WO2025087717A1

WO2025087717A1 - Use of a methane inhibitor in combination with a probiotic strain to reduce methane emissions from ruminants

Info

Publication number: WO2025087717A1
Application number: PCT/EP2024/078898
Authority: WO
Inventors: Nicola Walker
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-10-25
Filing date: 2024-10-14
Publication date: 2025-05-01
Anticipated expiration: 2026-04-25

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 probiotic strain and propanediol mononitrate to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

Description

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

Probiotics are live micro-organisms that when administered in adequate amounts, are associated with a health benefit on the host. Probiotics may defend against pathogens in the intestine to provide health benefits. Probiotics may compete against pathogens for the same essential nutrients, leaving less available for the pathogen to utilize. Alternatively, they may bind to adhesion sites, preventing pathogen attachment by reducing the surface area available for pathogen colonization. A postulated mechanism includes signaling of immune cells by probiotics may result in the secretion of cytokines, targeting the pathogen for destruction. Finally, some have proposed probiotics may attack pathogenic organisms by releasing bac- teriocins, killing them directly.

The present invention further relates to animal feed compositions and feed additives comprising propanediol mononitrate and at least one probiotic strain 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.

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.

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.

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).

O₂NO / ^OH formula (I)

Surprisingly, it has now been found, that the combined use of at least one probiotic strain and propanediol mononitrate leads to a synergistically reduced methane formation.

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

Therefore, in a first embodiment, the present invention provides the use of at least one probiotic and propanediol mononitrate for reducing the formation of methane emanating from the digestive activities of ruminants.

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 probiotic strain and propanediol mononitrate.

In a third embodiment, the present invention relates to a (ruminant) feed composition or feed additive comprising at least one probiotic strain and propanediol mononitrate.

It is well understood, that the probiotic 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 probiotic 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.

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

It is well understood, that in all embodiments of the present invention probiotic 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 probiotic and propanediol mononitrate.

Thus, the present invention also relates to the (combined) use of probiotic 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 probiotic 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 %.

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.

The one or more probiotic can be a probiotic yeast strain or yeast derivatives (for example Phileo live yeast) or a bacterial strain (for example lactic acid bacteria).

In a typical embodiment, the one or more probiotic can be selected from the group consisting of lactic acid bacteria, a Lactobacillus, a Bifidobacterium, a Saccharomyces, an Enterococcus, a Streptococcus, a Pediococcus, a Leuconostoc, a Lactococcus, an Oenococcus, a Bacillus, Carnobacterium, Propionibacterium, Clostridium, Megasphaera and an Escherichia or combinations thereof.

In a suitable embodiment, the animal feed composition comprises one or more probiotics selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus mega- terium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, and Propionibacteria sp.

In a further suitable embodiment, the Lactobacillus strain can be selected from the group consisting of Lactobacillus acidophilus; Lactobacillus casei; Lactobacillus gasseri; Lactobacillus rhamnosus; Lactobacillus salivarius; Lactobacillus reuteri, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus johnsonii and combinations thereof.

Preferred embodiments of Lactobacillus can be selected from the group consisting of Lactobacillus acidophilus; Lactobacillus casei; Lactobacillus reuteri, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus johnsonii and combinations thereof.

The Bifidobacterium may include various Gram positive non-motile anaerobic bacteria. Strains of the genus Bifidobacterium are also often used as probiotic bacteria as they are known for their variety of resistance mechanisms to bile salts, which is important since the beneficial effects of probiotic bacteria is often generated in the presence of this biological fluid. The Bifidobacteria may be selected from the group consisting of Bifidobacterium bifidum; Bifidobacterium animalis; Bifidobacterium infantis; Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium breve.

The Saccharomyces may be selected from the group consisting of Saccharomyces cere- visiae, Saccharomyces bayanus, Saccharomyces thermophilus and Saccharomyces bou- lardii, particularly Saccharomyces thermophilus and Saccharomyces boulardii.

A suitable Lactococcus is Lactococcus lactis.

In a suitable embodiment, the Enterococcus is selected from the group consisting of Enterococcus durans and Enterococcus faecium.

In a suitable embodiment, the Streptococcus is Streptococcus thermophilus. In a suitable embodiment, the Bacillus is selected from the group consisting Bacillus subtilis, Bacillus coagulans, Bacillus amyloliquefaciens, Bacillus lichenformis, Bacillus subtilis, Bacillus cereus. Bacillus cereus, namely Bacillus cereus NVH 75/95 is an efficient probiotic for animals.

In a typical embodiment, the composition, animal feed additive or animal feed comprises a probiotic from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA-6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B- 50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.

Suitably, the composition, animal feed additive or animal feed further comprises a probiotic from one or more of the following strains of Bacillus pumilus'. NRRL B-50016, ATCC 700385, NRRL B-50885 or NRRL B-50886.

In a further typical embodiment, the composition, animal feed additive or animal feed comprises a probiotic from one or more of the following strains of Bacillus lichenformis'. NRRL B 50015, NRRL B-50621 or NRRL B-50623.

In a further suitable embodiment, composition, animal feed additive or animal feed comprises a probiotic from one or more of the following strains of Bacillus amyloliquefaciens'. DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B- 50606, NRRL B-50013, NRRL B-50151, NRRL B-50141 , NRRL B-50147 or NRRL B-50888. In a suitable embodiment, the Escherichia is selected from the group consisting of Escherichia coli Nissle, such as Escherichia coli Nissle 1917.

As stated, in a typical embodiment, the composition, animal feed additive or animal feed comprises a combination of probiotics selected from the group consisting of

Lactobacillus, a Bifidobacterium, a Saccharomyces, an Enterococcus, a Streptococcus, a Pediococcus, a Leuconostoc, a Lactococcus, an Oenococcus, a Bacillus, Carnobacterium, Propionibacterium, Clostridium, Megasphaera and an Escherichia. Preferred combinations include a combination of one or more Lactobacillus and one or more Saccharomyces, one or more Lactobacillus and one or more Streptococcus, one or more Lactobacillus and one or more Bacillus, one or more Lactobacillus and one or more Bifidobacterium, one or more Saccharomyces and one or more Streptococcus, one or more Saccharomyces and one or more Bacillus, one or more Saccharomyces and one or more Bifidobacterium, one or more Saccharomyces and one or more Bacillus, one or more Streptococcus and one or more Bifidobacterium, one or more Streptococcus and one or more Bacillus, one or more Bacillus and one or more Bifidobacterium.

In another typical embodiment, the composition, animal feed additive or animal feed comprises a combination of Lactobacillus strains such as a combination of Lactobacillus acidophilus and L. johnsonif, Lactobacillus acidophilus and L. case/; Lactobacillus acidophilus and Lactobacillus rhamnosus; L. johnsonii and Lactobacillus acidophilus; L. johnsonii and L. casei; and L. johnsonii and Lactobacillus rhamnosus.

In a further embodiment, the composition, animal feed additive or animal feed comprises a a combination of S. thermophilus and L. bulgaricus. Another suitable combination can be selected from the group consisting of Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium bifidum and Bacillus coagulans.

In another embodiment, the one or more bacterial strains acting as probiotics are present in the form of a stable spore.

The bacterial count of each of the bacterial strains acting as probiotics in the animal feed composition is typically between 1x10⁴ and 1x10¹⁴ CFU/kg of dry matter, preferably between 1x10⁶ and 1x10¹² CFU/kg of dry matter, and more preferably between 1x10⁷ and 1x10¹¹ CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10⁸ and 1x10¹° CFU/kg of dry matter.

The bacterial count of each of the bacterial strains acting as probiotics in the animal feed composition is between 1x10⁵ and 1x10¹⁵ CFU/animal/day, preferably between 1x10⁷ and 1x10¹³ CFU/animal/day, and more preferably between 1x10⁸ and 1x10¹² CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1x10⁹ and 1x10¹¹ CFU/animal/day. In one embodiment, the amount of probiotics is 0.001 % to 10% by weight of the composition.

Examples of commercial products acting as probiotics are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme + probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik). 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 probiotic 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 probiotic 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.

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.

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.

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 probiotic or via drinking water.

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.

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.

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.

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.

In another preferred embodiment of the invention the probiotic 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 probiotic additive formulation).

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

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

Probiotic 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.

In one preferred embodiment of the invention propanediol mononitrate and probiotic are added to the feed as single (not premixed) ingredients.

In another preferred embodiment of the invention propanediol mononitrate and probiotic are added to the feed as premixed ingredients. Probiotic 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.

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

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.

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

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

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.

Probiotic 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.

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); 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%).

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.

Apart from the active ingredients of the invention (i.e. probiotic 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 probiotic together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

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

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

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

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.

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). 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.

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.

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.

As mentioned above a premix is an example of a feed additive which may comprise probiotic 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.

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 probiotic 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. 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.

It is also well understood that in all uses and methods according to the present invention probiotic 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.

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

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.

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 probiotic can be pre-mixed before the administration or can be added separately to the animal feed compositions and feed additives.

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. 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.

Further components that could be given together with the probiotic 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.

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.

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.

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.

In further embodiments, the present invention relates to the use of probiotic 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.

The present invention is further described by the following example which should not be construed as limiting the scope of the invention.

Claims

1. Use of at least one probiotic strain (probiotic) 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 the one or more probiotic is a probiotic yeast strain or yeast derivatives, for example Phileo live yeast or a bacterial strain, for example lactic acid bacteria.

3. The use according to claim 1 or 2, wherein the probiotic is selected from the group consisting of a Lactobacillus, a Bifidobacterium, a Saccharomyces, an Enterococcus, a Streptococcus, a Pediococcus, a Leuconostoc, a Lactococcus, an Oenococcus, a Bacillus, Carnobacterium, Propionibacterium, Clostridium, Megasphaera and an Escherichia or combinations thereof.

4. The use according to claim 1 and/ or 2, 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.

5. 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.

6. 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 probiotic and propanediol mononitrate, wherein the effective amount of propanediol mononitrate is selected in the range from 0.05 to 5 g PDMN/ animal/ day.

7. The method according to claim 5, 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.

8. A feed composition or feed additive comprising probiotic 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 probiotic in an amount sufficient to enhance the effect of propanediol mononitrate in reducing methane emission.

9. The ruminant feed according to claim 9, wherein the at least one or more probiotic is a probiotic yeast strain or yeast derivatives, for example Phileo live yeast or a bacterial strain, for example lactic acid bacteria.

10. The feed composition according to claim 10, wherein the feed composition or feed additive is a mineral premix, a vitamin premix or a bolus.

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