetone, ethyl acetate or an alcohol such as methanol, ethanol, propanol, isopropyl alcohol, or butanol.

In one embodiment, the pH of the aqueous solution is in a range of about 2 to 11, 2.5 to 10.5, 3 to 10, 3.5 to 9.5, 4 to 9, 4.5 to 8.5, 5 to 8, 5.5 to 7.5, 6 to 8, or about 7.

In one embodiment, the at least one oil comprises an edible oil. 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, 30 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.

In one embodiment, the edible oil is vegetable oil. In one embodiment, the edible oil is canola oil.

In one aspect, the anti-methanogenic oil composition is provided as an animal feed or supplement.

In one aspect, there is provided an animal feed or supplement comprising the anti- methanogenic oil composition according to any embodiments or examples thereof as described herein. Anti-methanogenic agents

In one embodiment, the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (w/w) is about, or at least about, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99 and 100 mg/g or a range comprising any of two of those integers.

In one embodiment, the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (w/w) is about 0.1 to 100 mg/g, about 0.1 to 95 mg/g, about 0.1 to 90 mg/g, about 0.1 to 85 mg/g, about 0.1 to 80 mg/g, about 0.1 to 75 mg/g, about 0.5 to 70 mg/g, about 0.5 to 65 mg/g, about 0.5 to 60 mg/g, about 0.5 to 55 mg/g, about 1 to 50 mg/g, about 1 to 45 mg/g, about 1 to 40 mg/g, about 1 to 35 mg/g, about 2 to 30 mg/g, about 2 to 25 mg/g, 3 to 20 mg/g, 3 to 15 mg/g 0.1 to 10 mg/g, about 0.5 to 9 mg/g, about 1 to 8 mg/g, about 1 to 7 mg/g, about 1 to 6 mg/g, about 2 to 5 mg/g.

In one embodiment, the one or more anti-methanogenic agents may include halomethanes, bromoform, dibromochloromethane, bromoiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichlorobromomethane, bromoiodomethane, diiodomethane, carbon tetrabromide or any combination thereof. In one example, the one or more anti-methanogenic agents comprises bromoform.

Contacting conditions

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise a duration of about 1 hour to 1 month, 6 hours to 1 month, 12 hours to 1 month, 1 day to 1 month, 2 days to 1 month, 3 days to 1 month, 4 days to 1 month, 5 days to 1 month, 6 days to 1 month, 1 week to 1 month, 1 week to 3 weeks, 10 days to 20 days, 8 days to 20 days, 9 days to 20 days, 10 days to 20 days, 11 days to 20 days, 12 days to 19 days, 13 days to 19 days, 14 days to 18 days, 14 days to 17 days, 14 days to 16 days, or about 15 days. In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise a temperature of about 1°C to 70°C, 2°C to 70°C, 3°C to 70°C, 4°C to 70°C, 5°C to 70°C, 6°C to 70°C, 7°C to 70°C, 8°C to 70°C, 9°C to 70°C, 10°C to 65°C, 11°C to 65°C, 12°C to 65°C, 13°C to 65°C, 14°C to 65°C, 15°C to

65°C, 16°C to 65°C, 17°C to 65°C, 18°C to 65°C, 19°C to 65°C, 20°C to 65°C, 21°C to

65°C, 22°C to 65°C, 23°C to 65°C, 24°C to 65°C, 25°C to 65°C, 26°C to 65°C, 27°C to

65°C, 28°C to 65°C, 29°C to 65°C, 30°C to 65°C, or about 35°C.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise agitation. In one embodiment, the agitation uses an agitator. In one embodiment, the agitator is a paddle agitator, an anchor agitator, a helical ribbon agitator, a propeller agitator, a turbine agitator, or an agitator with a screw impeller, a retreat curve impeller, a hydrofoil impeller, a dispersion blade impeller, and a coil impeller. In one embodiment, the agitation uses an anchor agitator. In one embodiment, the agitation uses an anchor agitator between 3 and 20 hertz, between 4 and 19 hertz, between 5 and 15 hertz, between 6 and 14 hertz or between 7 and 13 hertz.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise a maceration step of macerating the biomass of red marine algae in an aqueous solution before oil is added. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with water. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with water equal parts. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with water before oil being added. In one embodiment, a maceration step of macerating the biomass comprising mixing the biomass with water may attain a majority of biomass size between 5 and 100 mm, between 10 and 90 mm, between 15 and 85 mm, between 15 and 80 mm, between 15 and 75 mm, between 15 and 70 mm, between 15 and 65 mm, between 15 and 60 mm, between 10 and 65 mm, between 20 and 60 mm, between 20 and 55 mm, or between 20 and 50 mm.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise a maceration step of macerating the biomass of red marine algae in oil before an aqueous solution is added. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with oil. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with oil equal parts. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with oil before water being added. In one embodiment, a maceration step of macerating the biomass comprising mixing the biomass with oil may attain a majority of biomass size between 5 and 100 mm, between 10 and 90 mm, between 15 and 85 mm, between 15 and 80 mm, between 15 and 75 mm, between 15 and 70 mm, between 15 and 65 mm, between 15 and 60 mm, between 10 and 65 mm, between 20 and 60 mm, between 20 and 55 mm, or between 20 and 50 mm.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise a maceration step of macerating the biomass of red marine algae in the heterogeneous extraction liquid. In one embodiment, a maceration step of macerating the biomass comprises mixing the biomass with the heterogeneous extraction liquid. In one embodiment, a maceration step of macerating the biomass comprising mixing the biomass with the heterogeneous extraction liquid may attain a majority of biomass size between 5 and 100 mm, between 10 and 90 mm, between 15 and 85 mm, between 15 and 80 mm, between 15 and 75 mm, between 15 and 70 mm, between 15 and 65 mm, between 15 and 60 mm, between 10 and 65 mm, between 20 and 60 mm, between 20 and 55 mm, or between 20 and 50 mm.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise the biomass being in the form of fresh seaweed (red marine algae) with the time to contact with the heterogeneous extraction liquid being immediately on removal from cultivation media up to 0.5 hour, up to 1 hour, up to 1.5 hours, up to 2 hours, up to 2.5 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 15 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, up to 30 hours, up to 36 hours, up to 42 hours, or up to 48 hours. In one embodiment, the biomass of red marine algae is dried or freeze dried before being contacted with the heterogeneous extraction liquid. In one embodiment, the biomass of red marine algae is being cleaned or washed before being contacted with the heterogeneous extraction liquid. In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise introducing one or more enzymes. In one embodiment, the enzymes are micro-granulated enzymes. In one embodiment, the enzymes are cellulases and/or proteases. In one embodiment, the enzymes are EnartisZym Arom MP and/or EnartisZym Color Plus. In one embodiment, the enzymes remains in an aqueous phase after an oil phase is separated from the heterogeneous extraction liquid.

In one embodiment, the conditions of contacting the biomass of red marine algae with the heterogeneous extraction liquid comprise homogenization. As used herein, the term "homogenization" means to break up the biomass to facilitate release of one or more anti- methanogenic agents from the biomass. The homogenization can take place by any means known in the art, such as crushing, grinding, milling, blending, cutting, slicing, or dicing.

Separation of oil

In one aspect, the step of separating the oil from the heterogeneous extraction liquid to obtain an anti-methanogenic oil composition comprises partitioning and removing the oil from the heterogeneous extraction liquid.

In one aspect, the step of separating the oil from the heterogeneous extraction liquid to obtain an anti-methanogenic oil composition comprises a three-phase (solid -oil - aqueous) separation process. In one embodiment, the three-phase separation process uses a tricanter separator. In one embodiment, the tricanter separator is Flottweg Tricanter Z23. In one embodiment, the tricanter separator is set up with an unput flow rate of 1 - 8 m³.hr^-1, a bowl speed of 5300-5700 rpm, a scroll speed (differential) of 1-2 rpm, impeller angle of 145-165 degree and a centrifugal force of 3000 - 4200 G. Using this approach, the wet solid, oil and aqueous phases were successfully separated.

In one aspect, the step of separating the oil from the heterogeneous extraction liquid to obtain an anti-methanogenic oil composition comprises decanting. The decanting is a two-phase separation of the solids from the liquids. In one embodiment, the decanting comprises partitioning the oil and water together from the solids. Anti-methanogenic oil composition

In one aspect, an anti-methanogenic oil composition comprises one or more oils and one or more anti-methanogenic agents which include halome thanes, bromoform, dibromochloromethane, bromoiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichlorobromomethane, bromoiodomethane, diiodomethane, carbon tetrabromide or combination thereof, wherein the total amount of the one or more anti- methanogenic agents extracted into the anti-methanogenic oil composition (w/v) is about 1 to 100 mg/ml, about 1 to 95 mg/ml, about 1 to 90 mg/ml, about 1 to 85 mg/ml, about 1 to 80 mg/ml, about 1 to 75 mg/ml, about 0.5 to 70 mg/ml, about 0.5 to 65 mg/ml, about 0.5 to 60 mg/ml, about 0.5 to 55 mg/ml, about 1 to 50 mg/ml, 1 to 45 mg/ml, 1 to 40 mg/ml, 1 to 35 mg/ml, about 2 to 30 mg/ml, about 2 to 25 mg/ml, about 3 to 20 mg/ml, about 3 to 15 mg/ml, about 0.1 to 10 mg/ml, about 0.5 to 9 mg/ml, about 1 to 8 mg/ml, about 1 to 7 mg/ml, about 1 to 6 mg/ml, about 1 to 5 mg/ml, or about 2 to 5 mg/ml.

In one example the one or more anti-methanogenic agents comprise bromoform.

In other examples, the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (mg/ml) is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, or 20. In other examples, the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (mg/ml) is less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. The total amount of the one or more anti-methanogenic agents extracted into the anti- methanogenic oil composition may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/ml) of between about 1 to 100, about 1 to 50, about 1 to 30, about 2 to 20, about 2 to 15, about 3 to 12, or about 4 to 10.

In other examples, the total amount of bromoform extracted into the anti-methanogenic oil composition (mg/ml) is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, or 20. In other examples, the total amount of bromoform extracted into the anti- methanogenic oil composition (mg/ml) is less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. The total amount of bromoform extracted into the anti-methanogenic oil composition may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/ml) of between about 1 to 100, about 1 to 50, about 1 to 30, about 2 to 20, about 2 to 15, about 3 to 12, or about 4 to 10.

In one embodiment, an amount (mg) of one or more anti-methanogenic agents extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is at least 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20. In other examples, an amount (mg) of one or more anti-methanogenic agents extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In other examples, an amount (mg) of one or more anti- methanogenic agents extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/g/g) of between about 0.8 to 100, about 1 to 50, about 1 to 30, about 2 to 20, about 2 to 15, about 3 to 12, or about 4 to 10.

In one embodiment, an amount (mg) of bromoform extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is at least 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20. In other examples, an amount (mg) of bromoform extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. In other examples, an amount (mg) of bromoform extracted into per an amount (g) of oil per a dry weight (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/g/g) of between about 0.8 to 100, about 1 to 50, about 1 to 30, about 2 to 20, about 2 to 15, about 3 to 12, or about 4 to 10.

In one embodiment, an amount (mg) of one or more anti-methanogenic agents extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is at least 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2. In other examples, an amount (mg) of the one or more anti-methanogenic agents extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is less than about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5. In other examples, an amount (mg) of one or more anti-methanogenic agents extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/g/g) of between about 0.08 to 10, about 0.1 to 5, about 0.1 to 3, about 0.2 to 2, about 0.2 to 1.5, about 0.3 to 1.2, or about 0.4 to 1.

In one embodiment, an amount (mg) of bromoform extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is at least 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2. In other examples, an amount (mg) of bromoform extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) is less than about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5. In other examples, an amount (mg) of bromoform extracted into per an amount (g) of oil per a spun fresh weight (SFW) (g) of a biomass of red marine algae loaded to the extraction (mg/g/g) may be in any range provided by any two of these upper and/or lower amounts, for example in a range (mg/g/g) of between about 0.08 to 10, about 0.1 to 5, about 0.1 to 3, about 0.2 to 2, about 0.2 to 1.5, about 0.3 to 1.2, or about 0.4 to 1.

In one embodiment, the extraction efficiency of one or more anti-methanogenic agents of a biomass of red marine algae extracted into an anti-methanogenic oil composition, which is calculated based on a theoretical maximum in a spun fresh weight (SFW) or dry weight of the biomass, is at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

In one embodiment, the extraction efficiency of bromoform in a biomass of red marine algae extracted into an anti-methanogenic oil composition, which is calculated based on a theoretical maximum in a spun fresh weight (SFW) or dry weight of the biomass, is at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%. In one aspect, an anti-methanogenic oil composition is prepared from the process disclosed herein.

Animal feed or supplement

In one aspect, a ruminant animal feed or supplement comprises the anti-methanogenic oil composition for use in reducing methane production in a ruminant animal.

In one aspect, a method of reducing methane production in a ruminant animal comprises administering to the ruminant animal of the ruminant animal feed or supplement.

In one aspect, there is provided use of the ruminant animal feed or supplement for reducing methane production in a ruminant animal.

Typically, the animal feed supplement of the present disclosure may be in the form of a powder or compacted or granulated solid. In practice, livestock may typically be fed the animal feed supplement by adding it directly to the ration, e.g. as a so-called top-dress, or it may be used in the preparation or manufacture of products such as compounded animal feeds or a lick blocks. The disclosure is not particularly limited in this respect. A supplement according to the disclosure may be fed to an animal in an amount ranging from 0.5-2000 g/animal/day.

In one embodiment, a supplement according to the disclosure is administered at an amount based on actual individual animal intake (e.g. g /kg DM intake).

The present animal feed supplement may comprise the anti-methanogenic oil composition extracted from at least one species of red marine algae. In one embodiment, the present animal feed supplement may be formulated so that when added to feed, the anti- methanogenic oil composition may be present at least 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% of the organic matter of the feed.

In one embodiment, the present animal feed supplement may be formulated so that when added to feed, the anti-methanogenic oil composition may be present less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5% of the organic matter of the feed. In one embodiment, the present animal feed supplement may be formulated so that when added to feed, the anti-methanogenic oil composition may be be in any range provided by any two of these upper and/or lower amounts, for example in a range (%) of between 0.0001-50, 0.0005-45, 0.001-40, 0.005-35, 0.01-30, 0.05-25, 0.1-20, 0.2-20, 0.5-15 or 1-10.

It is within the skills of the trained professional to determine exactly the ideal amounts of the components to be included in the supplement and the amounts of the supplement to be used in the preparation of the ration or compounded animal feed, etc., taking into account the specific type of animal and the circumstances under which it is held.

The animal feed or supplements of the present disclosure may comprise any further ingredient without departing from the scope of the disclosure. It may typically comprise well- known excipients that are necessary to prepare the desired product form and it may comprise further additives aimed at improving the quality of the feed and/or at improving the performance of the animal consuming the supplement. Suitable examples of such excipients include carriers or fillers, such as lactose, sucrose, mannitol, starch crystalline cellulose, sodium hydrogen carbonate, sodium chloride and the like and binders, such as gum arabic, gum tragacanth, sodium alginate, starch, PVP and cellulose derivatives, etc. Examples of feed additives known to those skilled in the art include vitamins, amino acids and trace elements, digestibility enhancers and gut flora stabilizers and the like.

The term 'compounded animal feed composition' as used herein, means a composition which is suitable for use as an animal feed and which is blended from various natural or non-natural base or raw materials and/or additives. Hence, in particular, the term 'compounded' is used herein to distinguish the present animal feed compositions from any naturally occurring raw material. These blends or compounded feeds are formulated according to the specific requirements of the target animal. The main ingredients used in commercially prepared compounded feeds typically include wheat bran, rice bran, corn meal, cereal grains, such as barley, wheat, rye and oat, soybean meal, alfalfa meal, cottonseed meal, wheat powder and the like. A commercial compound feed will typically comprise no less than 15% of crude protein and no less than 70% digestible total nutrients, although the present disclosure is not particularly limited in this respect. Liquid, solid as well as semi-solid compounded animal feed compositions are encompassed within the scope of the present disclosure, solid and semi-solid forms being particularly preferred. These compositions are typically manufactured as meal type, pellets or crumbles. In practice, livestock may typically be fed a combination of compounded feed, such as that of the present disclosure, and silage or hay or the like. Typically a compounded animal feed may be fed in an amount within the range of 0.3-10 kg/animal/day. It is within the skills of the trained professional to determine proper amounts of these components to be included in the compounded animal feed, taking into account the type of animal and the circumstances under which it is held.

The compounded animal feed compositions of the present disclosure may comprise any further feed additive typically used in the art. As is known by those skilled in the art, the term 'feed additive' in this context refers to products used in animal nutrition for purposes of improving the quality of feed and the quality of food from animal origin, or to improve the animals' performance, e.g. providing enhanced digestibility of the feed materials. Nonlimiting examples include technological additives such as preservatives, antioxidants, emulsifiers, stabilising agents, acidity regulators and silage additives; sensory additives, especially flavours and colorants; (further) nutritional additives, such as vitamins, amino acids and trace elements; and (further) zootechnical additives, such as digestibility enhancers and gut flora stabilizers.

As will be clear to those skilled in the art, the present compounded animal feed compositions can comprise any further ingredient or additive, without departing from the scope of the present disclosure.

Examples

Materials and Methods

The red seaweed Asparagopsis armata was either harvested from natural populations from the East Coast of Tasmania or farmed biomass from longline cultivation in the Mercury Passage, East Coast, Tasmania (42°36'17.6"S 147°59'35.5"E). The biomass was placed into ambient seawater (Fish bin 1000 L - Aquasure) on collection and transported to facilities at Sea Forest Ltd, Triabunna before being processed.

The biomass harvested from natural populations or farmed was removed from the seawater and dewatered using a centrifuge (L’originale Dynamic) for 10 spins to reach a consistent fresh weight (SFW - spun fresh weight) prior to being weighed (Ohaus Pioneer PX224). Sub-samples of 30 grams of biomass were removed and weighed prior to being frozen and subsequently lyophilized using a freeze drier (Buchi Lyovapor L200) to determine a SFW: DW (spun fresh weight : dry weight) ratio and to provide a sample for the quantification of bromoform in the freeze dried biomass (see Quantification of bromoform).

Laboratory Tests

Following this preparation, weighed aliquots of biomass were added to a combination of potable water and canola oil (Macquarie Oil Cold Pressed GMO-free Canola oil) with the oil separating as a ‘capping’ layer on the water with the biomass. Laboratory trials (LT) were conducted with ratios of water : oil ranging from 5:1 to 10:1 and in these trials the biomass was steeped for 15 days without agitation.

After the 15 day period of steeping aliquots of water and oil were removed using a pipette and each analysed for the concentration of bromoform (mg.g’¹). These results were compared with the concentration of bromoform in the initial biomass using the concentration of bromoform in the freeze dried material (mg.g ¹ DW) and the SFW : DW ratio to determine the total amount of bromoform added to the oil on water (OoW) extraction mixture from the SFW.

A mass balance determination of the bromoform added as SFW (g bromoform) with the bromoform present in the oil (mg.g ¹ x g oil) and water (mg.g ¹ x g water) allowed for the determination of the extraction efficiency of bromoform from the SFW biomass to the oil and water fractions of the extraction mixture.

IBC Tests (industrial scale processing)

A similar process was executed at a larger scale to quantify the industrial utility of the oil on water (OoW) method. Following the process of harvesting the weighed SFW (determined as above) was added to an 1000 litre intermediate bulk container (IBC) with ratios of water : oil ranging from 5:1 to 10:1.

The biomass : water : oil was then steeped for 15 days both with and without mechanical agitation. Agitation was provided using a Fluidpro/SEW variable speed agitator (SEW Variable speed drive with custom anchor agitator plate and shaft) at a mixing rate of 4.0-8.5 hertz. After the 15 day period of steeping (with and without agitation) aliquots of water and oil were removed using a pipette and each analysed for the concentration of bromoform (mg.g’¹). These results were compared with the concentration of bromoform in the initial biomass using the concentration of bromoform in the freeze dried material (mg.g ¹ DW) and the SFW : DW ratio to determine the total amount of bromoform added to the oil on water (OoW) extraction mixture from the SFW. A mass balance determination was conducted as above.

A similar process was executed where following the process of harvesting the weighed SFW was macerated (Ultraflow Single Phase 750w cutter pump) to produce a mean size of the biomass between 10mm - 65mm. The maceration was executed as an in-line process where the macerated biomass was pumped directly into an intermediate bulk containers (IBC) with ratios of water : oil ranging from 5:1 to 10:1. In this process the IBC was also heated up to 65°C using a heating sleeve (Kuhlmann IBC heating jacket with triple digital controller) over a 24 - 72 hour period.

Industrial scale tests confirmed the suitability to separate the three-phases (solid - oil - aqueous) using a tricanter separator (Flottweg Tricanter Z23) with an unput flow rate of 1 - 8 m³.hr^-1, a bowl speed of 5300-5700 rpm, a scroll speed (differential) of 1-2 rpm, impeller angle of 145-165 degree and a centrifugal force of 3000 - 4200 G. Using this approach, the wet solid, oil and aqueous phases were successfully separated.

Quantification of bromoform

Bromoform was quantified following the extraction from samples (water and oil) where the sample of (0.1 -0.2 g) was added to a 20mL glass vial with PTFE-lined lid and lO.OmL of methanol added, with a surrogate (to assess extraction efficiency). The vials are then shaken for 1 minute and placed on a rotating tumbler for 30 mins prior to being centrifuged. Following the centrifuge, a portion (20-100 pL) of the extract was added to Ultra-high quality (UHQ) water in a 40mL headspace vial and analysed by Purge and Trap GC-MS, using a Teledyne Tekmar AquaTek-LVA sampler and Lumin concentrator coupled to an Agilent 8860 GC and 5977B Mass Spectrometer. Analysis was carried out in Selected Ion Monitoring (SIM) mode and quantitated against authentic standards. Results

Laboratory Tests

Examples of the laboratory tests are presented in Table 1 below using oil on water (OoW) with a water to oil ratio of 6.52:1 (LT1) and 5.43:1 (LT2).

Table 1. Examples of the laboratory tests

ND = not detected

¹ = total bromoform in SFW (mg) = concentration of bromoform in DW (mg.g ¹ ) x DW (g) = mg

² = maximum theoretical concentration of bromoform in oil = total bromoform in SFW (mg) / total weight of oil (g) = mg.g ¹ ³ = total bromoform in oil = concentration of bromoform in oil (mg.g ¹) x total weight of oil (g) = mg

⁴ = Mass balance of the percentage of total bromoform in SFW transferred to water = total bromoform in water / total bromoform in SFW

⁵ = Mass balance of the percentage of total bromoform in SFW transferred to oil = total bromoform in oil / total bromoform in SFW

For Lab Test 1, the OoW method resulted in a recovery of 81.4 % confirming the ability of the method to recover bromoform effectively. As a comparison to where an oil only extraction was used in a ratio of 1.2:1 with SFW (450 g SFW : 375 g oil) the oil at 100% extraction efficiency would result in a concentration of 0.74 mg.g-1 oil compared to the present OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 6.06 mg.g-1 and a measured concentration of 4.93mg.g-l at a measured extraction efficiency of 81.4%. Notably, the present OoW method provides for an improved concentration of bromoform of more than six times that of the method of Magnusson et al. (Magnusson et al. (2020). Using oil immersion to deliver a naturally-derived, stable bromoform product from the red seaweed Asparagopsis taxiformis. Algal Research, volume 51. https://doi.Org/10.1016/j.algal.2020.102065). For Lab Test 2, the OoW method resulted in a recovery of 97.8 % confirming the ability of the method to recover bromoform at a level close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (200 g SFW : 166.7 g oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 0.24 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 0.86 mg.g-1 and a measured concentration of 0.84mg.g-l at a measured extraction efficiency of 97.8 %. Notably, the OoW method provides for an improved concentration of bromoform of up to four times of the method of Magnusson et al. 2020.

IBC Test (Industrial scale tests)

Examples of industrial scale tests using intermediate bulk containers (IBC) are presented in Table 2 below using oil on water (OoW) with a water to oil ratio of 8.70:1 (IBC 3 and IBC 5) and 5.67:1 (IBC 24, IBC 30 and IBC 39). Notably, in these tests multiple harvests of biomass were combined and examples are provided of the OoW method being effective with two (IBC 3), three (IBC 5, IBC 30) and four (IBC24, IBC 39) harvests. Examples are also provided of heating (IBC 30 - 65°C), and maceration and heating (IBC 39 - 35°C). In all cases the transfer of bromoform to the oil exceeds 90% efficiency as quantified by mass balance.

Table 2. Examples of industrial scale tests

ND = not detected

IBC 3 combined two independent harvests of biomass and the OoW method resulted in a recovery of 96.9 % confirming the ability of the method to recover bromoform at a level close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (251.76 kg SFW : 209.8 kg oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 0.478 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 2.18 mg.g-1 and a measured concentration of 2.11 mg.g-1 at a measured extraction efficiency of 96.9 %. Notably, the OoW method provides for an improved concentration of bromoform of more than four times that of the method of Magnusson et al. 2020.

IBC 5 combined three independent harvests of biomass and the OoW method resulted in a recovery of 99.3 % confirming the ability of the method to recover bromoform at a level close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (235.32 kg SFW : 196.1 kg oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 0.16 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 0.685 mg.g-1 and a measured concentration of 0.68mg.g-l at a measured extraction efficiency of 99.3 %. Notably, the OoW method provides for an improved concentration of bromoform of more than four times that of the method of Magnusson et al. 2020.

IBC24 combined four independent harvests of biomass and the OoW method resulted in a recovery of 91.41 % confirming the ability of the method to recover bromoform at a level close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (230.19 kg SFW : 191.8 kg oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 1.15 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 3.18 mg.g-1 and a measured concentration of 2.91mg.g-l at a measured extraction efficiency of 91.41 %. Notably, the OoW method provides for an improved concentration of bromoform of more than 2.5 times that of the method of Magnusson et al. 2020.

IBC30 combined three independent harvests of biomass and was heated to 65°C over 72 hours as described above prior to quantification of bromoform in the oil and water. The OoW method resulted in a recovery of 97.65 % confirming the ability of the method to recover bromoform close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (259.83 kg SFW : 216.53 kg oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 1.22 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 3.34 mg.g-1 and a measured concentration of 3.26 mg.g-1 at a measured extraction efficiency of 97.65 %. Notably, the OoW method provides for an improved concentration of bromoform of more than 2.5 times that of the method of Magnusson et al. 2020.

IBC39 combined four independent harvests of biomass with maceration as described above and was heated to 35 °C for 72 hours prior to quantification of bromoform in the oil and water. The OoW method resulted in a recovery of 99.46 % confirming the ability of the method to recover bromoform close to the theoretical maximum (100%). As a comparison of a method where oil was used in a ratio of 1.2:1 with SFW (341.97 kg SFW : 284.98 kg oil) (Magnusson et al. 2020) the oil at 100% extraction efficiency would result in a concentration of 0.65 mg.g-1 oil compared to the OoW method with a maximum theoretical concentration (assuming 100% extraction efficiency) of 4.04 mg.g-1 and a measured concentration of 4.02 mg.g-1 at a measured extraction efficiency of 99.46 %. Notably, the OoW method provides for an improved concentration of bromoform of more than six times that of the method of Magnusson et al. 2020.

Claims

1. A process for preparing an anti-methanogenic oil composition, wherein the process comprises: contacting a biomass of red marine algae with a heterogeneous extraction liquid comprising an aqueous solution and at least one oil, and/or with at least one of the aqueous solution and the at least one oil prior to forming the heterogeneous extraction liquid, under conditions to extract one or more anti-methanogenic agents from the biomass into the heterogeneous extraction liquid; and separating the oil from the heterogeneous extraction liquid to obtain an anti- methanogenic oil composition comprising one or more anti-methanogenic agents.

2. The process of claim 1 , wherein the red marine algae is Asparagopsis including Asparagopsis taxiformis or Asparagopsis armata.

3. The process of claim 1 or claim 2, wherein the ratio of the biomass to the heterogeneous extraction liquid is in a range of about 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1.

4. The process of any one of claims 1 to 3, wherein the ratio of the aqueous solution to the oil in the heterogeneous extraction liquid is in a range of about 100:1 to 1:1, 50:1 to 2:1, 25:1 to 3:1, or 10:1 to 4:1.

5. The process of any one of claims 1 to 4, wherein the aqueous solution comprises or consists of water.

6. The process of claim 5, wherein the aqueous solution further comprises a polar solvent.

7. The process of any one of claims 1 to 6, wherein the pH of the aqueous solution is in a range of about 5 to 9, 6 to 8, or about 7.

8. The process of any one of claims 1 to 7, wherein the heterogeneous extraction liquid is a heterogeneous liquid mixture comprising or consisting of water and one or more oils.

9. The process of any one of claims 1 to 8, wherein the oil is an edible oil.

10. The process of any one of claims 1 to 9, wherein the anti-methanogenic oil composition is an animal feed or supplement.

11. The process of any one of claims 1 to 10, wherein the total amount of the one or more anti-methanogenic agents extracted into the anti-methanogenic oil composition (w/w) is about 0.1 to 100 mg/g, about 1 to 50 mg/g, or about 1 to 8 mg/g.

12. The process of any one of claims 1 to 11, wherein the one or more anti-methanogenic agents are selected from bromoform, dibromochloromethane, bromoiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichlorobromomethane, bromoiodomethane, diiodomethane, carbon tetrabromide or any combination thereof.

13. The process according to any one of claims 1 to 12, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise a duration of about 1 hour to 1 month, 1 day to 1 month, 1 week to 3 weeks, or 10 days to 20 days.

14. The process according to any one of claims 1 to 13, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise a temperature of about 5°C to 70°C, 10°C to 65°C, 20°C to 65°C, or 30°C to 65°C.

15. The process according to any one of claims 1 to 14, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise agitation.

16. The process according to any one of claims 1 to 15, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise a maceration step of macerating the biomass in the aqueous solution before the oil is added.

17. The process according to any one of claims 1 to 16, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise the biomass being in the form of fresh seaweed with the time to contact with the heterogeneous extraction liquid being immediately on removal from cultivation media up to about 3 hours, up to about 12 hours, or up to about 24 hours.

18. The process according to any one of claims 1 to 17, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise introducing one or more enzymes.

19. The process according to any one of claims 1 to 18, wherein the conditions of contacting the biomass with the heterogeneous extraction liquid comprise homogenization.

20. The process according to any one of claims 1 to 19, wherein the step of separating the oil from the heterogeneous extraction liquid to obtain an anti-methanogenic oil composition comprises partitioning and removing the oil from the heterogeneous extraction liquid.

21. The process according to any one of claims 1 to 19, wherein the step of separating the oil from the heterogeneous extraction liquid to obtain an anti-methanogenic oil composition comprises decanting.

22. An anti-methanogenic oil composition comprising one or more oils and one or more anti-methanogenic agents selected from bromoform, dibromochloromethane, bromoiodomethane, triiodomethane, bromochloroiodomethane, dibromomethane, dichlorobromomethane, bromoiodomethane, diiodomethane, carbon tetrabromide or combination thereof, wherein the total amount of the one or more anti-methanogenic agents extracted into the anti- methanogenic oil composition (w/v) is about 1 to 100 mg/ml, about 1 to 50 mg/ml, or about 1 to 8 mg/ml.

23. An anti- methanogenic oil composition prepared from the process of any one of claims 1 to 21.

24. A ruminant animal feed or supplement comprising the anti-methanogenic oil composition of claim 22 or claim 23 for use in reducing methane production in a ruminant animal.

25. A method of reducing methane production in a ruminant animal by administration to the ruminant animal of a ruminant animal feed or supplement of claim 24.

26. Use of the ruminant animal feed or supplement of claim 24 for reducing methane production in a ruminant animal.