Fucan and polyphenol complex formed from a brown alga and use of said complex in controlling methane metabolism in a ruminant

Abstract

The invention relates to a complex comprising at least one fucan and at least one polyphenol, characterised in that the at least one fucan is a natural fucan of a brown alga, and in that the at least one polyphenol is a natural polyphenol of a brown alga.

Description

Description Title of the invention: Fucan and Polyphenol complex formed from a Brown Algae and Use of this Complex in the Control of Methane Metabolism in a Ruminant

[0001] The invention relates to a complex comprising at least one fucan and at least one polyphenol formed from at least one brown algae. The invention also relates to any use of such a complex of at least one fucan and at least one polyphenol formed from a brown algae for controlling methane metabolism in a ruminant. But the invention also relates to such a complex of at least one fucan and at least one polyphenol formed from at least one brown algae for its use in controlling methane metabolism in a ruminant.

[0002] It has long been known that ruminant digestion generates methane, which is released into the atmosphere, and that this release of methane into the atmosphere could contribute to global warming by accentuating the "greenhouse effect." Solutions to limit methane production by ruminants are therefore being sought.

[0003] The invention therefore aims at such a solution.

[0004] Brown algae (Phaeophyceae) are a class of algae in the Ochrophyta phylum. The cell walls of brown algae are composed primarily of fucans and alginates. They also contain small amounts of cellulose.

[0005] Brown algae fucans are polymers based on fucose and fucose derivatives. These polymers are high molecular weight polysaccharides, i.e., those with a molecular weight that can reach several hundred thousand Daltons (Da). As fucose derivatives, the complex fucans naturally occurring in brown algae may include fucose sulfate, in particular α-1,2-L-fucose-4-sulfate, but also at least one saccharide monomer other than a fucose derivative, in particular uronic acids, D-xylose, D-galactose.

[0006] Brown algae polyphenols (or phlorotannins) are at least primarily phloroglucinol oligomers. Brown algae polyphenols have been identified in intracellular vesicles (physodes) in a soluble form and in the cell wall in an insoluble form.

[0007] WO2019 / 193556 in the name of the present applicant discloses a process for obtaining oligofucans with a molecular mass of less than 5,000 Da and enriched in sulfate, from a brown macroalgae chosen from Lamaniria dititata and / or Lamaniria hyperborea. The process of WO2019 / 193556 comprises: - a first step of soaking fresh brown algae in water acidified to pH 2.0 by adding sulfuric acid, for 2 to 3 hours at room temperature, - a second stage of recovery of the soaking juice, by sieving, - a third stage of adjusting the pH of the sieving juice to a value of 6.5 by adding a 33% sodium hydroxide solution, a fourth stage of clarification of the sieving juice adjusted to pH 6.5 by ultrafiltration with a filtration threshold of 100,000 Da, a fifth stage of fractionation of the permeate by ultrafiltration with a filtration threshold of 2000 Da, a sixth stage of concentration of the permeate at 20°Bx on a vacuum concentrator, a seventh stage of demineralization of the concentrate, by electrodialysis, - an eighth step of precipitation of the oligofucans by addition of 2 volumes of ethanol per volume of demineralized concentrate, a ninth step of washing the precipitate with ethanol at a rate of 1 mass of ethanol per mass of precipitate, then - drying and grinding of the precipitate.

[0008] The oligofucans of the low molecular weight oligofucan composition of WO2019 / 193556 have 85% to 95% sulfated fucose. The oligofucan composition of WO2019 / 193556 comprises 70% to 80% mono- or di-fucan oligomers, 20% to 30% tri- or tetra-fucans and is substantially free of oligofucan of higher degree of polymerization.

[0009] WO2019 / 193556 mentions the cosmetic use of such an oligofucan composition, for its anti-aging, anti-wrinkle and / or firming action on the skin, the use of such an oligofucan composition as a medicament for the treatment of inflammation, osteoarthritis, angiogenesis, cancer and / or obesity.

[0010] WO2019 / 193556 does not concern high molecular weight fucans.

[0011] Also known from WO90 / 15823 is the anticoagulant and antithrombotic activity of brown algae fucan fractions with intermediate molecular weights between 5 kDa and 40 kDa. The process of WO90 / 15823 for obtaining brown algae fucan fractions with molecular weights between 5 kDa and 40 kDa comprises grinding the thalli of a brown algae in a mixture of absolute ethanol containing 5% by weight of formalin, followed by a first extraction with a mixture of absolute ethanol containing 5% by weight of formalin and a second extraction with a mixture of acetone and toluene in a volume ratio (2 / 1). In the process of WO90 / 15823, the formalin has the function of making the polyphenols insoluble in water. The extracted fucans are then degraded and fractionated.

[0012] WO90 / 15823 does not concern high molecular weight fucans or complexes of at least one natural fucan and at least one polyphenol.

[0013] Also known from GB890207 is a process for extracting crude fucans in which air-dried brown seaweed Fucus vesiculosus is ground for 28 hours in a ball mill. The resulting powder is brought into contact with water and heated with stirring in a boiling water bath for about 18 hours. Toluene is added and the mixture is left at room temperature overnight. The supernatant is collected and filtered off any insoluble matter and then fractionated to yield a crude fucan extract.

[0014] The process of GB890207 and in particular precipitation by toluene does not in practice make it possible to obtain a complex comprising at least one fucan and at least one polyphenol according to the invention, nor such a complex which is free of toluene.

[0015] The above-mentioned documents aim to propose processes for the purification of crude fucans for their fractionation / depolymerization or for the fractionation / depolymerization of brown algae extract containing fucans.

[0016] None of the above-mentioned documents relates to a complex comprising at least one high molecular weight fucan and at least one brown algae polyphenol.

[0017] None of the above-mentioned documents concern the field of control of methane metabolism in a ruminant.

[0018] The invention aims to overcome this drawback.

[0019] The invention therefore aims to provide a complex of at least one fucan and at least one polyphenol.

[0020] The invention also aims to provide a complex of at least one fucan and at least one brown algae polyphenol which is standardized and suitable for being produced industrially and marketed.

[0021] The invention also aims to provide such a complex of at least one fucan and at least one polyphenol for use in controlling methane metabolism in ruminants.

[0022] But the invention also aims to provide such a complex of at least one fucan and at least one polyphenol for its use as medicaments for the control of excessive methane metabolism in ruminants.

[0023] To do this, the invention relates to a complex comprising at least one fucan and at least one polyphenol, characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural polyphenol from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed as a result of implementing a process for obtaining a decoction juice enriched with a complex of at least one fucan and at least one polyphenol, in which process at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C (at atmospheric pressure) for a period of between about 15 hours and about 20 hours, in particular about 16 hours, and so as to form the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol,the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol results in coprecipitation of said at least one fucan and said at least one polyphenol.,

[0024] Throughout the text, the term "natural" qualifying the fucan(s) and the polyphenol(s) specifies that the said fucan(s) and the said polyphenol(s) according to the invention are fucans and polyphenols present in brown algae in the natural state. In particular, the term "natural" specifies that the said fucan(s) and the said polyphenol(s) according to the invention are not not substantially depolymerized during the process of obtaining the decoction juice and have a molecular mass substantially identical to the molecular mass of said fucan(s) and said polyphenol(s) in the brown algae in its natural state.

[0025] According to certain advantageous embodiments, the aqueous composition is formed from drinking water obtained from a drinking water distribution network, without the voluntary addition of an additive separate from the water.

[0026] According to certain advantageous embodiments, the method for obtaining the decoction juice comprises a step of acidifying the decoction juice to a pH value of between about pH 1.8 and about pH 2.2, in particular between about pH 1.9 and about pH 2.1, preferably of about 2.0 so as to precipitate at least a portion - in particular all - of the alginates and at least a portion - in particular all - of the proteins of the decoction juice and form an acidified decoction juice. In these embodiments, the acidified decoction juice is rapidly separated from the precipitate of alginates and / or proteins by any suitable means for separating the liquid acidified decoction juice and a solid precipitate, for example by filtration, in particular by press filtration.In these embodiments, the complex of at least one fucan and at least one polyphenol present in the acidified decoction juice is preserved due to the rapid separation of the liquid acidified decoction juice and the precipitate of alginates and / or proteins.

[0027] According to certain advantageous embodiments, the method for obtaining the decoction juice comprises a step of neutralizing the acidified decoction juice to pH 6.5. The neutralization of the acidified decoction juice can be carried out by adding sodium hydroxide (NaOH) to the acidified decoction juice so as to form a neutralized decoction juice. In these embodiments, the neutralization step follows, as quickly as possible, the step of precipitation and liquid / solid separation so as to limit to a minimum the residence time of the complex of at least one fucan and at least one polyphenol in an acidic environment.

[0028] According to certain advantageous embodiments, the method for obtaining the decoction juice comprises a step of ultrafiltration of the neutralized decoction juice, on an ultrafiltration membrane having a cut-off threshold less than or equal to 100 kDa and under conditions capable of eliminating soluble minerals and low molecular weight organic compounds and forming a retentate comprising the complexes of at least one fucan and at least one polyphenol and substantially free of soluble minerals and low molecular weight organic compounds.

[0029] According to certain embodiments, the coprecipitation by ethanol of said at least one fucan and said at least one polyphenol of the complex according to the invention is accompanied by a formation of a precipitate of a darker color (than the color of a precipitate of said at least one fucan alone), indicating the coprecipitation of said at least one light-colored fucan and said at least one darker-colored polyphenol of the complex according to the invention.

[0030] The inventor has, in this regard, observed that soaking brown algae at room temperature in acidified water at pH 2.0 as implemented in the method of WO2019 / 193556 does not in reality allow the complex according to the invention to be formed, as revealed by coprecipitation in ethanol. He also observed that the fucan composition described in WO2019 / 193556 actually has no effect on the control of methane metabolism in ruminants.

[0031] The inventor further observed that tests of fractionation / extraction of fucans into oligofucans by depolymerization, without going through a step of maintaining at least one brown algae in an aqueous composition (at atmospheric pressure) at a temperature between 90°C and 100°C for a duration between around 15 hours and around 20 hours, said aqueous composition being neither a deliberately acidified aqueous composition, nor an aqueous composition comprising formaldehyde, do not in reality make it possible to form the complex of at least one fucan and at least one polyphenol according to the invention.

[0032] According to certain embodiments, at least one polyphenol of the complex of at least one fucan and at least one polyphenol is selected from the group consisting of polyphenols of the phlorotannin family. According to certain embodiments, at least one polyphenol is a phloroglucinol polymer.

[0033] According to certain embodiments, at least one fucan of the complex of at least one fucan and at least one polyphenol is a sulfated polysaccharide of high molecular weight, in particular of molecular weight greater than 100,000 Da. According to certain embodiments, a majority (by mass) of the fucans of the complex of at least one fucan and at least one polyphenol has a molecular weight greater than 100,000 Da. According to certain embodiments, at least of the order of 90% by mass of the fucans of the complex of at least one fucan and at least one polyphenol have a molecular weight greater than 100,000 Da. According to certain embodiments, at least of the order of 95% or at least of the order of 96%, or at least of the order of 97%, or at least of the order of 98%, or at least of the order of 99%, by mass of the fucans of the complex of at least one fucan and at least one polyphenol have a molecular weight greater than 100,000 Da.According to certain embodiments, 100% (by mass) of the fucans of the complex of at least one fucan and at least one polyphenol have a molecular weight greater than 100,000 Da. The molecular weight of the fucans is determined by any known means, in particular by multi-detection size exclusion chromatography.

[0034] According to certain preferred embodiments, the complex comprising at least one fucan and at least one polyphenol is substantially free - in particular totally free - of alginate. The process for obtaining the decoction juice comprising the complex comprising at least one fucan and at least one polyphenol according to the invention comprises a step of acidification of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol, said acidification step being a step of precipitating acidification of alginates.

[0035] According to certain preferred embodiments, the complex comprising at least one fucan and at least one polyphenol is substantially free - in particular totally free - of protein. The process for obtaining the decoction juice and the complex comprising at least one fucan and at least one polyphenol according to the invention comprises a step of acidifying the decoction juice enriched with a complex of at least one natural fucan and at least one po- natural lyphenol, said acidification step being a protein coagulating / precipitating acidification step.

[0036] According to certain advantageous embodiments, the aqueous composition for maintaining at least one brown algae at a temperature between 90°C and 100°C is water.

[0037] According to these embodiments, the process for obtaining the decoction juice does not require any organic solvent, in particular any organic solvent toxic to the environment. In particular, the process for obtaining the decoction juice does not require the use of toluene. The complex comprising at least one fucan and at least one polyphenol according to the invention is free from any trace of organic solvent, in particular toluene.

[0038] Advantageously and according to the invention, the aqueous composition for maintaining at least one brown algae at a temperature of between 90°C and 100°C for a period of between around 15 hours and around 20 hours is free from any acid compound for voluntary acidification of the aqueous composition, in particular free from any strong acid compound for voluntary acidification, in particular from any strong acid compound for voluntary acidification chosen from sulfuric acid (H2SO4) and hydrochloric acid (HCl).

[0039] Advantageously and according to the invention, the aqueous composition for maintaining at least one brown algae at a temperature between 90°C and 100°C for a period of between around 15 hours and around 20 hours is free of formaldehyde in aqueous solution (formalin).

[0040] The inventor also observed that the fucan fractions of brown algae obtained in WO90 / 15823 by initial grinding of thalli of a brown algae in a mixture of absolute ethanol containing 5% by weight of formalin, does not in reality allow the formation of the complex according to the invention, as revealed by a coprecipitation in ethanol. He also observed that the fucan composition described in WO90 / 15823 has in reality no effect on the control of methane metabolism in ruminants.

[0041] The inventor believes that, without this analysis being supported by any other scientific demonstration than that of coprecipitation in ethanol, maintaining at least one brown algae in an aqueous composition at a temperature between 90°C and 100°C and at atmospheric pressure, for a duration of between around 15 hours and around 20 hours actually makes it possible to form the complex according to the invention, soluble in said aqueous composition and having the effect on the control of methane metabolism in ruminants.

[0042] For the purposes of the present invention, the term "brown algae" means brown-colored algae. Advantageously and according to the invention, the brown algae is an algae of the Phaeophyceae class.

[0043] According to certain embodiments, at least one - in particular each - brown algae is in a fresh form or in a dry form or in a dehydrated form.

[0044] According to some embodiments, at least one brown alga is selected from the class Phaeophyceae. According to some of these embodiments, at least one brown alga is selected from the group consisting of Ascophyllum sp. - in particular Ascophyllum nodosum -, Fucus sp., Lamaniria sp., Euchema sp., Macrocystis sp., Alaria sp. and Undaria sp..

[0045] In some embodiments, at least one brown algae is a macroalga or macroscopic algae.

[0046] In some embodiments, at least one brown algae is a fresh brown algae. In some embodiments, at least one brown algae is used as harvested and rinsed with water after harvesting. In some embodiments, at least one brown algae is a thallus of the at least one brown algae.

[0047] According to certain embodiments, at least one brown algae is a dried brown algae. Dried brown algae means a brown algae from which only a portion of its free or non-constituent water has been removed. According to certain embodiments, at least one brown algae is used after drying, in particular after air drying, of at least one brown algae as harvested and rinsed with water after harvesting.

[0048] According to certain embodiments, at least one brown algae is a dehydrated brown algae. Dehydrated brown algae means a brown algae that has been stripped of substantially all of its free or non-constituent water. The invention also relates to any use of a complex according to the invention comprising at least one natural fucan from a brown algae and at least one natural polyphenol from a brown algae.

[0049] According to certain embodiments, the use of a complex according to the invention is a non-therapeutic use.

[0050] According to some of these embodiments, such non-therapeutic use of a complex according to the invention aims at regulation, in particular limiting regulation, of the productive metabolism of methane in a ruminant.

[0051] The non-therapeutic use according to the invention therefore aims to provide a complex according to the invention comprising at least one fucan and at least one polyphenol to limit methane production by a ruminant.

[0052] The non-therapeutic use according to the invention therefore aims to provide a complex according to the invention for the preservation of the environment.

[0053] According to some of these embodiments, the invention relates to a use of a complex comprising at least one fucan and at least one polyphenol for limiting the productive metabolism of methane in a ruminant, characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural polyphenol from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed due to the implementation of a process for obtaining a decoction juice enriched with a complex of at least one fucan and at least one polyphenol, process in which at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C (at atmospheric pressure) for a duration of between about 15 hours and about 20 hours, in particular about 16 hours,so as to form a decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol, the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least, a natural polyphenol causes a coprecipitation of said at least one fucan and said at least one polyphenol.

[0054] According to some of these embodiments, the complex according to the invention is a complex inhibiting the methyltransferase activity of N'-nicotinamide methyl transferase (NNMT) equivalent to the coM-methyltransferase of methanogenic archaebacteria present in the digestive system of ruminants.

[0055] According to certain embodiments, the complex according to the invention is a complex inhibiting the methyltransferase activity of N'-nicotinamide methyl transferase (NNMT) equivalent to the coenzyme M-methyltransferase of methanogenic archaebacteria present in the digestive system of ruminants.

[0056] According to certain other embodiments, the use of a complex according to the invention is a therapeutic use.

[0057] According to some of these embodiments, such therapeutic use of a complex according to the invention aims at regulation, in particular limiting regulation, of a metabolism producing excess methane in a ruminant.

[0058] According to some of these embodiments, the invention relates to a use of a complex comprising at least one fucan and at least one polyphenol for limiting the productive metabolism of excess methane in a ruminant, characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural polyphenol from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed due to the implementation of a process for obtaining a decoction juice enriched with a complex of at least one fucan and at least one polyphenol, process in which at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C (at atmospheric pressure) for a duration of between about 15 hours and about 20 hours, in particular about 16 hours,so as to form a decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol, the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol results in coprecipitation of said at least one fucan and said at least one polyphenol.,

[0059] According to some of these embodiments, the invention therefore relates to a complex comprising at least one natural fucan from a brown algae and at least one natural polyphenol from a brown algae for use in a treatment limiting excessive methane production in a ruminant, the complex being characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural polyphenol from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed due to the implementation of a method for obtaining a decoction juice enriched with complex of at least one fucan and at least one polyphenol, a process in which at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C (at atmospheric pressure) for a period of between about 15 hours and about 20 hours, in particular about 16 hours, so as to form the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol, the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol results in coprecipitation of said at least one fucan and said at least one polyphenol.

[0060] Other aims, characteristics and advantages of the invention will appear on reading the following description, the drawings and the illustrative examples of certain possible but non-limiting embodiments of the invention, in which:

[0061] [Fig 1] Figure 1 is an illustrative diagram of the metabolic pathway of methane production of a methanogenic archaea or archaeobacterium present in the digestive tract of a ruminant,

[0062] [Fig 2] Figure 2 is a detail of an illustrative diagram of the metabolic pathway of methane production of a methanogenic archaea or archaeobacterium present in the digestive tract of a ruminant,

[0063] [Fig 3] Figure 3 is a detail of an illustrative diagram of the metabolic pathway for methane production of a methanogenic archaea or archaeobacterium present in the digestive tract of a ruminant.

[0064] EXAMPLE

[0065] Complex of at least one fucan and at least one polyphenol according to the invention

[0066] Preparation Ascophyllum algae are dried and then reduced to larger flakes of around 10 mm. In a double-jacketed reactor equipped with an agitator, a quantity of water taken from the drinking water distribution network and corresponding to 7 times the mass of dry Ascophyllum algae to be treated is preheated to a temperature of +92°C. The mass of dry Ascophyllum algae flakes is added to the water previously heated to +92°C. From the moment the temperature of the suspension of dry Ascophyllum algae in the water reaches a temperature of +92°C, the suspension is maintained at a temperature of +92°C for 16 hours. The suspension is maintained at this temperature of +92°C for 16 hours with stirring sufficiently fast to allow a substantially homogeneous distribution of the algae flakes in the water but slow enough not to crush the algae flakes. A decoction juice is formed. After 16 hours of stirring, the decoction juice is subjected to sieving or liquid / solid separation by any known means available. The temperature of the resulting liquid decoction filtrate is then brought to +65°C. The pH of the liquid decoction filtrate cooled to +65°C is adjusted to pH 2.0 by adding a strong acid such as hydrochloric acid or sulfuric acid. Acidification of the filtrate The cooled decoction liquid is accompanied by coagulation / precipitation of alginates and / or proteins. The precipitate formed is separated from the acidified decoction liquid filtrate by any means. For example, the precipitate is separated from the acidified decoction liquid filtrate on a filter press or by centrifugation. The precipitate separated from the acidified decoction liquid filtrate is discarded. The pH of the acidified liquid decoction filtrate, separated from the precipitate, is neutralized to a pH value of 6.5 by the addition of a sodium hydroxide solution (NaOH). A decoction extract comprising the complex of at least one fucan and at least one polyphenol according to the invention is formed. The decoction extract comprising the complex according to the invention is subjected to a step of ultrafiltration of the decoction extract on an ultrafiltration membrane with a cut-off threshold of the order of 100 kDa so as to eliminate the soluble minerals and the low molecular weight organic compounds and form a retentate comprising the complexes of at least one fucan and at least one polyphenol according to the invention and substantially free of soluble minerals and low molecular weight organic compounds. The retentate is subjected to a step of rinsing and adjustment of the permeate to a °Brix value of less than 0.5, by diafiltration. The rinsed retentate is concentrated to a value of at least 10°Bx on an ultrafiltration recirculation loop or in a vacuum concentrator and brought back to room temperature.

[0067] Characterization of the complex of at least one fucan and at least one polyphenol An aliquot volume of the retentate is mixed with 2 volumes of ethanol. A dark-colored precipitate is formed, indicating coprecipitation of fucans and polyphenols.

[0068] Characterization of the molecular weight of fucans from the complex of at least one fucan and at least one polyphenol - Multi-detection size exclusion chromatography The molecular weights of the fucans from the complex of at least one fucan and at least one polyphenol are determined using a DIONEX (Ultimate 3000) assembly comprising: a degasser, a pump and a sample changer; - one precolumn and four Shodex OHpak 30 cm columns, models 802.5HQ, 804HQ, 806HQ and 807HQ with a separation range of 500 to 100,000 kDa; one OPTILAB rEX differential refractometer (Wyatt Technologies), and; one DAWN HELEOS II multi-angle light scattering detector (Wyatt Technologies). The flow rate is 0.5 mL / min and the mobile phase is formed of Millipore quality water with added sodium nitrate (NaNCh) at a concentration of 0.1 M and sodium azide (NaNs) at a concentration of 200 ppm.

[0069] Packaging The concentrate obtained is spray-dried and packaged in 10 and / or 20 kg bags.

[0070] Mother and daughter solutions of fucan / polyphenol complex according to the invention A stock solution is prepared by dissolving the water-soluble powder at a rate of 100 mg of water-soluble powder per mL of distilled water. The solubilization of the water-soluble powder in distilled water is obtained by vortexing and treatment with ultrasound. The stock solution is diluted 1 / 3 to obtain a concentration of 30 mg / mL. A daughter solution diluted 10 times (10 mg / mL) from the stock solution is made in distilled water. The daughter solution is subjected to 7 serial dilutions of 1 / 3 in distilled water. The concentrations are given in Table 1 below. [Table 1] Table 1

[0071] Methyltransferase inhibition test The inhibition of the activity of N'-nicotinamide methyltransferase (NNMT) by the complex according to the invention is measured using the measurement kit ("NNMT Inhibitor Screening Assay Kit", MAK299, Merck, Darmstadt, Germany) of the activity of N'-nicotinamide methyltransferase (NNMT). This measurement kit is based on the measurement of the activity of N'-nicotinamide methyltransferase (NNMT) catalyzing the methylation reaction of nicotinamide by transfer of a methyl group from S-adenosyl methionine (SAM) onto nicotinamide. As a result of this transfer, S-adenosyl methionine is demethylated to S-adenosyl homocysteine cooperating with a fluorophore of the kit. The fluorescence is detected at 485 nm, the excitation being carried out at 395 nm.

[0072] Methane metabolism (Cfh) in methanogenic archaea or archaeobacteria A summary diagram of a methane biosynthesis pathway in methanogenic archaea or archaea is shown in Figure 1. Methanogenic archaea or archaea are present in large quantities in the digestive system of ruminants and participate in the anaerobic methanogenic fermentation of carbon dioxide (CO2) produced by these ruminants. Methanofuran (MF) is formylated to formylmethanofuran (Formyl-MF) in a first step of methanogenesis. A formylmethanofuran dehydrogenase catalyzes this reaction from a carbon dioxide (CO2) molecule, which is the primary source of carbon for this metabolic pathway. Formylmethanofuran is converted to N5-methyltetrahydromethanopterin (N5-formyl-THM) by the action of formylmethanofuran dehydrogenase and the coenzyme tetrahydromethanopterin (THM) converted to methanofuran (MF).N5-Methyl tetrahydromethanopterin (N5-formyl-THM) is converted to N5-methyl tetrahydromethanopterin (N5-Methyl-THM) involving a coenzyme F420-dependent methylene-THMPT reductase. A transfer of the methyl group from N5-Methyl-THM to Coenzyme M (HS-CoM), involving coenzyme M methyltransferase, leads to Methyl Coenzyme M (C0M-S-CH3). The final step in this methane metabolic pathway involves the action of methyl-CoM reductase, using C0M-S-CH3 as the methyl donor and Coenzyme B (CoB-SH) as the hydrogen donor, resulting in the release of methane (CH4) by forming the Coenzyme B-Coenzyme M (C0B-SS-C0M) pair, or the CoM-SS-HTP pair in the case of another electron acceptor such as 7-mercaptoheptonylthreonine phosphate (HS-HTP), such as. décrit en figure 2. Les références bibliographiques citées ci-après complètent la description ci-dessus : Tobias Grâwert, J. Agric. Food Chem., 2014, 62(52), 12487-90. "'Inhibition of Methyl-CoM Reductase from Methanobrevibacter ruminantium by 2-Bromoethanesul- fonatef Hao Chen, Front. Microbiol., 2020, Volume 11 - 2020, “Methyl-Coenzyme M Reductase and Its Post-translational Modifications'' Prakash Divya, Dissertation, 2014, “Me- thyl-coenzyme M reductase: Elucidating the process of activation and study of the effect of the methanogenesis inhibitor 3 -nitrooxypropanol” and Todd D. Pihl, Journal of Bacteriology, 1994, 6384-6391, "Growth Phase-Dependent Transcription of the Genes That Encode the Two Methyl Coenzyme M Reductase Isoenzymes and N5-Methyltetrahydromethanop- terin: Coenzyme M Methyltransferase in Methanobacterium thermoautotrophicum AH”. In summary, methane is produced due to the activity of several enzymes including methyltransferase and methylreductase (methyl-CoM reductase) and the inhibition of this enzyme leads to a decrease in methane synthesis. The inhibition of the activity of N'-nicotinamide methyltransferase (NNMT) by the complex according to the invention foreshadows the inhibition of the above coenzyme M methyltransferase and an inhibitory effect on the synthesis of methane (CH4) produced by these archaea or archaebacteria by the complex according to the invention.

[0073] Inhibition of N'-nicotinamide methyltransferase (NNMT). The test is carried out in 96-well plates, in accordance with the manufacturer's instructions for use, each well containing: 58.5 pl NNMT Buffer 2.5 pl Enzyme NNMT 10 pl of an Enzyme I solution 2pl S-Adenosylmethionine (SAM) 2pl Enzyme II solution 50pl of the corresponding diluted complex sample 25 pl Nicotinamide. The 96-well plate is placed at a temperature of 37°C for 15 min. Then, 50 μl of isopropanol is added to each well to stop the reaction. The 96-well plate is placed at a temperature of +4°C for 5 min, then 50 μl of fluorescent probe revealing thiols is added to each well. The fluorescence development time is 5 min. The reading is carried out in a fluorimeter by excitation at the wavelength of 395 nm and analysis at the wavelength of 485 nm. The percentage inhibition of N'-nicotinamide methyltransferase (NNMT) activity by the complex according to the invention is given in comparison with the inhibition provided by a control solution in which 50 pL of sample are replaced by 50 pL of NNMT buffer. The percentage inhibition provided by each sample is calculated according to formula 1, below: [Math 1] Fluorescence control — Fluorescence sample % sample inhibition = - - - — - x 100 Fluorescence control Formula 1

[0074] The results are given in Table 2 below and represented in Figure 3. [Table 2] Table 2 The complex according to the invention is capable of inhibiting the activity of N'-nicotinamide methyl-transferase (NNMT) according to the dose response curve shown in Figure 3. An ICso value obtained is 36.56 pg. This quantity in pg corresponds to the quantity of complex per well, i.e. for a volume of 250 pL / well.

[0075] Evaluation of the enzymatic activity of N'-nicotinamide methyltransferase (NNMT) The enzymatic activity is defined on the international scale (IU) such that 1 unit of enzyme corresponds to the quantity of enzyme necessary to transform 1 pmolc (10 -6moles) of enzyme substrate per minute, under optimal enzymatic reaction conditions. The nicotinamide formed as a result of the reaction is measured and quantified by liquid chromatography coupled with tandem mass spectrometry (LC / MS / MS). To do this, in a 1.5 mL centrifuge tube, are added: 70.5 pl of NNMT buffer 2.5 μl of NNMT enzyme 2 μl of S-Adenosylmethionine (SAM) 25 μl of 1-methylnicotinamide. The 1.5 mL tube is placed at a temperature of 37°C for 15 min. Then, the reaction is stopped by adding 50 μl of isopropanol. 10 μL of the reaction medium is injected into LC / MS / MS. A calibration line is carried out with nicotinamide solutions at concentrations of 10 pg / mL; 5%g / mL; 1 g / mL; 0.5 g / mL and 0.1 pg / mL. The enzymatic activity of N'-nicotinamide methyltransferase (NNMT) is estimated to be 0.0408 U / mL. The inhibitory activity of the complex described above is 2.789 IU / g, corresponding to the amount of complex required to inhibit 50% of the NNMT enzyme.

[0076] The complex of at least one natural fucan from a brown algae and at least one natural polyphenol from a brown algae exhibits inhibitory activity on an enzyme involved in the methane metabolism of archaea of the ruminant digestive system.

[0077] The invention may be the subject of numerous variants and applications other than those described above. In particular, it goes without saying that unless otherwise indicated the different structural and functional characteristics of each of the embodiments described above- above should not be considered as combined and / or closely and / or inextricably linked to each other, but rather as mere juxtapositions. Furthermore, the structural and / or functional features of the various embodiments described above may be subject in whole or in part to any different juxtaposition or any different combination.

Claims

Claims

1. Complex comprising at least one fucan and at least one polyphenol, characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural phloroglucinol oligomer from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed as a result of implementing a process for obtaining a decoction juice enriched with a complex of at least one fucan and at least one polyphenol, in which process at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C for a period of between 15 hours and 20 hours, so as to form the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol,the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol results in coprecipitation of said at least one fucan and said at least one polyphenol.,

2. Complex according to claim 1, characterized in that the coprecipitation by ethanol of said at least one fucan and said at least one polyphenol is accompanied by the formation of a precipitate of a darker color than the color of a precipitate of said at least one fucan alone, indicating the coprecipitation of said at least one light-colored fucan and said at least one polyphenol.

3. Complex according to one of claims 1 or 2, characterized in that the aqueous composition for maintaining at least one brown algae at a temperature between 90°C and 100°C is water.

4. Complex according to one of claims 1 to 3, characterized in that the process for obtaining the complex of at least one fucan and at least one polyphenol does not require any organic solvent, in particular any organic solvent toxic to the environment, the complex of at least one fucan and at least one polyphenol being free from any trace of organic solvent.

5. Complex according to one of claims 1 to 4, characterized in that the aqueous composition for maintaining at least one brown algae at a temperature between 90°C and 100°C for a period between 15 hours and 20 hours is free from any acid compound for voluntary acidification of the aqueous composition.

6. Complex according to one of claims 1 to 5, characterized in that the aqueous composition for maintaining at least one brown algae at a temperature between 90°C and 100°C for a period between 15 hours and 20 hours is free of formaldehyde in aqueous solution.

7. Complex according to one of claims 1 to 6, characterized in that at least one brown alga is chosen from the group consisting of Ascophyllum sp., Fucus sp., Lamaniria sp., Euchema sp., Macrocystis sp., Undaria sp. and Alaria sp..

8. Complex according to one of claims 1 to 7, characterized in that at least one brown algae is in a fresh form or in a dry form or in a dehydrated form.

9. Non-therapeutic use of a composition comprising a complex according to one of claims 1 to 8.

10. Use according to claim 9, for regulation, in particular limiting regulation, of the productive metabolism of methane in a ruminant.

11. Complex comprising at least one natural fucan from a brown algae and at least one natural polyphenol from a brown algae for use in a treatment limiting excessive methane production in a ruminant, the complex being characterized in that said at least one fucan is a natural fucan from a brown algae, in that said at least one polyphenol is a natural polyphenol from a brown algae, and in that the complex comprising at least one fucan and at least one polyphenol is formed as a result of implementing a process for obtaining a decoction juice enriched with a complex of at least one fucan and at least one polyphenol, process in which at least one brown algae is maintained in an aqueous composition at a temperature of between 90°C and 100°C for a period of between 15 hours and 20 hours, so as to form the decoction juice enriched with a complex of at least one natural fucan and at least one polyphenol. less natural polyphenol,the complex being characterized in that precipitation by ethanol of said at least one fucan of the decoction juice enriched with a complex of at least one natural fucan and at least one natural polyphenol results in coprecipitation of said at least one fucan and said at least one polyphenol.,

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