Red macroalgae processing method

Abstract

The present invention relates to a process for obtaining a composition comprising halogenated compounds from red macroalgae. Said process comprises the steps: a) providing red macroalgae; b) mixing the red macroalgae of step a) with cyclodextrin to bring the cyclodextrin in contact with the halogenated compounds to obtain cyclodextrin-halogenated compound complexes; and c) separating the cyclodextrin-halogenated compound complexes to obtain a composition comprising halogenated compounds. In particular, the present invention relates to a process for extracting and stabilising a composition comprising halogenated compounds and the use thereof to reduce methane production in a ruminant animal.

Description

[0001] RED MACROALGAE PROCESSING METHOD

[0002] Technical field of the invention

[0003] The present invention relates to a process for obtaining a composition comprising halogenated compounds from red macroalgae. In particular, the present invention relates to a process for extracting and stabilising a composition comprising halogenated compounds and the use thereof to reduce methane production in a ruminant animal.

[0004] Background of the invention

[0005] Methane (CH4) is a gaseous compound naturally present, synthesized, and emitted throughout different ecosystems on earth, as well as being a greenhouse gas (GHG), capable of absorbing infrared radiation when present in the atmosphere. The synthesis of methane is performed, in part, by microbial activity found in ruminants, with methane emissions through eructation being a natural part of feed degradation. Methane emissions led into the atmosphere are increasing due to anthropogenic use of land and agriculture, resulting in methane concentrations in the atmosphere growing year on year. The increasing atmospheric concentration is impacting the global climate, as methane has a global warming potential 28 times greater when compared to CO2.

[0006] When seen in context, the impact on GHG impact from ruminating animals in agriculture is significant. In 2021, Denmark emitted 44.5mt CChe, with 3.3mt being directly from cattle-specific enteric fermentation processes. For this reason, impacting and reducing the GHG intensity of fermentation processes is paramount to sustain the current levels of cattle agriculture found both within Denmark and globally.

[0007] A biosolutions approach using red seaweed, which has been globally researched, presents a feed-based, non-therapeutic method of bringing down methane emissions from enteric fermentation significantly (e.g., EP3102219 Bl). The technology applies specific seaweed species, containing anti-microbial bioactives, which lower the number of methane-synthesising bacteria found in the cattle rumen. The seaweed can be introduced through feed, only requiring very small inclusion rates to be effective. When fed raw and in major quantities, the seaweed reduces appetite and overall consumption of feed in cattle. This reinforces the demand to have concentrated and minimal volumes of product, to eliminate negative impact on cattle production.

[0008] Other approaches involve administering synthetic bioactives to ruminants (WO23150832 Al). However, the seaweed-based product yields better methane inhibition results when compared to artificial concentrations of equal amounts of the bioactives, due to a complex distribution of adjacent compounds, all supporting the methane-inhibiting effect (Abbott DW, et al. (2020), Felix, R. et al. (2021), Ahmed, E. and Nishida, T (2024)). This supports the use of cultivated seaweed instead of chemically sourced additives.

[0009] A major challenge in applying the technology has been the stabilisation of the essential volatile bioactives, which is required for documented and reliable impact at scale. An effective but energy-intense method is freeze drying the seaweed. This process has been shown to stabilise the product but is both costly and counteracting the GHG emission reduction achieved by the product itself. Enabling the bioactive technology of the seaweed at scale thus requires a post-cultivation process with reduced financial and GHG impact.

[0010] WO24073575 Al discloses a seaweed feed product comprising a halogenated compound-diminished seaweed material and halogenated compounds stabilised in a binding agent. Including the seaweed material in the final product increases the risk of microbial degradation, which can shorten the shelf life of the product. Furthermore, it prevents the product from being in a liquid form, limiting its supply to intensive (barn or feedlot) and not grazing (water troughs) animals. Oil-based extractions have also been used to stabilize the essential bioactives (EP3890761 Al), which have demonstrated good qualities in isolating and stabilising the bioactive components and introduces a new product formulation on farm. An oil-based extract provides the correct function and potential for a new product formulation, but availability and on-farm applicability are limited. This is due to the additional resource required in the methane blocking product, when presented as an oil-based extract. Using an oil-based product alters the nutritional composition of the general feed that is given to the treated cattle. A significant financial cost is also introduced by using a solvent such as oil.

[0011] To counteract both the financial burden and to accommodate on-farm appliance, water-based extracts have been tested (Magnusson, M. et al. (2020)), but show lesser results when compared to oil, due to the physicochemical nature and extraction behaviour of the bioactive components.

[0012] Hence, an improved processing method for extracting bioactives from red macroalgae would be advantageous, and in particular a more efficient extraction process stabilising the bioactives would be advantageous.

[0013] Summary of the invention

[0014] Thus, an object of the present invention relates to providing an improved extraction process for extracting and stabilizing the bioactives, such as bromoform, from red macroalgae. In particular, it is an object of the present invention to provide a process that solves the above-mentioned problems of the prior art with stabilization, cost, GHG emission with freeze-drying, microbial degradation, and on-farm applicability.

[0015] Water-based solvent properties can be changed by addition of different chemical compounds, allowing to accommodate the loss of performance seen between oil and water. Cyclodextrins are a class of starch compounds which have been demonstrated to increase solubility of nonpolar substances in aqueous environments and are thus interesting in relation to the challenges presented here. Thus, the present invention provides a process for obtaining a composition comprising halogenated compounds from red macroalgae using cyclodextrin. Said composition is used to reduce methane production in a ruminant animal.

[0016] Thus, one aspect of the invention relates to a process for obtaining a composition comprising halogenated compounds from red macroalgae, said process comprising the steps: a) providing red macroalgae; b) mixing the red macroalgae of step a) with cyclodextrin to bring the cyclodextrin in contact with the halogenated compounds to obtain cyclodextrin-halogenated compound complexes; and c) separating the cyclodextrin-halogenated compound complexes to obtain a composition comprising halogenated compounds. Another aspect of the present invention relates to a composition comprising halogenated compounds obtained by or obtainable by the process according to the present invention.

[0017] Yet another aspect of the present invention is to provide a cyclodextrin- halogenated compound complex, wherein the cyclodextrin comprises an integer of a-D-glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D-glucopyranoside units, more preferably 7 or 8 a-D-glucopyranoside units, most preferably 7 a-D-glucopyranoside units, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of Ci-Cs hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0018] Still another aspect of the present invention relates to the use of the composition according to the present invention or the cyclodextrin-halogenated compound complex according to the present invention or the feed ingredient according to the present invention for reducing methane production in a ruminant animal.

[0019] Example 1 demonstrates that cyclodextrins, in particular a-, -, and y- cyclodextrin, can be used to extract and stabilise halogenated compounds, such as bromoform. Example 1 also shows that there is an indication that the different cyclodextrins perform differently based on size, with 0-cyclodextrin showing the greatest level of bromoform extraction. The cyclodextrin solvents performed equally well to oil-based solvents in terms of bromoform extraction. However, a feed comprising the bioactives extracted using cyclodextrin is preferred compared with a feed comprising bioactives extracted using oil, since using an oil-based product alters the nutritional composition of the general feed that is given to the ruminants. The oil is both energy-heavy and impacts digestion in the ruminants. Furthermore, a significant financial cost is also introduced by using a solvent such as oil. Hence, a process using cyclodextrins to extract bioactives from red macroalgae is preferred as opposed to using the oil-based extraction processes disclosed in the prior art. Example 2 demonstrates that the cyclodextrins can be modified by the addition of substituents. In particular, hydroxypropyl-modified p-cyclodextrin aqueous solvent outperformes organic rapeseed oil when extracting the bioactive component bromoform and also shows a superior methane inhibition in in vitro fermentation analysis. Thus, introducing modifications of cyclodextrins is an important tool to increase extraction performance.

[0020] Example 3 demonstrates that the degree of extraction is impacted to a large degree by both duration of, and temperature during, extraction. The temperatures and conditions of extraction are applied across three separate ratios of biomass and solvent. There is both an increased extraction rate, and a significant evaporation process happening due to the increased temperature. Correctly applying temperature at lower extraction durations increases yield efficiency.

[0021] Brief description of the figures

[0022] Figure 1 shows the concentration of bromoform, the bioactive constituent found inside the seaweed, for four different extracts. All extracts were prepared as described in Example 1. Included in the figure are three different aqueous solvents, with either Alpha, Beta or Gamma cyclodextrins as an additive, as well as an organic rapeseed oil solvent for reference.

[0023] Figure 2 shows the extraction efficiency (%) of bromoform in the solvents when compared to the freeze-dried seaweed, which is determined as the benchmark. Bromoform was quantified to be 3.74 mg per gram of freeze-dried biomass. All extracts were prepared as described in Example 1. Included in the figure are three different aqueous solvents, with either Alpha, Beta or Gamma cyclodextrins as an additive, as well as an organic rapeseed oil solvent for reference.

[0024] Figure 3 shows the concentration of bromoform, the bioactive constituent found inside the seaweed, for two different extracts. All extracts were prepared as described in Example 2. Included in the figure is an aqueous solvent with hydroxypropyl-modified Beta cyclodextrins as an additive, as well as an organic rapeseed oil solvent for reference. Figure 4 shows the In Vitro Fermentation performance, measured in % of methane inhibition, when compared to a base diet with no product addition. All extracts were prepared as described in Example 2. Included in the figure is an aqueous solvent with hydroxypropyl-modified Beta cyclodextrins as an additive, as well as an organic rapeseed oil solvent for reference.

[0025] Figure 5 shows the concentration of bromoform, the bioactive constituent found inside the seaweed, for similar extracts at different temperatures, durations and ratios of biomass and solvent. All extracts were prepared as described in Example 3. Included in the figure is an aqueous solvent with hydroxypropyl-modified 0- cyclodextrins as an additive.

[0026] The present invention will now be described in more detail in the following.

[0027] Detailed description of the invention

[0028] Definitions

[0029] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0030] Halogenated compounds

[0031] The term 'halogenated compounds' refers to any compound that comprises at least one halogen. The halogenated compounds preferably consist of carbon, hydrogen, at least one halogen and optionally oxygen. The halogenated compounds may comprise one or more halogens or a mixture of different halogens. 'Halogenated compounds' include but are not limited to bromoform, dibromomethane, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

[0032] Red macroalgae

[0033] Red macroalgae is a subgroup of red algae (used interchangeably with 'red seaweed'). Red algae are divided into the Cyanidiophyceae group, and two sister clades called SCRP (Stylonematophyceae, Compsopogonophyceae, Rhodellophyceae and Porphyridiophyceae) and BF Bangiophyceae and Florideophyceae) . The SCRP clade is microalgae, consisting of both unicellular forms and multicellular microscopic filaments and blades. The BF clade is macroalgae, i.e., seaweed that usually do not grow to more than about 50 cm in length, but a few species can reach lengths of 2 m. Thus, the term 'Red macroalgae' refers to any algae that is both a macroalgae and a red alga. Thus, 'red macroalgae' refers to any algae in the Bangiophyceae and Florideophyceae classes. In the present context, the red macroalgae is preferably in the Florideophyceae class, more preferably in the Bonnemaisoniaceae family.

[0034] Cyclodextrin

[0035] Cyclodextrins, abbreviated CDs, are a family of cyclic oligosaccharides consisting of glucopyranoside subunits linked by a-1,4 glycosidic bonds. Cyclodextrins are composed of 5 or more, typically 6-8, glucose monomers organized in a toroidal shape. The three main types of cyclodextrins comprising 6, 7 and 8 glucose subunits are alpha- (a-cyclodextrin), beta- (p-cyclodextrin) and gammacyclodextrins (y-cyclodextrin), respectively. The main types of cyclodextrins may be represented by formula (I) where n is selected from 6, 7 or 8 and R1, R2and R3is either H or a substituent.

[0036] Each a-D-glucopyranoside unit may comprise 0, 1, 2 or 3 substitutions, wherein said substitutions are located at position R1, R2or R3. The substitution pattern may be different between the a-D-glucopyranoside units, i.e., each a-D- glucopyranoside may have different substitutions or a different number of substituents compared with the other a-D-glucopyranoside units in the same cyclodextrin compound. The main types of cyclodextrins may also be represented by formula (II), wherein n is either 1 (a-cyclodextrin), 2 (p-cyclodextrin) or 3 (y-cyclodextrin).

[0037] Substitution may occur at any one of the alcohol groups in formula II. Thus, each a-D-glucopyranoside unit may comprise 0-3 substitutions.

[0038] Due to this arrangement, the interior of the toroids is less hydrophilic than the aqueous solvent environment and thereby able to host other hydrophobic molecules, thus making cyclodextrins and derivatives thereof suitable for solubilisation of hydrophobic compounds, such as the halogenated compounds mentioned in the present invention.

[0039] Cyclodextrin-halogenated compound complex

[0040] In the present context, the term 'cyclodextrin-halogenated compound complex' refers to the complexes that form when cyclodextrins host at least one halogenated compound. The cyclodextrins may also host more than one halogenated compound and optionally other secondary metabolites.

[0041] Halogen

[0042] The term 'halogen' refers to any of the elements occupying group VIIA (17) of the periodic table. Said elements include fluorine, chlorine, bromine, iodine, astatine, and tennessine. In the present context, the term 'halogen' is used to describe the halogenated compounds, and it is to be understood that the halogenated compounds may comprise one or more halogens or a mixture of different halogens. The preferred halogens in the present context are bromine, chlorine, iodine, and fluorine, with bromine and chlorine being more preferred. Substituent

[0043] Substitution relates to a chemical reaction during which one functional group in a chemical compound is replaced by another functional group. In the present context, substitution relates to the replacement of hydrogen in an alcohol (-OH) in a a-D-glucopyranoside unit with a substituent, wherein each substituent is independently selected from the group consisting of H, Ci-Cs hydroxyalkyl, Ci-Cs di hydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0044] When referring to -OH groups herein, the terms alcohol or alcohol group and hydroxy may be used interchangeably.

[0045] Ci-Ce alkyl

[0046] In the present context, the term 'Ci-Ce alkyl' refers to any alkane that comprises an integer of carbon atoms selected from the range of 1 to 6 and lacks at least one hydrogen. The Ci-Ce alkyl may be acyclic and have the general formula of - CnH2n+i or cyclic and have the general formula of -CnH2n-i. In the present context, the Ci-Ce alkyl is preferably acyclic. The Ci-Ce alkyl may be linear of branched, e.g., C4 alkyl may be butyl (also called n-butyl), 2-methylpropyl (also called 'isobutyl'), 2-butyl (also called 'sec-butyl') and tert-butyl. The Ci-Ce alkyl is preferably selected from the group consisting of methyl, ethyl, propyl, 2-propyl, butyl, 2-methylpropyl, 2-butyl, tert-butyl, pentyl, 2-methylbutan-2-yl, 2,2- dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl, 2- methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3- dimethylbutyl.

[0047] Cation

[0048] The term 'cation' refers to a positively charged ion with fewer electrons than protons. In the present context, the cation is preferably monovalent, i.e. the cation has a valency of one. Examples of such monovalent cations are Na+and K+.

[0049] Homogenisation

[0050] The term 'homogenisation' refers to a process of making things uniform or similar (homogenous). In the present context, 'homogenisation' refers to the process of making a mixture comprising red macroalgae and cyclodextrin uniform macroscopically. This ensures that the cyclodextrin is brought in contact with the halogenated compounds which would otherwise be "hidden" in the red macroalgae. The homogenising can take place by any means known in the art, such as crushing, grinding, milling, blending, cutting, slicing, or dicing.

[0051] Vigorous mixing

[0052] In the present context, 'vigorous mixing' refers to mixing the red macroalgae and cyclodextrin by cutting or in other means destroying the red macroalgae structure to free the halogenated compounds. Thus, the vigorous mixing is more rough than non-vigorous mixing and includes blending, pressure blasting, and ultrasound treating the mixture.

[0053] Non-vigorous mixing

[0054] In the present context, 'non-vigorous mixing' refers to mixing the red macroalgae and cyclodextrin to bring the cyclodextrins in contact with the halogenated compounds which were previously stored in the red macroalgae. Thus, the non- vigorous mixing is gentler that vigorous mixing and does not involve cutting or destroying the red macroalgae structure. The non-vigorous mixing includes inverting, stirring, vortexing, and ultrasound treating the mixture.

[0055] Oil

[0056] The term 'oil' refers to any nonpolar chemical substance that is composed primarily of hydrocarbons and is hydrophobic (does not mix with water) and lipophilic (mixes with other oils). The hydrophobic substance is typically a liquid at ambient temperature and pressure. In the present context, the oil is preferably an edible oil, such as rapeseed oil. The edible oil includes but is not limited to 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, and walnut oil. Also, the oil is preferably not a lipid. Lipid is the scientific term for the fatty acids, steroids and similar chemicals often found in the oils produced by living organisms. Ruminant animal

[0057] A ruminant animal is a mammal of the order Artiodactyla that acquires nutrients from plant-based food by initially softening and partially fermenting it within the animal's first stomach chambers, then regurgitating the semi-digested mass (known as cud) and chewing it again. The process of rechewing the cud to further break down plant matter and stimulate digestion is called "rumination". In the present context, the ruminant animals are preferably a member of the Ruminantia (ruminants), Suina (pigs), or Tylopoda (camels) subgroups. The ruminant animal includes but is not limited to cattle, sheep, goats, giraffes, bison, moose, elk, yaks, water buffalo, deer, alpacas, camels, llamas, wildebeest, antelope, pronghorn, and nilgai.

[0058] Reducing methane production

[0059] In the present context, 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. Fermentation in the rumen and the gut of a ruminant animal gives rise to production of gas, including methane. The present invention aims to reduce this production, 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. The reduction of methane production can be assessed by In Vitro Fermentation analysis.

[0060] WO23150832 Al discloses that cyclodextrin can be used to stabilise manufactured bromoform and that cyclodextrin-bromoform complexes can inhibit methane production in enteric fermentation. However, cyclodextrin is not used in a process for extracting bromoform or other bioactives from red macroalgae. Instead WO23150832 Al uses edible oil to extract said bioactives.

[0061] Example 1 demonstrates that a-, -, and y-cyclodextrins can be used to extract and stabilize bromoform and perform comparatively equal to an oil solvent.

[0062] Thus, an object of the present invention was to provide an improved extraction process for extracting and stabilizing the bioactives, such as bromoform, from red macroalgae. The bioactives that the present inventors extract are halogenated compounds and have an anti-methanogenic effect in ruminant animals. Thus, an aspect of the present invention relates to a process for obtaining a composition comprising halogenated compounds from red macroalgae, said process comprising the steps: a) providing red macroalgae; b) mixing the red macroalgae of step a) with cyclodextrin to bring the cyclodextrin in contact with the halogenated compounds to obtain cyclodextrin-halogenated compound complexes; and c) separating the cyclodextrin-halogenated compound complexes to obtain a composition comprising halogenated compounds.

[0063] Halogenated compounds

[0064] The halogenated compounds comprise as their name imply halogens. In an embodiment the halogenated compounds comprise at least one halogen, preferably at least two halogens, more preferably in the range of 1-4 halogens, most preferably three halogens. In another embodiment, the halogen is selected from bromine, chlorine, iodine, and fluorine, or a mixture thereof. Thus, the halogenated compounds may comprise one or more halogens either of the same element or a mixture of the elements (bromine, chlorine, iodine, and fluorine). In a preferred embodiment, the halogen is selected from bromine, chlorine, or a mixture thereof. In yet another embodiment, the halogenated compounds comprise 2 or 3 halogens, wherein the halogens are selected from bromine, chlorine, and a mixture thereof.

[0065] Several different halogenated compounds have been proven effective in reducing methane production in ruminant animals among these are bromoform, dibromomethane, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid. Thus, in an embodiment, the halogenated compounds are selected from the group consisting of bromoform, dibromomethane, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid, or a mixture thereof. However, the level of bromoform in red macroalgae is increased compared with any other halogenated compound with anti-methanogenic properties, and bromoform is therefore the most important halogenated compound extracted using the process of the present invention. Thus, in an embodiment the halogenated compound is bromoform. The present invention can be used to extract multiple different halogenated compounds that the red macroalgae may comprise. Thus, in an embodiment, the composition comprising halogenated compounds comprises a mixture of halogenated compounds. Red macroalgae

[0066] 'Red macroalgae' refers to any algae that is both a macroalgae and a red alga. Thus, 'red macroalgae' refers to any algae in the Bangiophyceae and Florideophyceae classes. In an embodiment, the red macroalgae is from the Florideophyceae class. In another embodiment, the red macroalgae is from the Bonnemaisoniaceae family, such as Asparagopsis taxiformis, such as Asparagopsis armata, such as Bonnemaisonia hamifera. In examples 1-3, the Asparagopsis taxiformis species have been applied. However, the present inventors have also demonstrated that Asparagopsis armata and Bonnemaisonia hamifera can be used as the red macroalgae in the present invention (data not shown). Thus, in yet another embodiment, the red macroalgae is selected from the group consisting of Asparagopsis taxiformis, Asparagopsis armata, and Bonnemaisonia hamifera, or a mixture thereof. In a preferred embodiment, the red macroalgae is Asparagopsis taxiformis. To avoid decomposition of the red macroalgae and the halogenated compounds therein, the red macroalgae is either used straight after harvest or frozen after collection. Thus, in an embodiment, the red macroalgae is frozen prior to step b).

[0067] Cyclodextrin

[0068] In the examples of the present invention, the cyclodextrins are dissolved in a liquid before using them in the present invention. Thus, in an embodiment, the cyclodextrin is a cyclodextrin solution. The cyclodextrins could be dissolved in any polar solvent but the solvent used in the examples is water. Hence, in an embodiment, the cyclodextrin solution comprises water, such as deionised water. In example 1, a- (6 a-D-glucopyranoside units), - (7 a-D-glucopyranoside units), and y-cyclodextrin (8 a-D-glucopyranoside units) was used in the process of the present invention to extract bioactives, such as bromoform, from red macroalgae. Thus, in an embodiment, the cyclodextrin comprises an integer of a-D- glucopyranoside units selected from the range of 5 to 9. In another embodiment, the cyclodextrin comprises 6, 7 or 8 a-D-glucopyranoside units. There is an indication that the different cyclodextrins perform differently based on size, with 0-cyclodextrin showing the greatest level of bromoform extraction. Thus, in a preferred embodiment, the cyclodextrin comprises 7 or 8 a-D-glucopyranoside units, preferably 7 a-D-glucopyranoside units. In yet another embodiment, the cyclodextrin comprises cyclodextrin selected from the group consisting of a- cyclodextrin, p-cyclodextrin, and y-cyclodextrin, or a mixture thereof, preferably p-cyclodextrin, y-cyclodextrin, or a mixture thereof, most preferably p- cyclodextrin.

[0069] In Example 2, the inventors demonstrate that hydroxypropyl-modified p- cyclodextrin outperforms the organic rapeseed oil in extracting bromoform from red macroalgae and the resulting product inhibited methane emissions from ruminal fermentation. Thus, in an embodiment, the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of Ci-Cs hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0070] In a preferred embodiment, the substituent is a Ci-Cs hydroxyalkyl, such as a Ci- Ce hydroxyalkyl, preferably a C1-C4 hydroxyalkyl, more preferably a C2-C4 hydroxyalkyl, most preferably C3 hydroxyalkyl.

[0071] In another embodiment, the substituent is a Ci-Cs dihydroxyalkyl, such as a Ci-Ce dihydroxyalkyl, preferably a C1-C4 dihydroxyalkyl, more preferably a C2-C4 dihydroxyalkyl, most preferably C3 dihydroxyalkyl.

[0072] In yet another embodiment, the substituent is a Ci-Cs alkyl, such as a Ci-Ce alkyl, preferably a C1-C4 alkyl, more preferably a C1-C4 alkyl, most preferably Ci alkyl.

[0073] In a further embodiment, the substituent is an aryl, such as phenyl, naphthyl, tolyl, or xylyl, preferably OH-substituted aryl. Each of phenyl, naphthyl, tolyl, and xylyl can be OH-substituted.

[0074] In an embodiment, the substituent is a Ci-Cs carboxylakyl, such as a Ci-Ce carboxylalkyl, preferably a C1-C4 carboxylalkyl, more preferably a C2-C4 carboxylalkyl.

[0075] In yet another embodiment, the substituent is a Ci-Cs sulfonylalkyl, such as a Ci- Ce sulfonylalkyl, preferably a C2-C6 sulfonylalkyl, more preferably a C3-C5 sulfonylalkyl, most preferably C4 sulfonylalkyl.

[0076] In an embodiment, cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of -(CH2)mR and - Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6, R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

[0077] In an embodiment, m is selected from the range of 1 to 5, preferably 1 to 4. In another embodiment, Z is a cation, preferably a monovalent cation. In another embodiment, R is -CH(OH)CH3 and m is selected from the range of 1 to 4, preferably 1 to 3, more preferably 1 to 2, most preferably 1. In yet another embodiment, the monovalent cation is selected from the group consisting of Na+and K+, preferably Na+.

[0078] The Ci-Ce alkyl can exist in multiple different isoforms. Thus, in an embodiment, the Ci-Ce alkyl is selected from the group consisting of methyl, ethyl, propyl, 2- propyl, butyl, 2-methylpropyl, 2-butyl, tert-butyl, pentyl, 2-methylbutan-2-yl,

[0079] 2.2-dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl, 2-methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and

[0080] 2.3-dimethylbutyl. In a preferred embodiment, the Ci-Ce alkyl is methyl. In another preferred embodiment, the substituent is selected from the group consisting of -CH3, -CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H. In example 2, the inventors demonstrated that 0- cyclodextrin modified with the hydroxypropyl-substituent was a particularly effective cyclodextrin to use in the process of the present invention. Thus, in a preferred embodiment, the substituent is -CH2CH(OH)CH3. In another embodiment, the cyclodextrin comprises 0-cyclodextrin with at least one substituent, wherein the substituent is -CH2CH(OH)CH3.

[0081] The cyclodextrin can comprise a mixture of different substituents. Thus, in an embodiment, the cyclodextrin comprises a mixture of substituents, wherein each substituent is independently selected from the group consisting of H, Ci-Cs hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0082] In an embodiment, the substituent is independently selected from the group consisting of -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6, R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H. The substituent replaces hydrogen in at least one alcohol (-OH) in a a-D- glucopyranoside unit in a cyclodextrin. Thus, in an embodiment, the cyclodextrin is of formula (I): n is an integer selected from 5-9,

[0083] R1, R2, and R3are independently selected from the group consisting of H, Ci-C8hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH- substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0084] In an embodiment, R1, R2, and R3are independently selected from the group consisting of -H, -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6,

[0085] R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

[0086] The inventors have tested different ratios between the red macroalgae and the cyclodextrin and the process of the present invention has been effective in all ratios tested. Thus, in an embodiment, the red macroalgae of step a) is mixed with cyclodextrin in a ratio selected from 400: 1 to 1: 1 (w / w), such as 300: 1 to 5: 1 (w / w). In a preferred embodiment, the red macroalgae of step a) is mixed with cyclodextrin in a ratio selected from 200: 1 to 10: 1 (w / w), more preferably 100: 1 to 20: 1 (w / w), most preferably 40: 1 (w / w). Mixing step b)

[0087] In order to bring the cyclodextrin in contact with the red macroalgae it is preferred to have a homogenisation step. Thus, in an embodiment, the mixing of step b) comprises a homogenisation step. In another embodiment, the homogenisation is performed by blending, stirring, inverting, vortexing, pressure blasting, or ultrasound treating the mixture of step b), or a mixture thereof, preferably by blending and inverting the mixture of step b). In the examples of the present invention, a blender is applied to homogenise the mixture. Thus, in an embodiment, the homogenisation is performed using a blender.

[0088] The present inventors have performed the mixing in step b) as two separate steps, wherein the first step is more vigorous than the second step, i.e., the red macroalgae structure is first cut or broken down to release the halogenated compounds in step bl) and subsequently gently mixed with the cyclodextrins in step b2). In an embodiment, the mixing of step b) comprises the steps: bl) a vigorous mixing; and b2) a non-vigorous mixing; wherein the vigorous mixing is selected from the group consisting of blending, pressure blasting, and ultrasound treatment, and the non-vigorous mixing is selected from the group consisting of inverting, stirring, vortexing, and ultrasound treatment.

[0089] In another embodiment, the vigorous mixing is blending and / or the non-vigorous mixing is inverting and / or stirring. In yet another embodiment, the non-vigorous mixing is performed for at least 10 minutes, such as at least 20 minutes, such as in the range of 30 minutes to 3 days, preferably in the range of 45 minutes to 2 days, more preferably in the range of 1 hour to 24 hours, such as 8 hours, such as 3 hours, preferably 1 hour.

[0090] Separation step c)

[0091] In step c), the cyclodextrin-halogenated compound complexes are separated from the remainder of the mixture to obtain a composition comprising halogenated compounds. Said separation can be performed using many different methods. Thus, in an embodiment, the separation in step c) is performed using a method selected from the group consisting of centrifugation, decantation, distillation, sedimentation and filtration. The present inventors used centrifugation to separate the cyclodextrin-halogenated compound complexes. Thus, in a preferred embodiment, the separation in step c) is performed using centrifugation. In another embodiment, the centrifugation is performed with a spinning velocity of less than 30,000 rpm, preferably less than 20,000 rpm, preferably in the range of 1,000 rpm to 15,000 rpm, such as in the range of 2,000 rpm to 10,000 rpm, more preferably less than 8,000 rpm, most preferably 5,000 rpm. In yet another embodiment, the centrifugation is performed with less than 65,000 x g, such as in the range of 1,000 x g to 50,000 x g, preferably less than 40,000 g x, such as less than 30,000 x g, such as less than 20,000 x g, preferably less than 10,000 x g, more preferably less than 5,000 x g, such as 4,500 x g, most preferably 3,200 x g. The separation is conducted relatively fast when using centrifugation. Thus, in an embodiment, the centrifugation is performed in less than 20 minutes, preferably less than 10 minutes, such as in the range of 30 seconds to 8 minutes, preferably in the range of 1 minute to 7 minutes, more preferably less than 6 minutes, most preferably 5 minutes.

[0092] Reaction conditions

[0093] As opposed to a commonly used extraction process in the prior art, the present invention does not add any oil during the process. Thus, in an embodiment, the process is performed with less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil. In a preferred embodiment, the process is performed essentially without oil. In another embodiment, the oil is an edible oil, such as rapeseed oil. In another embodiment, the 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, and walnut oil, or a mixture thereof. In yet another embodiment, the oil is not a lipid. In another embodiment, the lipid is a fatty acid or steroid.

[0094] In example 3, the inventors demonstrated that the present invention can be conducted at a multitude of different temperatures. Thus, in an embodiment, step b) and step c) is performed at a temperature of less than 80 °C, such as in a temperature range of 5 °C to 70 °C, preferably less than 75 °C, more preferably in the temperature range of 20 °C to 60 °C, most preferably 60 °C.

[0095] In another embodiment, the non-vigorous mixing is performed in the range of 1 hour to 24 hours and step b) and step c) is performed in a temperature range of 20 °C to 60 °C.

[0096] Product by process

[0097] An aspect of the present invention relates to a composition comprising halogenated compounds obtained by or obtainable by the process according to the present invention. The process disclosed herein is performed essentially without oil, however, the red macroalgae comprises in itself between 2 % and 7 % oil of which some might end up in the composition comprising halogenated compounds. Thus, in an embodiment, said composition comprises less than 7 % oil, such as less than 6 % oil, preferably less than 3 % oil, such as less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil. In another embodiment, said composition is essentially without oil.

[0098] Product claims

[0099] Another aspect of the present invention relates to a cyclodextrin-halogenated compound complex, wherein the cyclodextrin comprises an integer of a-D- glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D- glucopyranoside units, more preferably 7 or 8 a-D-glucopyranoside units, most preferably 7 a-D-glucopyranoside units, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of Ci-Cs hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

[0100] In a preferred embodiment, the substituent is a Ci-Cs hydroxyalkyl, such as a Ci- Ce hydroxyalkyl, preferably a C1-C4 hydroxyalkyl, more preferably a C2-C4 hydroxyalkyl, most preferably C3 hydroxyalkyl.

[0101] In another embodiment, the substituent is a Ci-Cs dihydroxyalkyl, such as a Ci-Ce dihydroxyalkyl, preferably a C1-C4 dihydroxyalkyl, more preferably a C2-C4 dihydroxyalkyl, most preferably C3 dihydroxyalkyl. In yet another embodiment, the substituent is a Ci-Cs alkyl, such as a Ci-Ce alkyl, preferably a C1-C4 alkyl, more preferably a C1-C4 alkyl, most preferably Ci alkyl. In a further embodiment, the substituent is an aryl, such as phenyl, naphthyl, tolyl, or xylyl, preferably OH-substituted aryl. Each of phenyl, naphthyl, tolyl, and xylyl can be OH-substituted. In an embodiment, the substituent is a Ci-Cs carboxylakyl, such as a Ci-Ce carboxylalkyl, preferably a C1-C4 carboxylalkyl, more preferably a C2-C4 carboxylalkyl. In yet another embodiment, the substituent is a Ci-Cs sulfonylalkyl, such as a Ci-Ce sulfonylalkyl, preferably a C2- Ce sulfonylalkyl, more preferably a C3-C5 sulfonylalkyl, most preferably C4 sulfonylalkyl.

[0102] In another embodiment, the substituent is independently selected from the group consisting of -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6, R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

[0103] In a preferred embodiment, the substituent is selected from the group consisting of -CH3, -CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H, more preferably -CH2CH(OH)CH3.

[0104] Yet another aspect relates to a feed ingredient comprising the composition according to the present invention or the cyclodextrin-halogenated compound complex according to the present invention. Another aspect relates to a feed comprising the feed ingredient according to the present invention.

[0105] Use claims

[0106] An aspect of the invention relates to the use of the composition according to the present invention or the cyclodextrin-halogenated compound complex according to the present invention as a feed, preferably a feed for a ruminant animal. Another aspect relates to the use of the composition according to the present invention or the cyclodextrin-halogenated compound complex according to present invention as a feed supplement, preferably a feed supplement for a ruminant animal.

[0107] The inventors demonstrate in example 2 that the halogenated compounds extracted using the process of the present invention can inhibit methane emissions from ruminal fermentation. Thus, an aspect of the invention relates to the use of the composition according to the present invention or the cyclodextrin- halogenated compound complex according to the present invention or the feed ingredient according to the present invention for reducing methane production in a ruminant animal. In an embodiment, the ruminant animal is selected from the members of the Ruminantia, Suina, and Tylopoda subgroups. In another embodiment, the ruminant animal is selected from the group consisting of cattle, sheep, goats, giraffes, bison, moose, elk, yaks, water buffalo, deer, alpacas, camels, llamas, wildebeest, antelope, pronghorn, and nilgai. In a preferred embodiment, the ruminant animal is cattle.

[0108] Another aspect of the present invention relates to the use of cyclodextrin to extract halogenated compounds from red macroalgae.

[0109] Yet another aspect relates to a method for reducing methane production in a ruminant animal, the method comprising feeding the ruminant animal the composition according to the present invention or the cyclodextrin-halogenated compound complex according to the present invention or the feed ingredient according to the present invention.

[0110] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0111] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0112] The invention will now be described in further details in the following non-limiting examples.

[0113] Examples

[0114] Example 1 - Producing cultivated seaweed products with non-modified cyclodextrins

[0115] Aim of study

[0116] The aim of this study was to investigate whether cyclodextrins in aqueous solvents could be used to extract and stabilize bioactives from red seaweed. At the same time, the individual performance of different cyclodextrins was investigated and compared to an oil-based extract solvent.

[0117] Materials and methods

[0118] Collection and preparation of seaweed biomass

[0119] A single culture of Asparagopsis taxiformis was cultivated in indoor, temperature- controlled saltwater tanks. The seaweed was removed from the cultivation tank and rinsed in saltwater, before being spun in a centrifuge (Thomas Centri 776 SEK) at 2000 rpm for 2 minutes and weighed out. The seaweed was frozen at -18 degrees Celsius after spinning and kept at frozen temperatures to maintain quality. A subset of the seaweed was freeze-dried (-55 °C at 0.992mbar for 72 hours, Christs Gamma 1-16 LSCpIus) and milled for quality control of subsequent product formulations.

[0120] Preparing solvents fit for seaweed bioactives extraction

[0121] Several solvents were applied to extract the target bioactives from the seaweed. These include water with added concentrations of a-cyclodextrins, water with added concentrations of 0-cyclodextrins, water with added concentrations of y- cyclodextrins. Cyclodextrins of a-, -, and y-configuration were supplied from Wacker Chemie (CAVAMAX, W6 through W8). The aqueous solutions of cyclodextrins were prepared by solubilising 5 grams of cyclodextrin in 1 L of demineralised water. All solutions were vacuum filtered before storage. All solutions were stored in airtight containers in a refrigerated area.

[0122] Initial mixing of solvents and seaweed

[0123] All solvents and biomass were mixed prior to any further processing. All mixtures consisted of biomass and solvent in a ratio of 0.1 gram biomass per mL solvent, resulting in a ratio of 1 : 10. Thus, the resulting ratio between biomass to cyclodextrin is 20: 1 The solvents were brought out of refrigeration and brought to room temperature before addition of frozen seaweed. Immediately after addition of frozen seaweed, the mixture was blended (NutriBullet PRO 900W) for improved efficiency of equipment later in the process.

[0124] Homogenisation and extraction of seaweed mixture The blended mixture of seaweed was homogenized (GEA PandaPlus 2000) at a working pressure of 1000 bar at room temperature. Before utilisation, the equipment was calibrated on and continuously processed room temperature water to ensure stability and mimic the solvent composition. Immediately after homogenisation, samples were stored in 15mL centrifuge tubes and put on a vertical rotor to provide continuous agitation during the extraction period. Extraction is performed for 24 hours at room temperature.

[0125] Centrifugation and storage of finished samples

[0126] After the full extraction period of 24 hours, all sample tubes were spun at 5000 rpm for 5 minutes to sediment seaweed from the solvents. All solvents were filtered (0.45um syringe filters) and put into new centrifuge tubes to ensure no particulate matter remained in liquid samples. The liquid samples were frozen at - 18 °C and stored prior to analysis.

[0127] Bromoform quantification

[0128] All liquid samples as well as a freeze-dried seaweed sample, serving as bromoform reference, were analysed via GC-FID equipment via an external commercial laboratory. The liquid samples were prepared for analysis by solubilization. The aqueous samples were prepared in ethyl acetate. The oilsolvent sample was prepared by chilled methanol. The freeze-dried sample was prepared by methanol.

[0129] Results

[0130] Cyclodextrins could be used to extract and stabilize bromoform. The concentrations of bromoform were quantified to be 0.052, 0.065, and 0.058 mg / mL for a-, -, and y-cyclodextrin extracts, respectively (Figure 1). The same extraction process performed with organic rapeseed oil, yielded concentrations of bromoform at 0.061 mg / mL. Thus, the individual cyclodextrins perform comparatively equal to the oil solvent. There is an indication that the different cyclodextrins perform differently based on size, with 0-cyclodextrin showing the greatest level of extraction. When comparing the bromoform yield efficiency for the cyclodextrin solvents and oil-based solvent with the freeze-dried seaweed (Figure 2), it is clear that said extraction processes are less efficient than freeze- drying seaweed in terms of extracting bromoform. However, said processes are also significantly more environmentally friendly with a reduced impact on greenhouse gas emission compared with the freeze-drying process.

[0131] Preliminary data indicates that the process of the present invention can also be used to extract bioactives from other red macroalgae species. In particular, the present inventors have demonstrated that the process of the present invention can extract bioactives from Asparagopsis armata and Bonnemaisonia hamifera in addition to the Asparagopsis taxiformis used in the present example.

[0132] Conclusion

[0133] Cyclodextrins can be used in an extraction process to obtain bioactives, such as bromoform. Solvents comprising a-, -, or y-cyclodextrin performed equally well to oil-based solvents in terms of bromoform extraction. However, a feed comprising the bioactives extracted using cyclodextrin is preferred compared with a feed comprising bioactives extracted using oil, since using an oil-based product alters the nutritional composition of the general feed that is given to the ruminants. The oil is both energy-heavy and impacts digestion in the ruminants. Furthermore, a significant financial cost is also introduced by using a solvent such as oil. Hence, a process using cyclodextrins to extract bioactives from red macroalgae is preferred as opposed to using the oil-based extraction processes disclosed in prior art. The results also indicated that the different cyclodextrins perform differently based on size, with 0-cyclodextrin showing the greatest level of bromoform extraction.

[0134] Example 2 - Producing wild seaweed products with substituted cyclodextrins

[0135] Aim of study

[0136] The aim of this study was to clarify whether there was an equivalent extraction potential for a hydroxypropyl-modified 0-cyclodextrin and an oil-based extract solvent.

[0137] Materials and methods

[0138] Collection and preparation of seaweed biomass

[0139] Asparagopsis taxiformis, in its gametophyte stage, was harvested from the Azores islands, Portugal. The seaweed was blotted dry after collection and put directly into a freezing environment and transported using air courier. Preparing solvents fit for seaweed bioactives extraction

[0140] Two solvents were applied to extract the target bioactives from the seaweed. These include water with added concentrations of hydroxypropyl p-cyclodextrins and store-bought organic rapeseed oil. The modified p-cyclodextrins were supplied from Wacker Chemie (CAVASOL W7 HP). The aqueous solutions of cyclodextrins were prepared by solubilising 100 grams of cyclodextrin in 1 L of demineralised water. All solutions were vacuum filtered before storage. All solutions were stored in airtight containers in a refrigerated area.

[0141] Blending and extraction of seaweed components

[0142] All solvents and biomass were mixed prior to any further processing. All mixtures consisted of biomass and solvent in a ratio of 1 gram biomass per mL solvent, resulting in a ratio of 1: 1. Thus, the ratio between biomass to cyclodextrin is 10: 1. The solvents were brought out of refrigeration and brought to room temperature before adding frozen seaweed. Immediately after addition of frozen seaweed, the mixture was blended (NutriBullet PRO 900W) and samples were stored in 15mL centrifuge tubes and put on a vertical rotor to provide continuous agitation during the extraction period. Extraction was performed for 24 hours at room temperature.

[0143] Centrifugation and storage of finished samples

[0144] After the full extraction period of 24 hours, all sample tubes were spun at 5000 rpm for 5 minutes to sediment seaweed from the solvents. All solvents were filtered (0.45um syringe filters) and put into new centrifuge tubes to ensure no particulate matter remained in liquid samples. The liquid samples were frozen at - 18 °C and stored prior to analysis.

[0145] Bromoform quantification

[0146] All liquid samples were analysed via GC-MS equipment at a public university. The liquid samples were prepared for analysis by solubilization. The aqueous sample was prepared in a 1: 1 mixture of dichloromethane and methanol. The oil-solvent sample was prepared by methanol.

[0147] In Vitro Fermentation analysis In vitro simulation of rumen fermentation

[0148] The impact of the bioactives on rumen fermentation characteristics was assessed in vitro by incubating maize silage (MS) as a standard feed in buffered rumen inoculum under anaerobic conditions, with or without the addition of bioactives extracted using either the hydroxypropyl p-cyclodextrin water-based solvent or the rapeseed oil solvent. The experiment was conducted at AU Viborg (Tjele, Denmark). The procedures involving rumen cannulation of cows and sampling of rumen fluid from these cows were approved by the Animal Experiments Inspectorate in accordance with the guidelines established by directive 2010 / 63 / EU and current Danish legislation (law no. 474, May 14, 2014), and were in compliance with ARRIVE guidelines. On the morning of each experiment, rumen fluid was collected half an hour before morning feeding from three rumen- cannulated nongestating dry Holstein cows housed at the experimental facility at Aarhus University, Foulum, Denmark. The handling and care of the cows complied with the guidelines set out by the Danish Ministry of Environment and Food (2020) (Act No. 2028, 2020) with respect to animal experimentation and care of animals under study. The cows were fed at maintenance level with a standard diet composed of straw, hay, and a concentrate mixture. The rumen fluid was immediately transferred to preheated thermo bottles and transported to the laboratory within 30 min after sampling, where it was filtered through two layers of moist cheesecloth, and the pH of the filtrate was measured. The final in vitro inoculum consisted of filtered rumen fluid and a buffer solution (redox indicator, reducing agent, buffer, and macro- and micromineral solutions as described by Menke and Steingass (1988)) mixed in a 2: 1 ratio. During the preparation of the buffer solution and final inoculum, the solution was continuously flushed with N2 to maintain anaerobic conditions. Incubations were conducted in Duran® bottles (capacity: 132 ± 1.1 mL) containing 0.5 g of MS, and 90 mL of buffered rumen fluid with or without 2 mL solution of bioactives extracted with either the waterbased or the oil-based solvent, to reach the concentration of 12% (w / w) of each bioactive solution on feed DM basis. The high inclusion rate was chosen to ensure that any effects on methane formation by the individual bioactives solutions were detectable. The MS was freeze-dried and milled through a 2-mm sieve on a centrifugal mill (Ultra Centrifugal Mill ZM 200, Verder Scientific, Hann, Germany). To account for possible effects of DMSO on fermentation, negative controls were included with bottles containing 2 mL of pure MilliQ water (MS) or 2 mL of pure DMSO (MS-DMSO) without bioactive solutions. The headspace of the bottles was flushed with N2 before the ANKOM pressure sensor module (AnkomTechnology, Macedon, NY, USA) was fitted on top of the bottle. All the bottles were incubated in an incubator shaker (New BrunswickTM Excella R E25R, Eppendorf, Hamburg, Germany) at 38.5 °C and 50 rpm oscillation for 48 h. All the treatments were tested in triplicate in each of two separate incubation runs. In each run, 3 blanks (bottles containing only buffered rumen fluid without MS) were also included. During the incubations, the pressure changes in the headspace of the bottles were continuously recorded every 10 min as a difference with respect to the atmospheric pressure. The produced gas was released from the headspace through the opening of a valve for 250 ms whenever the pressure inside the bottle reached 0.75 psi above ambient pressure, and the accumulated gas production was automatically calculated. The released gas was collected in a gas-tight 1 L Aluminium Bag CEK-1 (GL Sciences Inc., Tokyo, Japan) connected to the module. After 48 h of incubation, the gasbags were removed. Ten mL of gas was extracted from each gasbag using a gas-tight 10 mL 1010SL syringe (Hamilton, Bonaduz, Switzerland) and transferred into evacuated GC-vials (Labco Limited, Ceredigion, United Kingdom) for analysis of methane concentration.

[0149] Methane and VFA analyses using GC-TCD

[0150] Methane concentrations in gas samples were analysed using a Trace 1310 GC with a TCD detector and a TriPlus Headspace autosampler (Thermo Fisher Scientific, Waltham, MA, USA) as described by Jensen et al. (2022). VFA analyses were performed on the liquid flow-through collected during filtration by GC-MS as described by Olijhoek et al. (2022).

[0151] Chemical composition of the standard feed

[0152] The MS used as standard feed had the following chemical composition (g / kg DM): organic matter (OM), 965; neutral detergent fiber (NDF), 329; starch, 351; crude protein, 77.7.

[0153] Results

[0154] The hydroxypropyl-modified p-cyclodextrins could be used to extract and stabilize bioactives, such as bromoform, from red seaweed. The concentrations of bromoform were quantified to be 0.815 mg / mL, whereas the same extraction process performed with organic rapeseed oil yielded concentrations of bromoform at 0.596 mg / mL (Figure 3). The aqueous extract inhibited methane emissions from ruminal fermentation with 97.44% while the oil-based extract inhibited 87.73% when both samples were tested in an In Vitro Fermentation analysis (Figure 4).

[0155] Conclusion

[0156] The study demonstrates that the hydroxypropyl-modified p-cyclodextrin aqueous solvent outperforms the organic rapeseed oil when extracting the bioactive component. This is further supported by the superior methane inhibition seen for the aqueous extract when compared to the oil-based extract. Thus, introducing modifications of cyclodextrins is an important tool to increase extraction performance.

[0157] Example 3 - Quantifying the extraction efficiency as a function of temperature and duration

[0158] Aim of study

[0159] The aim of this study was to investigate the importance of the extraction conditions in aqueous solvents to extract bromoform efficiently. In this study three variables of extraction were tested: Temperature, duration, and ratio of biomass per solvent volume.

[0160] Materials and methods

[0161] Collection and preparation of seaweed biomass

[0162] A single culture of Asparagopsis taxiformis was cultivated in indoor, temperature- controlled saltwater tanks. The seaweed was removed from the cultivation tank and rinsed in saltwater, before being spun in a centrifuge (Thomas Centri 776 SEK) at 2000 rpm for 2 minutes and weighed out. The seaweed was taken directly after drying and put in the solvent for extraction process. A subset of the seaweed was freeze-dried (-55 °C at 0.992mbar for 72 hours, Christs Gamma 1- 16 LSCpIus) and milled for quality control of subsequent product formulations.

[0163] Preparing solvents fit for seaweed bioactives extraction

[0164] Only an aqueous solution of p-cyclodextrin was applied in this example. The p- cyclodextrin was supplied from Wacker Chemie (CAVAMAX W7). The aqueous solutions of cyclodextrins were prepared by solubilising 5 grams of p-cyclodextrin in 1 L of demineralised water. All solutions were vacuum filtered before storage. All solutions were stored in airtight containers in a refrigerated area.

[0165] Initial mixing of solvents and seaweed

[0166] All solvents and biomass were mixed prior to any further processing. Three different mixture ratios were applied in this study being 0.2, 0.33, and 1 gram per mL This resulted in ratios between biomass and solvent of 1:5, 1:3, and 1: 1 respectively, and therefore ratios between biomass and cyclodextrins of 40: 1, 66: 1 and 200: 1. Immediately after addition of fresh seaweed, the mixture was blended (NutriBullet PRO 900W).

[0167] Extraction of seaweed mixture

[0168] Immediately after blending, samples were stored in 15mL centrifuge tubes and put on a vertical rotor to provide continuous agitation during the extraction period. The rotor was placed inside of a heating cabinet (RCHT-1200B) to maintain a stable temperature profile during extractions. Temperatures of 25 °C, 40 °C and 60 °C were applied in this study. Extraction periods of 3, 8, and 24 hours were applied, with increasing durations for higher temperatures.

[0169] Centrifugation and storage of finished samples

[0170] After the full extraction period, all sample tubes were spun at 5000 rpm for 5 minutes to sediment seaweed from the solvents. All solvents were filtered (0.45um syringe filters) and put into new centrifuge tubes to ensure no particulate matter remained in liquid samples. The liquid samples were frozen at -18 °C and stored prior to analysis.

[0171] Bromoform quantification

[0172] All liquid samples as well as a freeze-dried seaweed sample, serving as a bromoform reference, were analysed via GC-FID equipment via an external commercial laboratory. The liquid samples were prepared for analysis by solubilization. The aqueous samples were prepared in ethyl acetate. The freeze- dried sample was prepared by methanol.

[0173] Results Temperature treatment was demonstrated to have both a positive impact on peak extraction yield, meaning the maximum amount of extractable bromoform, as well as having an evaporating effect when applied for long periods of time. The positive impact is seen at both 40 °C and 60 °C, with 60 °C showing most improvement on peak yield. The negative impact was also shown for both the 40 °C and 60 °C treatment, with all quantifiable bromoform being gone after 24 hours for all but one sample. The negative impact was shown to be most prominent in high-ratio samples. The highest possible extraction of bromoform was shown for the 1:5 ratio, 3-hour extraction at 60 °C. This supports the implementation of short and high temperature processes when extracting bromoform.

[0174] Conclusion

[0175] Temperature, duration of extraction, and ratio between biomass and solvent all impact extraction efficiencies. It has been shown that temperature and duration can be implemented to increase peak bromoform yield when applied correctly, in short durations with high temperatures. At the same time, it was shown that using raised temperature levels during extraction can evaporate the product that is being extracted.

[0176] References

[0177] • EP3102219 Bl

[0178] • WO23150832 Al

[0179] • Abbott DW, et al. -. Seaweed and Seaweed Bioactives for Mitigation of Enteric Methane: Challenges and Opportunities. Animals (Basel). 2020 Dec 18; 10(12):2432. doi: 10.3390 / anil0122432.

[0180] • Felix, R. et al. -. The biotechnological potential of Asparagopsis armata: What is known of its chemical composition, bioactivities and current market?, Algal Research, Volume 60, 2021, 102534.

[0181] • Ahmed, E. and Nishida, T. : The anti-methanogenic efficacy of Asparagopsis armata: Could it be attributable solely to its bromoform content?, Animal Feed Science and Technology, Volume 318, 2024, 116118.

[0182] • EP3890761 Al • Magnusson, M. et al.: Using oil immersion to deliver a naturally-derived, stable bromoform product from the red seaweed Asparagopsis taxiformis, Algal Research, Volume 51, 2020, 102065, ISSN 2211-9264

[0183] • Menke, K. H., Steingass, H. : Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7-55 (1988)

[0184] • Jensen, R. H. et al. Untargeted metabolomics combined with solid phase fractionation for systematic characterization of bioactive compounds in hemp with methane mitigation potential. Metabolites 12, 77 (2022). • Olijhoek, D. W. et al. Feeding up to 91% concentrate to Holstein and Jersey dairy cows: Effects on enteric methane emission, rumen fermentation and bacterial community, digestibility, production, and feeding behavior. J. Dairy Sci.

[0185] Items

[0186] 1. A process for obtaining a composition comprising halogenated compounds from red macroalgae, said process comprising the steps: a) providing red macroalgae; b) mixing the red macroalgae of step a) with cyclodextrin to bring the cyclodextrin in contact with the halogenated compounds to obtain cyclodextrin-halogenated compound complexes; and c) separating the cyclodextrin-halogenated compound complexes to obtain a composition comprising halogenated compounds.

[0187] 2. The process according to item 1, wherein the halogenated compounds comprise at least one halogen, preferably at least two halogens, more preferably in the range of 1-4 halogens, most preferably three halogens.

[0188] 3. The process according to item 2, wherein the halogen is selected from bromine, chlorine, iodine, and fluorine, or a mixture thereof, preferably bromine, chlorine, or a mixture thereof.

[0189] 4. The process according to any one of the preceding items, wherein the halogenated compounds comprise 2 or 3 halogens, wherein the halogens are selected from bromine, chlorine, and a mixture thereof.

[0190] 5. The process according to any one of the preceding items, wherein the halogenated compounds are selected from the group consisting of bromoform, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid, or a mixture thereof, preferably bromoform.

[0191] 6. The process according to any one of the preceding items, wherein the composition comprises a mixture of halogenated compounds.

[0192] 7. The process according to any one of the preceding items, wherein the red macroalgae is from the Bonnemaisoniaceae family, such as Asparagopsis taxiformis, such as Asparagopsis armata, such as Bonnemaisonia hamifera. 8. The process according to any one of the preceding items, wherein the red macroalgae is frozen prior to step b).

[0193] 9. The process according to any one of the preceding items, wherein the cyclodextrin is a cyclodextrin solution.

[0194] 10. The process according to item 9, wherein the cyclodextrin solution comprises water, such as deionised water.

[0195] 11. The process according to any one of the preceding items, wherein the cyclodextrin comprises an integer of a-D-glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D-glucopyranoside units, more preferably 7 a-D-glucopyranoside units.

[0196] 12. The process according to any one of the preceding items, wherein the cyclodextrin comprises cyclodextrin selected from the group consisting of a- cyclodextrin, p-cyclodextrin, and y-cyclodextrin, or a mixture thereof.

[0197] 13. The process according to any one of the preceding items, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6,

[0198] R is selected from the group consisting of -CH(OH)CH3, -COOH, and -SO3Z, wherein Z is selected from a cation and H.

[0199] 14. The process according to item 13, wherein m is selected from the range of 1 to 5, preferably 1 to 4.

[0200] 15. The process according to any one of items 13 or 14, wherein Z is a cation, preferably a monovalent cation.

[0201] 16. The process according to item 15, wherein the monovalent cation is selected from the group consisting of Na+and K+, preferably Na+. 17. The process according to any one of items 13-16, wherein the Ci-Ce alkyl is selected from the group consisting of methyl, ethyl, propyl, 2-propyl, butyl, 2- methylpropyl, 2-butyl, tert-butyl, pentyl, 2-methylbutan-2-yl, 2,2-dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl, 2-methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, preferably methyl.

[0202] 18. The process according to any one of items 13-17, wherein the substituent is selected from the group consisting of -CH3, -CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H, preferably -CH2CH(OH)CH3.

[0203] 19. The process according to any one of the preceding items, wherein the cyclodextrin comprises p-cyclodextrin with at least one substituent, wherein the substituent is -CH2CH(OH)CH3.

[0204] 20. The process according to any one of the preceding items, wherein the cyclodextrin is of formula (I) : n is an integer selected from 5-9,

[0205] R1, R2, and R3are independently selected from the group consisting of -H,

[0206] - (CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6, R is selected from the group consisting of -CH(OH)CH3, -COOH, and -SO3Z, wherein Z is selected from a cation and H.

[0207] 21. The process according to any one of the preceding items, wherein the red macroalgae of step a) is mixed with cyclodextrin in a ratio selected from 400: 1 to 1 : 1 (w / w), such as 300: 1 to 5: 1 (w / w), preferably 200: 1 to 10: 1 (w / w), more preferably 100: 1 to 20: 1 (w / w), most preferably 40: 1 (w / w).

[0208] 22. The process according to any one of the preceding items, wherein the mixing of step b) comprises a homogenisation step.

[0209] 23. The process according to item 22, wherein the homogenisation is performed by blending, stirring, inverting, vortexing, pressure blasting, or ultrasound treating the mixture of step b), or a mixture thereof, preferably by blending and inverting the mixture of step b).

[0210] 24. The process according to any one of items 22 or 23, wherein the homogenisation is performed using a blender.

[0211] 25. The process according to any one of the preceding items, wherein the mixing of step b) comprises the steps: bl) a vigorous mixing; and b2) a non-vigorous mixing; wherein the vigorous mixing is selected from the group consisting of blending, pressure blasting, and ultrasound treatment, and the non-vigorous mixing is selected from the group consisting of inverting, stirring, vortexing, and ultrasound treatment.

[0212] 26. The process according to item 25, wherein the vigorous mixing is blending and / or the non-vigorous mixing is inverting and / or stirring.

[0213] 27. The process according to any one of items 25 or 26, wherein the non-vigorous mixing is performed for at least 10 minutes, such as at least 20 minutes, such as in the range of 30 minutes to 3 days, preferably in the range of 45 minutes to 2 days, more preferably in the range of 1 hour to 24 hours, such as 8 hours, such as 3 hours, preferably 1 hour.

[0214] 28. The process according to any one of the preceding items, wherein the separation in step c) is performed using a method selected from the group consisting of centrifugation, decantation, distillation, sedimentation and filtration, preferably centrifugation.

[0215] 29. The process according to item 28, wherein the centrifugation is performed with a spinning velocity of less than 30,000 rpm, preferably less than 20,000 rpm, preferably in the range of 1,000 rpm to 15,000 rpm, such as in the range of 2,000 rpm to 10,000 rpm, more preferably less than 8,000 rpm, most preferably 5,000 rpm.

[0216] 30. The process according to item 28, wherein the centrifugation is performed with less than 65,000 x g, such as in the range of 1,000 x g to 50,000 x g, preferably less than 40,000 g x, such as less than 30,000 x g, such as less than 20,000 x g, preferably less than 10,000 x g, more preferably less than 5,000 x g, such as 4,500 x g, most preferably 3,200 x g.

[0217] 31. The process according to any one of items 28-30, wherein the centrifugation is performed in less than 20 minutes, preferably less than 10 minutes, such as in the range of 30 seconds to 8 minutes, preferably in the range of 1 minute to 7 minutes, more preferably less than 6 minutes, most preferably 5 minutes.

[0218] 32. The process according to any one of the preceding items, wherein the process is performed with less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil or essentially without oil.

[0219] 33. The process according to item 32, wherein the oil is an edible oil, such as rapeseed oil.

[0220] 34. The process according to any one of the preceding items, wherein step b) and step c) is performed at a temperature of less than 80 °C, such as in a temperature range of 5 °C to 70 °C, preferably less than 75 °C, more preferably in the temperature range of 20 °C to 60 °C, most preferably 60 °C.

[0221] 35. The process according to any one of items 25-27, wherein the non-vigorous mixing is performed in the range of 1 hour to 24 hours and step b) and step c) is performed in a temperature range of 20 °C to 60 °C.

[0222] 36. A composition comprising halogenated compounds obtained by or obtainable by the process according to any one of items 1-35.

[0223] 37. The composition according to item 36, wherein said composition comprises less than 7 % oil, such as less than 6 % oil, preferably less than 3 % oil, such as less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil.

[0224] 38. The composition according to any one of items 36 or 37, wherein said composition is essentially without oil.

[0225] 39. A cyclodextrin-halogenated compound complex, wherein the cyclodextrin comprises an integer of a-D-glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D-glucopyranoside units, more preferably 7 a-D- glucopyranoside units.

[0226] 40. A cyclodextrin-halogenated compound complex according to item 39, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6,

[0227] R is selected from the group consisting of -CH(OH)CH3, -COOH, and -SO3Z, wherein Z is selected from a cation and H, preferably the substituent is selected from the group consisting of -CH3, - CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H, more preferably -CH2CH(OH)CH3. 41. A feed ingredient comprising the composition according to any one of items 36-38 or the cyclodextrin-halogenated compound complex according to any one of items 39 or 40.

[0228] 42. A feed comprising the feed ingredient according to item 41.

[0229] 43. Use of the composition according to any one of items 36-38 or the cyclodextrin-halogenated compound complex according to any one of items 39 or 40 as a feed, preferably a feed for a ruminant animal.

[0230] 44. Use of the composition according to any one of items 36-38 or the cyclodextrin-halogenated compound complex according to any one of items 39 or 40 as a feed supplement, preferably a feed supplement for a ruminant animal.

[0231] 45. Use of the composition according to any one of items 36-38 or the cyclodextrin-halogenated compound complex according to any one of items 39 or 40 or the feed ingredient according to item 41 for reducing methane production in a ruminant animal.

[0232] 46. Use according to any one of items 43-45, wherein the ruminant animal is selected from the members of the Ruminantia, Suina, and Tylopoda subgroups.

[0233] 47. Use according to any one of items 43-46, wherein the ruminant animal is selected from the group consisting of cattle, sheep, goats, giraffes, bison, moose, elk, yaks, water buffalo, deer, alpacas, camels, llamas, wildebeest, antelope, pronghorn, and nilgai, preferably cattle.

[0234] 48. Use of cyclodextrin to extract halogenated compounds from red macroalgae.

[0235] 49. A method for reducing methane production in a ruminant animal, the method comprising feeding the ruminant animal the composition according to any one of items 36-38 or the cyclodextrin-halogenated compound complex according to any one of items 39 or 40 or the feed ingredient according to item 41.

Claims

Claims1. A process for obtaining a composition comprising halogenated compounds from red macroalgae, said process comprising the steps: a) providing red macroalgae; b) mixing the red macroalgae of step a) with cyclodextrin to bring the cyclodextrin in contact with the halogenated compounds to obtain cyclodextrin-halogenated compound complexes; and c) separating the cyclodextrin-halogenated compound complexes to obtain a composition comprising halogenated compounds.

2. The process according to claim 1, wherein the halogenated compounds comprise at least one halogen, preferably at least two halogens, more preferably in the range of 1-4 halogens, most preferably three halogens.

3. The process according to claim 2, wherein the halogen is selected from bromine, chlorine, iodine, and fluorine, or a mixture thereof, preferably bromine, chlorine, or a mixture thereof.

4. The process according to any one of the preceding claims, wherein the halogenated compounds comprise 2 or 3 halogens, wherein the halogens are selected from bromine, chlorine, and a mixture thereof.

5. The process according to any one of the preceding claims, wherein the halogenated compounds are selected from the group consisting of bromoform, dibromomethane, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid, or a mixture thereof, preferably bromoform.

6. The process according to any one of the preceding claims, wherein the composition comprises a mixture of halogenated compounds.

7. The process according to any one of the preceding claims, wherein the red macroalgae is from the Bonnemaisoniaceae family, such as Asparagopsis taxiformis, such as Asparagopsis armata, such as Bonnemaisonia hamifera.

8. The process according to any one of the preceding claims, wherein the red macroalgae is frozen prior to step b).

9. The process according to any one of the preceding claims, wherein the cyclodextrin is a cyclodextrin solution.

10. The process according to claim 9, wherein the cyclodextrin solution comprises water, such as deionised water.

11. The process according to any one of the preceding claims, wherein the cyclodextrin comprises an integer of a-D-glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D-glucopyranoside units, more preferably 7 or 8 a-D-glucopyranoside units, most preferably 7 a-D-glucopyranoside units12. The process according to any one of the preceding claims, wherein the cyclodextrin comprises cyclodextrin selected from the group consisting of a- cyclodextrin, p-cyclodextrin, and y-cyclodextrin, or a mixture thereof, preferably p-cyclodextrin, y-cyclodextrin, or a mixture thereof, most preferably p- cyclodextrin.

13. The process according to any one of the preceding claims, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of Ci-Cs hydroxyalkyl, Ci-Cs di hydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-C8sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

14. The process according to claim 13, wherein the substituent is a Ci-Cs hydroxyalkyl, such as a Ci-Ce hydroxyalkyl, preferably a C1-C4 hydroxyalkyl, more preferably a C2-C4 hydroxyalkyl, most preferably C3 hydroxyalkyl.

15. The process according to claim 13, wherein the substituent is a Ci-Cs di hydroxyalkyl, such as a Ci-Ce dihydroxyalkyl, preferably a C1-C4 dihydroxyalkyl, more preferably a C2-C4 dihydroxyalkyl, most preferably C3 dihydroxyalkyl.

16. The process according to claim 13, wherein the substituent is a Ci-Cs alkyl, such as a Ci-Ce alkyl, preferably a C1-C4 alkyl, more preferably a C1-C4 alkyl, most preferably Ci alkyl.

17. The process according to claim 13, wherein the substituent is an aryl, such as phenyl, naphthyl, tolyl, or xylyl, preferably OH-substituted aryl.

18. The process according to claim 13, wherein the substituent is a Ci-Cs carboxylakyl, such as a Ci-Ce carboxylalkyl, preferably a C1-C4 carboxylalkyl, more preferably a C2-C4 carboxylalkyl.

19. The process according to claim 13, wherein the substituent is a Ci-Cs sulfonylalkyl, such as a Ci-Ce sulfonylalkyl, preferably a C2-C6 sulfonylalkyl, more preferably a C3-C5 sulfonylalkyl, most preferably C4 sulfonylalkyl.

20. The process according to claim 13, wherein the substituent is independently selected from the group consisting of -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6,R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

21. The process according to claim 20, wherein m is selected from the range of 1 to 5, preferably 1 to 4.

22. The process according to claim 20, wherein R is -CH(OH)CH3 and m is selected from the range of 1 to 4, preferably 1 to 3, more preferably 1 to 2, most preferably 1.

23. The process according to any one of claims 20 or 21, wherein Z is a cation, preferably a monovalent cation.

24. The process according to claim 23, wherein the monovalent cation is selected from the group consisting of Na+and K+, preferably Na+.

25. The process according to claim 20, wherein the Ci-Ce alkyl is selected from the group consisting of methyl, ethyl, propyl, 2-propyl, butyl, 2-methylpropyl, 2- butyl, tert-butyl, pentyl, 2-methylbutan-2-yl, 2,2-dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl, 3-methylbutan-2-yl, 2-methylbutyl, hexyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, preferably methyl.

26. The process according to any one of claims 20-25, wherein the substituent is selected from the group consisting of -CH3, -CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H, preferably -CH2CH(OH)CH3.

27. The process according to any one of the preceding claims, wherein the cyclodextrin comprises p-cyclodextrin with at least one substituent, wherein the substituent is -CH2CH(OH)CH3.

28. The process according to any one of the preceding claims, wherein the cyclodextrin is of formula (I) :n is an integer selected from 5-9,R1, R2, and R3are independently selected from the group consisting of H, Ci-Cs hydroxyalkyl, Ci-Cs dihydroxyalkyl, Ci-Cs alkyl, aryl, such as OH- substituted aryl, Ci-Cs carboxylakyl, and Ci-Cs sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

29. The process according to claim 28, wherein R1, R2, and R3are independently selected from the group consisting of -H, -(CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6, R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

30. The process according to any one of the preceding claims, wherein the red macroalgae of step a) is mixed with cyclodextrin in a ratio selected from 400: 1 to 1: 1 (w / w), such as 300: 1 to 5: 1 (w / w), preferably 200: 1 to 10: 1 (w / w), more preferably 100: 1 to 20: 1 (w / w), most preferably 40: 1 (w / w).

31. The process according to any one of the preceding claims, wherein the mixing of step b) comprises a homogenisation step.

32. The process according to claim 31, wherein the homogenisation is performed by blending, stirring, inverting, vortexing, pressure blasting, or ultrasound treating the mixture of step b), or a mixture thereof, preferably by blending and inverting the mixture of step b).

33. The process according to any one of claims 31 or 32, wherein the homogenisation is performed using a blender.

34. The process according to any one of the preceding claims, wherein the mixing of step b) comprises the steps: bl) a vigorous mixing; and b2) a non-vigorous mixing; wherein the vigorous mixing is selected from the group consisting of blending, pressure blasting, and ultrasound treatment, and the non-vigorous mixing is selected from the group consisting of inverting, stirring, vortexing, and ultrasound treatment.

35. The process according to claim 34, wherein the vigorous mixing is blending and / or the non-vigorous mixing is inverting and / or stirring.

36. The process according to any one of claims 34 or 35, wherein the non- vigorous mixing is performed for at least 10 minutes, such as at least 20 minutes, such as in the range of 30 minutes to 3 days, preferably in the range of 45 minutes to 2 days, more preferably in the range of 1 hour to 24 hours, such as 8 hours, such as 3 hours, preferably 1 hour.

37. The process according to any one of the preceding claims, wherein the separation in step c) is performed using a method selected from the group consisting of centrifugation, decantation, distillation, sedimentation and filtration, preferably centrifugation.

38. The process according to claim 37, wherein the centrifugation is performed with a spinning velocity of less than 30,000 rpm, preferably less than 20,000 rpm, preferably in the range of 1,000 rpm to 15,000 rpm, such as in the range of 2,000 rpm to 10,000 rpm, more preferably less than 8,000 rpm, most preferably 5,000 rpm.

39. The process according to claim 37, wherein the centrifugation is performed with less than 65,000 x g, such as in the range of 1,000 x g to 50,000 x g, preferably less than 40,000 g x, such as less than 30,000 x g, such as less than 20,000 x g, preferably less than 10,000 x g, more preferably less than 5,000 x g, such as 4,500 x g, most preferably 3,200 x g.

40. The process according to any one of claims 37-39, wherein the centrifugation is performed in less than 20 minutes, preferably less than 10 minutes, such as in the range of 30 seconds to 8 minutes, preferably in the range of 1 minute to 7 minutes, more preferably less than 6 minutes, most preferably 5 minutes.

41. The process according to any one of the preceding claims, wherein the process is performed with less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil or essentially without oil.

42. The process according to claim 41, wherein the oil is an edible oil, such as rapeseed oil.

43. The process according to any one of the preceding claims, wherein step b) and step c) is performed at a temperature of less than 80 °C, such as in a temperature range of 5 °C to 70 °C, preferably less than 75 °C, more preferably in the temperature range of 20 °C to 60 °C, most preferably 60 °C.

44. The process according to any one of claims 34-36, wherein the non-vigorous mixing is performed in the range of 1 hour to 24 hours and step b) and step c) is performed in a temperature range of 20 °C to 60 °C.

45. A composition comprising halogenated compounds obtained by or obtainable by the process according to any one of claims 1-44.

46. The composition according to claim 45, wherein said composition comprises less than 7 % oil, such as less than 6 % oil, preferably less than 3 % oil, such as less than 2 % oil, such as less than 1.5 % oil, preferably less than 1 % oil, more preferably less than 0.5 % oil, most preferably less than 0.1 % oil.

47. The composition according to any one of claims 45 or 46, wherein said composition is essentially without oil.

48. A cyclodextrin-halogenated compound complex, wherein the cyclodextrin comprises an integer of a-D-glucopyranoside units selected from the range of 5 to 9, preferably 6, 7 or 8 a-D-glucopyranoside units, more preferably 7 or 8 a-D- glucopyranoside units, most preferably 7 a-D-glucopyranoside units, wherein the cyclodextrin comprises at least one substituent, wherein each substituent is independently selected from the group consisting of Ci-Cs hydroxyalkyl, Ci-Cs di hydroxyalkyl, Ci-Cs alkyl, aryl, such as OH-substituted aryl, Ci-Cs carboxylakyl, and Ci-C8sulfonylalkyl, or mixtures thereof, preferable Ci-Cs hydroxyalkyl.

49. The cyclodextrin-halogenated compound complex according to claim 48, wherein the substituent is a Ci-Cs hydroxyalkyl, such as a Ci-Ce hydroxyalkyl, preferably a C1-C4 hydroxyalkyl, more preferably a C2-C4 hydroxyalkyl, most preferably C3 hydroxyalkyl.

50. The cyclodextrin-halogenated compound complex according to claim 48, wherein the substituent is independently selected from the group consisting of - (CH2)mR and -Ci-Ce alkyl, wherein m is an integer selected from the range of 1 to 6,R is selected from the group consisting of -CH(OH)CH3, -OH, -COOH, and - SO3Z, wherein Z is selected from a cation and H.

51. The cyclodextrin-halogenated compound complex according to any one of claims 48-50, wherein the substituent is selected from the group consisting of - CH3, -CH2CH(OH)CH3, CH2COOH, and (CH2)4 SO3Z, wherein Z is selected from a cation or H, more preferably -CH2CH(OH)CH3.

52. A feed ingredient comprising the composition according to any one of claims 45-47 or the cyclodextrin-halogenated compound complex according to any one of claims 48-51.

53. A feed comprising the feed ingredient according to claim 52.

54. Use of the composition according to any one of claims 45-47 or the cyclodextrin-halogenated compound complex according to any one of claims 48- 51 as a feed, preferably a feed for a ruminant animal.

55. Use of the composition according to any one of claims 45-47 or the cyclodextrin-halogenated compound complex according to any one of claims 48- 51 as a feed supplement, preferably a feed supplement for a ruminant animal.

56. Use of the composition according to any one of claims 45-47 or the cyclodextrin-halogenated compound complex according to any one of claims 48- 51 or the feed ingredient according to claim 52 for reducing methane production in a ruminant animal.

57. Use according to any one of claims 54-56, wherein the ruminant animal is selected from the members of the Ruminantia, Suina, and Tylopoda subgroups.

58. Use according to any one of claims 54-57, wherein the ruminant animal is selected from the group consisting of cattle, sheep, goats, giraffes, bison, moose, elk, yaks, water buffalo, deer, alpacas, camels, llamas, wildebeest, antelope, pronghorn, and nilgai, preferably cattle.

59. Use of cyclodextrin to extract halogenated compounds from red macroalgae.

60. A method for reducing methane production in a ruminant animal, the method comprising feeding the ruminant animal the composition according to any one of claims 45-47 or the cyclodextrin-halogenated compound complex according to any one of claims 48-51 or the feed ingredient according to claim 52.

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