The present invention relates to novel fatty acid esters as well as the use thereof as for inhibiting the methane production in 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. It has, however, been found that the methane reducing activity of an agent is strongly time-dependent, i.e. the inhibition rate is normally high at initial dosing but decreases quickly over time. Consequently, the methane inhibiting agent generally has to be fed in short time intervals to maintain an appropriate methane reduction - which, however, often does not comply with the feeding regime.

Thus, there is an ongoing need for methane inhibiting agent, which exhibit a longterm methane reducing effect. Surprisingly, it has now been found that certain novel fatty acid esters are able to effectively inhibit the methane formation in the rumen fluid over an extended period of time, which makes it more compatible with general feeding regimes.

Thus, in a first embodiment, the present invention relates to fatty acid esters of formula (I)

formula (I)

wherein

n is an integer selected in the range from 1 to 15, and

R is a Cg-19-alkyl or a C ₉ -i ₉ -alkenyl group,

with the proviso that when n is > 3 the resulting hydrocarbon chain may be interrupted by -O- or -NH-.

In a preferred embodiment, the present invention relates to fatty acid esters of formula (I), wherein n is an integer selected in the range from 1 to 15, and R is C ₉ - Cig-alkyl group or C ₉ -Ci ₉ -alkenyl group.

The term C ₉ -i ₉ -alkyl as used herein refers to linear or branched alkyl groups having from 9 to 19 carbon atoms and which are saturated.

The term C ₉ -i ₉ -alkenyl as used herein refers to linear or branched alkyl chains having from 9 to 19 carbon atoms and which have at least one carbon-carbon double bond, preferably one or two double bonds, which may be (independently from each other) in (£) or (^-configuration.

In all embodiments of the present invention n is preferably an integer selected in the range from 3 to 9, more preferably in the range from 3 to 6. Most preferably in all embodiments of the present invention n is 3.

In all embodiments of the present invention R is preferably a Ci ₀ -i7-alkyl group or a Cio-17-alkenyl group, more preferably a Ci ₀ -i5-alkyl group or a Ci ₅ -i7-alkenyl group, most preferably a Cn-15-alkyl group such as in particular a Cn-alkyl group. In all embodiments of the present invention preferably the alky or alkenyl groups are linear alkyl or linear alkenyl groups.

Particularly advantageous R groups in all embodiments of the present invention are undecanyl-, tridecanyl- and pentadecanyl-groups as well as (6Z,9Z)- heptadeca-6,9- dienyl- and (9Z)-heptadeca-9-enyl-groups. Most preferred in all embodiments of the present invention R is a linear Cio-i7-alkyl group, more preferably a linear Ci ₀ -i5-alkyl group, most preferably a linear Cn-15-alkyl group, such as in particular a linear Cnalkyl group.

Particularly advantageous fatty acid esters of formula (I) are listed in table 1.

Table 1

The fatty acid esters of the present invention can be manufactured according to standard methods in the art known for the preparation of nitrooxy organic molecules as well as esters. The nitrooxy group may e.g. be introduced in a reaction of the respective alcohol with nitrosulfuric acid. The esters may e.g. be prepared by esterification of the fatty acid, respectively an acid chloride or anhydride thereof, with a nitrooxyalkanol or a diol.

Thus, the present invention also relates to a process for the manufacture of a fatty acid ester of formula (I), said process encompassing the step of reacting a fatty acid of formula (II), respectively an acid chloride or an anhydride thereof, with a nitrooxyalcohol of formula (III).

(II) (III) (I) Alternatively, in a first step the fatty acid can be reacted with the respective diol to form a fatty acid monoester, followed by reacting the respective monoester with nitrosulfuric acid.

Thus, in another embodiment, the present invention relates to a process for the manufacture of a fatty acid ester of formula (I), said process encompassing the step of reacting a fatty acid of formula (II), respectively an acid chloride or an anhydride thereof, with an alcohol of formula (IV), followed by reacting the obtained fatty acid monoester (V) with nitrosulfuric acid.

(II) (IV) (V) (I)

It is well understood, that all the definitions and preferences as given herein also apply to the process according to the present invention. Particularly suitable fatty acids (II) include saturated fatty acids such as preferably dodecanoic acid, tetradecanoic acid, hexadecenoic acid and stearic acid, unsaturated fatty acids such as preferably co-7 unsaturated fatty acids, e.g. 5-dodecenoic acid, 7-tetradecenoic acid, 9-hexadecenoic acid and 1 1 -octadecenoic acid as well as co-9 unsaturated fatty acids such as preferably trans-oleic acid and cis-oleic acid as well as co-6 fatty acids such as preferably linoleic acid.

The most preferred fatty acids in the processes according to the present invention are selected from the group of dodecanoic acid, tetradecanoic acid, palmitic acid, oleic acid and linoleic acid.

The most preferred alcohol of formula (III), respectively the diol of formula (IV), in the processes according to the present invention are 3-nitrooxypropanol respectively 1 ,3-propandiol.

In a further embodiment the present invention relates to the use of at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein as an active compound in animal feeding for reducing the formation of methane emanating from the digestive activities of ruminants and/or for improving ruminant performance.

The invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants and/or for improving ruminant animal performance, said method comprising the oral administration of a sufficient amount of at least one fatty acid ester as defined by formula (I) with all the definitions and preferences as given herein to the animal. It is to be understood by oral administration a simple feeding, or manual administration of a bolus.

The fatty acid esters according to the present invention are particularly suitable to act over an extended period of time, i.e. over a period of at least 10 hours, preferably at least 16 hours, most preferably at least 20 such as a period of 24 hours after administration.

Thus, the present invention also relates to uses or methods according to the present invention, wherein the administered doses are separated in time from each other by at least 10 hours, preferably by at least 16 hours, more preferably by at least 20 hours, most preferably by at least 24 hours.

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.

The present invention also relates to the use of at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein, wherein the methane production in ruminants calculated in liters per kilogram of dry matter intake is reduced by at least 10 % when measured in metabolic chambers. Preferably, methane reduction is at least 15 %, more preferably, at least 20 %, even more preferably, at least 25 %, most preferably, at least 30 %. Alternative methane emission measurements may also be used like using a laser beam or for dairy ruminants, correlating methane production to the volatile fatty acid (VFA) profile in milk.

The present invention also relates to the use at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein, wherein the amount of the at least one fatty acid ester administered to the ruminant animal is selected in the range from 1 mg to 10 g per kg of feed, preferably from 10 mg to 1 g per kg of feed, more preferably, from 50 mg to 500 mg per kg of feed.

As indicated above, the fatty acid esters of the present invention are useful as compounds for feed additives and animal feed compositions for ruminants, and accordingly are useful as the active ingredients in such feed to reduce methane formation in the digestive tract of the animal, and/or to improve ruminant performance.

For the realisation of their use as such ingredients for the feed of ruminants the at least one fatty acid ester as defined by formula (I) with all the definitions and preferences as given herein may be incorporated in the feed by methods known per se in the art of feed formulation and processing.

Further aspects of the present invention are therefore formulations, i.e. feed additives and animal feed compositions containing at least one fatty acid ester as defined by formula (I) with all the definitions and preferences as given herein.

The present invention therefore also relates to a feed composition or a feed additive comprising at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein. Preferably, the feed composition or feed additive is a ruminant base mix. In a preferred embodiment, the composition is a mineral premix, a vitamin premix including vitamins and minerals or a bolus.

The normal daily dosage of a fatty acid ester according to the invention provided to an animal by feed intake depends upon the kind of animal and its condition. Normally this dosage should be in the range of from about 1 mg to about 10 g, preferably from about 10 mg to about 1 g, more preferably, 50 mg to 500 mg compound per kg of feed.

The at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein may be used in combination with conventional ingredients present in an animal feed composition (diet) such as calcium carbonates, electrolytes such as ammonium chloride, proteins such as soya bean meal, wheat, starch, sunflower meal, corn, meat and bone meal, amino acids, animal fat, vitamins and trace minerals.

Particular examples of compositions of the invention are the following:

- An animal feed additive comprising (a) at least one compound selected from table 1 and (b) at least one fat-soluble vitamin, (c) at least one water-soluble vitamin, (d) at least one trace mineral, and/or (e) at least one macro mineral;

- An animal feed composition comprising at least one compound selected from table 1 and a crude protein content of 50 to 800 g/kg feed.

Therefore, in a preferred embodiment, the present invention relates to a ruminant feed composition or feed additive 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 microingredients in a larger mix.

Apart from the fatty acid esters of the invention, 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 at least one compound according to the invention 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 the fatty acid esters of the invention together 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, and cobalt.

- Examples of macro minerals are calcium, phosphorus 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 timothy, ryegrasses, fescues. Legumes include among others clover, lucerne or alfalfa, peas, beans and vetches. Whole cereals include among others barley, maize (corn), oat, sorghum. Other forage crops include sugarcane, 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) whereby material with a high water content is treated with a controlled anaerobic fermentation process (naturally-fermented or additive treated).

Concentrate is largely made up of cereals (such as barley including brewers grain and distillers grain, maize, wheat, sorghum), but also often contain protein-rich feed ingredients such as soybean, rapeseed, palm kernel, cotton seed and sunflower.

Cows may also be fed total mixed rations (TMR), where all the dietary components, e.g. forage, silage and concentrate, are mixed before serving.

As mentioned above a premix is an example of a feed additive which may comprise the at least one fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein. It is understood that the fatty acid ester according to the present invention may be administered to the animal in different other forms. For example, the fatty acid esters can also be included in a bolus that would be placed in the rumen and that would release a defined amount of the fatty acid ester continuously in well-defined dosages over a specific period of time.

The present invention further relates to a method for reducing the production of methane emanating from the digestive activities of ruminants and/or for improving ruminant animal performance, comprising orally administering a sufficient amount of at least one the fatty acid ester as defined by formula (I) and with all the definitions and preferences as given herein.

Moreover, the invention further relates to a method as described above, wherein the fatty acid ester of formula (I) is administered to the animal in combination with at least one additional active substance selected from the group consisting of diallyl disulfide, garlic oil, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid and derivatives thereof.

The invention also relates to a method as described above, wherein the ruminant animal is selected from the group consisting of: cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai, and more preferably from the group consisting of: cattle, goats and sheep.

The invention also relates to a method as described above, wherein the amount of the at least one fatty acid ester as defined in formula (I) and with all the definitions and preferences as given herein administered to the ruminant animal is from about 1 mg to about 10 g per kg feed, preferably from about 10 mg to about 1 g, more preferably from 50 mg to 500 mg compound per kg of feed.

The invention also relates to a method as described above, wherein the methane production in ruminants calculated in liters per kilogram of dry matter intake is reduced by at least 10 % when measured in metabolic chambers. Preferably, methane reduction is at least 15 %, more preferably, at least 20 %, even more preferably, at least 25 %, most preferably, at least 30 %. Alternative methane emission measurements may also be used like using a laser beam or for dairy ruminants, correlating methane production to the VFA profile in milk.

The invention also relates to a method as described above, wherein the ruminant feed conversion ratio is reduced by at least 1 % when measured in conventional performance trial. Preferably, the feed conversion ratio is reduced by at least 2 %, more preferably, by at least 2.5 %, even more preferably, by at least 3 %, most preferably, by at least 3.5 %.

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

Example 1: Synthesis of 3-nitrooxy-propyl fatty acid esters

2.446 g 3-nitrooxypropanol (3NOP) was dissolved in tert-butylmethylether (MTBE) (2 ml per mmol 3NOP) and triethylamine (1.1 equiv) was added. The resulting solution was cooled to 5°C with an ice/water bath. Then a solution of the respective fatty acid chloride (1 equiv.) in MTBE (1 ml per mmol 3NOP) was added dropwise within 10 minutes. In the case of linoleic acid, the acid instead of the chloride was used. The resulting suspension was stirred at r.t. for 2 hours, after which time only traces of the starting alcohol were visible in tic (cyclohexane/ethyl acetate 3:1 , staining with permanganate). The reaction mixture was then poured into 100ml water and the organic phase was washed 4 times with water (50ml each). The organic phase was dried with magnesium sulfate. During suction a precipitate formed in the filtrate. This precipitate was filtered off. The slightly yellow filtrate was evaporated to dryness under reduced pressure yielding the compounds in the amount and purities as outlined in table 2.

Table 2

Example 2: In vitro test for methane production

A modified version of the“Hohenheim Forage value Test (HFT)” was used for testing the effect of specific compounds on the rumen functions mimicked by this in-vitro system.

Principle: Feed (i.e. a TMR) (300 mg) is given into a syringe with a composition of rumen liquor and an appropriate mixture of buffers (i.e. rumen-fluid buffer mix: 25 ml) and the substances to be tested in the concentrations as outlined in table 2 (the inhibitors to be tested are diluted in ethanol to reach the respective concentration of dry matter in 50mI). The solution is incubated at 39°C for 24h, while analyzing the gas produced after 8 and 24 hours. The quantity of produced gas is measured and put into a formula for conversion. After the incubation the composition of gas is measured with a GC.

Reagents:

Mass element solution:

6.2 g potassium dihydrogen phosphate (KH2PO4)

0.6 g magnesium sulfate heptahydrate (MgS0 _{4 *} 7H 0)

9 ml concentrated phosphoric acid (1 mol/l)

dissolved in distilled water to 1 I (pH about 1.6)

Buffer solution:

35.0 g sodium hydrogen carbonate (NaHC0 ₃ )

4.0 g ammonium hydrogen carbonate ((NH ₄ )HC0 ₃ )

dissolved in distilled water to 1 I

Trace element solution:

13.2 g calcium chloride dihydrate (CaCI _{2 *} 2H ₂ 0)

10.0 g manganese(ll) chloride tetrahydrate (MnCI _{2 *} 4H ₂ 0)

1.0 g cobalt(ll) chloride hexahydrate (CoCI _{2 *} 6H ₂ 0)

8.0 g iron(lll) chloride (FeCI _{3 *} 6H ₂ 0)

dissolved in distilled water to 100 ml

Sodium salt solution:

100 mg sodium salt

dissolved in distilled water to 100 ml

Reduction solution:

first 3 ml sodium hydroxide (c = 1 mol/l), then 427.5 mg sodium sulfide hydrate (Na ₂ S * H ₂ 0) are added to 71.25 ml H ₂ 0

solution must be prepared shortly before it is added to the medium solution Procedure:

Sample weighing:

TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage) is sieved to 1 mm and weighed exactly into the syringes. One run contains 4 repetitions, each with 16 syringes and comprises substrate controls, which display the gas production without the effect of the tested compounds, carrier controls, which display the gas production in the presence of the carrier (solvent) only (i.e. EtOH used to dissolve the test compounds), and test samples (in the carrier), which contain the test substances in a concentration of 100pmol/l.

Preparation of the medium solution:

The components are mixed in a Woulff bottle in following order:

71 1 ml water

0.18 ml trace element solution

355.5 ml buffer solution

355.5 ml mass element solution

The completed solution is warmed up to 39 °C followed by the addition of 1.83 ml sodium salt solution and the addition of reduction solution at 36 °C.

The rumen liquor (750 ml) is added, when the indicator turns colorless under continued agitation and C0 -gassing.

Filling the syringes, incubation and determining gas volumes:

The rumen-fluid-buffer-mix is added to the glass syringe prepared as outlined above containing the TMR and the active to be tested. The syringes are then incubated for 24 hours at 39 °C under gentle agitation. After 8, respectively 24 hours the volume of gas produced is measured, and the percentage of methane in the gas phase is determined by gas chromatography.

Results

Table 3 outlines the methane inhibiting effect of various fatty acid diesters after 8 hours and 24 hours. Table 3: Methanogenese inhibition

As can be retrieved from table 3, the fatty acid esters according to the present invention are particularly suitable to reduce the methane formation, even over an extended period of time, while the saturated Cn-i ₅ fatty acid esters were particularly advantageous.