Ruminant feed additive composition

Abstract

Provided herein, inter alia, are ruminant feed or feed additive compositions, and methods of using these feed or feed additive compositions to reduce methane emissions in ruminants while improving animal performance, these feed or feed additive compositions comprise one or more compounds that increase hydrogen production in ruminants, such as, but not limited to, 3-nitroxypropanol (3NOP) and one or more acetogens.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 462,697, filed April 28, 2023, the disclosure of which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] Provided herein, inter alia, are methods and compositions for reducing methane emissions in ruminants while improving animal performance on one or more metrics. BACKGROUND

[0003] The primary end products of rumen fermentation are volatile fatty acids (VFAs), microbial protein, and gases (methane (CH4), carbon dioxide (CO2), and hydrogen (H2)). VFAs and microbial protein are absorbed in the digestive system and incorporated into the metabolism of the animal host. However, some fermentation end products (CO2 and H2) cannot be absorbed in the rumen. In this case, CO2 and H2 are consumed by methanogenic archaea (MAs) to produce methane, which is released by the animal into the atmosphere, where it acts as a greenhouse gas. It is believed that about 2% to 12% of the total energy consumed by ruminants can be metabolized as CH4. The environmental harm of methane release into the atmosphere combined with the potential loss of energy to the animal represents two major global ruminant agricultural challenges.

[0004] Approaches to achieve a sustainable animal production system by mitigating rumen methane emissions and increasing livestock productivity currently rely on: (1) rumen inhibitors of the methane generation pathway and (2) rumen modulators.

[0005] Among the rumen inhibitors, 3-nitrooxypropanol (abbreviated as 3NOP), developed by DSM (Bovaer®; U.S. Patent Nos. 9,365,489 and 10,154,981, which are hereby incorporated by reference in their entirety), is an organic compound having the formula HOCH2CH2CH2ONO2 (a mononitrate of 1,3 propanediol) that inhibits the enzyme that is methyl coenzyme M reductase (MCR). MCR catalyzes the last step in the methane generation pathway, and when fed to ruminants, its methane excretion is reduced by 30%.

[0006] While successful in reducing methane, a meta-analysis of all available animal data after supplementation with 3NOP showed only modest increases in animal growth and yield. Thus, what is needed is a method of reducing or inhibiting methane excretion from ruminants while improving production performance in ruminants.

[0007] The subject matter disclosed herein addresses this need and provides additional benefits as well. SUMMARY

[0008] Provided herein, inter alia, are ruminant feed or feed additive compositions comprising one or more compounds that increase hydrogen production in a ruminant (such as, but not limited to, 3-nitrooxypropanol (3NOP)) and one or more acetogenic bacteria, and methods of using these feed or feed additive compositions to reduce methane emissions in ruminants while improving animal performance.

[0009] Accordingly, in some aspects, provided herein are ruminant feed additives or feed compositions comprising (a) one or more rumen-inhibitor compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen. In some embodiments, the rumen-inhibitor compounds comprise one or more of nitrooxy compounds, coenzyme M analogs, pterin compounds, hydroxymethylglutaryl-CoA reductase inhibitors, antimicrobial peptides, or plant secondary metabolites. In some of any of the embodiments disclosed herein, the rumen-inhibitor compounds comprise 3-nitrooxypropanol (3NOP) or a coenzyme-M analog. In some of any of the embodiments disclosed herein, the one or more acetogenic bacteria are one or more bacteria selected from the group consisting of Acetobacterium bakii, Blautia hydrogenotrophica, Blautia producta, Blautia schinkii, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskii, Clostridium ljungdahlii, Clostridium scatologenes, Sporomusa acidovorans, Eubacterium limosum, Acetobacterium woodii, Sporomusa silvacetica, Moorella thermoacetica, Acetitomaculum ruminis, Ruminococcus productus, and Clostridium magnum. In some of any of the embodiments disclosed herein, the one or more compounds that promote hydrogen consumption in the rumen comprise one or more of nitrate, sulfate, nitro compounds, or propionate precursors. In some of any of the embodiments disclosed herein, the feed or feed additive composition further comprises one or more enzymes. In some embodiments, the enzymes comprise one or more of glucoamylase, xylanase, amylase, phytase, beta-glucanase, fucosidase, and / or protease.

[0010] In other aspects, provided herein is a method for reducing the production of methane originating from the digestive activity of a ruminant animal and / or for improving the performance of a ruminant animal, the method comprising orally administering to the animal an effective amount of any of the feed additives or feed compositions disclosed herein. In some embodiments, the ruminant animal is selected from the group consisting of beef cattle, dairy cattle, veal, goats, sheep, giraffes, bison, European bison, yaks, water buffalo, deer, camels, llamas, alpacas, pronghorn, and nilgai. In some of any of the embodiments disclosed herein, the concentration of hydrogen gas in the rumen of the animal is less than 5%. In some of any of the embodiments disclosed herein, the feed additive or feed composition is not pretreated using wet explosion pretreatment.

[0011] In further aspects, provided herein is a method for producing a feed additive or feed composition, the method comprising combining: (a) one or more rumen inhibitor compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen. In some embodiments, the rumen inhibitor compounds comprise one or more of a nitrooxy compound, a coenzyme M analog, a pterin compound, a hydroxymethylglutaryl-CoA reductase inhibitor, an antimicrobial peptide, or a plant secondary metabolite. In some of any of the embodiments disclosed herein, the rumen inhibitor compounds comprise 3-nitrooxypropanol (3NOP) or a coenzyme-M analog. In some of any of the embodiments disclosed herein, the one or more acetogenic bacteria is one or more bacteria selected from the group consisting of Butyrivibrio fibrisolvens, Blautia hydrogenotrophica, Blautia longisporous, Blautia coccoides, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium nealii, Clostridium scatologenes, Endomycosphaera acetoacidivorans, Eubacterium limosum, Eubacterium yurii, Endomycosphaera forestomacii, Moorella thermautotrophica, Ruminococcus albus, Streptococcus productus, and Clostridium magnum. In some of any of the embodiments disclosed herein, the one or more compounds that promote hydrogen consumption in the rumen comprise one or more of a nitrate salt, a sulfate salt, a nitro compound, or a propionate precursor. In some of any of the embodiments disclosed herein, the method further comprises combining one or more enzymes with the feed additive or feed composition. In some embodiments, the enzymes comprise a glucoamylase and / or a phytase. In some of any of the embodiments disclosed herein, the method further comprises packaging the feed additive or feed composition. In some of any of the embodiments disclosed herein, the feed additive or feed composition is not pretreated using wet explosion pretreatment.

[0012] Each of the aspects and embodiments described herein can be used together, unless expressly or clearly excluded from the context of the embodiment or aspect.

[0013] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Graphs depicting the effect of B. prod., freeze-dried Asparagopsis taxiformis (FDAT), and 3NOP (alone or in combination) on hydrogen, methane, total gas, and VFA production from in vitro rumen cultures. Different superscript letters indicate statistical significance.

[0015] Figure 2 Graphs depicting the effect of A. woo., freeze-dried Asparagopsis taxiformis (FDAT), and 3NOP (alone or in combination) on hydrogen, methane, total gas, and VFA production from in vitro rumen cultures. Different superscript letters indicate statistical significance. DETAILED DESCRIPTION

[0016] Ruminants are mammals of the order Artiodactyla that digest plant-based food by first softening it in the animal's first stomach, called the rumen, and then regurgitating the semi-digested material, now called cud, and chewing it again. The process of chewing the cud to further break down the plant matter and stimulate digestion is called "ruminating" or "rumination."

[0017] Methane is produced as a natural result of the anaerobic fermentation that occurs during rumination. Methane production represents a loss of energy for the host animal (i.e., carbon that could be used to produce glucose for metabolic needs is expelled into the atmosphere as CH4). Carbohydrates make up 70-80% of the dry matter in the typical ruminant diet, and yet, absorption of carbohydrates from the gastrointestinal tract of ruminants is often very limited. The reason for this is the extensive fermentation of carbohydrates in the rumen, resulting in the production of acetate, propionate, and butyrate as the main products. These products are called volatile fatty acids (VFAs).

[0018] In addition to energy loss, methane is a greenhouse gas that is many times more potent than carbon dioxide (CO2). Its concentration in the atmosphere has doubled in the last century and continues to increase dramatically. Ruminants are the main contributors to biogenic methane formation. Disrupting or limiting methane production that occurs during rumen fermentation is accompanied by the prospect of reducing greenhouse emissions that contribute negatively to climate change.

[0019] Unfortunately, studies have shown that disruption of methane production has a negative impact on overall rumen fermentation and that the H2 scavenging function of methanogens is important for stable rumen function (Murali et al., 2021). Inhibition of methane production in artificial rumen bioreactors has been extensively studied, primarily for the purpose of avoiding energy loss in animals (Van Kessel, 1996). These studies have shown that an increase in H2 partial pressure also inhibits the overall conversion of the rumen to some extent (Ahring et al., 2018). This can explain why inhibition of methane production by rumen inhibitors such as 3NOP has failed to significantly capture the energy from CH4 production inhibition and convert it into animal performance such as improved weight gain (Anuraga Jayanegara et al., 2018).

[0020] Without being bound by theory, it is hypothesized that rumen inhibitors (e.g., 3NOP) disrupt the methane production cycle, while affecting the hydrogen cycle in the rumen, leading to a detrimental accumulation of H2 against rumen fermentation. Thus, the inventors of the present application have surprisingly found that a combination of a rumen inhibitor (such as but not limited to 3NOP or a coenzyme-M analog) and a hydrogen scavenger (such as but not limited to one or more acetogenic microorganisms) is able to maximize the potential of the rumen inhibitor by minimizing / balancing the side effects of blocking methane production (i.e., increasing production of H2). It is believed, again without being bound by theory, that such a combination will restore a balanced H2 cycle and optimal fermentation within the rumen.

[0021] Thus, the present application provides a significant reduction in energy loss, which is manifested by a significant enhancement of performance of animal production, as compared to supplementation of a rumen inhibitor alone, as well as similar or better CH4 reduction.

[0022] I. DEFINITIONS

[0023] As used herein, the term "ruminant" or "ruminant animal" refers to a mammal (both male and female) that is capable of deriving nutrients from plant-based foods by fermentation (primarily by bacterial action) in a specialized stomach compartment prior to digestion. This process typically requires regurgitation of the fermented ingesta (called cud) and re-chewing. The re-chewing of the cud to further break down plant matter and stimulate the process of digestion is called "rumination." The main difference between ruminants and non-ruminants is that ruminants have three or four stomach compartments. This group includes deer, antelope, buffalo, cattle, sheep, camels, and goats, among others.

[0024] As used herein, the term "lactating ruminant" refers to a ruminant that is capable of producing milk after parturition.

[0025] As used herein, the term "dairy ruminant" refers to a ruminant whose milk is used for commercial purposes, including periods of time when the animal is not producing milk.

[0026] As used interchangeably herein, the terms "rumen inhibitor" or "rumen methane inhibitor" relate to all compounds suitable for reducing methane emissions in ruminants. Suitable rumen inhibitors include, but are not limited to: garlic extract, allicin, chloroform, bromoform, nitrates, nitroethane, lauric acid, dodecanic acid dihydroxypropyl ester, seaweed (such as Hawaiian micro-algae Chaetoceros), one or more compounds shown in Table 1 or Table 4, or one or more compounds further described below, or disclosed in U.S. Patent Nos. 9,365,489 and 10,154,981 (incorporated herein by reference). In other embodiments, the rumen inhibitor is 3-nitrooxypropanol (3NOP) or a coenzyme-M analog.

[0027] As used herein, the term "reducing" with respect to a particular trait, characteristic, property, biological process, or phenomenon means a decrease in the particular trait, characteristic, property, biological process, or phenomenon. The trait, characteristic, property, biological process, or phenomenon can be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%.

[0028] As used herein, "administer" or "administering" means the act of introducing one or more rumen inhibitors, one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen to an animal, such as by feeding or by gavage. In one embodiment, the composition is administered to the subject via a water line from which the animal drinks.

[0029] As used herein, “effective amount” means the amount of a substance (e.g., one or more rumen inhibitors, one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) used to improve one or more indicators in an animal. Improvements in one or more indicators in an animal, such as, but not limited to, any of the following: reduced methane production, increased production of volatile fatty acids, improved feed conversion ratio (FCR); improved milk production; improved weight gain; improved feed efficiency; improved intestinal microbiome status; and / or improved carcass quality, can be measured as described herein or by other methods known in the art. An effective amount can be administered to an animal by providing ingested feed and / or water containing the substance. Additionally, the substance (e.g., one or more rumen inhibitors, one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) can also be administered in one or more doses. Furthermore, in some embodiments, some of the substance (e.g., a rumen inhibitor and / or one or more compounds that promote hydrogen consumption in the rumen) can be administered in the feed or as a component of the feed, and other substances (e.g., one or more acetogenic bacteria) can be administered through a water line.

[0030] “Feed” and “food” mean, respectively, any natural or artificial diet, meal, or the like, or a component of such a meal, intended or suitable for consumption, ingestion, digestion, respectively, by a non-human animal and a human. As used herein, the term “food” is used in a broad sense and encompasses food and food products for humans as well as food for non-human animals (i.e., feed).

[0031] As used herein, the term “feed” is used synonymously herein with “feedstuff.” Feed broadly refers to a liquid or solid material used to nourish an animal and to maintain normal or accelerated growth of an animal, including newborn or young and developing animals. The term includes a compound, preparation, mixture, or composition suitable for ingestion by an animal, such as, for example, poultry, such as quail, duck, turkey, and chicken. In some embodiments, a feed or feed composition comprises a base food composition and one or more feed additives or feed additive compositions.

[0032] As used herein, the term "feed additive" refers to a component included for the purpose of fortifying a base feed with additional components to promote feed intake, treat or prevent disease, or alter metabolism. However, in some embodiments, the feed additive is formulated for delivery to an animal via a water line, rather than being added directly to the feed. Feed additives include premixes. As used herein, the term "feed additive" also refers to a substance added to a feed or water administered with a feed. Feed additives can be added to a feed for a variety of reasons. For example, to increase the digestibility of the feed, to supplement the nutritional value of the feed, to improve the immune defenses of the recipient, and / or to increase the shelf life of the feed. In some embodiments, the feed additive supplements the nutritional value of the feed and / or improves the immune defenses of the recipient. In some embodiments, the feed additive is not for administration to a human.

[0033] As referred to herein, a "premix" can be a composition consisting of micronutrients such as, but not limited to, one or more of vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are generally compositions suitable for blending into commercial diets.

[0034] As used herein, the term "animal performance" can be determined by feed efficiency and / or milk production and / or weight gain of the animal and / or by feed conversion and / or by digestibility of nutrients in the feed and / or digestible or metabolizable energy in the feed and / or by nitrogen retention and / or by the animal's ability to avoid the negative effects of disease or by the immune response of the subject. Performance characteristics can include, but are not limited to: body weight; weight gain; mass; percent body fat; height; body fat distribution; growth; growth rate; milk production; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily gain (ADG); retention and / or secretion of any one or more of copper, sodium, phosphorus, nitrogen, and calcium; amino acid retention or absorption; mineralization, bone mineralization carcass yield and carcass quality. Animal performance can also be measured by increased rumen acetate / propionate ratio, decreased feed conversion, increased feed intake, increased weight gain, increased carcass yield, or increased milk or milk protein content and / or yield, or increased milk fat content and / or yield.

[0035] As used herein, the term "feed efficiency" refers to the amount of milk production or animal weight gain that occurs when an animal is fed ad libitum or a specified amount of food over a period of time. "Increased feed efficiency" means that the use of the feed additive composition in the feed or drinking water delivered via a water line results in increased milk production or weight gain per unit of feed intake compared to an animal fed in the absence of the feed additive composition according to the present application.

[0036] As used herein, "feed conversion ratio" refers to a measure of the efficiency of a subject in converting mass of feed into an increase in a desired output, and is calculated by dividing the mass of food consumed over a specified period of time by that output. For example, if the animal is being raised for meat (e.g., beef), that output can be the increased mass of the animal. If the animal is being raised for another intended purpose (e.g., milk production), that output will be different, such as the amount of milk produced. The term "feed conversion ratio" can be used interchangeably with the terms "feed conversion rate" or "feed conversion efficiency." A "lower feed conversion ratio" or "improved feed conversion ratio" means that the use of the feed additive composition in the feed results in a lower amount of feed required to be fed to the animal for increasing a specified amount of animal body weight or for increasing a specified amount of milk production (as compared to the amount of feed required to increase the same amount of animal body weight or to increase the same amount of milk production when the feed does not include the feed additive composition).

[0037] As used herein, the term "direct fed microbial" ("DFM") is a source of live (viable) microorganisms that, when applied in sufficient numbers, can impart a benefit to its recipient, i.e., a probiotic. A DFM can comprise one or more such microorganisms, such as a bacterial strain. Non-limiting classes of DFMs include Bacillus, Bifidobacterium, Propionibacterium, lactic acid bacteria, fungi, and yeast. Thus, the term DFM encompasses one or more of the following: direct fed bacteria, direct fed yeast, and combinations thereof. The class Bacilli is unique among the spore-forming, gram-positive rods. These spores are very stable and can withstand environmental conditions such as heat, moisture, and a range of pH. These spores germinate into vegetative cells that are viable when ingested by an animal and can be used in meal and pellet diets. Lactic acid bacteria are gram-positive cocci that produce lactic acid that has antagonistic effects on some pathogens. Species of lactic acid bacteria include, but are not limited to, Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. In some embodiments, the DFM is an acetogenic bacteria. In other embodiments, the DFM is a Megasphaera species, for example M. elsdenii. The terms "probiotic," "probiotic culture," and "DFM" are used interchangeably herein and define a live microorganism (including, for example, a bacterium or yeast) that beneficially affects the host organism (i.e., by imparting one or more demonstrable benefits (e.g., health benefits or balanced gut microbiota benefits) to the host organism (such as digestive and / or performance benefits (such as improved growth and reduced methane emissions))) when ingested or topically applied, for example, in sufficient numbers. While there is no lower or upper limit to probiotic intake, it has been shown that at least 10 6 -10 12 , for example at least 10 6 -10 10 , for example 10 8 -10 10 colony forming units (cfu) as a daily dose is generally effective in achieving a beneficial effect in a subject.

[0038] As used herein, the term "acetogenic bacteria" refers to microorganisms that are capable of the Wood-Ljungdahl pathway and thus capable of converting CO, CO2, and / or hydrogen to acetate. These microorganisms include those that do not have the Wood-Ljungdahl pathway in their wild-type form but have acquired this trait due to genetic modification. These microorganisms can also be referred to as carboxydotrophic bacteria. There are currently 21 different genera of acetogenic bacteria known, and these can also include some Clostridia. These bacteria are capable of using carbon dioxide or carbon monoxide as a carbon source, and hydrogen as an energy source. In addition, alcohols, aldehydes, carboxylic acids, and many hexoses can also be used as carbon sources. The reduction pathway that leads to acetate formation is referred to as the acetyl-CoA or Wood-Ljungdahl pathway.

[0039] As used herein, the term "CFU" means "colony forming unit" and is a measure of viable cells, where a colony represents a collection of cells derived from a single progenitor cell.

[0040] As used herein, "animal feed acceptable salt" means any non-toxic salt that is capable of providing, directly or indirectly, a compound or derivative of a compound described herein when administered to a recipient. Acids commonly employed to form acceptable salts include inorganic acids such as hydrosulfurous acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, benzene sulfonic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, p-brombenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, as well as related inorganic and organic acids. Thus, such animal feed acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [beta]-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred animal feed acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and with organic acids such as maleic acid. Suitable cations for forming feed acceptable salts include ammonium, sodium, potassium, calcium, magnesium, and aluminum cations, among others.

[0041] Certain ranges are presented herein with numerical values that include the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is close to or approximately the number that the term precedes. When interpreting this term in the context of a specific value, "about" should be interpreted to mean that the value of the specific element or parameter is within 10% of the stated value, preferably within 1% of the stated value, and more preferably within 0.1% of the stated value.

[0042] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0043] It is further noted that the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "alone," or the like in

[0044] It is still further noted that, as used herein, the phrase "consisting essentially of means a composition wherein the recited one or more components are present in a total amount of less than 30% by weight of the total composition, in the presence of other known components, and do not affect or interfere in the action or activity of the one or more components.

[0045] It is further noted that, as used herein, the term "comprising" means including but not limited to, the components following the term "comprising." The components following the term "comprising" are essential or mandatory, but the composition including the components can further include other non-essential or optional components.

[0046] It is further noted that, as used herein, the term "consisting of means including and limited to, the components following the term "consisting of." Thus, the components following the term "consisting of are essential or mandatory, and no other components are present in the composition.

[0047] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification is intended to include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0049] Other definitions of terms can appear throughout the specification.

[0050] II. Compositions

[0051] Provided herein are compositions comprising: one or more rumen-inhibitor compounds; and one or more acetogenic bacteria; and / or one or more compounds that promote hydrogen consumption in the rumen. These compositions can be used to reduce methane production and improve animal (e.g., ruminant) performance.

[0052] A. Rumen inhibitor compounds

[0053] In some embodiments of the application, the rumen-inhibitor compound can be any of the compounds disclosed in Table 1.

[0054] In other embodiments, the rumen-inhibitor compound can be any organic molecule, or a salt thereof, substituted with at least one nitrooxy group at any position, defined by the following formula (I):

[0055]

[0056] wherein Y is an organic molecule having the following composition: C a H b O d N e S g wherein

[0057] a is comprised between 1 and 25, in some embodiments, a is comprised between 1 and 10

[0058] b is comprised between 2 and 51, in some embodiments, b is comprised between 2 and 21

[0059] d is comprised between 0 and 8, in some embodiments, d is comprised between 0 and 6

[0060] e is comprised between 0 and 5, in some embodiments, e is comprised between 0 and 3

[0061] g is comprised between 0 and 3, in some embodiments, g is comprised between 0 and 1.

[0062] In other embodiments, the organic molecule having formula (I) has the following composition: C a H b O d N e S g wherein

[0063] a is comprised between 1 and 10

[0064] b is comprised between 2 and 21

[0065] d is comprised between 0 and 6

[0066] e is comprised between 0 and 3

[0067] g is comprised between 0 and 1.

[0068] In another embodiment, the compound having formula (I) according to the application is a compound wherein b is comprised between 3 and 51, and in other embodiments, b is comprised between 3 and 21.

[0069] In another embodiment, the compound of formula (I) according to the present application is a compound of formula (II),

[0070]

[0071] wherein

[0072] n is comprised between 0 and 12, in some embodiments n is comprised between 0 and 6, and wherein, if n≠0, the carbon chain is a linear, cyclic or branched aliphatic carbon chain, which can be unsubstituted or substituted with up to 3 hydroxy-, alkoxy-, amino-, alkylamino-, dialkylamino- or nitrooxy groups; or an alkenyl or alkynyl carbon chain (mono- or polyunsaturated and in any isomeric form),

[0073] R4 is independently hydrogen or a saturated linear, cyclic chain or branched alkyl or alkenyl group containing 1 to 12, preferably 1 to 6 carbon atoms,

[0074] X is hydrogen, R5, R5≡N, -OR5, -OCOR5, -NR5R6, -ONO2, -COOR5, - CONR5R6, -NHSO2R5 or -SO2NHR5,

[0075] R5 and R6 are independently hydrogen, a C1-C12 linear, branched or cyclic alkyl chain (which is unsubstituted or substituted with up to 3 hydroxy-, alkoxy-, amino-, alkylamino-, dialkylamino- or nitrooxy groups), an alkenyl or alkynyl carbon chain (which can be mono- or polyunsaturated and in any isomeric form).

[0076] For all embodiments of the present application, it is understood that the compounds of formula (I) and the compounds of formula (II) can be in any isomeric form.

[0077] It is understood that in the above definition of the compounds of formula (II), when n > 2, the carbon chain can be linear or branched at any position along the carbon chain. Furthermore, the carbon chain can be branched at different positions along the carbon chain with multiple branches. Furthermore, when n > 3, the aliphatic carbon chain can form a cyclic moiety. This cyclic moiety can carry a nitrooxy moiety at any position (2,3,4) and it can also be branched at multiple positions with any aliphatic group. The branched aliphatic group is preferably a methyl, ethyl or propyl group. Furthermore, the carbon chain can be further substituted with up to 3 hydroxy-, alkoxy-, amino-, alkylamino-, dialkylamino- or nitrooxy groups.

[0078] In the above definition of the derivatives of formula (II), preferred alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, pentyl, neopentyl, hexyl, cyclohexyl and 2-ethyl-hexyl and octyl. Furthermore, any alkyl or alkenyl group containing three or more carbon atoms can be linear, branched or cyclic. Furthermore, for linear or branched C2-Cio-alkenylene groups, this is understood to encompass alkenylene groups having one or, from C4, multiple double bonds; examples of such alkenylene groups are those of the formula -CH=CH-, -CH=CH-CH2-, -CH=CH-(CH2)3- and -(CH=CH)2-.

[0079] In another embodiment, the compound of formula (I) is selected from the list of compounds of formula (I) listed in Table 4 together with their chemical formula and salts thereof.

[0080] Table 4: Compounds of formula I

[0081]

[0082]

[0083]

[0084] In another embodiment, even more preferred compounds of formula (I) are selected from the list comprising 3-nitrooxypropanol, ethyl 3-nitrooxypropanoate, methyl 3-nitrooxypropanoate and 3-nitrooxypropanoic acid and salts thereof.

[0085] The compounds of formula (I) of the present application can also comprise salts of nitrooxy organic molecules. Preferred cations for the salt preparation can be selected from the group consisting of sodium (Na + ), potassium (K + ), lithium (Li + ), magnesium (Mg 2+ ), calcium (Ca 2+ ), barium (Ba 2+ ), strontium (Sr 2+ ) and ammonium (NH 4+ ). The salts can also be prepared from alkali or alkaline earth metals.

[0086] In principle, the compounds of formula (I) according to the present application can be manufactured according to methods known per se for the synthesis of nitrooxy organic molecules, and / or based on methods as described in PCT / EP2010 / 069338 and European patent application No. 10 195 857.7, which are incorporated herein by reference.

[0087] In all these cases, the appropriate method for purifying the product (compound of formula (I)) can be selected by the person skilled in the art, either by column chromatography, or the compound of formula (I) can be isolated and purified by methods known per se, for example by adding a solvent such as diethyl ether or ethyl acetate to induce the separation of the crude product after the reaction from the mixture, and drying the collected crude product over Na2S04.

[0088] B. acetogenic bacteria

[0089] Acetogenic bacteria are microorganisms that produce acetate (CH3COO - ) as the end product of anaerobic respiration or fermentation. However, the term is often used in a narrower sense, only for those bacteria and archaea that carry out both anaerobic respiration and carbon fixation via the reductive acetyl-CoA pathway (also known as the Wood-Ljungdahl pathway). These true acetogenic bacteria are also called "homoacetogens", and they can produce acetyl-CoA from two molecules of carbon dioxide (CO2) and four molecules of molecular hydrogen (H2) (and in most cases, from acetyl-CoA, acetate as the end product, starting). This process is called acetogenesis, and unlike acetate fermentation, although both occur in the absence of molecular oxygen (O2) and produce acetate. Although it was previously thought that only bacteria were acetogenic, some archaea can be considered acetogenic.

[0090] In some embodiments, the acetogenic bacteria can be from one or more of the following genera: Acetitomaculum, Acetobacterium, Acetohalobium, Acetonema, Alkalibaculum, Blautia, Butyribacterium, Carboxydothermus, Clostridium, Desulfotomaculum, Eubacterium, Fuchsiella, Holophaga, Marvinbryantia, Moorella, Oxobacter, Sporomusa, Terrisporobacter, Terriacetogenum, Thermoanaerobacter, Ruminococcus, and / or Treponema.

[0091] In particular, acetogenic bacteria suitable for use in the compositions and methods disclosed herein can be one or more of the bacteria listed in Table 2 and / or selected from the group consisting of Acetoanaerobium notera (ATCC 35199), Acetonema longum (DSM 6540), Acetobacterium carbinolicum (DSM 2925), Acetobacterium malicum (DSM 4132), Acetobacterium sp. No. 446 (Morinaga et al., 1990, J. Biotechnol.[ Technologies, Vol. 14, pp. 187-194), Acetobacterium wieringae (DSM 1911), Acetobacterium woodii (DSM 1030), Alkalibaculum bacchi (DSM 22112), Archaeoglobus fulgidus (DSM 4304), Blautia producta (DSM 2950, formerly known as Ruminococcus productus, Peptostreptococcus productus), Butyribacterium methylotrophicum (DSM 3468), Clostridium aceticum (DSM 1496), Clostridium autoethanogenum (DSM 10061, DSM 19630, and DSM 23693), Clostridium carboxidivorans (DSM 15243), Clostridium coskatii (ATCC No. PTA-10522), Clostridium drakei (ATCC BA-623), Clostridium formicoaceticum (DSM 92), Clostridium glycolicum (DSM 1288), Clostridium ljungdahlii (DSM 13528), Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii ERI-2 (ATCC 55380), Clostridium ljungdahlii O-52 (ATCC 55989), Clostridium mayombei (DSM 6539), Clostridium methoxybenzovorans (DSM 12182), Clostridium neopropionicum species, Clostridium ragsdalei (DSM 15248), Clostridium scatologenes (DSM 757), Clostridium sp. ATCC 29797 (Schmidt et al., 1986, Chem. Eng. Commun.[Chem. Eng. Commun.], Vol. 45, pp. 61-73), Desulfotomaculum kuznetsovii (DSM 6115), Desulfotomaculum thermobezoicum subsp. thermosyntrophicum (DSM 14055), Eubacterium limosum (DSM 20543), Methanosarcina acetivorans C2A (DSM 2834), Moorella sp. HUC22-1 (Sakai et al., 2004, Biotechnol. Let. [Biotechnol. Let.], Vol. 29, pp. 1607-1612), Moorella thermoacetica (DSM 521, formerly Clostridium thermoaceticum), Moorella thermoautotrophica (DSM 1974), Oxobacter pfennigii (DSM 322), Sporomusa aerivorans (DSM 13326), Sporomusa ovata (DSM 2662), Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Sporomusa termitida (DSM 4440), and Thermoanaerobacter kivui (DSM 2030, formerly Acetogenium kivui).

[0092] In particular, the acetogenic microbial cell used according to any aspect of the application can be selected from the group consisting of Clostridium ljungdahlii and Clostridium autoethanogenum. In one example, the suitable bacterium can be Clostridium ljungdahlii. In particular, the strain is selected from the group consisting of Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium ljungdahlii COL and Clostridium ljungdahlii O-52, which can be used to convert syngas to hexanoic acid. These strains are described, for example, in WO 98 / 00558, WO 00 / 68407, ATCC 49587, ATCC 55988 and ATCC 55989. In another example, the acetogenic bacteria selected bacterium can be Clostridium autoethanogenum.

[0093] The expression of butyryl-CoA:acetate-CoA transferase (cat3) is increased in the cells according to any aspect of the application relative to the wild type cell. This enzyme is particularly advantageous in the acetogenic cells according to any aspect of the application as it has a broad substrate specificity (Stadtman ER (1953). J Biol Chem 203: 501-512 and Stadtman ER (1953) Fed Proc 12: 692-693.) and is able to catalyze the conversion of acyl CoA to form at least one fatty acid (Seedorf et al., (2007) PNAS 105 (6): 2128-2133). Due to the presence of the cat3 enzyme, it is possible to produce at least one acid from a carbon source comprising CO and / or CO2 in the presence of the acetogenic cells according to any aspect of the application. The acid can be produced from the carbon source via acetate production, wherein acetate can be used as a CoA acceptor. This can therefore allow the cells according to any aspect of the application to be more efficient and effective in the production of fatty acids.

[0094] In one embodiment, one or more acetogenic bacteria can be included in any of the feed additive formulations disclosed herein, and can optionally be formulated as a liquid, dry powder, or granule. In one embodiment, one or more acetogenic bacteria in any of the feed additive formulations disclosed herein can be formulated as a single mixture. In another embodiment, one or more acetogenic bacteria in any of the feed additive formulations disclosed herein can be formulated as separate mixtures. In still another embodiment, the separate mixtures of one or more acetogenic bacteria in any of the feed additive formulations disclosed herein can be administered at the same time or at different times. In still another embodiment, the separate mixtures of one or more acetogenic bacteria in any of the feed additive formulations disclosed herein can be administered simultaneously or sequentially. In yet another embodiment, a first mixture comprising one or more acetogenic bacteria can be administered, followed by a second mixture comprising any of the feed additive formulations disclosed herein. In still another embodiment, a first mixture comprising any of the feed additive formulations disclosed herein can be administered, followed by a second mixture comprising one or more acetogenic bacteria.

[0095] Dry powders or granules can be prepared by means known to those skilled in the art, such as in a top-spray fluid bed coater, in a Wurster bottom-spray fluid bed, or by rotary drum granulation (e.g., high shear granulation), extrusion, pan coating, or in a micro ingredient mixer.

[0096] The acetogenic bacteria can be prepared as one or more cultures and one or more carriers (if used), and can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the time can be increased or decreased. The components are blended so that a uniform mixture of the culture and carrier is obtained. In one embodiment, the final product can be a dry, flowable powder. In other embodiments, the final product can be encapsulated (particularly when anaerobic bacteria are used as the DFM). The one or more acetogenic bacteria comprising one or more bacterial strains can then be added to an animal feed or feed premix, added to water for an animal, or administered in other ways known in the art. The proportion of individual strains included in the one or more acetogenic bacteria mixtures can vary from 1% to 99%, preferably from 25% to 75%. Suitable dosage ranges for the DFM in an animal feed can be from about 1 x 10 3 CFU / g feed to about 1 x 10 10 CFU / g feed, suitably between about 1 x 10 4 CFU / g feed to about 1 x 10 8 CFU / g feed, suitably between about 7.5 x 10 4 CFU / g feed to about 1 x 10 7CFU / g feed, suitably more than about 1 x 10 3 CFU / g feed, suitably more than about 1 x 10 4 CFU / g feed, suitably more than about 1 x 10 4 CFU / g feed, or suitably more than about 1 x 10 5 CFU / g feed.

[0097] The dosage of the one or more acetogenic bacteria in the feed additive composition can be between about 1 x 10 3 CFU / g composition and about 1 x 10 13 CFU / g composition, such as 1 x 10 5 CFU / g composition and about 1 x 10 13 CFU / g composition, such as between about 1 x 10 6 CFU / g composition and about 1 x 10 12 CFU / g composition, such as between about 3.75 x 10 7 CFU / g composition and about 1 x 10 11 CFU / g composition, such as more than about 1 x 10 5 CFU / g composition, such as more than about 1 x 10 6 CFU / g composition, such as more than about 3.75 x 10 7 CFU / g composition, such as more than about 2 x 10 5 CFU / g composition, such as more than about 2 x 10 6 CFU / g composition, suitably more than about 3.75 x 10 7 CFU / g composition.

[0098] C. Compounds that promote hydrogen consumption in the rumen

[0099] In further aspects, any of the feed additives or feed compositions disclosed herein can further comprise one or more compounds that promote hydrogen consumption (i.e., reducing power consumption) in the rumen. These compounds can include, but are not limited to, nitrates, sulfates, nitro compounds (e.g., 3-nitropropionate, 2-nitropropanol, 2-nitroethanol, and / or nitroethane), and / or propionate precursors (e.g., malate, acrylate, oxalacetate, and / or fumarate).

[0100] In some embodiments, the one or more compounds that promote hydrogen gas consumption in the rumen are administered to the ruminant at a concentration of any of 1 mg / kg body weight (bw), 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 6 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, or more.

[0101] D. Essential oils

[0102] In one embodiment, the present disclosure includes a ruminant feed additive or feed composition comprising (a) one or more rumen depressant compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen gas consumption in the rumen, and further containing at least one essential oil (e.g., cinnamaldehyde and / or thymol). Essential oils are concentrated volatile oils having the characteristic odor of the plant from which they are derived. Typically, essential oils are obtained by distillation of plants and contain a mixture of component compounds. The component compounds of these essential oils include anethole, beta-ionone, capsaicin, carvacrol, cinnamaldehyde, citral, cresol, eugenol, guaiacol, limonene, thymol, tannins, and vanillin.

[0103] The animal feed or feed additive compositions disclosed herein can comprise at least 1 g of cinnamaldehyde per 1000 kg of animal feed, at least 2 g of cinnamaldehyde per 1000 kg of animal feed, at least 3 g of cinnamaldehyde per 1000 kg of animal feed, at least 4 g of cinnamaldehyde per 1000 kg of animal feed, at least 5 g of cinnamaldehyde per 1000 kg of animal feed.

[0104] The animal feed or feed additive compositions disclosed herein can comprise at least 1 mg of cinnamaldehyde per kg of animal feed, at least 2 mg of cinnamaldehyde per kg of animal feed, at least 3 mg of cinnamaldehyde per kg of animal feed, at least 4 mg of cinnamaldehyde per kg of animal feed, at least 5 mg of cinnamaldehyde per kg of animal feed.

[0105] The animal feed or feed additive compositions disclosed herein can comprise less than 6 g of cinnamaldehyde per 1000 kg of animal feed; such as, for example, less than 5.9 g of cinnamaldehyde. The animal feed can comprise less than 18 g of cinnamaldehyde per 1000 kg of animal feed, such as, for example, less than 17 g of cinnamaldehyde per 1000 kg of animal feed, less than 16 g of cinnamaldehyde per 1000 kg of animal feed, less than 15 g of cinnamaldehyde per 1000 kg of animal feed, less than 14 g of cinnamaldehyde per 1000 kg of animal feed.

[0106] When the animal feed or feed additive composition disclosed herein comprises thymol, the animal feed can comprise at least 1 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 2 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 3 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 4 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 5 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 6 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 7 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 8 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 9 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 10 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 11 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 12 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 13 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 14 g of thymol per 1000 kg of the animal feed or feed additive composition, at least 15 g of thymol per 1000 kg of the animal feed or feed additive composition. The animal feed or feed additive composition can comprise less than 50 g of thymol per 1000 kg of the animal feed or feed additive composition.

[0107] E. Enzymes

[0108] In one embodiment, the present disclosure includes a ruminant feed additive or feed composition comprising (a) one or more rumen-inhibiting compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen, and further containing at least one enzyme (e.g., a glucoamylase and / or a phytase). Suitable enzymes for use in the methods according to the present disclosure include, but are not limited to, glucoamylases, xylanases, amylases, phytases, fucosidases, beta-glucanases, and proteases.

[0109] 1. Glucoamylase

[0110] Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts.

[0111] In one embodiment, the present disclosure includes a ruminant feed additive or feed composition comprising (a) one or more rumen-inhibitor compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen, and further comprising one or more glucoamylases. The glucoamylase can be any commercially available glucoamylase. Suitably, the glucoamylase can be a 1,4-alpha-D-glucan glucohydrolase (EC 3.2.1.3). The glucoamylase can be encapsulated, coated to prevent or limit degradation in the rumen environment, or otherwise in particulate form. All E.C. enzyme classifications referred to herein relate to the classification provided in Enzyme Nomenclature— Recommendations (1992) - ISBN 0-12-226164-3 of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, which is incorporated herein.

[0112] Glucoamylases have been successfully used in commercial applications for many years. Additionally, various mutations have been introduced in fungal glucoamylases (e.g., Trichoderma reesei glucoamylase (TrGA)) to enhance thermostability and specific activity. See, e.g., WO 2008 / 045489; WO 2009 / 048487; WO 2009 / 048488; and U.S. Patent No. 8,058,033. Glucoamylase activity can be assessed using any means known in the art, including those described in the Example section below.

[0113] The glucoamylase can be derived from any suitable source, for example from a microorganism or a plant. The glucoamylase can be from a fungal or bacterial source, selected from the group consisting of Aspergillus glucoamylases, such as Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or variants thereof, such as those disclosed in WO 92 / 00381, WO 00 / 04136 and WO 01 / 04273 (from Novozymes, Denmark); A. awamori glucoamylase disclosed in WO 84 / 02921, Aspergillus oryzae glucoamylase (Hata et al., 1991, Agric. Biol. Chem. 55(4): 941-949), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E / Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulfide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704); and introduction of Pro residues at positions A435 and S436 (Li et al., 1997, Protein Eng. 10: 1199-1204).

[0114] Other glucoamylases include Athelia rolfsii (previously known as Corticium rolfsi) glucoamylase (see U.S. Patent No. 4,727,026 and Nagasaka et al., 1998, Appl. Microbiol. Biotechnol. 50: 323-330), particularly derived from Talaromyces duponti, Talaromyces emersonii (WO 99 / 28448), Talaromyces leycettanus (U.S. Patent No. Re. 32,153), and Talaromyces thermophilus (U.S. Patent No. 4,587,215).

[0115] Bacterial glucoamylases include those from Clostridium, particularly C. thermoamylolyticum (EP 135138) and C. thermohydrosulfuricum (WO 86 / 01831), Trametes cingulata, Pachykytospora papyracea, and Leucopaxillus giganteus (all disclosed in WO 2006 / 069289); or Peniophora rufomarginata disclosed in WO 2007 / 124285 or PCT / US2007 / 066618; or mixtures thereof. Hybrid glucoamylases can be used in the present application. Examples of hybrid glucoamylases are disclosed in WO 2005 / 045018. Particular examples include the hybrid glucoamylases disclosed in Tables 1 and 4 of Example 1 (these hybrids are hereby incorporated by reference).

[0116] The glucoamylase can have a high degree of sequence identity to any of the above-mentioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the above-mentioned mature enzyme sequences.

[0117] Commercially available glucoamylase compositions include AMG 200L; AMG 300L; SAN™ SUPER, SAN™ EXTRAL, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME ULTRA, SPIRIZYME™ EXCEL, and AMG™ E (from Novozymes A / S, Denmark); OPTIDEX™ 300, GC480™, and GC147™ (from Genencor Int., USA); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™, and G990 ZR (from Genencor Int.).

[0118] 2. Phytase

[0119] In another embodiment, the present disclosure includes a ruminant feed additive or feed composition comprising (a) one or more rumen-inhibitor compound; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compound that promotes hydrogen consumption in the rumen, and further comprising one or more phytase. Phytases for use in the present invention can be classified as 6-phytase (classified as E.C. 3.1.3.26) or 3-phytase (classified as E.C. 3.1.3.8). Phytases can be encapsulated, coated to prevent or limit degradation in the rumen environment, or otherwise in particulate form. In one embodiment, phytases for use in the present invention can be one or more phytases in one or more of the commercial products in Table 5 below:

[0120] Table 5: Representative commercial phytases

[0121]

[0122] In one embodiment, the phytase is a Citrobacter phytase, derived from, for example, Citrobacter freundii, in some embodiments, for example, Citrobacter freundii NCIMB 41247 and variants thereof as disclosed in WO 2006 / 038062 (incorporated herein by reference) and WO 2006 / 038128 (incorporated herein by reference), Citrobacter braakii YH-15 as disclosed in WO 2004 / 085638, Citrobacter braakii ATCC 51113 as disclosed in WO 2006 / 037328 (incorporated herein by reference), and variants thereof, for example, as disclosed in WO 2007 / 112739 (incorporated herein by reference) and WO 2011 / 117396 (incorporated herein by reference); Citrobacter amalonaticus, in some embodiments, Citrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC 25407 as disclosed in WO 2006 037327 (incorporated herein by reference); Citrobacter gillenii, in some embodiments, Citrobacter gillenii DSM 13694 as disclosed in WO 2006 037327 (incorporated herein by reference); or Citrobacter intermedius, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae; a Citrobacter species polypeptide or variant thereof.

[0123] In some embodiments, the phytase is an E. coli phytase sold under the name Danisco A / S Phyzyme XP™. Alternatively, the phytase can be a Buttiauxella phytase, such as a Buttiauxella agrestis phytase, such as the phytases taught in WO 2006 / 043178, WO 2008 / 097619, WO 2009 / 129489, WO 2008 / 092901, PCT / US2009 / 41011, or PCT / IB2010 / 051804, all of which are incorporated by reference herein. Alternatively, the phytase can be an engineered, robust high-Tm clade phytase polypeptide, such as the phytases disclosed in WO 2020 / 106796 (incorporated by reference herein).

[0124] In one embodiment, the phytase can be a phytase from Hafnia (e.g., from Hafnia alvei), such as one or more phytases taught in US 2008263688, which is incorporated by reference herein. In one embodiment, the phytase can be a phytase from Aspergillus (e.g., from Apergillus orzyae). In one embodiment, the phytase can be a phytase from Penicillium (e.g., from Penicillium funiculosum).

[0125] In some embodiments, the phytase is present in the feed or feed additive composition in a range of about 1000 FTU / kg of feed to about 8000 FTU / kg of feed. In some embodiments, about 2000 FTU / kg of feed to about 7000 FTU / kg of feed. In some embodiments, about 3000 FTU / kg of feed to about 6000 FTU / kg of feed. In one embodiment, the phytase is present in the feedstuff in excess of about 200 FTU / kg of feed, suitably in excess of about 300 FTU / kg of feed, suitably in excess of about 400 FTU / kg of feed. In one embodiment, the phytase is present in the feedstuff in less than about 1000 FTU / kg of feed, suitably in less than about 750 FTU / kg of feed. In some embodiments, the phytase is present in the feed additive composition in a range of about 40 FTU / g of composition to about 40,000 FTU / g of composition, about 80 FTU / g of composition to about 20,000 FTU / g of composition, about 100 FTU / g of composition to about 10,000 FTU / g of composition, and about 200 FTU / g of composition to about 10,000 FTU / g of composition. In one embodiment, the phytase is present in the feed additive composition in excess of about 40 FTU / g of composition, suitably in excess of about 60 FTU / g of composition, suitably in excess of about 100 FTU / g of composition, suitably in excess of about 150 FTU / g of composition, suitably in excess of about 200 FTU / g of composition. In one embodiment, the phytase is present in the feed additive composition in less than about 40,000 FTU / g of composition, suitably in less than about 20,000 FTU / g of composition, suitably in less than about 15,000 FTU / g of composition, suitably in less than about 10,000 FTU / g of composition.

[0126] It is understood that, as used herein, 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from substrate in one minute under the reaction conditions defined in ISO 2009 Phytase Assay (standard assay for determining phytase activity), and 1 FTU can be found in International Standard ISO / DIS 30024: 1-17, 2009. In one embodiment, the enzyme is classified using the E.C. classification scheme described above, and the E.C. classification scheme designates enzymes having that activity when tested in the assay taught herein to determine 1 FTU.

[0127] F. Feed and feed additive formulations

[0128] Animal feed can include plant material, such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of these plant materials or plant protein sources for poultry, swine, ruminants, aquaculture, and pets. The terms "animal feed", "feedstuff", and "roughage" are used interchangeably and can comprise one or more feed materials selected from the group comprising a) roughage: such as pasture grass, grass, hay, alfalfa, silage grass, silage corn; b) cereals: such as small grain cereals (e.g. wheat, barley, rye, oats and combinations thereof) and / or large grain cereals (such as maize or sorghum); c) cereal by-products: such as corn protein meal, distillers dried solubles (DDGS, especially corn based distillers dried solubles (cDDGS)), wheat bran, coarse wheat meal, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; d) proteins obtained from: soybean, sunflower, peanut, lupin, pea, fava bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from plant and animal sources; and / or f) minerals and vitamins.

[0129] When used as or for the preparation of a feed, such as a functional feed, the feed or feed additive composition of the present application can be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. For example, at least one component selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben can be mentioned.

[0130] In one embodiment, the feed or feed additive composition of the present application is mixed with a feed component to form a feedstuff. As used herein, "feed component" means all or part of a feedstuff. Part of a feedstuff can mean one ingredient of a feedstuff or more than one (e.g. 2 or 3 or 4 or more) ingredients of a feedstuff. In one embodiment, the term "feed component" encompasses a premix or a premix ingredient.

[0131] In some embodiments, the feed can be roughage or a premix thereof, a compound feed or a premix thereof. The feed additive composition according to the present application can be mixed with a compound feed, a component of a compound feed or mixed into a premix of a compound feed or into roughage, a component of roughage or a premix of roughage.

[0132] As used herein, the term "roughage" means any foodstuff provided to or consumed by an animal. Further, roughage includes silage, compressed and pelleted feed, oil and mixed rations, and also includes sprouted grains and legumes.

[0133] Roughage can be obtained from one or more of the plants selected from the group consisting of grasses, corn (zea mays), alfalfa (Medicago sativa), barley, birdsfoot trefoil, brassica plants, chau moellier, collard greens, canola (brassica napus), rutabaga (swedish turnip), radish, clover, hybrid clover, red clover, subterranean clover, white clover, fescue, vetch, millet, oats, sorghum, soybean, trees (pruned branches of trees used as tree hay), wheat, and legumes.

[0134] The term "compound feed" means commercial feed in the form of meal, pellets, nut, cake or crumb. Compound feed can be blended from a variety of raw materials and additives.

[0135] These blends are formulated according to the specific needs of the target animal.

[0136] Compound feed can be complete feed providing all the daily required nutrients, concentrate providing a part of the ration (protein, energy) or supplement providing only additional nutrients such as minerals and vitamins. The main ingredients used in compound feed are feed grains, which include corn, wheat, canola meal, rapeseed meal, lupin, soybean, sorghum, oat and barley.

[0137] Suitably, "premix" as referred to herein can be a composition made up of micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. Premix is generally a composition suitable for blending into commercial rations.

[0138] In another aspect, when the feed additive composition is formulated into granules, the granules comprise a hydrating barrier salt coated on a protein core. Such a salt coating has the advantage of improved heat resistance, improved shelf stability and protection from other feed additives that would otherwise adversely affect the feed additive. Preferably, the salt used for the salt coating has an aw greater than 0.25 or a constant humidity greater than 60% at 20°C. In some embodiments, the salt coating comprises Na2S04.

[0139] The skilled person will appreciate that different animals require different feedstuffs, and even the same animal can require different feedstuffs depending on the purpose for which the animal is being kept. Optionally, the feedstuff can also contain additional minerals (such as, for example, calcium) and / or additional vitamins. In some embodiments, the feedstuff is corn-soybean meal, wheat or a mixed grain mixture.

[0140] Feedstuffs are typically produced in a feed mill, where the raw materials are first ground to the appropriate particle size and then mixed with the appropriate additives. The feedstuffs can then be produced as a mash or pellets; the latter typically involves a process by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a specific size. The pellets are allowed to cool. Subsequently, liquid additives such as fats and enzymes can be added. The production of feedstuffs can also involve an additional step comprising extrusion or puffing prior to pelleting, particularly by extrusion or puffing by suitable techniques that can include the use of steam.

[0141] The feed additive composition and / or feedstuffs comprising the same can be used in any suitable form. The feed additive composition can be used in the form of a solid or liquid formulation or alternatives thereof. Examples of solid formulations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules, which can be wettable, spray-dried, or freeze-dried. Examples of liquid formulations include, but are not limited to, aqueous, organic, or aqueous-organic solutions, suspensions, and emulsions.

[0142] In some applications, the feed additive composition can be mixed with the feed or administered in the drinking water (e.g., drinking water from a water well, a spring, a dug well, a semi- artesian well, and an artesian well, a municipal water supply, a lake, or a creek). In other embodiments, one or more components of the feed additive composition (e.g., (a) one or more rumen-inhibitor compounds; and (b) (1) one or more acetogenic bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen) are administered in the drinking water, and one or more components of the feed additive composition are administered in the feed, either concurrently or simultaneously. When water line delivery is contemplated, administration of the feed additive composition can include, but is not limited to, one or more of the following: mixing the composition into the water, rehydrating the composition components (e.g., acetogenic bacteria), adding the hydrated composition into a drench applicator, and administering it to the water line via a dosatron or other pumping means.

[0143] The feedstuffs and / or feed additive composition can be mixed with at least one mineral and / or at least one vitamin. The composition thus derived can be referred to herein as a premix. The feedstuffs can comprise at least 0.0001% by weight of the feed additive. Suitably, the feedstuffs can comprise at least 0.0005%, at least 0.0010%, at least 0.0020%, at least 0.0025%, at least 0.0050%, at least 0.0100%, at least 0.020%, at least 0.100%, at least 0.200%, at least 0.250%, at least 0.500% by weight of the feed additive.

[0144] Preferably, the food or feed additive composition can further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is preferably selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na2S04, talc, PVA and mixtures thereof. In further embodiments, the food or feed additive can further comprise a metal ion chelator. The metal ion chelator can be selected from EDTA or citric acid.

[0145] Formulations comprising any of the enzymes as described herein can be prepared in any suitable manner to ensure that the formulation comprises active enzyme. Such formulations can be in liquid, dry powder or granules. Preferably, the feed additive composition is in solid form, suitable for addition on or to feed pellets.

[0146] Dry powders or granules can be prepared by means known to those skilled in the art, such as high shear granulation, drum granulation, extrusion, spheronization, fluid bed agglomeration, fluid bed spray drying.

[0147] The feed additive compositions described herein can be formulated as dry powders or granules as described in WO 2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645 (each incorporated herein by reference).

[0148] In one embodiment, an animal feed can be formulated into a granule of a feed composition comprising: a core; an active agent; and at least one coating, the active agent of the granule remaining at least 50% active, at least 60% active, at least 70% active, at least 80% active after being subjected to one or more conditions selected from: a) a feed pelleting process, b) a steam heated feed pre-treatment process, c) storage, d) storage as an ingredient in an unpelleted mix, and e) storage as an ingredient in a feed base mix or feed premix comprising at least one compound selected from: a trace mineral, an organic acid, a reducing sugar, a vitamin, choline chloride, and a compound that produces an acidic or basic feed base mix or feed premix.

[0149] In relation to the granule, the at least one coating can comprise a moisture hydrating material that is at least 55% w / w of the granule; and / or the at least one coating can comprise two coatings. The two coatings can be a moisture retaining coating and a moisture barrier. In some embodiments, the moisture retaining coating can be from 25% w / w to 60% w / w of the granule and the moisture barrier can be from 2% w / w to 15% w / w of the granule. The moisture retaining coating can be selected from inorganic salts, sucrose, starch and maltodextrin and the moisture barrier can be selected from polymers, gums, whey and starch.

[0150] A feed additive composition can be formulated into a pellet for animal feed, the pellet comprising: a core; an active agent that remains at least 80% active after storage and after a steam heat pelleting process in which the pellet is used as an ingredient; a moisture barrier layer; and a moisture retaining coating that comprises at least 25% w / w of the pellet, the pellet having a water activity of less than 0.5 prior to the steam heat pelleting process.

[0151] III. Methods

[0152] A. Methods for reducing methane production and improving animal performance

[0153] The present disclosure relates to a method for reducing the production of methane originating from the digestive activity of a ruminant animal and / or for improving the performance of a ruminant animal, the method comprising orally administering to the animal an effective amount of any of the feed additives or feed compositions disclosed herein.

[0154] In some embodiments, administering to a ruminant animal an effective amount of a ruminant feed or feed additive composition (containing one or more rumen inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in reduced methane production (such as any of a reduction of about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction in production, including all values between these percentages) (as compared to the amount of methane produced in a ruminant animal that has not been administered an effective amount of a feed or feed additive composition disclosed herein).

[0155] In other embodiments, administering to a ruminant animal an effective amount of a ruminant feed or feed additive composition (containing one or more rumen inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in increased rumen acetate production (such as any of an increase of about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125% or more increase in production, including all values between these percentages) (as compared to rumen acetate production in a ruminant animal that has not been administered an effective amount of a feed or feed additive composition disclosed herein or that has been administered only one or more rumen inhibitor compounds).

[0156] In some embodiments, administering to a ruminant an effective amount of a ruminant feed or feed additive composition (containing one or more rumen-inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in decreased feed conversion ratio (FCR; such as any of about a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in FCR, including all values between these percentages) (as compared to the FCR of a ruminant that was not administered an effective amount of a feed or feed additive composition disclosed herein or that was administered only one or more rumen-inhibitor compounds).

[0157] In other embodiments, administering to a ruminant an effective amount of a ruminant feed or feed additive composition (containing one or more rumen-inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in increased feed intake (such as any of about a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125% or more increase in feed intake, including all values between these percentages) (as compared to the feed intake of a ruminant that was not administered an effective amount of a feed or feed additive composition disclosed herein or that was administered only one or more rumen-inhibitor compounds).

[0158] In other embodiments, administering to a ruminant an effective amount of a ruminant feed or feed additive composition (containing one or more rumen-inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in increased weight gain (such as any of about a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125% or more increase in weight gain, including all values between these percentages) (as compared to the weight gain of a ruminant that was not administered an effective amount of a feed or feed additive composition disclosed herein or that was administered only one or more rumen-inhibitor compounds).

[0159] In other embodiments, administering to a ruminant an effective amount of a ruminant feed or feed additive composition (containing one or more rumen-inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in increased carcass yield (such as any of about a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125% or more increase in carcass yield, including all values between these percentages) (as compared to the carcass yield of a ruminant that was not administered an effective amount of a feed or feed additive composition disclosed herein or that was administered only one or more rumen-inhibitor compounds).

[0160] In other embodiments, administering to a ruminant an effective amount of a ruminant feed or feed additive composition (containing one or more rumen-inhibitor compounds, and one or more acetogenic bacteria, and / or one or more compounds that promote hydrogen consumption in the rumen) results in increased milk, or milk fat content and / or yield, or milk protein content and / or yield (such as any of about a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or 125% or more increase in milk, or milk fat content and / or yield, or milk protein content and / or yield, including all values between these percentages) (as compared to the milk, or milk fat content and / or yield, or milk protein content and / or yield of a ruminant that was not administered an effective amount of a feed or feed additive composition disclosed herein or that was administered only one or more rumen-inhibitor compounds).

[0161] B. Methods for producing a feed additive or feed composition

[0162] Also provided herein are methods for producing a feed additive or feed composition, the method comprising combining: one or more rumen-inhibitor compounds (such as any of the rumen-inhibitor compounds disclosed herein; and one or more acetogenic bacteria (such as any of the acetogenic bacteria disclosed herein); and / or one or more compounds that promote hydrogen consumption in the rumen (such as any of those disclosed herein). In further embodiments, the method further comprises packaging the feed additive or feed composition.

[0163] The application can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation. Example

[0164] Example 1 : Use of a composition that causes hydrogen production in the rumen in combination with acetogenic bacteria in the feed of a ruminant to reduce methane production and increase VFA production Experimental design:

[0165] This example describes a specific combination of rumen inhibitors with the rumen sausage bacteria DSM 5522, acetogen Clostridium woodii DSM 1030 (originating from the German Collection of Microorganisms and Cell Cultures, DSMZ). Several treatments were tested in separate fermentation vials.

[0166] Without being bound by theory, it is believed that the addition of one or more rumen inhibitor compounds that cause H2 accumulation in the rumen in combination with one or more acetogenic bacteria will result in a double benefit of reduced methane production and increased total volatile fatty acid (VFA, e.g., short-chain fatty acids - end products of rumen fermentation, mainly acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids) production in ruminants. This double benefit is only achieved by the combination of a ruminant inhibitor compound and acetogenic bacteria.

[0167] Results

[0168] • Control: no additives added to the feed

[0169] • Part 1 : Rumen inhibitor compounds that cause H2 accumulation in the rumen, e.g., a compound selected from Table 1 or any rumen inhibitor compound disclosed herein. Several addition levels in the range of 0.0 to 0.5 mg / g dry matter (DM) substrate were tested

[0170] • Part 2: One or more acetogenic bacterial species were added in the range of 0 to 10 9 CFU / ml.

[0171] • Part 1 + Part 2: Both a rumen inhibitor and acetogenic bacteria were added, each in the same range specified above.

[0172] Table 1 : Non-limiting examples of ruminant inhibitor compounds that cause H2 accumulation in the rumen

[0173]

[0174] Table 2: Non-limiting examples of acetogenic bacteria

[0175]

[0176] Table 3: Non-limiting examples of compounds or microorganisms that promote hydrogen consumption in the rumen

[0177]

[0178] Substrate is added in all cases. The substrate is dried at 60°C and ground through a 1 mm sieve. The appropriate amount of substrate is added in each fermentation vial (e.g. in a 120 ml fermentation vial, 500 mg dry substrate for 50 ml medium).

[0179] Filtered rumen fluid (SRF) is collected and maintained anaerobically at 39°C.

[0180] Fermentation buffer is prepared, deoxygenated (e.g. by continuous sparging with C02) and heated to 39°C. SRF is added in the appropriate ratio, e.g. 1 part SRF / 4 parts buffer, to make the medium. Fermentation vials are dispensed with medium in an anaerobic manner (e.g. 50 ml).

[0181] Immediately after dispensing of the medium, additives are added to the medium in the levels specified above (if applicable). Gas pressure in the vial headspace is recorded by regular manual readings using a pressure sensor or, alternatively, an automated (continuous) system. At several time points during the fermentation (e.g. 6 h, 12 h, 18 h, 24 h), gas is sampled and analysed for H2, CH4 and C02 in a gas chromatograph following standard methods. Gas can be analysed directly at the time of collection or, alternatively, can be collected into vacuum tubes for later analysis.

[0182] At several time points during the fermentation (e.g. 6 h, 12 h, 18 h, 24 h), fermentation medium is sampled for later analysis of VFAs and other metabolites. Samples for VFA analysis are deproteinated (e.g. 0.8 ml sample is mixed with 0.5 ml solution containing 20 g / l of metaphosphoric acid in 0.5 N HCI and centrifuged at 15000 x g for 15 min at 4°C) and the supernatant is stored by freezing prior to analysis. An internal standard can also be added (e.g. 4 g / l of crotonic acid). Samples are analysed for VFAs following standard methods, e.g. by gas chromatography or liquid chromatography.

[0183] After the appropriate time (e.g. 24 h), gas pressure is read and fermentation is stopped (e.g. by placing the vials on ice). A gas sample is collected (e.g. into a vacuum tube). Alternatively, gas can be analysed directly. The H2, CH4 and C02 concentrations of the gas are analysed. The vials are then opened and liquid samples are taken for VFA analysis as described above.

[0184] Finally, the whole vial content is processed to estimate substrate disappearance, e.g. by filtration through a sintered crucible of pore size 1. The residue is oven-dried at 60 °C for 48 h and then the dry residue is decontaminated from microorganisms by detergent washing (e.g. by weighing into an F57 ANKOM filter bag and washing with neutral detergent at 100 °C for 1 h in an ANKOM fiber analyzer). The residue remaining in the bag is weighed to assess DM and neutral detergent fiber (NDF) disappearance.

[0185] Example 2: Effect of a combination of a methane inhibitor and acetogenic bacteria on gas production and composition and VFA production of in vitro rumen incubations.

[0186] The concentration and / or absolute volume of CH4 produced, and / or the relative volume of CH4 produced (per unit of fermentation end product (e.g. VFA) or per unit of fermentation substrate) is assessed.

[0187] The change in concentration and / or absolute accumulation of VFAs, in particular acetic acid, and / or the change in relative accumulation of VFAs, in particular acetic acid (per unit of fermentation gas or per unit of fermentation substrate) is assessed.

[0188] The change in absolute disappearance of substrate, or in relative disappearance per unit of fermentation gas or fermentation end product (e.g. VFA) is assessed.

[0189] Figure 1 Figure 2

[0190] Trinitroxypropanol (3NOP) was sourced from Cymit Química, S.L. (C / Pamplona 96-104, local 15, 08018 Barcelona, Spain). Freeze-dried Asparagopsis taxiformis (FDAT) was provided by seaExpert - Consultoria na Areas das Pescas Lda. (Travessa do Farrobim 15, 9900-361 Horta-Açores, Portugal). Acetogenic bacterial strains (Blautia producta (DSM 2950) and Acetobacterium woodii (DSM 1030)) were sourced from the Leibniz Institute DSMZ GmbH (Inhoffenstraße 7B, 38124 Braunschweig, Germany).

[0191] In vitro rumen incubations were established as follows: In the morning prior to feeding, rumen contents were obtained from one of five rumen cannulated sheep and filtered through double cheesecloth. Rumen fluid was diluted 1 :2 in buffer (Menke and Steingass, 1988) and 50 ml aliquots were added anaerobically to 120 ml Wheaton bottles containing 0.5 g of a 50:50 roughage: concentrate diet (previously ground to pass 1-mm 2 sieves). Treatments consisted of: (1) control incubations (diet only), and diet plus (2) 5 ml live acetogen strain culture incubations, (3) FDAT + autoclaved acetogen culture, (4) 3NOP + autoclaved acetogen culture, (5) FDAT in combination with 5 ml live acetogen culture, and (6) 3NOP in combination with 5 ml live acetogen culture. Autoclaved acetogen cultures were added to treatments 3 and 4 to correct for any potential impact of culture products added with live strains in treatments 2, 5, and 6. Additionally, one blank bottle (rumen fluid + buffer only) was included per sheep to correct for basal gas and VFA production. In all cases, 3NOP was dosed at 0.1% in diet dry matter (0.5 mg / bottle), and FDAT was added at a concentration of 2.0% in diet dry matter (10 mg FDAT with 490 mg diet).

[0192] Bottles were sealed and incubated at 39°C. Live or autoclaved acetogen cultures were inoculated 3 h after the start of incubation. Gas was measured for release, and samples were taken for methane and hydrogen analysis after 6 h and 24 h of incubation. At 24 h, bottles were opened, pH was measured, and subsamples were collected for determination of volatile fatty acids (VFAs) by gas chromatography (Stewart and Duncan, 1985, Journal of General Microbiology 131 : 427-435).

[0193] Two separate experiments were run, each involving one of the two acetogenic bacterial strains and were analyzed separately. Each experiment included 5 replicates (5 sheep). Treatment effects were analyzed by randomized block ANOVA with individual sheep as the blocking term. When a significant effect was detected across treatments, mean values were compared by Fisher's protected LSD test and P values were required for the following contrasts: (1) control versus acetogenic bacterial strain, (2) FDAT versus FDAT + acetogenic bacterial strain, and (4) 3NOP versus 3NOP + acetogenic bacterial strain. Genstat 23rd edition (VSN International, Hemel Hempstead, UK) was used.

[0194] Incubation results for B. prolongus and the respective combinations (Experiment 1) are reported in Table 1, while the results for W. succinogenes (Experiment 2) are reported in Table 2. ​ ​

[0195] Experiment 1: Methane production was significantly reduced by both the methane inhibitors FDAT (-92% after 24 h) and 3NOP (-77% after 24 h), with FDAT showing greater inhibition than 3NOP in this experiment. Consequently, the increase in accumulated hydrogen was also greater for FDAT (+190% after 24 h) compared to 3NOP (+81% after 24 h). Total VFA decreased: -21% with FDAT and -11% with 3NOP, mainly at the expense of acetate. B. prolongus (alone) did not affect any of the fermentation results. However, when combined with FDAT, B. prolongus did not affect the inhibition of methane caused by FDAT, while causing a significant increase in acetate proportion (+5% calculated total acetate) and a decrease in accumulated hydrogen after 6 h of incubation of -23%. No interaction of B. prolongus with 3NOP was observed, possibly because 3NOP had a lower effect on methane than FDAT in this experiment, as mentioned previously.

[0196] ​​Experiment 2: Similar to Experiment 1, W. succinogenes (alone) did not cause significant changes in fermentation parameters, but when combined with FDAT, the acetate ratio increased while methane inhibition did not change. No statistical impact on individual gas production was found for the combination relative to FDAT alone, but total gas was reduced by -10% when W. succinogenes was added on top of FDAT. In contrast to Experiment 1, in this experiment, FDAT and 3NOP alone had similar impact on methane production (after 24 h -88% to -89%) and hydrogen accumulation (after 24 h 112%-157%), and interestingly, when W. succinogenes was added to either FDAT or 3NOP cultures, the acetate ratio increased or tended to increase to a similar extent.

Claims

1. A ruminant feed additive or feed composition comprising (a) one or more rumen inhibitor compounds; and (b) (1) one or more acetic acid-producing bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen.

2. The feed additive or feed composition of claim 1, wherein the rumen inhibitor compound comprises one or more of a nitrooxy compound, a coenzyme M analog, a pterin compound, a hydroxymethylglutaryl-CoA reductase inhibitor, an antimicrobial peptide, or a plant secondary metabolite.

3. The feed additive or feed composition according to claim 1 or claim 2, wherein the rumen inhibitor compound comprises 3-nitrooxypropanol (3NOP) or a coenzyme M analog.

4. The feed additive or feed composition according to any one of claims 1-3, wherein the one or more acetic acid-producing bacteria are one or more bacteria selected from the group consisting of: *Acetobacterium bakii*, *Blautia hydrogenotrophica*, *Blautia producta*, *Blautia schinkii*, *Clostridium autoethanogenum*, *Clostridium carboxidivorans*, *Clostridium coskii*, *Clostridium ljungdahlii*, *Clostridium scatologenes*, *Sporomusa acidovorans*, *Eubacterium limosum*, *Acetobacterium woodii*, and *Sporomusa forestica*. Silvacetica, Moorella thermoacetica, Acetitomaculum ruminis, Ruminococcus productus, and Clostridium magnum.

5. The feed additive or feed composition according to any one of claims 1-4, wherein the one or more compounds that promote hydrogen consumption in the rumen comprise one or more of nitrates, sulfates, nitro compounds or propionate precursors.

6. The feed or feed additive composition according to any one of claims 1-5, wherein the feed or feed additive composition further comprises one or more enzymes.

7. The feed or feed additive composition of claim 6, wherein the enzyme comprises glucosylamylase and / or phytase.

8. A method for reducing methane production from the digestive activity of ruminants and / or for improving ruminant performance, the method comprising orally administering to the animal an effective amount of a feed additive or feed composition as described in any one of claims 1-7.

9. The method of claim 8, wherein the ruminant is selected from the group consisting of: beef cattle, dairy cattle, veal calves, goats, sheep, giraffes, bison, European bison, yaks, buffalo, deer, camels, alpacas, llamas, wildebeest, antelopes, pronghorn, and blue antelope.

10. The method of claim 8 or claim 9, wherein the hydrogen concentration in the rumen of the animal is less than 5%.

11. The method of any one of claims 1-10, wherein the feed additive or feed composition is not pretreated using wet explosion pretreatment.

12. A method for producing a feed additive or feed composition, the method comprising combining: (a) one or more rumen inhibitor compounds; and (b) (1) one or more acetic acid-producing bacteria; and / or (2) one or more compounds that promote hydrogen consumption in the rumen.

13. The method of claim 12, wherein the rumen inhibitor compound comprises one or more of a nitrooxy compound, a coenzyme M analog, a pterin compound, a hydroxymethylglutaryl-CoA reductase inhibitor, an antimicrobial peptide, or a plant secondary metabolite.

14. The method of claim 12 or claim 13, wherein the rumen inhibitor compound comprises 3-nitrooxypropanol (3NOP) or a coenzyme M analog.

15. The method of any one of claims 12-14, wherein the one or more acetic acid-producing bacteria are one or more bacteria selected from the group consisting of: *Acetobacter beyeris*, *Brutobacter hygroscopicus*, *Brutobacter elongatus*, *Brutobacter channerii*, *Clostridium ethanolophilum*, *Clostridium carbon monoxideophilum*, *Clostridium korskinense*, *Clostridium yunnanense*, *Clostridium scutellarioides*, *Clostridium scutellarioides*, *Clostridium scutellarioides*, *Clostridium myxobolum*, *Acetobacter wulliganum*, *Clostridium foresticum*, *Clostridium thermophilum*, *Rumen acetozoonum*, *Rumen cocci*, and *Clostridium macrocarpa*.

16. The method of any one of claims 12-15, wherein the one or more compounds that promote hydrogen consumption in the rumen comprise one or more of nitrates, sulfates, nitro compounds, or propionate precursors.

17. The method of any one of claims 12-16, the method further comprising combining one or more enzymes with the feed additive or feed composition.

18. The method of claim 17, wherein the enzyme comprises one or more of glucosylamylase, xylanase, amylase, phytase, β-glucanase, fucosidase, and / or protease.

19. The method of any one of claims 12-18, the method further comprising packaging the feed additive or feed composition.

20. The method of any one of claims 12-19, wherein the feed additive or feed composition is not pretreated using wet explosion pretreatment.

Images