Use of a methane inhibitor in combination with a strain of acetobacter to reduce methane emissions from ruminants

Abstract

The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to the administration of at least one strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter and propanediol mononitrate to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

Description

[0001] Use of a methane inhibitor in combination with a strain of Acetobacter to reduce methane emissions from ruminants

[0002] The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to the administration of at least one strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter and propanediol mononitrate to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

[0003] Acetobacter is a genus of acetic acid bacteria. Acetic acid bacteria are characterized by the ability to convert ethanol to acetic acid in the presence of oxygen. Of these, the genus Acetobacter is distinguished by the ability to oxidize lactate and acetate into carbon dioxide and water. Bacteria of the genus Acetobacter have been isolated from industrial vinegar fermentation processes and are frequently used as fermentation starter cultures.

[0004] Methanobrevibacter is a genus of archaeans in the family Methanobacteriaceae. The species within Methanobrevibacter are strictly anaerobic archaea that produce methane, for the most part through the reduction of carbon dioxide via hydrogen. Most species live in the intestines of larger organisms, such as termites and are responsible for the large quantities of greenhouse gases that they produce. Mbr. smithii, found in the human intestine, may play a role in obesity.

[0005] The present invention further relates to animal feed compositions and feed additives comprising propanediol mononitrate and at least strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter as well as the use of said feed composition or feed additives for reducing the production of methane emanating from the digestive activities of ruminants.

[0006] The temperature of the air surrounding the earth is increasing, a process referred to as global warming. One of the main focuses to reduce this warming effect is to reduce the amount of greenhouse gases emitted into the atmosphere. Greenhouse gases are emitted from several different sources, both natural and artificial; however, the two sources with the most emphasis are the agricultural and fossil fuel industries. Within agriculture, ruminants and in particular cattle are the major contributors to the biogenic methane formation, and it has been estimated that the prevention of methane formation from ruminants would almost stabilize atmospheric methane concentrations.

[0007] Methane emission from the ruminant livestock sector — a by-product from enteric fermentation of plant biomass in the ruminant digestive system — is produced by methanogenic archaea. Various attempts have been made in the last decade to mitigate methane production from ruminant animals. Although the approaches vary, the most popular method so far are feed additives which act in the rumen fluid by reducing respectively inhibiting the methane production by methanogenic archaea.

[0008] Propanediol mononitrate (also referred to herein as PDMN or 3-Nitrooxypropanol (3-NOP)) having the chemical structure of formula (I) has been reported to significantly reduce the methane production in ruminants (see e.g. WO2012 / 084629).

[0009] 02N0XZ\ / 0H formula (I)

[0010] Surprisingly, it has now been found, that the combined use of at least one strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter ( below called bacterial strain) and propanediol mononitrate leads to a synergistically reduced methane formation.

[0011] Thus, the combined use of a propanediol mononitrate and a strain of the bacterial genus Acetobacter has a great potential in the mitigation of climate change by significantly reducing the methane emissions emanating during the digestive activities of ruminants.

[0012] Therefore, in a first embodiment, the present invention provides the use of at least one strain of the bacterial genus Acetobacter_ and propanediol mononitrate for reducing the formation of methane emanating from the digestive activities of ruminants.

[0013] In a second embodiment, the invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the animal an effective amount of at least one bacterial strain (in the following referred to a strain of the bacterial genus Acetobacter') and propanediol mononitrate. In a third embodiment, the present invention relates to a (ruminant) feed composition or feed additive comprising at least one bacterial strain and propanediol mononitrate.

[0014] It is well understood, that the bacterial strain and propanediol mononitrate may be administered admixed together (i.e. pre-mixed) or separately to the ruminant, while in the latter case it is well understood that the administration of propanediol mononitrate and the at least one bacterial strain occurs within a certain time window, i.e. within at most 6 h, preferably within 3 h, more preferably within 1 h, such as within 0.5 h. Most preferably, if administered separately, both ingredients are concomitantly administered to the ruminant e.g. by concomitant addition thereof into the animal’s feed (diet) or into the feed rack.

[0015] Preferably, in all embodiments of the present invention the at least one bacterial strain and propanediol mononitrate are administered together in form of a feed composition or feed additive comprising the at least one bacterial strain and propanediol mononitrate.

[0016] It is well understood, that in all embodiments of the present invention bacterial strain and propanediol mononitrate are administered to the ruminant in an effective amount, i.e. in an amount which results in a methane reduction, preferably of at least 10 %, more preferably of at least 20 %, most preferably of at least 30 %, when compared to the control, i.e. to ruminants not supplemented with bacterial strain and propanediol mononitrate.

[0017] Thus, the present invention also relates to the (combined) use of bacterial strain and propanediol mononitrate, wherein the methane production in ruminants is reduced by at least 10 % when compared to a control (i.e. in the absence of bacterial strain and propanediol mononitrate). Preferably, the methane reduction is at least 15 %, more preferably at least 20 %, even more preferably at least 25 %, most preferably at least 30 %.

[0018] Propanediol mononitrate [CAS No: 100502-66-7] is a known compound which can e.g. be manufactured as outlined in W02004043898 or WO2012084629 and which is available under the trademark Bovaer® at DSM Nutritional Products Ltd.

[0019] The term ‘an effective amount’ as used herein refers to an amount necessary to obtain a reduction of the methane emissions resulting from the digestive activities of a ruminant. It is well understood, that said reduction may be achieved by one single (daily) dose or by repeated (daily) doses. Furthermore, it is well understood by a person skilled in the art, that the effective amount of bacterial strain and propanediol mononitrate in the uses, methods and compositions according to the invention may vary depending upon known factors, such as the characteristics of the particular composition and its mode and route of administration, the bacterial strain content of the respective product, the age, health and weight of the ruminant, the frequency of treatment, all of which can be determined by the expert in the field with normal trials or with the usual considerations regarding the intake regime and / or the formulation.

[0020] Preferably, in all embodiments of the present invention, the effective amount of propanediol mononitrate to be administered to the ruminants is selected in the range from 0.05 to 5 g PDMN / animal / day, more preferably in the range from 0.1 to 4 g PDMN / animal / day, most preferably in the range from 0.25 to 3 g PDMN / animal / day. Further suitable effective amounts are selected in the range from 0.5 to 3 g PDMN / animal / day or from 1 to 3 g PDMN / animal / day.

[0021] With regard to the feed, preferably, in all embodiments of the present invention, the effective amount of propanediol mononitrate in the feed is selected in the range from 10 mg to 300 mg PDMN / kg DM / day, more preferably in the range from 25 to 150 g PDMN / kg DM / day, most preferably in the range from 50 to 100 g PDMN / kg DM / day.

[0022] Propanediol mononitrate is preferably administered in the form of a powderous formulation thereof. PDMN can also be administered in liquid form, optionally in a mixture with bacterial strain or via drinking water.

[0023] Preferably, said powderous formulation is a powderous formulation comprising PDMN and a carrier material. Suitable carrier includes any carrier well known in the food and feed industry such as silicone dioxide (silica) without being limited thereto.

[0024] Powderous formulations comprising PDMN and a carrier material are usually prepared by PDMN being sprayed onto or admixed with the carrier material by standard methods in the art, e.g. by diluting PDMN in an organic solvent suitable for the preparation of food or feed products such as e.g. dichloromethane, spraying or admixing said solution with / onto the carrier followed by evaporation of the organic solvent. Alternatively, PDMN can be diluted in a suitable edible oil before being sprayed onto or admixed with the carrier material. In the latter cases the respective edible oil is generally not removed. The powderous formulation may in addition contain usual additives used in the preparation of powderous formulations for feed application.

[0025] The amount of PDMN in the powderous formulation according to the present invention is preferably selected in the range of 1 to 20 wt.-%, preferably in the range of 2 to 15 wt.-%, most preferably in the range of 4 to 12 wt.-%, based on the total weight of the composition.

[0026] Particularly suitable powderous formulation to be used in all embodiments of the present invention consists essentially of PDMN, propylene glycol and silica as e.g. outlined in WO2018149756 and WO2018149755 and are commercially available as Bovaer® 10 from DSM Nutritional Products Ltd.

[0027] In another preferred embodiment of the invention the bacterial strain of the present invention is added to the feed (diet) of the animal in the form of a single feed additive formulation (also referred to herein as bacterial strain additive formulation).

[0028] The term feed additive formulation as used herein refers to liquid bacterial strain or concentrated bacterial strain, or a powderous formulation, which can either be added directly into an animal feed (diet) or into an premix.

[0029] In all embodiments of the present invention, it is further preferred if propanediol mononitrate and bacterial strain are administered in the absence of (i.e. not concomitantly with) any antibiotic.

[0030] Bacterial strain and propanediol mononitrate in all uses and methods according to the present invention are preferably administered concomitantly to the ruminant, either by prior admixing thereof or by separate addition to the diet of the animal.

[0031] In one preferred embodiment of the invention propanediol mononitrate and bacterial strain are added to the feed as single (not premixed) ingredients.

[0032] In another preferred embodiment of the invention propanediol mononitrate and bacterial strain are added to the feed as premixed ingredients. Bacterial strain and propanediol mononitrate are preferably administered via a (ruminant) feed composition or feed additive to the ruminant, e.g. by admixing the individual components with the ruminant’s feed.

[0033] Thus, preferably, in all uses and methods according to the present invention propanediol mononitrate as well as the bacterial strain with all the definitions and preferences as given herein are administered to the ruminant incorporated into a (ruminant) feed composition or feed additive.

[0034] The term feed composition or feed additive as used herein means any preparation, mixture, or composition suitable for, or intended for oral intake by an animal. Exemplary feed for ruminants such as cows include forage (grass, legumes, silage), hay, grass, grain as well as soy without being limited thereto.

[0035] Said feed compositions or feed additives may be prepared by methods known per se in the art of feed formulation and processing.

[0036] Said feed compositions and feed additives are still novel. Thus, further aspects of the present invention are (ruminant) feed compositions and feed additives comprising bacterial strain and propanediol mononitrate with all the definitions and preferences as given herein.

[0037] In one preferred embodiment, the feed compositions and feed additives are a mineral premix, a vitamin premix including vitamins and optionally minerals or a bolus.

[0038] Bacterial strain and propanediol mononitrate may be used in combination with conventional ingredients present in an animal feed composition (diet) such as forages (raw, grass, hay, silages), co-products from industry (citrus pulp, soybean hulls, distillers dried or wet grains with solubles, minerals (calcium carbonates, electrolytes such as ammonium chloride, macro and trace minerals and in all forms, inorganic and organic), proteins such as soya bean meal, sunflower meal, meat and bone meal, fish meal, amino acids and others, energy ingredients such as grains, wheat, starch, barley, millet, sorghum, corn, animal and plants fat or oils, and vitamins without being limited thereto.

[0039] Particular examples of feed compositions of the invention are the following: An animal feed additive comprising (a) propanediol mononitrate and (b) one or more of (b-1) fat-soluble vitamin(s), (b-2) water-soluble vitamin(s), (b-3) trace mineral(s) and (b- 4) macro mineral(s);

[0040] An animal feed composition comprising (a) propanediol mononitrate and (b) one or more of (b-1) a crude protein content of 50 to 800 g / kg feed (50-80%), (b-2) fat from 5 - 100 g / kg feed (5-10%), (b-3) NDF from 150 - 700 g / kg feed (15-70%), (b-4) TDN from 300 - 800 (30 - 80%) and (b-5) starch from 150 - 700 g / kg feed (15 - 70%).

[0041] The so-called premixes are examples of animal feed additives of the invention. A premix designates a preferably uniform mixture of one or more micro-ingredients with diluents and / or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.

[0042] Apart from the active ingredients of the invention (i.e. bacterial strain and propanediol mononitrate), the premix of the invention preferably contains at least one fat-soluble vitamin, and / or at least one water soluble vitamin, and / or at least one trace mineral, and / or at least one macro mineral. In other words, the premix of the invention comprises the propanediol mononitrate and bacterial strain together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

[0043] Macro minerals may be separately added to the feed. Therefore, in a particular embodiment, the premix comprises bacterial strain and propanediol mononitrate with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace-minerals.

[0044] The following are non-exclusive lists of examples of these components:

[0045] Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.

[0046] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate. Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, manganese, and cobalt.

[0047] Examples of macro minerals are calcium, phosphorus, potassium, magnesium and sodium. As regards feed compositions for ruminants such as cows, as well as ingredients thereof, the ruminant diet is usually composed of an easily degradable fraction (named concentrate) and a fiber-rich less readily degradable fraction (named hay, forage, or roughage).

[0048] Hay is made of dried grass, legume or whole cereals. Grasses include among others temperate or tropical grasses, timothy, ryegrasses, fescues, brachiaria, panicum, tifton. Legumes include among others clover, lucerne or alfalfa, peas, beans and vetches. Whole cereals include among others barley, maize (corn), oat, wheat, sorghum. Other forage crops include sugarcane, sugarcane bagasse, citrus pulp, kales, rapes, and cabbages. Also root crops such as turnips, swedes, mangles, fodder beet, and sugar beet (including sugar beet pulp and beet molasses) are used to feed ruminants. Still further crops are tubers such as potatoes, cassava and sweet potato. Silage is an ensiled version of the fiber-rich fraction (e.g. from grasses, legumes or whole cereals) and grains (e.g. high moisture corns silage) whereby material with a high water content is treated with a controlled anaerobic fermentation process (naturally- fermented or additive treated) without being limited thereto.

[0049] Concentrate feed is largely made up of cereals grains (such as barley including brewers grain and distillers grain, maize, wheat, sorghum), but also often contain protein-rich feed ingredients such as soybean meal, rapeseed meal, palm kernel and sunflower meal without being limited thereto.

[0050] Ruminants (males, females and in all growth stages and adults) may also be fed total mixed rations (TMR), where all the dietary components, e.g. forage, silage, other feed ingredients and concentrate, are mixed before serving.

[0051] As mentioned above a premix is an example of a feed additive which may comprise bacterial strain and propanediol mononitrate. It is understood that the compounds may be administered to the animal in different other forms. For example the compounds can also be included in a bolus that would be placed in the rumen and that would release a defined amount of the active compounds continuously in well-defined dosages over a specific period of time.

[0052] In a particular advantageous embodiment, the feed composition according to the present invention is a ruminant feed (often also referred to as ruminant diet) where all the dietary components, e.g. macro and micro ingredients, forage, silage and concentrate feed and additives including bacterial strain and propanediol mononitrate are included. Such ruminant feed or diet is also often referred to as total mixed ration (TMR) or Partial mixed ration (PMR) or nutritional supplement for grazing animals.

[0053] Preferably, in all embodiments of the present invention, in said ruminant feed the amount of the propanediol mononitrate is selected in the range from 1 mg to about 25 g per kg dry matter feed, preferably from about 1 mg to about 10 g per kg dry matter feed, more preferably from about 10 mg to about 1 g per Kg dry matter feed, most preferably from 20 mg to 500 mg per Kg of dry matter feed, such as from about 20 mg to 250 mg per Kg of dry matter feed, or even mor preferably from 10 mg to 300 mg per dry matter kg feed such as in the range from 50 mg to 150 mg per kg dry matter feed or 60 mg to 100 mg per kg dry matter feed.

[0054] It is also well understood that in all uses and methods according to the present invention bacterial strain and the propanediol mononitrate have to be supplemented timely together to excerpt the synergistic effect, can however been added separately to the diet of the animal, e.g. can be added separately into the respecting feeding rack.

[0055] Thus, the present invention relates to a method of supplementing bacterial strain and propanediol mononitrate to a ruminant, said method encompassing the step of adding bacterial strain and propanediol mononitrate with all the definitions and preferences as given herein concomitantly to a feed rack.

[0056] The amount of ruminant feed administered to a ruminant may vary dependent on the kind and age. Generally, the amount of dry matter fed to beef cattle or dairy cows is selected in the range of 1 .5% to 3.5% of live weight, such as for an animal of 500 kg of live weigh it means to feed 7.5 to 17.5 kg dry matter / d.

[0057] In all embodiments of the present invention, it is to be understood by oral administration, a simple feeding, or manual administration of a bolus. It is also well understood, that propanediol mononitrate and the bacterial strain can be pre-mixed before the administration or can be added separately to the animal feed compositions and feed additives.

[0058] Methane emission by ruminants can easily be measured in individual animals in metabolic chambers by methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755- 2766). Moreover, it can also be assessed at barn level by an emerging technology using laser beam (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802) or Sulfur hexafluoride or just SF6 or GreenFeed system. Alternatively, methane produced by a dairy ruminant can also be assessed by measurement of fatty acid profiles in milk according to WO 2009 / 156453.

[0059] The present invention also relates to the use of a feed composition or feed additive according to the present invention which feed composition or feed additive comprises one or more additional active substance which shows similar effects with regard to methane formation in the rumen and which is selected from the group consisting of diallyl disulfide, garlic oil, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid and derivatives thereof.

[0060] Further components that could be given together with the bacterial strain and the propanediol mononitrate are for example yeasts, oregano extracts, tannins and tannic acids and essential oils e.g., thymol, 3-methylphenol, vaniline, guajacol and eugenol.

[0061] It is at present contemplated that diallyl disulfide, garlic oil, allyl isothiocyanate deoxycholic acid, chenodeoxycholic acid and derivatives thereof are independently administered in dosage ranges of for example 0.01-500 mg active substance per kg feed (ppm). These compounds are either commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art.

[0062] Ruminating mammals according to the present invention include cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and Nilgai.

[0063] For all embodiments of the present invention, domestic cattle, sheep and goat are the more preferred species. For the present purposes most preferred species are domestic cattle. The term includes all races of domestic cattle, and all production kinds of cattle, in particular dairy cows and beef cattle. It is well understood that the term dairy cows and beef cattle encompasses animals in all ages and physiological stage of life and production systems such as confined, semi-confined and grazing.

[0064] In further embodiments, the present invention relates to the use of bacterial strain to synergistically enhance the methane reducing properties of propanediol mononitrate in ruminants. It is well understood that all the definitions and properties as defined herein also apply to said use.

[0065] The present invention is further described by the following example which should not be con- strued as limiting the scope of the invention.

Claims

Claims1 . Use of at least one strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter and propanediol mononitrate (PDMN) for reducing the formation of methane emanating from the digestive activities of ruminants.

2. The use according to claim 1 , wherein propanediol mononitrate is administered to the ruminant in an amount selected in the range from .05 to 5 g PDMN / animal / day, preferably 0.1 to 4 g PDMN / animal / day, most preferably from 0.25 to 3 g PDMN / ani-mal / day.

3. The use according to anyone or more of the preceding claims, wherein the ruminant animal is selected from the group of domestic cattle, most preferably from beef cattle or dairy cows.

4. A method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the ruminant an effective amount of bacterial strain and propanediol mononitrate, wherein the effective amount of propanediol mononitrate is selected in the range from 0.05 to 5 g PDMN / animal / day.

5. The method according to claim 4, wherein the effective amount of propanediol mononitrate is selected in the range from 0.1 to 4 g PDMN / animal / day, most preferably from 0.25 to 3 g PDMN / animal / day.

6. A feed composition or feed additive comprising at least one strain of the bacterial genus Acetobacter or the archaeal genus Methanobrevibacter and propanediol mononitrate, wherein the feed composition is a ruminant feed comprising propanediol mononitrate in an amount selected in the range from 20 mg to 250 mg propanediol mononitrate per kg dry matter feed and bacterial strain in an amount sufficient to enhance the effect of propanediol mononitrate in reducing methane emission.

7. The feed composition according to claim 6, wherein the feed composition or feed additive is a mineral premix, a vitamin premix or a bolus.

8. Use of bacterial strain, preferably liquid bacterial strain (LW) or concentrated bacterial strain (CW), or a powderous bacterial strain (WP), to synergistically enhance the methane reducing properties of propanediol mononitrate in ruminants.* * *