Methods of reducing enteric methane emission

Abstract

The invention relates to a feed composition comprising at least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum. Also provided use of the feed composition in the reduction of enteric methane emissions and / or increase of milk production and / or quality in ruminants. Furthermore, the invention relates to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (ANI) of at least 98.1% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM I-6138.

Description

[0001] METHODS OF REDUCING ENTERIC METHANE EMISSION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a feed composition for use in reducing enteric methane emissions and increasing milk yield and / or quality in ruminants. Also provided is methods of using the feed composition.

[0004] BACKGROUND

[0005] It is known the agricultural industry contributes to the production of greenhouse gases, particularly methane, from the rearing of livestock; for example, from ruminant animals such as cattle in the beef and dairy industries.

[0006] Enteric methane is produced by microbial fermentation in the rumen of the cattle during digestion and through belching, result in the emission of methane, a potent greenhouse gas into the atmosphere. To reduce cattle methane emissions a number of alternative feeds have been developed.

[0007] Feed additives like red seaweed (e.g. Asparagopsis taxiformis), plant extracts i.e. phytogenic feeds, and 3-Nitrooxypropanol (3-NOP) have proven effective in reducing enteric methane emissions when fed regularly as part of an animal's diet. Such feed additives are commercially available. For example, Bovaer™ (DSM Nutritional Products Ltd., Basel, Switzerland) comprises 3-NOP and on average, reduces enteric methane emissions by 30% from dairy cows and 45% from beef cattle. Other feed ingredients based on nitrate containing compounds (Silvair™- Cargill USA) have also been shown to have a methane mitigating effect when included in ruminant diets.

[0008] Despite the positive impact of existing feed additives such as Bovaer™ on enteric methane emissions, the additional energy available through the reduction in losses via methane production (which is between 2 and 12% of gross energy intake lost due to methane production), is not redirected to other metabolic pathways leading to more productivity, i.e. , milk production, in dairy cows. That is, the supplementation of animal feed with Bovaer™ had no effect on animal productivity.

[0009] Livestock farming contributes about 14.5% of total anthropogenic emissions of greenhouse gases, in which enteric methane (CH4), has been stated as the primary source (39.1 %). Hence, it is important to engage different practices to reduce the carbon footprint from livestock production. Bacteria-based interventions could be regarded as a safe option for mitigating the CH4 emission in cattle. Some bacteria species in cow's rumen are predominantly producing propionate as their end-fermentation product. Eventually the produced propionate would be used by the animals for their energy requirements such as milk yield.

[0010] Enteric methane emission represents an energy loss for ruminants (e.g., dairy cattle), varying between 2 and 12% of gross energy intake. It is believed that by reducing the CH4 synthesis in rumen, those spilled energy could be used to some level by cows for improving their productivity.

[0011] Current CH4 inhibitor feed additives and ingredients in the market such as 3-NOP or Nitrate, despite showing evident reduction of CH4 in cows are not able to increase the productivity in animals e.g., milk yield in dairy cows (van Gastelen et al., 2022; DOI 10.3168 / jds.2O21 -20782, Olijhoek et al., 2016; DOI 10.3168 / jds.2O15-10691 ). Direct-fed microbials (DFM) are defined, according to US Food and Drug administration, as the feed additive products containing only a source of live naturally existing microbes. DFMs can be divided as two main categories i.e., bacterial and fungal.

[0012] Bacterial strains isolated from cow's rumen as a solution to compete with methanogens and redirect the energy, via navigating the hydrogen away from methane synthesis, for potentially improving animal production (e.g., milk or meat) is quite compelling. More specifically, bacteria strains that are able to trigger the propionate pathway would be of interest as propionate production is an alternative route for hydrogen sink than methane synthesis by methanogenic archaea. The Propionibacterium family are among these groups of bacteria where propionate is among their main fermentation product. The outcome from this family of bacteria on methane emission reduction is not consistent and depends on the nature of cow's diet. In-vitro work using Propionibacterium thoenii T15 strain showed reduction of methane concentration, where the diet encompassed higher levels of neutral-detergent fiber (NDF) (Chen et al., 2020; DOI 10.1080 / 09064702.2020.1737215). Moreover, Propionibacterium jensenii in combination with a bacteria strain from genus Lactobacillus reduced methane emission in dairy cows fed only high NDF diet (Berger et al., 2012;

[0013] WO2012147044A1 ). Conversly, feeding dairy cows with certain dosage of Propionibacterium freudenreichii 53-\N in which dairy cows were fed high-starch content diet led to higher CH4 intensity compared with the negative control group (Jeyanathan et al., 2019; DOI 10.1186 / s40104-019-0342-9). However, a recent field dairy cow study using a combination of a methane inhibitor product (3-NOP) and mixture of two bacteria products, containing Bacillus subtilis and licheniformis and Propionibacterium acidipropionici did not result in synergistic reduction of CH4 nor higher milk yield in animals. Therefore, the results obtained previously have been very variable in their outcome.

[0014] There is therefore a need to simultaneously reduce methane emissions and increase animal productivity, while maintaining animal welfare. The present invention addresses this need.

[0015] Object of the invention

[0016] The objective of the present invention is to improve the state of the art and in particular to provide to simultaneously methane emissions reduction and increase animal productivity, while maintaining animal welfare. More particularly, the objective of the invention is to provide a feed composition comprising least one probiotic bacterium to provide these benefits. Summary of the invention

[0017] The present invention relates a feed composition comprising at least one probiotic bacterium which reduces methane emission from ruminants.

[0018] According to one aspect, the present invention relates to a feed composition comprising least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum.

[0019] According to a second aspect, the present invention relates to use of the feed composition according to the first aspect of invention for reducing enteric methane emissions and / or increasing milk production and / or quality in ruminants.

[0020] In a further aspect, the present invention relates to a method of reducing enteric methane emissions and / or increasing milk production and / or quality in ruminants, the method comprising administering the feed composition of any of to the first aspect of the invention a ruminant.

[0021] In an additional aspect, the invention relates to a method of improving the nutritional value of milk obtained from a ruminant, the method comprising administering the feed composition according to the first aspect of the invention and obtaining the milk.

[0022] In an additional aspect, the invention relates to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (ANI) of at least 98.1 % to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM 1-6138.

[0023] According to the invention, a new species of the Propionibacterium family with methane mitigation capacity and capture of hydrogen for propionate production has been found. The identified bacteria strain is genetically different and novel from the recent publication identifying a Propionibacterium ruminifibrarum isolate from cow rumen (Vaidya et al., 2019; DOI 10.1099 / ijsem.0.003544). Vaidya et al., only characterized their Propionibacterium ruminifibrarum isolate and no data has been shown regarding its methane reduction capability.

[0024] Without wishing to be bound by theory, the present results show that the novel bacteria strain (genus Propionibacterium of the species ruminifibrarum) is able to redirect the hydrogen away from methane production towards specific metabolite pathways such as propionate production, especially when the bacteria is combined with a methane inhibitor compound such as 3-NOP. This has not been observed by other species within the Propionibacterium family.

[0025] Furthermore, it has been found that the bacteria strain (genus Propionibacterium of the species ruminifibrarum) is able to reduce methane concentration and more importantly navigate the hydrogen into certain metabolite pathways which is beneficial for improving animal productivity. This was demonstrated in two different diets i.e. , diets high in neutral detergent fiber content (NDF) or starch, see e.g. Examples 1 and 2. The positive results on few of the Propionibacterium strains were obtained when the diet provided in the in-vitro test or fed to the animal had high NDF content. Positive results on both high NDF or high starch content diets shows that the bacteria strain (genus Propionibacterium of the species ruminifibrarum) is impactful in larger range of dairy cattle production systems.

[0026] Brief Description of the Drawings

[0027] These and other aspects of the invention will now be described, by way of example only, with reference to the accompanying figures in which:

[0028] Figure 1 shows the impact of Propionibacterium bacteria, 3-NOP, and their combination on gas production (selected and total), volatile fatty acids (selected and total) and in-vitro dry matter disappearance (IVDMD) after 24h in-vitro batch fermentation culture using high neutral detergent fibre (NDF) diet as feed substrate. The impact is shown proportionally to the control (100%), except for hydrogen (H2) that is proportionally to the 3-NOP, and statistical significance is visualized using Fisher’s Least Significant Difference post-hoc procedure (LSD, a=5%).

[0029] Figure 2 shows the impact of Propionibacterium bacteria, 3-NOP, and their combination on gas production (selected and total), volatile fatty acids (selected and total) and in-vitro dry matter disappearance (IVDMD) after 24h in-vitro batch fermentation culture using high starch diet as feed substrate. The impact is shown proportionally to the control (100%), except for hydrogen (H2) that is proportionally to the 3-NOP, and statistical significance is visualized using Fisher’s Least Significant Difference post-hoc procedure (LSD, a=5%).

[0030] Figure 3 shows the impact of bacteria strain A, and 3-NOP on gas production (total and selected) after 24 and 48h in-vitro batch fermentation culture using a 50:50 forage to concentrate ratio diet as feed substrate. The impact is shown proportionally to the control (100%), except for hydrogen (H2) that is proportionally to the 3-NOP, and statistical significance is visualized using Fisher’s Least Significant Difference post-hoc procedure (LSD, a=5%).

[0031] Figure 4 shows the in-vitro experimental set-up.

[0032] Figure 5 shows the serum bottles encompassing rumen digesta and Ankom bags containing the feed as substrate for the in-vitro batch fermentation culture.

[0033] Detailed description of the invention

[0034] Some example implementations of the present invention will now be further described. In the following passages, different aspects and embodiments of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. As discussed above, the present invention relates to a feed composition comprising least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum.

[0035] It has the surprising and unexpected been found that a feed composition comprising least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum has an effect on increasing milk yield, and / or reducing methane emissions, and / or increasing milk quality and / or increasing health of the ruminate.

[0036] Advantageously, the feed composition according to the invention comprises a methane inhibitor. Methane inhibitors may be selected from the group consisting of 3-nitroxypropanol, bromoform-containing seaweed species, Iodoform, halogenated compounds (natural or synthetic) such as chloroform, Iodoform, bromoform and their metabolites or a combination thereof. Preferably, the methane inhibitor comprises 3-nitroxypropanol.

[0037] In a preferred embodiment of the invention, the probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (ANI) of at least 98.1% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM 1-6138.

[0038] Alternatively, in the feed composition according to the invention the probiotic bacterium strain has an ANI of at least 99% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited under deposit number CNCM 1-6138.

[0039] Advantageously, the feed composition according to the invention is a Propionibacterium ruminifibrarum is a strain deposited under deposit number CNCM 1-6138. The present invention is based, at least in part, on a novel Propionibacterium ruminifibrarum strain. This Propionibacterium ruminifibrarum strain is referred to herein CNCM 1-6138, and was deposited with the Collection Nationale de Cultures de Micro-organisms (CNCM), Institute Pasteur by SOCIETE DES PRODUITS NESTLE S.A according to Budapest Treaty on the 06 November 2024 receiving the deposit number CNCM 1-6138.

[0040] In some embodiments, the Propionibacterium ruminifibrarum strain has an AN I of at least at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, compared to the Propionibacterium ruminifibrarum strain deposited with the CNCM under deposit number CNCM 1-6138.

[0041] Preferably, the Propionibacterium ruminifibrarum strain has an AN I of at least 99.9% compared to the Propionibacterium ruminifibrarum strain deposited with the CNCM under deposit number CNCM 1-6138.

[0042] Suitably, the Propionibacterium ruminifibrarum strain has an AN I of at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, of at least 98.5%, of at least 98.6%, of at least 98.6 %, of at least 98.7 %, of at least 98.8 %, of at least 98.9 %, of at least 99 %, of at least 99.1 %, of at least 99.2 %, of at least 99.3 %, of at least 99.4 %, of at least 99.5 %, of at least 99.6 %, of at least 99.7 %, of at least 99.8 %, or of at least 99.9 % compared to the Propionibacterium ruminifibrarum strain deposited with the CNCM under deposit number CNCM I- 6138.

[0043] Furthermore, according to the present invention, a surprising and unexpected discovery has been found in that combinations of 3-nitrooxypropanol and at least one propionate-producing probiotic bacterium according to the invention has an increased effect, specifically a synergistic effect on increasing milk yield, and / or reducing methane emissions, and / or increasing milk quality and / or increasing health of the ruminate. According to the invention, it has been found that combining an existing methane reducing feed additive, Bovaer™ with another feed supplement can enhance animal productivity by redirecting additional energy obtained from the inhibition of methane production as well as generate a cumulative effect on the enteric methane reduction in dairy cows.

[0044] It has been found that probiotics can be used as an alternative to redirect the additional energy available through the reduction in losses via methane production.

[0045] Accordingly, in one aspect of the invention there is provided a feed composition comprising a coenzyme-B sulfoethylthiotransferase (Methyl coenzyme reductase) inhibitor, preferably 3-nitrooxypropanol, and at least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium selected from the genus Propionibacterium.

[0046] A used herein “a feed composition” may be considered to be a combination of ingredients and their proportions that comprise an animal feed. A feed composition may also be referred to as a nutritional supplement. A nutritional supplement is a composition that can be added to the diet to provide macro and / or micro-nutrients.

[0047] According to the invention the propionate-producing bacterium is Propionibacterium ruminifibrarum Propionibacterium ruminifibrarum is a genus of gram-positive, anaerobic, rod-shaped bacteria able to synthesize propionate via transcarboxylase enzymes.

[0048] By 3-nitrooxypropanol may be meant a compound with the following structure: In one embodiment, the feed composition comprises Bovaer™.

[0049] The feed composition may further comprise at least one additional propionate- producing probiotic bacterium and / or at least one additional bacterium that increases the abundance of propionate producing bacteria. Preferably, the at least one additional bacterium is selected from the Bacillus genus.

[0050] Bacillus is a genus of gram-positive, anaerobic, rod-shaped bacteria. Species of Bacillus can be aerobes or facultative anaerobes. The species of Bacillus selected may also be able to produce propionate, for example through the propionate fermentation pathway from succinate. Examples of suitable species of Bacillus include Bacillus anthraci, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Bacillus coagulans, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus mycoides, Bacillus sphaericus, Bacillus thuringiensis israelensis, and Bacillus brevis. In one embodiment, the feed composition additionally comprises Bacillus subtilis. In another embodiment, the feed composition additionally comprises Bacillus licheniformis.

[0051] Preferably, the feed composition comprises Bacillus subtilis and Bacillus licheniformis. In one embodiment, the feed composition comprises Bovacillus™.

[0052] In a preferred embodiment of the invention the feed composition comprises 3- nitrooxypropanol and three probiotic bacterium, wherein the probiotic bacteria are Bacillus subtilis, Bacillus licheniformis and Propionibacterium acidipropionici.

[0053] The feed composition may comprise 30-150 mg / kg, preferably 60, 70 or 80 mg / kg of

[0054] 3-nitrooxypropanol (e.g. 30 - 150 mg / kg Bovaer™, preferably 60 - 80 mg / kg Bovaer™). The feed composition may comprise at least one probiotic bacterium comprising bacteria from the Propionibacterium genus in the feed at a dose of 5 - 20 mg / kg dry matter.

[0055] The feed composition may comprise at least one probiotic bacterium comprising at least one bacteria from the Bacillus genus is in the feed at a dose of 50 - 230 mg / kg dry matter, preferably at a dose of 120 - 160 mg / kg dry matter.

[0056] The feed composition may additionally comprise one or more carriers. Typical components of animal feed are cereal grains such as maize, sorghum, wheat, rice, oats, barley, corn and millet; brans, such as wheat bran, maize bran and deoiled rice bran; protein meals / cakes such as rapeseed meal / cake, soybean meal, cottonseed meal / cake, groundnut meal / cake, coconut meal / cake, palm kernel meal / cake, sesame cake, linseed cake, maize germ oil cake, maize gluten meal, sunflower meal, kardi meal and guar meal; and vitamins and minerals, such as calcite powder, salt, di-calcium phosphate and vitamins A, D3 and E. Preferably, carriers used in the present invention are spring barley and mineral mix.

[0057] The coenzyme-B sulfoethylthiotransferase inhibitor and the at least one probiotic bacterium may be part of the same composition, and as such, both the coenzyme-B sulfoethylthiotransferase inhibitor, such as 3-nitrooxypropanol, and the at least one probiotic bacterium are administered to the animal at the same time. Alternatively, the coenzyme-B sulfoethylthiotransferase inhibitor, such as 3- nitrooxypropanol, and the at least one probiotic bacterium are separate compositions. In this example, the separate compositions may be administered concurrently or sequentially.

[0058] In one embodiment, the feed composition comprises Bovaer™, Omni-Bos™ P169 and Bovacillus™. In one embodiment, the feed composition comprises 60 to 80mg / kg dry matter of Bovaer™, 120-160 mg / kg dry matter of Bovacillus™ and 5 to 20mg / Kg dry matter of Omni-Bos™.

[0059] In another embodiment of the invention, the composition comprises a yeast, preferably a yeast selected from the group consisting of Saccharomyces cerevisiae, Kluyvermyces species or Pichia jadinii (formerly Candida utilus), or one probiotic bacterium being Megasphaera elsdenii, or one probiotic bacterium being Lactobacillus, or a combination thereof. The yeast when supplemented in the feed entering the rumen has the effect of altering the rumen microbiome to promote microbial protein synthesis, stabilise rumen pH, increase fibre degradation, increase volatile fatty acid (VFA) production and cause minor shifts within the fermentation profile. Megasphaera elsdenii is able to utilize lactate for propionate production and further reduce the methane emission.

[0060] In another aspect of the invention, the feed composition increases the production of volatile fatty acids such as propionate in a ruminant.

[0061] In a further aspect, of the invention there is provided a method of increasing the production of volatile fatty acids such as propionate in a ruminant.

[0062] In another aspect of the invention, there is provided the use of the feed composition of the invention in reducing enteric methane emissions and / or increasing milk production and / or quality in ruminants.

[0063] In another aspect of the invention, there is provided a method of reducing enteric methane emissions and / or increasing milk production and / or quality in an animal, such as a ruminant, wherein the method comprises administering the feed composition of the invention to the animal.

[0064] In an embodiment the feed composition has a synergistic effect on increasing milk yield, and / or reducing methane emissions, and / or increasing milk quality of a ruminant.

[0065] In one embodiment, methane emissions are reduced by at least 16%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more compared to the level of methane emissions in an animal not fed the feed composition of the invention. Preferably, methane emissions are reduced by more than 30%. There are several ways to measure methane emissions from animals known in the art, these include: respiration chambers, the SF6- technique, breath sampling during milking and feeding, the GreenFeed method and use of a laser methane detector.

[0066] The respiration chamber method is the gold standard technique for measuring methane in ruminants. A single animal (or more) is confined in a chamber for between 2 and 7 days. Concentration of methane is measured at the air inlet and outlet vents of the chamber. The difference between outlet and inlet concentrations is multiplied by airflow to indicate methane emissions rate. Respiration chambers are available in a variety of materials with different chamber sizes and air flow rates.

[0067] In the SF6 tracer gas technique air is sampled near the animal’s nostrils through a tube attached to a halter and connected to an evacuated canister worn around the animal’s neck or on its back. A capillary tube or orifice plate is used to restrict airflow through the tube so that the canister is between 50% and 70% full after approximately 24 hours. A permeation tube containing SF6 is placed into the rumen of each animal. The pre-determined release rate of SF6 is multiplied by the ratio of methane to SF6 concentrations in the canister to calculate methane emission rate.

[0068] In methods to measure methane concentration in the breath of cows during milking and / or feeding (also known as “sniffer methods”) air is sampled near the animal’s nostrils through a tube fixed in a feed bin and connected directly to a gas analyser. The feed bin may be in an automatic milking station or in a concentrate feeding station. A variety of different gas analysers may be used such as: Nondispersive Infrared (NDIR), Fourier-transform infrared (FTIR) or photoacoustic infrared (PAIR). Methane concentration measured during a sampling visit of typically between 3 and 10 min may be specified as the overall mean, or the mean of eructation peaks. CO2 can be used as a tracer gas daily methane output can be calculated according to the ratio of methane to CO2 and daily CO2 output predicted from performance of the animal. GreenFeed is a method of measuring methane through breath samples, whereby breath samples are provided when animals visit a bait station. Samples of breath from individual animals are taken several times per day for short periods (3 to 7 min). GreenFeed is a portable standalone system used in barn and pasture applications, and incorporates an extractor fan to ensure active airflow and head position sensing for representative breath sampling. Measurements are pre- processed by the manufacturer, and data are available in real time through a web-based data management system. As GreenFeed captures a high proportion of emitted air and measures airflow, which can be calibrated using a tracer gas, methane emission is estimated as a flux at each visit. Providing visits occur throughout the 24 h, methane emission can be estimated directly as g / day (Garnsworthy PC, Difford GF, Bell MJ, Bayat AR, Huhtanen P, Kuhla B, Lassen J, Peiren N, Pszczola M, Sorg D, Visker MHPW, Yan T. Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle. Animals (Basel). 2019 Oct 21 ;9(10):837).

[0069] The laser methane detector (LMD) is a highly responsive, hand-held device that is pointed at an animal’s nostrils and measures methane column density along the length of the laser beam. Typically, animals are restrained either manually or in head yokes at a feed fence for the required length of time. The operator has to stand at the same distance (1 to 3 m) from each animal every time throughout. The LMD can be used in the animal’s normal environment, although for consistency restraint is required during measurement.

[0070] The term “increasing milk production” may include increasing milk volume yield; and / or increasing milk solids yield. In preferred embodiments, the term “increase in milk yield” means an increase in milk volume compared to the average volume produced by a ruminant which is not fed a feed composition of the invention. Milk yield can be measured as a volume over a given time. Typically, milk yield is measured as litres per day.

[0071] Milk yield may be increased by at least 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more compared to the yield of milk in an animal that is not fed a feed composition of the invention. Preferably, milk yield may be increased by 5 to 30%, more preferably by 5 to 15%.

[0072] Good-quality raw milk is free of debris and sediment, free of off-flavours, low in bacterial count, and low in somatic cell count (SCC). The total bacterial count is a measure of the number of bacteria in milk. A high total bacterial count can indicate that the milk is not fresh or that it has been contaminated. The SCC is a measure of the number of white blood cells in milk. A high SCC can indicate that the cow is infected with mastitis, a bacterial infection of the mammary gland. High quality milk is a rich source of protein, fat, calcium and other nutrients such as branched chain fatty acids (BCA’s). BCA’s include but are not limited to iso 13:0, iso 15:0, iso 17:02, iso 14:0, iso 16:0 iso 18:0, anteiso 13:0, anteiso 15:0 and anteiso 17:02.

[0073] In one embodiment, increasing the quality of milk may comprise increasing the content of at least one of milk fat and / or the fat to protein ratio of the milk. In a preferred embodiment milk fat is increased by around 1 - 20%, preferably 5 - 8%; and / or milk branched chain fatty acids are increased by around 1 - 20%, preferably around 5%; and / or the fat to protein ratio of the milk is increased by 1 - 30%, preferably around 10%, more preferably the fat to protein ratio of the milk is around 1 .2.

[0074] Any one of the above parameters may be increased by at least 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more compared to the parameters in an animal that is not fed a feed composition of the invention.

[0075] The quality of the milk may also be increased or improved by increasing the amount of branched chain fatty acids in the milk, such as 13:0, iso 15:0, iso 17:02, iso 14:0, iso 16:0 iso 18:0, anteiso 13:0, anteiso 15:0 and anteiso 17:02. Branched chain fatty acids are known to have a number of health benefits including, reducing inflammation, aiding weight maintenance, energy homeostasis and improved insulin sensitivity.

[0076] Accordingly, in a further aspect of the invention, there is provided a method of improving the nutritional value of milk obtained from a ruminant, the method comprising administering the feed composition of the invention and obtaining the milk.

[0077] In another aspect of the invention, there is provided a ruminate tissue or milk from a ruminate, where the tissue, milk or milk-derived product is obtained or obtainable by the method of the invention.

[0078] In another aspect of the invention there is provided a milk or milk-derived product, wherein the milk or milk-derived product comprises approximately a 1 - 20% increase, preferably a 5 - 8% increase in the concentration of branched chain fatty acids, compared to milk or milk derived products that are derived from cows that have not consumed the feed composition of the invention.

[0079] In all aspects described herein the animal is preferably a ruminant, and the ruminant is preferably a cow. Alternatively, the ruminant is a sheep or a goat.

[0080] Where the term “administered” is used herein it is also meant fed. Although other routes of administration are included in the scope of the present invention.

[0081] In another aspect of the invention, there is provided a method of improving the health of an animal, preferably a ruminant, wherein the method comprises administering the feed composition of the invention to an animal.

[0082] By “health of an animal” is meant any one of feed efficiency, milk somatic cell counts and / or health of the microbiome. Any one of these parameters may be increased when the feed composition of the invention is administered to the animal. Feed efficiency refers to the conventional measure of livestock milk production efficiency. Animals that have a low feed efficiency are considered to be efficient users of feed in the art. The formula used to calculate feed efficiency is:

[0083] Dry Matter Intake Feed Efficiency = - - - - -

[0084] Milk Production

[0085] In one embodiment, the feed efficiency is decreased by at least 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more compared to the feed efficiency is animals not administered the feed composition of the invention.

[0086] Somatic cell count (SCC) is a measure of the number of white blood cells in milk. A high SCC can indicate that the cow is infected with mastitis, a bacterial infection of the mammary gland. SCC is an indicator of udder health, and a low SCC is associated with good udder health.

[0087] In any of the methods described above, the feed composition may be administered for at least 4 weeks, preferably at least 10 weeks, more preferably around 12 weeks.

[0088] As discussed above, the invention relates also relates to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (ANI) of at least 98.1 % to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM 1-6138.

[0089] Furthermore, the invention relates to a probiotic bacterium strain which has an ANI of at least 99% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited under deposit number CNCM 1-6138. Additionally, the invention relates to a probiotic bacterium strain being a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum deposited with Collection nationale de cultures de micro- organismes (CNCM) under deposit number CNCM 1-6138.

[0090] In the present context, 16S rRNA genes sequencing is the most preferable method for identification, and classification of the bacteria strain, in which the reads from next-generation sequencing will be BLASTED against curated databases for bacteria characterization.

[0091] Advantageously, the bacteria strain is in the range of 1 * 10A8 CFU - 1 * 10A11 CFU, preferably 1 * 10A8 CFU - 1 * 10A10 CFU, more preferably 1 * 10A8 CFU - 1 * 10A9 CFU, most preferably 1 * 10A8 CFU depicting significant methane mitigation and re-direction of hydrogen in favour of propionate synthesis pathway.

[0092] The invention is now set out in the following non-limiting example.

[0093] Example 1 - Identification of Propionibacterium bacteria strain and efficacy on ruminal methane emission and fermentation in high neutral detergent fibre content feed in-vitro

[0094] The bacteria strain was isolated from the rumen content of a ruminant animal. Based on the 16S rRNA gene sequencing, the bacteria strain was classified within the Genus Propionibacterium. More specifically, the bacteria strain was identified as Propionibacterium ruminifibrarum.

[0095] In-vitro rumen fermentation culture it was found that the bacteria strain showed efficacy on reducing methane emission and redirection of hydrogen to certain metabolite pathways, particularly propionate production. Inoculation of the bacteria strain (1* 10A8 CFU) decreased methane concentration by 24% compared with the control group. The acetate / propionate ration was significantly lower (by 20%) in the bacteria group compared with control. The propionate concentration was significantly higher, whereas the butyrate concentration was lower in the bacteria group in comparison with the control. This indicates that the bacteria strain is changing the fermentation profile more favourable for propionate production. Furthermore, when the bacteria strain was combined with a methane inhibitor (e.g., 3-NOP), the bacteria was able to redirect the accumulated hydrogen (due to methanogenesis inhibition by the methane inhibitor compound) into production of propionate, which the animal could use as source of energy for productivity. This was evident in which concentration of hydrogen in the bacteria and methane inhibitor combination group was 26% lower in comparison to the methane inhibitor alone after 24h in-vitro fermentation culture test. Moreover, the concentration of propionate was significantly (by 32%) higher compared with the methane inhibitor alone. The ratio of acetate / propionate in the bacteria and methane inhibitor combination was also lower than the methane inhibitor alone

[0096] Example 2 - Propionibacterium efficacy on ruminal methane emission and fermentation in high starch content feed in-vitro

[0097] An additional 24h in-vitro rumen fermentation culture batch was carried out in which the feed used as substrate for bacterial fermentation was a high starch content diet. Results from the in-vitro test indicated that inoculation of the bacteria strain (1* 10A8 CFU) was able to significantly (by 16%) decrease the methane concentration than the control group. In the bacteria and methane inhibitor combination group, the hydrogen concentration was clearly reduced by 18% in comparison with the methane inhibitor (e.g., 3-NOP) alone.

[0098] Example 3 - Bacteria strain A efficacy on ruminal methane emission reduction in-vitro

[0099] Bacteria strain A was isolated from the rumen content of a ruminant animal. Based on the 16S rRNA gene sequencing, the bacteria strain has been classified within the Genus Clostridium. A 24h and 48h in-vitro rumen fermentation culture was conducted using a diet containing 50:50 forage to concentrate ratio as substrate for microbial fermentation in the bottles containing rumen digesta. The experiment contained a negative control (fermentation bottles containing feed as substrate without any treatment), Bacteria Strain A treatment, and positive control (fermentation bottles containing feed with treatment of a methane inhibitor compound i.e. , 3-NOP). Total gas production was not significantly different in the bacteria strain A group compared with negative control group. The methane concentration in the bacteria strain A group did not significantly differ from the negative control. The hydrogen concentration hardly changed after treatment with bacteria strain A in comparison with the negative control group.

[0100] (Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)

[0101] FOR RECEIVING OFFICE USE ONLY

[0102] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS:1 . A feed composition comprising least one probiotic bacterium, wherein the at least one probiotic bacterium is a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum.

2. Feed composition of claim 1 , wherein the composition comprises a methane inhibitor.

3. Feed composition of claim 2, wherein the methane inhibitor comprises 3- nitroxypropanol.

4. Feed composition according to claims 1 - 3, wherein the probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (AN I) of at least 98.1 % to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM 1-6138.

5. Feed composition according to claim 4, wherein the probiotic bacterium strain has an AN I of at least 99% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited under deposit number CNCM I- 6138.

6. Feed composition according to claims 1 -3, wherein the Propionibacterium ruminifibrarum is a strain deposited under deposit number CNCM 1-6138.

7. Feed composition according to claims 1 -6, wherein the composition comprises at least one further probiotic bacterium selected from the genus Bacillus.

8. Feed composition according to any of claims 1-7, wherein the at least one probiotic bacterium is Bacillus.

9. Feed composition according to any of claims 1-8, wherein the composition comprises a yeast, preferably a yeast selected from the group consisting of Saccharomyces cerevisiae, or one probiotic bacterium being Megasphaera elsdenii, or one probiotic bacterium being Lactobacillus, or a combination thereof.

10. Feed composition according to any of claims 1 -9, wherein the feed composition further comprises a carrier, wherein the carrier comprises a cereal grain and / or at least one vitamin.11 . Use of the feed composition according to any of claims 1 -10 in reducing enteric methane emissions and / or increasing milk production and / or quality in ruminants.

12. A method of reducing enteric methane emissions and / or increasing milk production and / or quality in ruminants, the method comprising administering the feed composition of any of claims 1 -10 to a ruminant.

13. Method according to claim 12, wherein enteric methane emissions are reduced by at least 16%, preferably at least 35%.

14. Method according to any of claims 12-13, wherein the methane inhibitor, preferably 3-nitrooxypropanol, and at least one propionate-producing bacterium are administered concurrently or separately and sequentially.

15. Method according to any of claims 12-14 wherein the ruminant is a cow.

16. A method of improving the nutritional value of milk obtained from a ruminant, the method comprising administering the feed composition according to claim 1-10 and obtaining the milk.

17. Milk obtained by the method of claim 16.

18. A probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum which has an Average Nucleotide Identity (ANI) of at least 98.1 % to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited with the CNCM under deposit number CNCM 1-6138.

19. A probiotic bacterium strain according to claim 18 which has an ANI of at least 99% to a probiotic bacterium strain being a propionate-producing bacterium strain of the genus Propionibacterium of the species ruminifibrarum deposited under deposit number CNCM 1-6138.

20. A probiotic bacterium strain being a propionate-producing bacterium of the genus Propionibacterium of the species ruminifibrarum deposited with Collection Nationale de Cultures de Microorganismes (CNCM) under deposit number CNCM 1-6138.

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