Use of lactic acid bacteria for improving feed efficiency

Abstract

The present invention relates to probiotic strains or derivatives thereof for increasing feed efficiency, enhancing growth and / or productivity, improving body weight or body composition, and / or increasing milk yield in ruminants, inhibiting growth of methanogenic bacteria and / or archaea bacteria in the forestomach of ruminants, reducing the ability of rumen microbiota to produce methane, and / or increasing milk yield in ruminants. Use for reducing methane emissions from ruminants, and / or reducing the footprint of greenhouse gas emissions from ruminants. Ruminant feed compositions are also provided.

Description

Technical Field

[0001] The present invention relates to the use of lactic acid bacteria strains for increasing feed efficiency, enhancing growth and / or productivity, improving the body weight or body composition of ruminants, and / or increasing milk production of ruminants, inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, reducing the ability of rumen microbiota to produce methane, reducing methane emissions from ruminants, and / or reducing the greenhouse gas emission footprint of ruminants. A ruminant feed composition is also provided. Background Art

[0002] Lactic acid bacteria (LAB) have been used as probiotics in humans with a variety of benefits. LAB have also been used in animals in an attempt to improve animal health and nutrition, with mixed results. They have also been studied as an alternative to antibiotics used as growth promoters.

[0003] On farm, LAB can be used as direct-fed microorganisms (DFM), probiotics, and silage inoculants. They act in a strain- and host-specific manner. Some studies have reported a variety of benefits, depending on the specific strain and host used, including reduced incidence of diarrhea, improved rumen development, improved feed efficiency, increased weight gain, and reduced morbidity. However, their effects on performance have been mixed, and the mode of action is unclear. (Krehbiel et al., 2003).

[0004] The use of LAB has also been proposed to reduce methane emissions from ruminants, with limited success.

[0005] The main source of methane emissions is the fermentation of organic matter by methanogenic bacteria and archaea. A common source of anthropogenic methane emissions is in agriculture, where methane is produced by enteric fermentation in the digestive tract of ruminants and from manure. These sources accounted for about 30% of total global anthropogenic methane emissions in 2017 (Jackson et al., 2020).

[0006] Furthermore, methanogenesis in ruminants not only contributes to greenhouse gas emissions, but is also energetically wasteful for the animals. It has long been recognized that methane production in ruminants significantly affects the efficiency with which these animals convert feed into metabolizable energy. Since methane represents a heat loss to ruminants of approximately 5-10% of their total caloric intake, this results in reduced efficiency. However, research into the potential use of LAB to reduce methane emissions has been limited to date.

[0007] Therefore, there is still a need for methods and compositions that can be used to increase feed efficiency, enhance growth and / or productivity, improve body weight or body composition of ruminants, and / or increase milk production of ruminants. It is also desirable to inhibit the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, reduce the ability of rumen microbiota to produce methane, reduce methane emissions from ruminants, and / or reduce the greenhouse gas emission footprint of ruminants.

[0008] It is an object of the present invention to go some way to fulfilling one or more of these desires, or at least to provide the public with a useful choice. Summary of the invention

[0009] In a first aspect, the present invention provides an isolated Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the NMIA accession number of the strain being V21 / 015448, dated August 2, 2021.

[0010] In one embodiment, the Lactobacillus rhamnosus strain FNZ142 is a biologically pure culture.

[0011] In a second aspect, the present invention provides a food or feed composition comprising Lactobacillus rhamnosus strain FNZ142 or its derivatives, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0012] In a third aspect, the present invention provides a ruminant feed composition for:

[0013] a. Increase feed efficiency of ruminants,

[0014] b. Enhance the growth and / or productivity of ruminants,

[0015] c. Improve the body weight and / or body composition of ruminants,

[0016] d. increasing the yield of milk and / or milk components produced by ruminants,

[0017] e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants,

[0018] f. Reduce the ability of the rumen microbiota to produce methane,

[0019] g. Reduce methane emissions from ruminants,

[0020] h. delivering microorganisms to ruminants, and / or

[0021] i. Reduce the greenhouse gas emissions footprint of ruminants,

[0022] A feed composition comprising Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or its derivative, the strain having NMIA accession number V21 / 015448 and dated August 2, 2021.

[0023] In some embodiments, the ruminant feed composition is a cattle feed composition. In some embodiments, the ruminant feed composition is a goat feed composition. In some embodiments, the ruminant feed composition is a sheep feed composition. In some embodiments, the ruminant feed composition is a bison feed composition. In some embodiments, the ruminant feed composition is a yak feed composition. In some embodiments, the ruminant feed composition is a buffalo feed composition. In some embodiments, the ruminant feed composition is a deer feed composition. In some embodiments, the ruminant feed composition is a camel feed composition. In some embodiments, the ruminant feed composition is an alpaca feed composition. In some embodiments, the ruminant feed composition is a llama feed composition. In some embodiments, the ruminant feed composition is a wildebeest feed composition. In some embodiments, the ruminant feed composition is an antelope feed composition. In some embodiments, the ruminant feed composition is a tailless feed composition.

[0024] In another aspect, the present invention provides a method for increasing feed efficiency of a ruminant, the method comprising the step of administering to the animal the food or feed composition according to the second aspect or the ruminant feed composition according to the third aspect.

[0025] In another aspect, the present invention provides a method for enhancing the growth and / or productivity of a ruminant, the method comprising the step of administering to the animal the food or feed composition according to the second aspect or the ruminant feed composition according to the third aspect.

[0026] In another aspect, the present invention provides a method for improving body weight and / or body composition of a ruminant, the method comprising the step of administering to the animal the food or feed composition according to the second aspect or the ruminant feed composition according to the third aspect.

[0027] In another aspect, the present invention provides a method for increasing the yield of milk and / or milk components produced by a ruminant, the method comprising the step of administering to the animal a food or feed composition according to the second aspect or a ruminant feed composition according to the third aspect.

[0028] In another aspect, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of a ruminant, the method comprising the step of administering to the animal the food or feed composition according to the second aspect or the ruminant feed composition according to the third aspect.

[0029] In another aspect, the present invention provides a method for reducing the ability of the rumen microbiota of a ruminant to produce methane, the method comprising the step of administering to the animal a food or feed composition according to the second aspect or a ruminant feed composition according to the third aspect.

[0030] In another aspect, the present invention provides a method for reducing methane emissions from ruminants, the method comprising the step of administering to the animal a food or feed composition according to the second aspect or a ruminant feed composition according to the third aspect.

[0031] In another aspect, the present invention provides a method for delivering a microorganism to a ruminant, the method comprising the step of administering to the animal the food or feed composition according to the second aspect or the ruminant feed composition according to the third aspect.

[0032] In another aspect, the present invention provides a method for reducing greenhouse gas emissions from ruminants, the method comprising the step of administering to the animal a food or feed composition according to the second aspect or a ruminant feed composition according to the third aspect.

[0033] On the other hand, the present invention provides a method for increasing feed efficiency of ruminants, wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0034] On the other hand, the present invention provides a method for enhancing the growth and / or productivity of ruminants, comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0035] On the other hand, the present invention provides a method for improving the weight and / or body composition of ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0036] On the other hand, the present invention provides a method for increasing the yield of milk and / or milk components produced by ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0037] On the other hand, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, wherein the method comprises administering to the ruminant an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0038] On the other hand, the present invention provides a method for reducing the ability of rumen microbiota to produce methane, wherein the method comprises administering to a ruminant an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0039] On the other hand, the present invention provides a method for reducing methane emissions from ruminants, wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0040] On the other hand, the present invention provides a method for delivering microorganisms to ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having NMIA accession number V21 / 015448, dated August 2, 2021.

[0041] On the other hand, the present invention provides a method for reducing the greenhouse gas emission footprint of ruminants, comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0042] On the other hand, the present invention provides a method for improving the absorption capacity of the digestive tract, such as increasing the absorption capacity of volatile fatty acids (VFA), wherein the method comprises administering to an animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0043] On the other hand, the present invention provides a method for improving the absorption capacity of the forestomach, such as increasing the absorption capacity of volatile fatty acids (VFA), wherein the method comprises administering to an animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0044] On the other hand, the present invention provides a method for improving the absorption capacity of the lower digestive tract, such as increasing the absorption capacity of volatile fatty acids (VFAs), wherein the method comprises administering to an animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0045] On the other hand, the present invention provides a method for enhancing the physical and / or functional development of the rumen or other cavities of the forestomach of a ruminant (e.g., a young ruminant, such as a young ruminant before weaning), wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0046] In some embodiments, the method increases the absorptive capacity of the forestomach and / or lower digestive tract. In some embodiments, the method increases the absorptive capacity of the forestomach and / or lower digestive tract by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or at least about 40% compared to untreated animals.

[0047] In one embodiment, the method enhances the anatomical development of the rumen. For example, the method enhances the development and / or muscularization of the rumen epithelium, such as increasing the growth of rumen masses, the growth of rumen papillae, an increase in papilla density (e.g., dorsal papilla density), and / or the total surface area of ​​the rumen wall of an animal.

[0048] In one embodiment, the method increases rumen weight, rumen wall thickness, rumen muscularization, and / or rumen thickness per cm, for example, compared to untreated animals. 2 Rumen papilla density of the rumen wall.

[0049] In one embodiment, the method increases rumen papilla length, width and / or surface area. For example, in some embodiments, the method increases rumen papilla length, width and / or surface area to at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.22, 1.24, 1.26, 1.28, 1.30, 1.32, 1.34, 1.36, 1.38, or 1.40 times that of an untreated animal.

[0050] In one embodiment, the method reduces rumen size. For example, in some embodiments, the method reduces rumen size by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26%, 28%, or 30% compared to untreated animals.

[0051] In one embodiment, the method enhances rumen function, or promotes maturation of the forestomach. For example, the method stimulates rumination, enhances dry matter intake (DMI), enhances absorptive capacity, and / or promotes maturation toward a mature physiology.

[0052] On the other hand, the present invention provides a method for changing the average residence time of digesta in the rumen or other chambers of the forestomach of a ruminant (e.g., a young ruminant, such as a young ruminant before weaning), wherein the method comprises administering to the ruminant an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0053] In some embodiments, the method reduces the mean residence time (MRT) of the digestate (e.g., pellets or liquid digestate) in the rumen. For example, the method reduces the mean residence time in the rumen by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or at least 40% compared to untreated animals. In some embodiments, the mean retention time is less than 30 hours, such as less than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 hours.

[0054] In some embodiments, the method inhibits the growth or reduces the abundance of methylotrophic methanogens in the digestive tract (e.g., forestomach and / or lower digestive tract) of an animal. In some embodiments, the method inhibits the growth or reduces the abundance of Methanosphaeria methanogens in the digestive tract (e.g., forestomach and / or lower digestive tract) of an animal.

[0055] In some embodiments, the methods reduce the abundance of Lachnospiraceae and / or Ruminococcaceae in the digestive tract (eg, forestomach and / or lower digestive tract) of an animal.

[0056] In some embodiments, Lactobacillus rhamnosus FNZ142 or a derivative thereof is administered in the form of a composition, which is a food, a beverage, a food additive, a beverage additive, an animal feed, an animal feed additive, an animal feed supplement, a dietary supplement, a carrier, a vitamin or mineral premix, a nutritional product, an enteral feeding product, a soluble, a slurry, a supplement, a medicine, a lick block, a drench pill, a tablet, a capsule, a pill or an intraruminal product, such as a bolus.

[0057] In another aspect, the present invention provides a composition comprising Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having NMIA accession number V21 / 015448, dated August 2, 2021. In some embodiments, the composition is a food, a beverage, a food additive, a beverage additive, an animal feed, an animal feed additive, an animal feed supplement, a dietary supplement, a carrier, a vitamin or mineral premix, a nutritional product, an enteral feeding product, a soluble, a slurry, a supplement, a medicament, a lick block, a draught pill, a tablet, a capsule, a pill, a bolus or an intraruminal product, or Lactobacillus rhamnosus FNZ142 is encapsulated in, for example, liposomes, microvesicles, microparticles or microcapsules.

[0058] In some embodiments, Lactobacillus rhamnosus FNZ142 or a derivative thereof is administered in drinking water, milk, milk powder, milk replacer, milk fortifier, whey, whey powder, part or total mixed ration (TMR), corn, soybeans, forage, grains, distiller's grains, sprouted grains, legumes, vitamins, amino acids, minerals, fiber, forage, grass, hay, straw, silage, nuts, leaves, meal, solubles, slurry, supplement, powdered feed, meal, fruit pulp, vegetable pulp, fruit or vegetable pulp, citrus meal, wheat middlings, corn cob meal, molasses, sucrose, maltodextrin, rice hulls, vermiculite, zeolite or crushed limestone.

[0059] In some embodiments, the method comprises administering to the animal an amount of at least about 10 Lactobacillus rhamnosus FNZ142 per kilogram of dry weight of carrier feed. 4 colony forming units, for example at least about 10 per kg dry weight of carrier feed 5 , at least about 10 6 , at least about 10 7 , at least about 10 8 , at least about 10 9 , at least about 10 10 , at least about 10 11 , at least about 10 12 or at least about 10 13 In some embodiments, the method comprises administering to the animal an amount of 10 4 Up to 10 13 In one embodiment, the method comprises administering to the animal Lactobacillus rhamnosus FNZ142 in an amount of 10 8 Up to 10 12 Colony forming units / kg dry weight of carrier feed.

[0060] In some embodiments, the derivative of Lactobacillus rhamnosus FNZ142 is a cell lysate of Lactobacillus rhamnosus FNZ142, a cell suspension of Lactobacillus rhamnosus FNZ142, a metabolite of Lactobacillus rhamnosus FNZ142, or a culture supernatant of Lactobacillus rhamnosus FNZ142, or killed Lactobacillus rhamnosus FNZ142. In some embodiments, the derivative of Lactobacillus rhamnosus FNZ142 is killed and / or does not replicate, for example, heat killed, lysed, pressure killed, irradiated and / or UV treated.

[0061] In some embodiments, the method comprises further administering at least one additional microorganism of a different species or strain, a vaccine that inhibits methanogens or methanogenesis, and / or a natural or chemically synthesized inhibitor of methanogenesis and / or an inhibitor of methanogens. An example of a useful methanogenic inhibitor is bromoform, which acts by inhibiting the efficiency of methyltransferases by reacting with the reduced vitamin B12 cofactor required for the penultimate step of methanogenesis.

[0062] In one embodiment, the method further comprises administering at least one different species or strain of microorganism, a vaccine that inhibits methanogens or methanogens, and / or a natural or chemically synthesized inhibitor of methanogens and / or a methanogen inhibitor that targets hydrogenotrophic methanogens (e.g., methanogens from the genus Methanobacterium).

[0063] In some embodiments, Lactobacillus rhamnosus FNZ142 or a derivative thereof is administered separately, simultaneously or sequentially with one or more agents selected from one or more prebiotics, one or more probiotics, one or more postbiotics, one or more dietary fiber sources, one or more galacto-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, one or more fructo-oligosaccharides, one or more short-chain fructo-oligosaccharides, one or more long-chain fructo-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, one or more milk-derived oligosaccharides (e.g., 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose) or any mixture of any two or more thereof.

[0064] In some embodiments, the method also enhances the growth or productivity of an animal, for example, the method increases the yield of milk and / or milk components produced by a ruminant. In some embodiments, the method increases the yield of milk fat, milk protein or milk solids in milk produced by an animal.

[0065] In some embodiments, the methods also increase body weight and / or improve body composition of the ruminant, such as altering the ratio of muscle to fat.

[0066] In some embodiments, the methods also increase wool growth in ruminants.

[0067] In some embodiments, the ruminant is a cow, goat, sheep, bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope or bison. In one embodiment, the ruminant is a cattle or sheep. In one embodiment, the ruminant is a cattle. In one embodiment, the ruminant is a lactating animal. In another embodiment, the ruminant is a pre-weaned animal, such as a calf or lamb.

[0068] In some embodiments, the ruminant feed composition is or includes part or total mixed ration (TMR), corn, soybeans, forage, grains, distiller's grains, sprouted grains, legumes, fiber, forage, grass, hay, straw, silage, nuts, leaves, meal, mash feed, fruit pulp, vegetable pulp, fruit or vegetable pulp, citrus meal, wheat semolina, corn cob meal, lick block, or molasses.

[0069] In some embodiments, the method comprises further administering at least one additional microorganism of a different species or strain, a vaccine that inhibits methanogens or methanogenesis, and / or a natural or chemically synthesized methanogenesis inhibitor and / or methanogen inhibitor.

[0070] In one embodiment, the method further comprises administering at least one different species or strain of microorganism, a vaccine that inhibits methanogens or methanogens, and / or a natural or chemically synthesized inhibitor of methanogens and / or a methanogen inhibitor that targets hydrogenotrophic methanogens (e.g., methanogens from the genus Methanobacterium).

[0071] In some embodiments, the ruminant feed composition further comprises one or more agents selected from the group consisting of one or more prebiotics, one or more probiotics, one or more postbiotics, one or more dietary fiber sources, one or more galacto-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, one or more fructo-oligosaccharides, one or more short-chain fructo-oligosaccharides, one or more long-chain fructo-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

[0072] In another aspect, the present invention provides a ruminant to which the method of the preceding aspect has been applied.

[0073] In another aspect, the present invention provides a method for producing an animal product with a reduced greenhouse gas emission footprint, the method comprising:

[0074] a. Provide the ruminant of the aforementioned aspect, and

[0075] b. Producing animal products from animals.

[0076] In some embodiments, the animal product includes dairy, meat, or wool.

[0077] In another aspect, the present invention provides a use of Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or a derivative thereof, the strain having NMIA accession number V21 / 015448, dated August 2, 2021, for preparing a composition, the composition being used for:

[0078] a. Increase feed efficiency of ruminants,

[0079] b. Enhance the growth and / or productivity of ruminants,

[0080] c. Improve the body weight and / or body composition of ruminants,

[0081] d. increasing the yield of milk and / or milk components produced by ruminants,

[0082] e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants,

[0083] f. Reduce the ability of the rumen microbiota to produce methane,

[0084] g. Reduce methane emissions from ruminants,

[0085] h. delivering microorganisms to ruminants, and / or

[0086] i. Reduce the greenhouse gas emissions footprint of ruminants.

[0087] In some embodiments, the composition is or comprises a ruminant feed composition according to the third aspect.

[0088] In another aspect, the present invention provides a Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having NMIA accession number V21 / 015448, dated August 2, 2021, for use in:

[0089] a. Increase feed efficiency of ruminants,

[0090] b. Enhance the growth and / or productivity of ruminants,

[0091] c. Improve the body weight and / or body composition of ruminants,

[0092] d. increasing the yield of milk and / or milk components produced by ruminants,

[0093] e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants,

[0094] f. Reduce the ability of the rumen microbiota to produce methane,

[0095] g. Reduce methane emissions from ruminants,

[0096] h. delivering microorganisms to ruminants, and / or

[0097] i. Reduce the greenhouse gas emissions footprint of ruminants.

[0098] The invention may also be described broadly as present in the parts, elements and features mentioned or indicated in the specification of the invention, either individually or collectively, in any or all combinations of two or more parts, elements or features, and where specific integers are mentioned herein that have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0099] Numerical ranges disclosed herein (e.g., 1 to 10) also include all rational numbers within the referenced ranges (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any range of rational numbers within the ranges (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be expressly stated in this application in a similar manner.

[0100] The term "comprising" used in this specification means "consisting at least in part of..." When interpreting each expression including the term "comprising" in this specification, features other than the term or those beginning with the term may also exist. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

[0101] In this specification where reference has been made to patent specifications, other external documents or other sources of information, this is generally to provide a context for discussing the features of the invention. Unless expressly stated otherwise, reference to such external documents should not be construed as an admission that such documents or such sources of information are prior art or form part of the common general knowledge in the art in any area of ​​rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The present invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0103] Figure 1 Body weights of heifers treated with FNZ142 (squares) or control treatment (triangles) for the first 14 weeks of life and subsequently after moving to pasture are shown. * indicates a significant difference between FNZ142 and control treatments (p<0.05). DETAILED DESCRIPTION

[0104] The present invention is based on the discovery that the rhamnosus lactobacillus strain FNZ142 and its derivatives increase the feed efficiency of ruminants. It is also shown herein that FNZ142 and its derivatives inhibit or suppress the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants and / or reduce the ability of rumen microbial population to produce methane. Inhibiting or curbing the growth of methanogenic bacteria and / or archaea can reduce methane emissions and can change the volatile fatty acid (VFA) spectrum, total VFA concentration, residual feed intake (RFI) and / or fermentation rate in the rumen and forestomach, which can serve as an energy source for driving increased feed efficiency, increased weight gain and / or increased productivity (such as milk, meat or wool production), and can stimulate rumen development, such as rumen papilla development.

[0105] Therefore, in a first aspect, the present invention provides an isolated Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0106] In a second aspect, the present invention provides a food or feed composition comprising the Lactobacillus rhamnosus strain FNZ142 or its derivatives, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0107] In a third aspect, the present invention provides a ruminant feed composition for:

[0108] a. Increase feed efficiency of ruminants,

[0109] b. Enhance the growth and / or productivity of ruminants,

[0110] c. Improve the body weight and / or body composition of ruminants,

[0111] d. increasing the yield of milk and / or milk components produced by ruminants,

[0112] e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants,

[0113] f. Reduce the ability of the rumen microbiota to produce methane,

[0114] g. Reduce methane emissions from ruminants,

[0115] h. delivering microorganisms to ruminants, and / or

[0116] i. Reduce the greenhouse gas emissions footprint of ruminants,

[0117] A feed composition comprising Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or its derivative, the strain having NMIA accession number V21 / 015448 and dated August 2, 2021.

[0118] On the other hand, the present invention provides a method for increasing feed efficiency of ruminants, wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0119] On the other hand, the present invention provides a method for enhancing the growth and / or productivity of ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0120] On the other hand, the present invention provides a method for improving the weight and / or body composition of ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0121] On the other hand, the present invention provides a method for increasing the yield of milk and / or milk components produced by ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0122] On the other hand, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, wherein the method comprises administering to the ruminant an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0123] In another aspect, the present invention provides a method for reducing the ability of rumen microbiota to produce methane, wherein the method comprises administering to a ruminant an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0124] On the other hand, the present invention provides a method for reducing methane emissions from ruminants, wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0125] On the other hand, the present invention provides a method for delivering microorganisms to ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having NMIA accession number V21 / 015448, dated August 2, 2021.

[0126] On the other hand, the present invention provides a method for reducing the greenhouse gas emission footprint of ruminants, the method comprising the step of administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0127] On the other hand, the present invention provides a method for improving the absorption capacity of the forestomach, such as increasing the absorption capacity of volatile fatty acids (VFA), wherein the method comprises administering to an animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0128] On the other hand, the present invention provides a method for enhancing the physical and / or functional development of the rumen of a ruminant (e.g., a young ruminant, such as a young ruminant before weaning), wherein the method comprises administering to the animal an effective amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof, the strain having an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

[0129] In one embodiment, the methods and compositions enhance the anatomical development of the rumen. For example, the methods enhance the development and / or muscularization of the rumen epithelium, such as increasing the growth of rumen masses, the growth of rumen papillae, the increase in papilla density (e.g., dorsal papilla density), and / or the total surface area of ​​the rumen wall of an animal.

[0130] In one embodiment, the methods and compositions disclosed herein increase rumen weight, rumen wall thickness, or 2 Rumen papilla density of the rumen wall.

[0131] In one embodiment, the method reduces rumen size. For example, in some embodiments, the method reduces rumen size by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26%, 28%, or 30% compared to untreated animals.

[0132] In one embodiment, the methods and compositions disclosed herein enhance rumen function. For example, the methods stimulate rumination and / or enhance dry matter intake (DMI).

[0133] In one embodiment, the methods and compositions disclosed herein alter the abundance of heterofermentative anaerobes in the rumen microbiota. In one embodiment, the methods and compositions disclosed herein increase the abundance of heterofermentative anaerobes in the rumen microbiota.

[0134] In one embodiment, the methods and compositions disclosed herein increase rumen turnover, post-rumen digestion, post-rumen absorption, or any combination of any two or more of these. Without wishing to be bound by theory, it has been hypothesized that microorganisms capable of rapid heterofermentative growth on soluble sugars that produce less hydrogen (which results in less methane formation) select for higher rumen turnover. For example, Kamke et al. (2016) noted that lactate is converted to butyrate rather than propionate, producing 2 mol of hydrogen per hexose, which can produce 0.5 mol of methane through a hydrogenotrophic pathway, and assuming that direct fermentation of hexose into butyrate and acetate by, for example, members of the Ruminococcaceae will produce 2.66 mol of hydrogen and allow the formation of 0.66 mol of methane. Therefore, it is predicted that lower hydrogen production via the lactate to butyrate pathway reduces methane production.

[0135] In some embodiments, the methods and compositions disclosed herein reduce the mean residence time (MRT) of a digestate (e.g., a pellet or liquid digestate) in the rumen. For example, the methods reduce the mean retention time by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or at least 40% compared to untreated animals.

[0136] The term "administering" refers to the action of introducing an effective amount of the Lactobacillus rhamnosus strain FNZ142 or a derivative thereof into the forestomach of a ruminant. More specifically, administration is by the oral route. Administration can be carried out in particular by supplementing the strain to the animal's feed or drink; the animal then ingests the supplemented feed or drink.

[0137] The term "effective amount" is an amount of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof sufficient to produce the desired effect, i.e., inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of an animal, reducing methane emissions from an animal, or increasing feed efficiency of an animal, compared to a reference. The desired effect (e.g., inhibiting the growth of methanogenic bacteria and / or archaea and / or reducing methane production or emissions) can be measured in vitro or in vivo. For example, the methods described herein can be used, e.g., in the examples below, in an artificial rumen system, e.g., in T. Hano (1993) J. Gen. Appl. Microbiol., 39, 35-45, or by oral administration to ruminants in vivo.

[0138] An effective amount may be administered to the ruminant in one or more doses.

[0139] The terms "reducing methane production" and "reducing methane emissions", such as "reducing methane production by an animal" and "reducing methane emissions by an animal" refer to reducing methane production or emissions by any mechanism and from any ruminant-related source. For example, the term may refer to a reduction in methane produced in the forestomach of a ruminant, or it may refer to a reduction in methane produced or emitted by the feces of a ruminant.

[0140] It is expected that the reduction in methane production may be due to a variety of mechanisms. These may include, for example, killing methanogens (i.e., bactericidal / archaeocidal effects), inhibiting the growth of methanogens (i.e., bacteriostatic / archaeocidal effects), and / or inhibiting the ability of the forestomach or rumen microbiome to produce methane. Inhibiting the ability of the forestomach or rumen microbiome to produce methane may be through a variety of mechanisms, including, for example, physical and / or chemical changes in the forestomach or rumen environment, changes in the microbiome, inhibition of one or more methanogenic pathways, and / or cross-feeding (or disruption of cross-feeding) of intermediates between members of the microbiome.

[0141] It should be understood that it is desirable to reduce greenhouse gas (GHG) emissions, such as methane emissions. GHG emissions can be reduced directly, such as by reducing the ability of rumen microbial populations to produce methane and / or by reducing methane emissions of ruminants, or indirectly. An example of indirectly reducing GHG emissions is by changing land use and / or land retirement. Animals with improved feed efficiency (such as animals to which the method or composition of the present invention has been applied) may require less pasture feed and / or less imported feed. Alternatively or additionally, more animals can be raised on a given land area, thereby achieving the same output when land use is reduced. In either case, the reduced land demand can allow, for example, to retire unused pastures for carbon sequestration by planting trees or other vegetation. Such land use changes can further reduce the GHG emissions of each farm, resulting in a lower GHG emission footprint for each animal and / or per kg of animal products (such as milk, meat or wool).

[0142] The GHG emissions footprint of animals and / or animal products can be determined using techniques known in the art. It should be understood that some GHGs produce more global warming potential than others. For example, 1 kg of methane emissions produces a global warming impact approximately equal to 25 kg of CO2. With this in mind, GHG emissions are typically reported as CO2 equivalents (CO 2e ), which is the amount of CO2 that has an equivalent global warming impact. The GHG emissions footprint can be calculated per animal or per amount of animal product (e.g. per kg milk solids, per kg meat or per kg wool). As mentioned above, the GHG emissions footprint should take into account changes in land use, such as planting trees or other vegetation for carbon sequestration.

[0143] The term "animal product" refers to any product produced by or from an animal, or any product containing any component of animal origin. The term is intended to include products produced directly from animals (e.g., milk, meat and wool) and products that include or are made from animal ingredients, which have optionally undergone further processing, optionally together with other ingredients. For example, the term is intended to include foods and beverages containing animal ingredients, such as various dairy products (including buttermilk, cheese, cream, formula, ice cream, milk, milk powder, pudding, milkshakes, smoothies and yogurt), meat products (e.g., French fries, minced meat, hamburgers, sausages, sausage meat and steak) and other products containing animal ingredients.

[0144] The term "feed efficiency" refers to the relationship between feed intake and muscle weight gain or milk yield. Microbial fermentation in the forestomach or rumen produces volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. These fatty acids are directly absorbed from the rumen wall and used as raw materials for the growth and development of animals, milk components and other final digestive products. Fatty acids and other nutrients can also be absorbed in the lower digestive tract such as the small intestine and / or large intestine. Most of the energy consumed by body tissues is used to produce milk or milk components, or muscle. Therefore, when increasing energy absorption and / or utilization, milk production, such as milk yield, and / or milk fat, milk protein and / or milk solids can be increased. It is also possible to achieve an increase in muscle and / or an improvement in body composition, such as changing the muscle / fat ratio of an animal.

[0145] Feed efficiency can be calculated by dividing the weight of the milk produced by the animal by the weight of the dry matter consumed by the animal. Therefore, when given the same nutrient input, an animal with a higher feed efficiency will produce more milk, milk with higher content of milk components such as but not limited to fat and protein, and / or will show increased weight gain compared to an animal with a lower feed efficiency. Feed efficiency can be measured by differences in animal growth by any of the following parameters: average daily weight gain, total weight gain, feed conversion ratio, which includes two feeds: gain and gain: feed, mortality and feed intake. That is, increased feed efficiency can mean that the ratio of feed intake / muscle weight increase is reduced. Increased feed efficiency can also mean that the ratio of muscle weight increase / feed intake is increased. The term feed efficiency can also refer to feed intake / weight gain or weight gain / feed intake. Feed efficiency can be standardized to take into account differences in protein and fat content by using energy-corrected milk (ECM) yield instead of milk weight. This can be measured using the following formula (Tyrrell and Reid, 1965):

[0146] ECM = (12.82 x fat weight in pounds) + (7.13 x protein weight in pounds) + (0.323 x milk weight in pounds).

[0147] "Feed conversion ratio" and "residual feed intake (RFI)" are also common measures of feed efficiency, and these terms are often used almost interchangeably. In animal husbandry, feed conversion ratio or feed conversion rate is a ratio or rate measure of the efficiency of the livestock body in converting the animal's feed into the desired output. RFI is defined as the difference between the animal's actual dry matter intake (DMI) and the expected DMI required for maintenance and growth.

[0148] The main advantage of improving feed efficiency (i.e., improving feed conversion or reducing RFI) is to reduce DMI in animals without compromising growth performance, since feed-related costs usually represent the largest production expense in beef or dairy production. Any reduction in DMI per unit of beef or dairy product produced will minimize feed costs, thereby maximizing the overall profitability of the beef or dairy industry.

[0149] In one embodiment, feed efficiency in a ruminant is increased to at least about 1.01×, e.g., at least about 1.02×, 1.03×, 1.04×, 1.05×, 1.06×, 1.07×, 1.08×, 1.09×, 1.10×, 1.12×, 1.14×, 1.16×, 1.18×, e.g., at least about 1.20×, of the feed efficiency of an untreated animal.

[0150] Increased feed efficiency may be due to changes in volatile fatty acid (VFA) distribution, total VFA concentration and / or fermentation rate in the rumen and forestomach. Alternatively or additionally, increased feed efficiency may also result from improved nutrient absorption due to improved digestive tract absorptive capacity. In some embodiments, the method improves the absorptive capacity of the digestive tract (e.g., forestomach and / or lower digestive tract), such as increasing the absorptive capacity of volatile fatty acids (VFA).

[0151] Alternatively or additionally, increased feed efficiency may also result from improved digestion. This may allow treated animals to digest food sources (such as poor quality forage) more efficiently, thereby obtaining an increased amount of energy compared to untreated animals.

[0152] In some embodiments, Lactobacillus rhamnosus FNZ142 or its derivatives transfer hydrogen metabolism from methane generation to short-chain / volatile fatty acid (VFA) production, such as conversion to propionic acid production. Propionate is mainly used as a glucose precursor in ruminants, and more propionate formation will likely lead to more efficient utilization of feed energy. Maximizing the flow of metabolic hydrogen away from methane and toward VFA (mainly propionate) in the forestomach or rumen will increase the efficiency of ruminant production and reduce its environmental impact, and will enhance rumen development and / or rumen papilla development.

[0153] Acetate is the major substrate for mammary gland lipid synthesis, as well as β-hydroxybutyrate produced during butyrate absorption. Therefore, a high acetate fermentation pattern would provide substrate to maintain or increase milk fat.

[0154] Thus, in some embodiments, Lactobacillus rhamnosus strain FNZ142 or a derivative thereof results in an increase in milk fat, milk protein, total milk volume and / or milk solids as a result of increased VFAs in the forestomach or rumen, which may serve as an increased energy source driving increased production.

[0155] In some embodiments, the yield of milk and / or milk components produced from the animal is preferably increased by 1.5% or more, more preferably by 3.0% or more, by 4.5% or more, or by 6.0% or more.

[0156] In some embodiments, Lactobacillus rhamnosus strain FNZ142 or a derivative thereof results in an increase in live weight, muscle mass and / or fat deposition, and / or an improvement in body composition, such as an altered muscle / fat ratio in animals due to increased VFAs in the forestomach or rumen, which can serve as an increased energy source driving increased production.

[0157] In some embodiments, the live weight of the animal is preferably increased by 1% or more, more preferably 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more compared to a reference animal.

[0158] It is expected that the present invention can also be used to extend the lactation cycle of lactating ruminants (e.g., cows). Cows direct most of their energy to milk production during lactation. After long-term lactation, their physical condition will be worse. Therefore, the lactation period is usually shortened or reduced to prevent excessive deterioration of physical condition. It is expected that the methods and ruminant feed compositions disclosed herein will increase the feed efficiency of ruminants and therefore reduce the impact of milk production on physical condition. As a result, cows can be made to last longer.

[0159] It is also expected that the present invention can also be used to reduce or improve the physical condition deterioration caused by lactation. It is expected that the method disclosed herein and ruminant feed composition will increase the feed efficiency of ruminants, and therefore cause ruminants to have improved physical condition at the end of lactation. For example, when animals enter the dry period, animals have a higher body condition score (BCS). As a result, ruminants need less dry matter intake during the off-season to obtain physical condition. Alternatively or additionally, the method disclosed herein and ruminant feed composition can be used to improve the physical condition of animals before lactation. For example, the method disclosed herein and composition can improve the body composition of mother and / or fetus or newborn. For example, the method disclosed herein and composition can improve the body composition and / or body weight of newborns at birth.

[0160] It is also contemplated that the present invention may be similarly used to reduce or ameliorate deterioration in body condition during other stressful times, such as calving, drought or inadequate feed intake.

[0161] Live weight and body condition scores are commonly used in the industry as measures of animal growth and performance.

[0162] Live weight is an objective measure used to assess animal growth and is one of the best measures of animal performance. It is the primary measure used by the dairy industry to indicate how well grown dairy heifers are. Significant research has focused on the relationship between live weight and performance, particularly heifer (dairy cow) performance, and has led to the identification of live weight targets. Achieving live weight targets will optimize lifetime performance of heifers, increase stock life and return on investment for agribusinesses.

[0163] In New Zealand, heifer liveweight targets are 30, 60 and 90 percent of mature liveweight at 6, 15 (before mating) and 22 (before grazing) months of age. The 22-month target includes adjustments for pregnancy. Cows that grow to liveweight targets are also more likely to meet body condition score (BCS) targets at calving, which contributes to better milk production in the first lactation.

[0164] Body Condition Score (BCS) is a subjective measure used to assess animal performance and ensure animal welfare is maintained. The industry standard dairy cow BCS scale is for heifers over 20 months of age. The BCS target for cows at 22 months of age (pre-calibrated) is 5.5.

[0165] There is a well-established relationship between live weight and milk yield. Therefore, one of the main benefits of achieving a live weight target is increased milk yield.

[0166] Liveweight of heifers prior to calving has been shown to have a significant effect on milk production in various studies in New Zealand (Handcock et al., 2019; McNaughton, LR and T Lopdell. 2013; MacDonald et al., 2005; van der Waaij et al., 1997) and overseas (Carson et al., 2002; Dobos et al., 2001).

[0167] If a 9% milk solids test is assumed, the expected response is approximately 2 kg milk solids per lactation for each 1% increase in target live weight achieved. For heifers with a pre-conditioned live weight target of 500 kg, 5 kg equals 1% live weight. Van der Waaij et al. (1997) reported a response of 6 L milk and 0.43 kg milk solids / kg live weight, while Dobos et al. (2001) reported a response of 5.35 L milk and 0.42 kg milk solids / kg live weight at first calving. For a 5 kg advantage, multiplying these values ​​by 5 gives 26.8-30 L milk and 2.1-2.15 kg milk solids for each 1% increase in live weight. McNaughton, LR and T Lopdell (2013) reported a similar response where in precalibrated cows, each 1% increase in percent target liveweight achieved was associated with an increase in milk yield of 23 ± 0.6 L in first lactation and 24 ± 0.9 L in second lactation (P < 0.001).

[0168] Thus, in some embodiments, the methods and compositions disclosed herein increase milk production, such as the yield of milk and / or milk components produced by ruminants. In some embodiments, the methods and compositions disclosed herein increase the yield of milk fat, milk protein, or milk solids in milk produced from animals.

[0169] In some embodiments, the methods and compositions disclosed herein increase first lactation milk production. In some embodiments, the methods and compositions disclosed herein increase the cumulative milk production of multiple lactations, such as the first two or three lactations. In some embodiments, the methods and compositions disclosed herein increase the cumulative milk production of all lactations of an animal.

[0170] In some embodiments, the milk production of the animal is preferably increased by 1% or more, more preferably by 2% or more, 3% or more, 4% or more, or 5% or more, compared to untreated animals.

[0171] In some embodiments, the milk production of the animal is preferably increased by 5 kg or more milk solids per lactation, more preferably by 6 kg or more, 7 kg or more, 8 kg or more, 9 kg or more, 10 kg or more, 11 kg or more, 12 kg or more, or 13 kg or more milk solids per lactation, compared to untreated animals.

[0172] In some embodiments, the milk production of the animal is preferably increased by 60 L or more milk solids per lactation, more preferably by 70 L or more, 80 L or more, 90 L or more, 100 L or more, 110 L or more, 120 L or more, or 130 L or more per lactation compared to untreated animals.

[0173] As described above, the methods and compositions disclosed herein enhance the physical and / or functional development of the rumen, particularly in the early life of young or pre-weaned ruminants. The development of the rumen involves three distinct processes: (i) anatomical development (e.g., growth of rumen mass and growth of rumen papillae), (ii) functional realization (e.g., fermentation capacity and enzyme activity), and (iii) microbial colonization (bacteria, fungi, methanogens, and protozoa).

[0174] The anatomical development of the rumen is a process that occurs in three stages: non-rumination (0-3 weeks), transition (3-8 weeks), and rumination (starting at 8 weeks). During the transition phase, the growth and development of the rumen absorptive surface area (papillae) is necessary to be able to absorb and utilize digestion end products, especially rumen volatile fatty acids. The presence and absorption of volatile fatty acids stimulate rumen epithelial metabolism and may be key to initiating rumen epithelial development. Continuous exposure to volatile fatty acids maintains rumen papilla growth, size, and function. Different volatile fatty acids stimulate this development differently, with butyrate being the most stimulating, followed by propionate. Therefore, it is expected that shifting hydrogen metabolism from methane formation to short-chain / volatile fatty acid (VFA) production, such as to propionate production, will enhance rumen epithelial growth and development.

[0175] Ruminants

[0176] Ruminants are a group of herbivores with a stomach comprising multiple compartments, which digest their food by the first microbial fermentation in the rumen to form a cud, regurgitate and chew the cud, and then swallow the chewed cud for further digestion. The group includes but is not limited to ruminants and the suborder Mastiopoda, and includes several domesticated livestock. In one embodiment, the ruminant is a cow, goat, sheep, bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope or bison. In a preferred embodiment, the ruminant is a cow or sheep.

[0177] The term "digestive tract" refers to the entire tract of the digestive system leading from the mouth to the anus. In ruminants, the digestive tract includes (but is not limited to) the mouth, esophagus, multi-compartment stomach, small intestine, cecum, large intestine and anus.

[0178] The ruminant stomach is divided into the non-glandular forestomach (rumen, reticulum, omasum) and the terminal glandular stomach, abomasum. The term "fore stomach" refers to the non-glandular part of the multi-compartment stomach of ruminants, including the rumen, reticulum and stomach, but excluding the terminal glandular stomach (abomasum).

[0179] The term "lower gastrointestinal tract" refers to the portion of the gastrointestinal tract after the stomach, including, but not limited to, the small intestine and the large intestine.

[0180] In one embodiment, the ruminant is a lactating animal. In another embodiment, the ruminant is a pre-weaned animal, such as a calf or a lamb.

[0181] In some embodiments, the ruminant is newborn, newborn or young. For example, in some embodiments, the age of the ruminant is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or 2 months.

[0182] In some embodiments, Lactobacillus rhamnosus strain FNZ 142 or a derivative thereof is administered to a ruminant before weaning. In some embodiments, Lactobacillus rhamnosus FNZ 142 or a derivative thereof is administered to a ruminant after weaning. In some embodiments, Lactobacillus rhamnosus FNZ 142 or a derivative thereof is administered to a ruminant before and after weaning. For example, in some embodiments, Lactobacillus rhamnosus strain FNZ 142 or a derivative thereof is administered throughout the life of the ruminant.

[0183] For example, Lactobacillus rhamnosus FNZ142 or its derivatives are administered to ruminants at or about the 0th day of birth, for example, the 0th day, the 1st day or the 2nd day of birth. Administration can then be at least once a day, for example, multiple times a day, enough to obtain a lasting effect. For example, administration can last for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 10 weeks or 3 months from birth. In some embodiments, the administration of Lactobacillus rhamnosus strain FNZ142 or its derivatives lasts for 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, or the life of the continuous ruminant.

[0184] Lactobacillus rhamnosus FNZ142

[0185] A culture of Lactobacillus rhamnosus FNZ142 (Lactobacillus rhamnosus FNZ142) was deposited on August 2, 2021 at the National Measurement Institute of Australia (NMIA), 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207, with accession number V21 / 015448. This is an international depositary unit for the international recognition of the deposit of microorganisms for patent procedures under the Budapest Treaty. The terms Lactobacillus rhamnosus strain FNZ142, Lactobacillus rhamnosus FNZ142, Lactobacillus rhamnosus FNZ142, and Lactobacillus rhamnosus FNZ142 are used interchangeably herein.

[0186] The whole genome sequencing using a combination of short read (Illumina) and long read (PacBio) sequencing technologies was used to generate a hybrid genome assembly. The final hybrid combination contained two overlapping groups. The total length was 2980199bp (2.98Mb). The species ID of the FNZ142 strain was confirmed to be Lactobacillus rhamnosus using the taxonomic sequence classifier program Kraken.

[0187] All W6S and related bioinformatics were performed in accordance with EFSA guidance (available at https: / / efsa.onlinelibrary.wiley.com / doi / pdf / 10.2903 / j.efsa.2018.5206) in conjunction with the latest EFSA statement (July 2021) (available at: https: / / efsa.onlinelibrary.wiley.com / doi / full / 10.2903 / j.efsa.2021.6506)

[0188] Morphological characteristics

[0189] The morphological properties of Lactobacillus rhamnosus FNZ142 are described as follows.

[0190] Short to medium rods with square ends in chains, typically 0.7 x 1.1 x 2.0-4.0 μm when grown in MRS broth. Gram-positive, non-motile, non-spore-forming, catalase-negative facultative anaerobic bacillus.

[0191] Other manifestations

[0192] It will be appreciated that a variety of methods known and available to those skilled in the art can be used to confirm the identity of Lactobacillus rhamnosus FNZ142, where exemplary methods include DNA fingerprinting, genomic analysis, sequencing and related genomic and proteomic technologies.

[0193] Lactobacillus rhamnosus strain FNZ142 and its derivatives

[0194] As herein, certain embodiments of the invention utilize live Lactobacillus rhamnosus strain FNZ 142. In other embodiments, derivatives of Lactobacillus rhamnosus strain FNZ 142 are utilized.

[0195] As used herein, the term "derivative" and grammatical equivalents thereof, when used with respect to bacteria (including with respect to specific strains of bacteria such as Lactobacillus rhamnosus (FNZ142) includes mutants and homologs of or derived from bacteria, killed or attenuated bacteria, such as but not limited to heat-killed, lysed, fractionated, pressure-killed, irradiated and UV- or light-treated bacteria, and materials derived from bacteria, including but not limited to bacterial cell wall compositions, bacterial cell lysates, lyophilized bacteria, antimethanogenic factors from bacteria, bacterial metabolites, bacterial cell suspensions, bacterial culture supernatants, and the like, wherein the derivative retains antimethanogenic activity. Transgenic microorganisms engineered to express one or more antimethanogenic factors are also contemplated. Methods of producing such derivatives (such as but not limited to one or more mutants of Lactobacillus rhamnosus strain FNZ142 or one or more antimethanogenic factors), particularly derivatives suitable for administration to ruminants (e.g., in compositions) are well known in the art.

[0196] It will be appreciated that methods suitable for identifying Lactobacillus rhamnosus strain FNZ142, such as those described above, are also suitable for identifying derivatives of Lactobacillus rhamnosus strain FNZ142, including for example mutants or homologues of Lactobacillus rhamnosus strain FNZ142, or for example bacterial metabolites from Lactobacillus rhamnosus strain FNZ142.

[0197] The term "antimethanogen factor" refers to a bacterial molecule responsible for mediating antimethanogen activity, including but not limited to bacterial DNA motifs, RNA including mRNA and miRNA, proteins, exosomes, bacteriocins, bacteriocin-like molecules, antimicrobial peptides, antibiotics, antimicrobials, small molecules, polysaccharides or cell wall components such as lipoteichoic acid and peptidoglycan, or any mixture of two or more thereof. Although, as mentioned above, these molecules have not been clearly identified, and do not wish to be bound by any theory, their presence can be inferred by the presence of antimethanogen activity.

[0198] The term "anti-methanogenic activity" refers to the ability of certain microorganisms to inhibit the growth or reduce the abundance of methanogenic bacteria and / or archaea, and / or reduce the ability of methanogenic bacteria and / or archaea to produce methane. This ability may be limited to inhibiting the growth and / or the ability to produce methane of certain groups of methanogenic bacteria and / or archaea, such as inhibiting the growth of hydrogenotrophic methanogens, inhibiting the ability of hydrogenotrophic methanogens to produce methane, inhibiting the growth of methylotrophic methanogens, inhibiting the ability of methylotrophic methanogens to produce methane, inhibiting the growth of certain species of methanogens, or inhibiting the ability of certain species of methanogens to produce methane.

[0199] By retaining anti-methanogenic activity, it is meant that a derivative of a microorganism, such as a mutant or homolog of a microorganism or an attenuated or killed microorganism, or a cell culture supernatant, still possesses useful anti-methanogenic activity, or a composition comprising a microorganism or its derivative still possesses useful anti-methanogenic activity. Although the bacterial molecules responsible for mediating anti-methanogenic activity have not been clearly identified, molecules that have been proposed as possible candidates include bacterial DNA motifs, RNA including mRNA and miRNA, proteins, exosomes, bacteriocins, antibiotics, surface proteins, small organic acids, polysaccharides, and cell wall components such as lipoteichoic acid and peptidoglycan. It has been hypothesized that these interact with components of methanogens and / or archaea to produce growth inhibition. Preferably, the activity retained is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the activity of an untreated (i.e., live or non-attenuated) control, and useful ranges can be selected between any of these values ​​(e.g., from about 35 to about 100%, from about 50 to about 100%, from about 60 to about 100%, from about 70 to about 100%, from about 80 to about 100%, and from about 90 to about 100%).

[0200] Using conventional solid substrates and liquid fermentation techniques well known in the art, Lactobacillus rhamnosus strain FNZ142 can be grown in sufficient amounts to allow for use as contemplated herein. For example, Lactobacillus rhamnosus strain FNZ142 can be used in large quantities for formulation using nutrient film or submerged culture growth techniques, such as under the conditions described in WO99 / 10476. In short, growth is carried out under aerobic conditions at any temperature satisfactory for organism growth. For example, for Lactobacillus rhamnosus strain FNZ142, a temperature range of 30 to 40° C., preferably 37° C., is preferred. The pH of the growth medium is slightly acidic, preferably about 6.0-6.5. The incubation time is sufficient to allow the isolate to reach a stable growth phase.

[0201] Bacterial cells can be harvested by methods well known in the art, such as by conventional filtration or precipitation methods (e.g., centrifugation) or drying using a cyclone system. The bacterial cells can be used immediately or stored, preferably freeze-dried or frozen at -20°C to 6°C, preferably -4°C, as long as standard techniques are used. As known in the art, cryoprotectants, cryopreservatives and / or lyoprotectants can also be used to enhance the stability and / or viability of bacterial cells when dried and / or frozen.

[0202] Supernatant

[0203] Other embodiments of the present invention utilize supernatants from cell cultures comprising Lactobacillus rhamnosus strain FNZ142 or derivatives thereof. These embodiments include methods of preparing bacterial culture supernatants, the methods comprising culturing bacterial cells and isolating supernatants from the cultured cells, thereby obtaining supernatants. The methods are also capable of further isolating bacterial molecules that are responsible for mediating anti-methanogen activity that are obtainable from the supernatant.

[0204] Those skilled in the art will appreciate that the supernatant that can be used in the present invention includes the supernatant from such culture, and / or a concentrate of such supernatant and / or a portion of such supernatant.

[0205] The term "supernatant" herein refers to culture medium from a bacterial culture, after which the bacteria have been removed from the culture medium, for example by centrifugation or filtration.

[0206] The supernatant that can be used in the present invention can be easily obtained by a simple method for preparing a bacterial culture supernatant, the method comprising:

[0207] a) culturing cells of Lactobacillus rhamnosus strain FNZ142,

[0208] b) optionally releasing active compounds and / or extracellular components of the cells by various cell treatments such as, but not limited to, acid or base modification, sonication, detergents such as sodium dodecyl sulfate (SDS) and / or Triton X, cell wall degrading enzymes such as mutases and / or lysozymes, salts and / or alcohols; and

[0209] c) separating the supernatant from the cultured cells,

[0210] Thus, a supernatant was obtained.

[0211] In a preferred embodiment of the method, the supernatant composition is further subjected to a drying step to obtain a dried culture product.

[0212] The drying step may conveniently be freeze drying or spray drying, but any drying method suitable for drying antimethanogenic factors such as bacteriocins is contemplated, including vacuum drying and air drying.

[0213] Although the content of the supernatant produced by Lactobacillus rhamnosus strain FNZ142 has not been described in detail, it is known that certain bacterial strains may produce bacteriocins, which are small heat-stable proteins, and therefore, without being bound by theory, it is expected that even drying methods that result in moderate heating of the culture eluate product, including spray drying, will produce active compositions, as shown in the examples described herein.

[0214] Lysate

[0215] The fluid containing the contents of the lysed cells is called a lysate. The lysate contains the active components of the bacterial cells and can be crude, thus containing all cellular components, or partially and / or completely separated into separate fractions, such as extracellular components, intracellular components, proteins, etc.

[0216] Methods for producing bacterial cell lysates are well known in the art. Such methods may include, but are not limited to, mechanical lysis (such as mechanical shearing, grinding, milling or ultrasonic treatment), enzymatic lysis (such as by enzymes that degrade the bacterial cell wall), chemical lysis (such as using detergents, denaturants, pressure changes and / or osmotic shock) and combinations of the above methods.

[0217] Therefore, other embodiments of the present invention utilize a lysate of Lactobacillus rhamnosus strain FNZ142 or a derivative thereof.

[0218] Cell suspension

[0219] In some embodiments, the present invention may also utilize a cell suspension comprising Lactobacillus rhamnosus strain FNZ142 or a derivative thereof.

[0220] In the present context, the term "cell suspension" relates to a number of cells of the Lactobacillus rhamnosus strain FNZ142 or a derivative thereof dispersed or suspended in a liquid, such as a liquid nutrient medium, a culture medium or a saline solution.

[0221] The cells may be present in the form of a cell suspension in a solution suitable for dispersion. The cell suspension may be dispersed, for example, by spraying, dipping or any other application method.

[0222] The cells may be viable, but the suspension may also include inactivated or killed cells or lysates thereof. In one embodiment, the suspension of the invention includes viable cells. In another embodiment, the suspension of the invention includes inactivated, killed or lysed cells.

[0223] Bacteriocin

[0224] Bacteriocins are antimicrobial compounds produced by bacteria to inhibit other bacterial strains and species.

[0225] Lactic acid bacteria (LAB) are known to produce bacteriocins, and these compounds are of global interest to the food industry because they inhibit the growth of many spoilage and pathogenic bacteria, thereby extending the shelf life and safety of food. Bacteriocins are generally considered to be narrow-spectrum antibiotics. In addition, bacteriocins, especially those of LAB, show extremely low human toxicity and have been consumed in fermented foods for thousands of years.

[0226] Another aspect of the present invention provides an isolated antimicrobial compound obtainable from Lactobacillus rhamnosus strain FNZ142 or a derivative thereof.Such an antimicrobial compound may be obtained, for example, from a supernatant or a lysate obtained from a method herein, the method further comprising an isolation step.

[0227] As shown in the examples disclosed herein, it has been found that Lactobacillus rhamnosus strain FNZ142 and / or compositions comprising Lactobacillus rhamnosus strain FNZ142 and / or culture supernatant of Lactobacillus rhamnosus strain FNZ142 are useful as antimicrobial compounds, particularly for inhibiting the growth of methanogenic bacteria and / or inhibiting the ability of methanogens.

[0228] As used herein, the term antimicrobial compound utilizes compounds that kill microorganisms, impair their survival or inhibit their growth.

[0229] Antimicrobial compounds can be grouped according to the microorganisms they primarily act on. For example, antibacterials are used against bacteria, and antifungals are used against fungi. They can also be classified according to their function. Compounds that kill microorganisms are called microbicidal, while compounds that only inhibit their growth are called microbial-static.

[0230] In one embodiment, the present invention relates to antimicrobial compounds that are microbicidal. In another embodiment, the present invention relates to antimicrobial compounds that are microbistatic. In another embodiment, the present invention relates to antimicrobial compounds that are antibacterial agents.

[0231] Ruminant feed or carrier composition

[0232] The ruminant feed composition that can be used for the present invention can be formulated into food, beverage, food additive, beverage additive, animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral feeding product, soluble, slurry, supplement, medicine, lick block, dunking medicine, tablet, capsule, pellet or rumen product, such as bolus. Those skilled in the art can prepare suitable formulations according to the teachings of technology and this specification.

[0233] The composition can be applied as a top dressing on standard feed materials such as diets, or mixed into standard feed materials such as diets. In addition, the strain can be part of a or total mixed ration (TMR), a pelleted feed, mixed with a liquid feed or beverage, mixed with a protein premix, or administered via a vitamin and mineral premix.

[0234] In one embodiment, compositions that can be used herein include any edible feed products that can carry bacteria or bacterial derivatives. As used in the present application, the term "feed" or "animal feed" refers to the material consumed by animals and contributes energy and / or nutrients to the diet of animals. Animal feed generally includes many different components, which can exist in the form of, for example, concentrates, premixes, co-products or particles. The example of feed and feed components includes partially or completely mixed diets (TMR), corn, soybeans, forage, cereals, distiller's grains, sprouted cereals, beans, vitamins, amino acids, minerals, fibers, forage, grass, hay, straw, silage, nuts, leaves, meal, solubles, slurries, supplements, powdered feeds, pulp, vegetable pulp, fruit or vegetable residues, citrus meal, wheat flour, corncob meal and molasses. Other compositions that can be used as carriers include milk, milk powder, milk substitutes, milk fortifiers, whey, whey powder, sucrose, maltodextrin, rice husks.

[0235] In certain embodiments, the feed composition is formed by growing Lactobacillus rhamnosus strain FNZ142 using a milk-based carrier (e.g., heated milk) or a non-milk-based carrier to produce a fermented yogurt-type composition. The method of producing such a fermented yogurt-type composition is well known in the art and may include, for example, incubating milk at a suitable temperature using a warm water bath or other heating means until sufficient cell density is reached, such as for more than 12 hours. In one embodiment, the temperature is 25-30°C. Optionally, milk may include other additives that promote bacterial growth, such as yeast extract. In certain embodiments, the method is carried out on site, for example, on a farm to be supplemented with probiotic feed. The fermented yogurt-type composition can be administered orally, such as by soaking. In some embodiments, the fermented yogurt-type composition is administered at a dosage of 1-100 ml per day, such as 2-50, 5-30 or 10-20 ml per day.

[0236] Other suitable feed formulations for ruminants are described in EW Crampton et al., Applied Animal Nutrition, WH Freeman and Company, San Francisco, CA., 1969 and DC C Hurch, Livestock Feeds and Feeding, O&B Books, Corvallis, Oreg., 1977, both of which are incorporated herein by reference.

[0237] In one embodiment, the compositions useful in the present invention include any non-feed carrier for animal consumption, such as vermiculite, zeolite, or crushed limestone, etc., to which the bacteria or bacterial derivatives are added.

[0238] In certain embodiments, the compositions of the present invention include live Lactobacillus rhamnosus strain FNZ 142. Methods for preparing such compositions are well known in the art.

[0239] In some embodiments, the compositions of the present invention include one or more derivatives of Lactobacillus rhamnosus strain FNZ142. In addition, methods for preparing such compositions are well known in the art and can utilize standard microbiological and pharmaceutical practices. In some embodiments, the compositions include dried culture products, such as supernatants or cell lysates herein.

[0240] It should be understood that a wide range of additives or carriers can be included in such compositions, for example to improve or maintain bacterial viability or to increase the antimethanogenic activity of Lactobacillus rhamnosus strain FNZ142 or its derivatives. For example, additives such as surfactants, wetting agents, humectants, adhesives, dispersants, stabilizers, permeants, and so-called stress additives (such as potassium chloride, glycerol, sodium chloride and glucose) that increase bacterial cell viability, growth, replication and survival, and cryoprotectants such as maltodextrin can be included. Additives can also include compositions that help maintain microbial viability during long-term storage, such as unrefined corn oil, or "conversion" emulsions containing a mixture of oil and wax on the outside and water, sodium alginate and bacteria on the inside.

[0241] In some embodiments, Lactobacillus rhamnosus FNZ142 or a derivative thereof is encapsulated. Methods for producing such encapsulated bacteria are well known in the art. In some embodiments, Lactobacillus rhamnosus FNZ142 or a derivative thereof is encapsulated in liposomes, microbubbles, microparticles or microcapsules, etc. Such encapsulating agents may include natural, semi-synthetic or synthetic polymers, waxes, lipids, fats, fatty alcohols, fatty acids and / or plasticizers, such as alginate, gum, K-carrageenan, chitosan, starch, sugar, gelatin, etc.

[0242] In certain embodiments, the Lactobacillus rhamnosus strain FNZ142 is in a form and amount that is reproductively active.

[0243] The composition may include a carbohydrate source, such as a disaccharide, including, for example, sucrose, fructose, glucose or dextrose.Preferably, the carbohydrate source is a carbohydrate source that can be utilized aerobically or anaerobically by the Lactobacillus rhamnosus strain FNZ142.

[0244] In such embodiments, the composition is preferably capable of supporting the reproductive viability of Lactobacillus rhamnosus strain FNZ142 for a period of greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months, and most preferably at least about 2 years to about 3 years or longer.

[0245] In certain embodiments, the oral composition is formulated to allow administration of an effective amount of Lactobacillus rhamnosus strain FNZ142 to establish a population in the digestive tract of an animal upon ingestion. The established population may be a transient or permanent population.

[0246] While various routes and methods of administration are contemplated, oral administration of Lactobacillus rhamnosus strain FNZ142, for example in a composition suitable for oral administration, is presently preferred. It will of course be appreciated that other routes and methods of administration may be utilizable or preferred in certain circumstances.

[0247] The term "oral administration" includes oral, buccal, enteral, intraruminal and intragastric administration.

[0248] In theory, one colony forming unit (cfu) should be sufficient to establish a population of Lactobacillus rhamnosus strain FNZ142 in an animal, but in practice, a minimum number of units is required to do so. Therefore, for therapeutic mechanisms that rely on a population of live, viable probiotics, the number of units administered to a subject will affect efficacy.

[0249] In one embodiment, the composition formulated for administration will be sufficient to provide at least about 6×10 9 cfu / day of Lactobacillus rhamnosus strain FNZ142, for example at least about 6×10 11 In another embodiment, the composition formulated for administration will be sufficient to provide at least about 10 cfu / day. 12 cfu of Lactobacillus rhamnosus strain FNZ142.

[0250] Methods for determining the presence of intestinal and / or rumen flora, such as Lactobacillus rhamnosus strain FNZ142, in the digestive tract of a subject are well known in the art, and examples of such methods are given herein. In certain embodiments, the presence of a population of Lactobacillus rhamnosus strain FNZ142 can be determined directly, for example by analyzing one or more samples obtained from an animal and determining the presence or amount of Lactobacillus rhamnosus strain FNZ142 in the samples. In other embodiments, the presence of a population of Lactobacillus rhamnosus strain FNZ142 can be determined indirectly, for example by observing a reduction in methane emissions or methane production, a reduction in hydrogen production, or a reduction in the number of other intestinal and / or rumen flora in a sample obtained from an animal. Combinations of these methods are also contemplated.

[0251] The efficacy of compositions useful according to the present invention can be evaluated in vitro and in vivo. See, for example, the following examples. Briefly, the compositions can be tested for their ability to inhibit the growth of methanogens and / or archaea or reduce their abundance, or to reduce the ability of methanogens and / or archaea to produce methane. For in vivo studies, the compositions can be fed to ruminants or injected into ruminants, and then evaluated for their effects on methane emissions from rumen methanogenic bacteria and / or archaea. Based on the results, appropriate dosage ranges and routes of administration can be determined.

[0252] The method of calculating the appropriate dose may depend on the nature of the active agent in the composition. For example, when the composition includes live bacteria, the dose may be calculated with reference to the number of live bacteria present. For example, as described in the Examples herein, the dose may be determined with reference to the number of colony forming units (CFU) administered per day or with reference to the number of CFU per kilogram of dry feed weight.

[0253] As a general example, it is estimated that approximately 1×10 6 cfu is about 1×10 12 cfu of Lactobacillus rhamnosus strain FNZ142, preferably about 1×10 6 cfu is about 1×10 11 cfu / kg / day, about 1×10 6 cfu is about 1×10 10 cfu / kg / day, about 1×10 6 cfu is about 1×10 9 cfu / kg / day, about 1×10 6 cfu is about 1×10 8 cfu / kg / day, about 1×10 6 cfu is about 5×10 7 cfu / kg / day, or about 1×10 6 cfu is about 1×10 7 cfu / kg / day. Preferably, it is expected that about 5×10 6 cfu is about 5×10 8 cfu of Lactobacillus rhamnosus strain FNZ142, preferably about 5×10 6 cfu is about 4×10 8 cfu / kg / day, about 5×10 6 cfu is about 3×10 8 cfu / kg / day, about 5×10 6 cfu is about 2×10 8 cfu / kg / day, about 5×10 6 cfu is about 1×10 8 cfu / kg / day, about 5×106 cfu is about 9×10 7 cfu / kg / day, about 5×10 6 cfu is about 8×10 7 cfu / kg / day, about 5×10 6 cfu is about 7×10 7 cfu / kg / day, about 5×10 6 cfu is about 6×10 7 cfu / kg / day, about 5×10 6 cfu is about 5×10 7 cfu / kg / day, about 5×10 6 cfu is about 4×10 7 cfu / kg / day, about 5×10 6 cfu is about 3×10 7 cfu / kg / day, about 5×10 6 cfu is about 2×10 7 cfu / kg / day, or about 5×10 6 cfu is about 1×10 7 cfu / kg / day.

[0254] In certain embodiments, the periodic dosage need not vary with the subject's body weight, dry feed weight, or other characteristics. In such an example, it is contemplated that approximately 1×10 6 cfu is about 1×10 13 cfu of Lactobacillus rhamnosus strain FNZ142, preferably about 1×10 6 cfu is about 1×10 12 cfu / day, about 1×10 6 cfu is about 1×10 11 cfu / day, about 1×10 6 cfu is about 1×10 10 cfu / day, about 1×10 6 cfu is about 1×10 9 cfu / day, about 1×10 6 cfu is about 1×10 8 cfu / day, about 1×10 6 cfu is about 5×10 7 cfu / day, or about 1×10 6 cfu is about 1×10 7 cfu / day.

[0255] In certain embodiments, it is contemplated that about 5×10 7 cfu is about 5×10 10cfu of Lactobacillus rhamnosus strain FNZ142, preferably about 5×10 7 cfu is about 4×10 10 cfu / day, about 5×10 7 cfu is about 3×10 10 cfu / day, about 5×10 7 cfu is about 2×10 10 cfu / day, about 5×10 7 cfu is about 1×10 10 cfu / day, about 5×10 7 cfu is about 9×10 9 cfu / day, about 5×10 7 cfu is about 8×10 9 cfu / day, about 5×10 7 cfu is about 7×10 9 cfu / day, about 5×10 7 cfu is about 6×10 9 cfu / day, about 5×10 7 cfu is about 5×10 9 cfu / day, about 5×10 7 cfu is about 4×10 9 cfu / day, about 5×10 7 cfu is about 3×10 9 cfu / day, about 5×10 7 cfu is about 2×10 9 cfu / day, or about 5×10 7 cfu is about 1×10 9 cfu / day. Preferably, 1×10 8 Up to 1×10 9 The dose was calculated as cfu / kg body weight.

[0256] It should be understood that in certain embodiments, it is not necessary to administer the dose every day. For example, the composition can be formulated for administration every two days, twice a week, once a week, once every two weeks, or once a month. Alternatively, in certain embodiments, the composition can be formulated for administration 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day, per meal, or per mouthful.

[0257] It should be understood that the composition is preferably formulated to allow for administration of an effective dose of Lactobacillus rhamnosus strain FNZ142 and / or one or more derivatives thereof. The dose of the composition administered, the time of administration and the general dosing regimen may vary between animals, depending on variables such as the selected mode of administration and the age, sex, weight and species of the animal. In addition, as described above, the appropriate dose may depend on the nature and formulation of the active agent in the composition.

[0258] In addition, the dosage of the composition can vary over time. For example, in some embodiments, the initial dosage regimen may be followed by a maintenance dosage regimen. It should be understood that a higher dosage may be required to establish a colony of Lactobacillus rhamnosus FNZ142 in an animal and a lower dosage may be sufficient to maintain a colony. Therefore, in some embodiments, the initial dosage regimen includes administering a higher dosage and / or a more frequent dosage than the maintenance dosage regimen. Preferably, the initial dosage regimen effectively establishes a colony of Lactobacillus rhamnosus FNZ142 in an animal, and preferably the maintenance dosage regimen effectively maintains a colony of Lactobacillus rhamnosus FNZ142 in an animal. In some embodiments, the maintenance dosage regimen includes administering a dosage once a day, every two days, twice a week, every week, every two weeks, or every month.

[0259] In some embodiments, the effect of the methods herein lasts after the administration of lactobacillus rhamnosus FNZ142. Without wishing to be bound by theory, it is expected that the administration of lactobacillus rhamnosus FNZ142 as described herein can cause long-term or even permanent changes in the forestomach and / or rumen of ruminants. In some embodiments, the effect lasts for 2 days after the last administration of lactobacillus rhamnosus FNZ142, for example, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years or 7 years after the last administration of lactobacillus rhamnosus FNZ142. In a preferred embodiment, the effect lasts in the life of the animal.

[0260] In the example where the composition includes one or more derivatives of Lactobacillus rhamnosus strain FNZ142, the dosage can be calculated by reference to the amount or concentration of the derivative administered per day. For example, when the bacteria are inactivated, the above amount is calculated before inactivation. For a composition comprising a culture supernatant of Lactobacillus rhamnosus strain FNZ142, the dosage can be calculated by reference to the concentration of the culture supernatant present in the composition. The concentration of the culture supernatant present in the composition can be calculated, for example, based on the cfu of the culture. For example, equivalent to 1×10 9 The dose of culture supernatant in cfu / day can be calculated from the total productivity of the culture and the total volume of the culture supernatant.

[0261] It should be understood that preferred compositions are formulated to provide effective doses in convenient forms and amounts. In certain embodiments, such as but not limited to those embodiments where periodic doses do not need to vary with the weight or other characteristics of the animal, the composition can be formulated as a unit dose. It should be understood that administration can include administration of a single daily dose or appropriate multiple discrete divided doses. For example, an effective dose of Lactobacillus rhamnosus strain FNZ142 can be formulated as a feed for oral administration.

[0262] However, as a general example, the inventors contemplate administration of about 1 mg to about 1000 mg of a composition useful herein per day, preferably about 50 to about 500 mg / day, or about 150 to about 410 mg / day or about 110 to about 310 mg / day. In one embodiment, the inventors contemplate administration of about 0.05 mg to about 250 mg of a composition useful herein per kg body weight.

[0263] In one embodiment, the compositions useful herein comprise at least about 0.1 wt%, 0.2 wt%, 0.5 wt%, 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 99 wt%, 99.5 wt%, 99.8 wt% or 99.9 wt% of Lactobacillus rhamnosus strain FNZ142 and / or derivatives thereof, or consist essentially of and / or derivatives thereof, and useful The range of can be selected from any of these aforementioned values ​​(e.g., from about 0.1 to about 50%, from about 0.2 to about 50%, from about 0.5 to about 50%, from about 1 to about 50%, from about 5 to about 50%, from about 10 to about 50%, from about 15 to about 50%, from about 20 to about 50%, from about 25 to about 50%, from about 30 to about 50%, from about 35 to about 50%, from about 40 to about 50%, from about 45 to about 50%, from about 0.1 to about 60%, from about 0.2 to about 60%, from about 0.5 to about 60%, from about 1 to about 60%, from about 5 to about 60%, from about 10 to about 60%, from about 15 to about 60%, from about 20 to about 60%, from about 2 from about 5 to about 70%, from about 10 to about 70%, from about 15 to about 70%, from about 20 to about 70%, from about 25 to about 70%, from about 30 to about 70%, from about 35 to about 70%, from about 40 to about 70%, from about 45 to about 70%, from about 0.1 to about 70%, from about 0.2 to about 70%, from about 0.5 to about 70%, from about 1 to about 70%, from about 5 to about 70%, from about 10 to about 70%, from about 15 to about 70%, from about 20 to about 70%, from about 25 to about 70%, from about 30 to about 70%, from about 35 to about 70%, from about 40 to about 70%, from about 45 to about 70%, from about 0.1 to about 80%, from about 0.2 to about 80%, from about 0.5 to about 80%, from about 1 to about 80%, from about 5 to about 80%, from about 10 to about 80%, from about 15 to about 80%, from about 20 to about 80%, from about 25 to about 80%, from about 30 to about 80%, from about 35 to about 80%, from about 40 to about 80%, from about 45 to about 80%, from about 0.1 to about 90%, from about 0.2 to about 90%, from about 0.5 to about 90%, from about 1 to about 90%, from about 5 to about 90%, from about 10 to about 90%, from about 15 to about 90%, from about 20 to about 90%, from about 25 to about 90%, from about 30 to about 90%, from about 35 to about 90%, from about 40 to about 90%, from about 45 to about 90%, from about 0.1 to about 99%, from about 0.2 to about 99%, from about 0.5 to about 99%, from about 1 to about 99%, from about 5 to about 99%, from about 10 to about 99%, from about 15 to about 99%, from about 20 to about 99%, from about 25 to about 99%, from about 30 to about 99%, from about 35 to about 99%, from about 40 to about 99%, and from about 45 to about 99%).

[0264] In one embodiment, the compositions useful herein include at least about 0.001 grams, 0.01 grams, 0.05 grams, 0.1 grams, 0.15 grams, 0.2 grams, 0.3 grams, 0.4 grams, 0.5 grams, 1 gram, 2 grams, 3 grams, 4 grams, 5 grams, 6 grams, 7 grams, 8 grams, 9 grams, 10 grams, 11 grams, 12 grams, 13 grams, 14 grams, 15 grams, 16 grams, 17 grams, 18 grams or 19 grams of Lactobacillus rhamnosus strain FNZ142 and and / or derivatives thereof, or consisting essentially of the same and / or derivatives thereof and useful ranges can be selected from any of these aforementioned values ​​(e.g., from about 0.01 gram to about 1 gram, from about 0.01 gram to about 10 grams, from about 0.01 gram to about 19 grams, from about 0.1 gram to about 1 gram, from about 0.1 gram to about 10 grams, from about 0.1 gram to about 19 grams, from about 1 gram to about 5 grams, from about 1 gram to about 10 grams, from about 1 gram to about 19 grams, from about 5 grams to about 10 grams, from about 5 grams to about 19 grams).

[0265] In certain embodiments, the compositions used herein comprise, consist essentially of, or consist of at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 rhamnosus strain FNZ142 per kg dry weight of the composition, and a useful range may be selected between any of these values ​​(e.g., from about 10 5 to about 10 13 cfu, from about 10 6 to about 10 12 cfu, from about 10 7 to about 10 12 cfu, from about 10 8 to about 10 11 cfu, from about 10 8 to about 10 10 cfu and from about 10 8 to about 10 9 cfu).

[0266] It will be apparent that the concentration of Lactobacillus rhamnosus strain FNZ142 and / or one or more derivatives thereof in a composition formulated for administration may be less than in a composition formulated for, for example, distribution or storage, and that the concentration of the composition formulated for storage and subsequently formulated into a composition suitable for administration must be sufficient to allow the composition for administration to also be sufficiently concentrated to be able to be administered in an effective dose.

[0267] Compositions useful herein can be used alone or in combination with one or more other therapeutic agents. The therapeutic agent can be a food, beverage, food additive, beverage additive, food component, beverage component, dietary supplement, vitamin or mineral premix, oil, oil blend, feed supplement rich in oil, nutritional product, medical food, nutrition, medicament or medicine. The therapeutic agent can be a probiotic or probiotic factor, and preferably effectively inhibits the growth of methanogens and / or archaea or reduces its abundance, or reduces the methane production of methanogens and / or archaea. In some embodiments, oil, oil blend or feed supplement rich in oil is palm oil residue (PKE) and / or PROLIQ.

[0268] When used in combination with another therapeutic agent, administration of the compositions useful herein and the other therapeutic agent can be simultaneous or sequential. Simultaneous administration includes administration of a single dosage form comprising all components or administration of separate dosage forms substantially simultaneously. Sequential administration includes administration according to different schedules, preferably such that there is overlap during the provision of the compositions useful in the present invention and the other therapeutic agent. Examples of other treatment agents include at least one different species or strain of microorganisms, vaccines that inhibit methanogens or methanogenesis, and / or natural or chemically synthesized inhibitors of methanogenesis and / or methanogen inhibitors, such as bromoform.

[0269] Suitable agents for use in the compositions herein that may be administered separately, simultaneously or sequentially include one or more prebiotic agents, one or more probiotic agents, one or more postbiotic agents, one or more phospholipids, one or more gangliosides, other suitable agents known in the art, and combinations thereof.

[0270] Generally, the term prebiotic refers to a substance that stimulates the growth and / or activity of bacteria in a biologically active animal digestive system. Prebiotics can be a selectively fermented ingredient that allows specific changes in the composition and / or activity of gastrointestinal microflora, which confer health benefits to the host. Probiotics generally refer to a microorganism that contributes to the balance of intestinal microorganisms, and intestinal microorganism balance works in maintaining health or providing other biological activities. Many types of lactic acid bacteria (LAB) such as lactobacillus and bifidobacterium are generally considered to be probiotics, but some types of bacillus and some yeasts are also found to be suitable candidates. Postbiotics refer to inactive bacterial products or metabolic byproducts from microorganisms such as probiotics that have biological activity in the host.

[0271] Useful prebiotics include galacto-oligosaccharides (GOS), short-chain GOS, long-chain GOS, fructo-oligosaccharides (FOS), short-chain FOS, long-chain FOS, inulin, galactans, fructans, lactulose, and any mixture of any two or more thereof. Some prebiotics are reviewed by Boehm G and Moro G (Structural and Functional Aspects of Prebiotics Usedin Infant Nutrition, J. Nutr. (2008) 138 (9): 1818S-1828S), which is incorporated herein by reference. Other useful agents may include dietary fiber, such as completely or partially insoluble or indigestible dietary fiber.

[0272] Therefore, in one embodiment, Lactobacillus rhamnosus strain FNZ142 and / or its derivatives can be administered separately, simultaneously or sequentially with one or more agents selected from the following: one or more probiotics, one or more prebiotics, one or more dietary fiber sources, one or more galacto-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, one or more fructo-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

[0273] In certain embodiments, the composition comprises Lactobacillus rhamnosus strain FNZ142 and / or its derivatives and one or more prebiotics, one or more probiotics, one or more postbiotics, one or more dietary fiber sources. In certain embodiments, the prebiotics comprise one or more fructooligosaccharides, one or more galacto-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

[0274] Without wishing to be bound by theory, it is believed that co-cultivation and / or co-administration of two or more lactic acid bacteria strains, such as three lactic acid bacteria strains, can reduce the incidence of culture failure due to phage infection. Therefore, in certain embodiments, the composition comprises Lactobacillus rhamnosus FNZ142 and one or more other lactic acid bacteria strains, preferably two or more other lactic acid bacteria strains. In other embodiments, the composition comprises Lactobacillus rhamnosus FNZ142 and one or more other compositions comprising one or more other lactic acid bacteria strains, preferably two or more other lactic acid bacteria strains, administered simultaneously or sequentially.

[0275] It should be understood that different compositions of the present invention can be formulated for administration to specific ruminant subject groups. For example, the formula of the composition suitable for application to cattle can be different from the formula suitable for application to different ruminants such as sheep. It should also be understood that the composition of the present invention can be formulated differently to be suitable for application to ruminants of different ages. For example, the formula of the composition suitable for application to calves or lambs can be different from the formula suitable for application to adult cows or sheep. In certain embodiments, the first composition can be formulated for application to young animals, such as pre-weaning animals, in the initial dosage regimen, and the second composition can be formulated for application to the same animal in the maintenance dosage regimen. In some embodiments, the first composition is formulated for pre-weaning animals, and the second composition is formulated for post-weaning animals.

[0276] Preparation of Lactobacillus rhamnosus strain FNZ142

[0277] Direct fed microorganisms (DFM) and their use in methods for modulating rumen function and improving performance in ruminants are known in the art, as are methods for their production.

[0278] In brief, the Lactobacillus rhamnosus strain FNZ142 can be cultivated using conventional liquid or solid fermentation techniques. In at least one embodiment, the strain is grown to the level of forming the highest number of spores in a liquid nutrient broth. The strain is produced by fermenting a bacterial strain, which can be started by expanding a seed culture. This includes repeatedly and aseptically transferring the culture to an increasingly larger volume to be used as an inoculum for fermentation, which can be carried out in a large stainless steel fermentor tank in a medium containing proteins, carbohydrates and minerals required for optimal growth. Non-limiting exemplary media are MRS or TSB. However, other media may also be used. After the inoculum is added to the fermentation vessel, the temperature is controlled and stirred to allow maximum growth. Once the culture reaches maximum population density, the culture is harvested by separating cells from the fermentation medium. This is usually carried out by centrifugation.

[0279] In one embodiment, the Lactobacillus rhamnosus strain FNZ142 was prepared by fermenting the strain to 1×10 8CFU / ml is about 1×10 9 The bacteria are collected by centrifugation and the supernatant is removed. The precipitated bacteria are then used to produce the DFM. In at least some embodiments, the precipitated bacteria are freeze-dried and then used to form the DFM. However, it is not necessary to freeze-dry the strains before using them. The strains may also be used in concentrated, unconcentrated or diluted form with or without preservatives.

[0280] The count of the culture can then be determined. CFU or colony forming unit is the viable cell count of a sample obtained by standard microbiological plating methods. The term is derived from the fact that individual cells will grow and become viable colonies in an agar medium when plated on a suitable medium.

[0281] Because multiple cells can give rise to one visible colony, the term colony forming unit is a more useful unit measure than cell number.

[0282] Example

[0283] 1. Example 1 - Plate screening of bacteriocin extracts against indicator methanogen strains

[0284] 1.1 Materials and methods

[0285] 1.1.1 Bacteriocin extraction

[0286] Bacteriocin extracts from cultures of Lactobacillus rhamnosus FNZ142 were prepared and their effects were determined against the methanogenic indicator strains Methanobrevibacterium bovis JJH1 ('JH1'), Methanosphaeroides WGK6 ('WGK6'), Methanobrevibacterium rumenii M1 ('M1') and Methanobrevibacterium gottschalkii D5 ('D5').

[0287] Lactobacillus rhamnosus FNZ142 was preserved from -80°C and obtained by plating on De Man-Rogosa-Sharpe agar (MRS, De Man et al., 1960) + lactose (2g / L). Using a small inoculation loop, the glycerol stock was streaked onto an MRS agar plate to obtain isolated colonies. The plate was incubated at 37°C for 48 hours in a sealed container. After growth, a single colony was selected, picked and streaked again on an agar plate, and incubated at 37°C to obtain isolated colonies. After 48 hours, a single colony from the re-streaked plate was inoculated into MRS liquid culture medium and incubated at 37°C for 48 hours. The inoculum (1mL) of each revived strain was then subcultured into 16mL MRS+nisin liquid culture medium (1ng / mL final concentration). Nisin was included in these culture media at very low levels to induce bacteriocin production. The culture was incubated overnight at 37°C. Overnight grown culture of Lactobacillus rhamnosus FNZ142 was used for bacteriocin extraction. One drop of the culture was used to prepare wet mount slides to examine the cells using phase contrast microscopy and to prepare Gram stains to check the culture purity.

[0288] The remaining culture (~16 mL) was transferred to a 50 mL Falcon tube for bacteriocin extraction according to Gaspar et al. (2018) with the following modifications. The pH of the culture was adjusted to ~6.8 with 6 M NaOH. 0.3 mL of catalase (2 mg / L) was then added to the culture and incubated at 37 °C for 30 min and then at 70 °C for 45 min. The culture was then centrifuged at 12,000 × g for 20 min at 4 °C, the supernatant was decanted, and the cell pellet was resuspended in 8 mL of 0.9% NaCl, pH 2. The pH of the resuspended pellet was checked and adjusted to pH 2 with 1 M HCl if necessary. The cells were incubated at 4 °C for 2 h with slow agitation on a shaking platform. The cells were then centrifuged at 12,000 × g for 20 min at 4 °C and the supernatant was collected into a fresh 15 mL Falcon tube. The pH of the supernatant was adjusted to pH 6.8 with 1 M NaOH and filtered under sterile conditions using a 10 mL syringe and needle through a sterile filter (Millex-GP 0.22 μm, 25 mm diameter, Millipore, Merck, Sigma-Aldrich NZ) into a sterile N2-flushed Hungate tube. The filtered supernatant was frozen at -20°C until use.

[0289] 1.1.2 Culture of Methanobacterium bovis JH1

[0290] To identify potential candidate LAB strains with anti-methanogen activity, a microtiter plate-based methanogen growth inhibition bioassay was used using a model methanogen strain, Methanobrevibacter boviskoreani JH1 (Li et al., 2019). Methanobrevibacter boviskoreani JH1 has an unusual growth ability to reduce CO2 to CH4 using ethanol as a source of reducing power, allowing JH1 to grow in microtiter plates incubated under anaerobic conditions without the need for H2 supply via 1 atmosphere of H2:CO2 (80:20) overpressure. This allows a high-throughput JH1 screening approach to identify inhibitory activity from LAB strains.

[0291] Using the anaerobic technique, cultures of Methanobacterium bovis JH1 for inoculation plate assays were grown by syringe in Balch tubes (anaerobic tubes, 18×150 mm, butyl rubber septum plugs, aluminum crimps, Bellco Glass, Vineland, NJ, USA) containing 9 mL of BY medium (Joblin, 1995) supplemented with (final concentrations) 60 mM sodium formate, 200 mM ethanol, 0.1 mL of vitamin solution (1×) and 0.1 mL of coenzyme M solution (10 μM). The tubes were incubated at 39° C. without shaking until visible turbidity appeared after 3 to 5 days and when they reached an OD between 0.8 and 1.0 relative to a distilled water blank. 600 Inoculation for microtiter plate assays Prior to removal of the inoculum, the overpressure in the JH1 culture tube was released by inserting a needle through the butyl rubber septum and allowing the accumulated gas to escape.

[0292] Freshly grown cultures were examined using wet mounts under a fluorescent microscope, and Methanobacterium bovis JH1 showed short oval rods that emitted green fluorescence under ultraviolet (UV) irradiation. Culture contamination was checked by inoculating culture samples into 9mL BY medium supplemented with 5mM glucose and incubating at 39°C for one day. If turbidity was not observed after 1 day, it was considered that the culture was not contaminated. Further culture verification was performed from time to time by extracting genomic DNA from culture and using conventional bacterial 16S primers (27f-GAGTTTGATCMTGGCTCAG, 1492r-GGYTACCTTGTTACGACTT) and archaebacterial specific 16S primers (915af-AGGAATTGGCGGGGGAGCAC, 1386r-GCGGTGTGTGCAAGGAGC) PCR amplification 16S rRNA genes. Culture purity was verified using the presence of a band with an archaebacterial primer set and the absence of a band with a bacterial primer set, and the sequencing results of the PCR product.

[0293] 1.1.3 Methanosphaera sp. WGK6 culture

[0294] Members of the genus Methanosphaeria account for approximately 8% of rumen methanogens (Henderson et al., 2015) and are typically H2-dependent methylotrophs, using H2 to reduce methanol to methane. Methanosphaeria WGK6 is an H2-utilizing methylotrophic methanogen isolated from the digestive tract of an Australian kangaroo, but it is also able to use ethanol as a source of reducing power to reduce methanol to methane (Hoedt, 2017). Similar to Methanobrevibacterium bovis JH1, this metabolic ability would theoretically allow WGK6 to grow on ethanol without the need for an overpressure of H2, thus enabling its growth in microtiter plates. Growth of Methanosphaeria WGK6 was tested in Hungate tubes using BRN-RF10 medium (Balch et al., 1979; Hoedt, 2017), in both cases with H2 (180 kPa overpressure of H2 + CO2; ) or ethanol as the energy source and methanol as the terminal electron acceptor. Attempts to grow WGK6 on ethanol + methanol were unsuccessful, but WGK6 was able to grow on methanol + H2 in Hungate tubes. Our initial attempts were to grow Methanosphaeria WGK6 with methanol in a microtiter plate format in a pressurized gas tank under an H2 + CO2 atmosphere (180 kPa overpressure), and almost no growth was detected after a week. However, after increasing the concentration of cysteine ​​added to the BRN-RF10 medium, Methanosphaeria WGK6 grew better. Therefore, a plate assay development was performed using a stainless steel gas tank capable of pressurization (H2 + CO2; 80:20).

[0295] Using anaerobic techniques and 180 kPa H2 + CO2 overpressure (80:20, BOC Gases NZ), the Methanosphaeroides WGK6 culture used for the assay was grown in 9 mL BRN-RF10 medium supplemented with (final concentration) 60 mM sodium formate, 1% methanol, 0.1 mL vitamin solution (1×) and 0.1 mL coenzyme M solution (10 μM) in Balch tubes via syringe. The tubes were incubated at 39°C without shaking until visible turbidity appeared after 3 to 5 days and they reached an OD between 0.8 and 1.0 relative to a distilled water blank. 600 Inoculation for plate assays Prior to removal of the inoculum, the overpressure in the WGK6 culture tube was released by inserting a needle through the butyl rubber septum and allowing the accumulated gas to escape.

[0296] 1.1.4 Rumen cultures of Methanobacterium M1 and Methanobacterium gottschalkii D5

[0297] The procedure for growing Methanobacterium rumenii M1 and Methanobacterium gottschalkii D5 was identical to the WGK6 protocol described in 1.1.3 above, except that it used BY medium for growth. The cultures for the assay were grown in 9 mL BY medium supplemented with (final concentration) 60 mM sodium formate, 0.1 mL vitamin solution (1×) and 0.1 mL coenzyme M solution (10 μM) in Balch tubes using anaerobic techniques and an H2+CO2 overpressure of 180 kPa (80:20, BOC Gases NZ). The tubes were incubated at 39°C without shaking until visible turbidity appeared after 3 to 5 days and used for inoculation of microtiter plate assays.

[0298] 1.1.5 Growth inhibition assay of Methanobacterium bovis JH1

[0299] The bacteriocin extract from Lactobacillus rhamnosus FNZ142 stored frozen in Hungate tubes under anaerobic conditions was thawed at room temperature. Except for the JH1 inoculum, all the assay components of each assay were added to the 3.75mL BY+ formate medium in a sterile 7.5mL Hungate tube by a syringe and needle flushed with CO2 in the ratio shown in Table 1. Each tube was then inoculated with a freshly grown JH1 culture, incubated at 39°C for 1 hour, then moved into an anaerobic chamber (98%CO2-2%H2 atmosphere; Coy Laboratory Products, USA) and distributed to the wells of a multi-well 96-well plate. The filled plate was placed in an AnaeroPack 2.5L rectangular wide-mouth bottle with MCG Anaeropack-Anaero (Ngaio Diagnostics, Nelson, NZ), the lid was sealed, and the bottle was taken out from the anaerobic chamber and incubated at 39°C. The plates were observed daily through a clear jar until the M. bovis JH1 control wells showed visible turbidity (usually within 5 to 6 days). The plates were then measured at 595 nm (OD) after shaking for 5 seconds in a Multiskan FC microplate photometer (Thermo Scientific, Auckland, NZ). 595 ) The optical density of each well was recorded. The absorbance readings of the medium control wells were subtracted as background and the % inhibition of M. bovis JH1 growth caused by the bacteriocin extract samples was calculated relative to the JH1 positive growth control wells (containing buffer only).

[0300] Table 1. Microtiter plate setup for M. bovis JH1 growth inhibition assay.

[0301]

[0302]

[0303] 1.1.6 Methanosphaeria WGK6 growth inhibition assay

[0304] Each assay component for the assay, except the WGK6 inoculum, was added to 3.75 mL of BRN-RF10 medium in a Hungate tube supplemented with 1% methanol (247 mM, final concentration) via a CO2-flushed syringe and needle, as described in Table 2. The tube was then moved into the chamber along with the inoculum tube. The medium containing all components except the inoculum was distributed to the plates in the chamber, and the inoculum was then added to the appropriate wells. The plates were placed in a horizontally positioned stainless steel gas tank to hold up to 4 microtiter plates at a time. Two anaerobic bladders (MCG Anaeropack-Anaero, NgaioDiagnostics, Nelson, NZ) were added, the container was sealed, the container was removed from the anaerobic chamber, and H was added. a +CO2 (80:20, BOC Gases NZ) pump to a pressure of 180kPa, then incubate at 39 ° C for 1 week. Check the container regularly to ensure that the overpressure is maintained, and if necessary, re-pressurize with H2+CO2. After incubation for 1 week, open the container and remove the plate. The contents of each well are evenly resuspended by using a multichannel pipette. Then shake in a Multiskan FC microplate photometer (Thermo Scientific, Auckland, NZ) for 5 seconds and immediately at 595nm (OD 595 ) The optical density of each well was recorded. The absorbance readings of the medium control wells were subtracted as background and the % inhibition of Methanosphaera WGK6 growth caused by the bacteriocin extract samples was calculated relative to the WGK6 positive growth control wells (containing only buffer instead of bacteriocin extract).

[0305] Table 2. Microtiter plate setup for Methanosphaera WGK6 growth inhibition assay.

[0306]

[0307] 1.1.7 Growth inhibition assay of Methanobacterium rumenicum M1 and Methanobacterium gottschalkii D5

[0308] Cultures of Methanobrevibacterium rumenii M1 and Methanobrevibacterium gottschalkii D5 were prepared as described above in 1.1.4. The overpressure in the tube was released before removing the inoculum.

[0309] Assay components were added to 3.5 mL of sterile BY medium in 7.5 mL Hungate tubes via a CO2-flushed syringe and needle as described in Table 3. Each tube was then inoculated with a freshly grown culture, incubated at 39°C for 1 hour, then moved into an anaerobic chamber and dispensed into wells of a 96-well multiwell plate. The plates were sealed and incubated in a stainless steel gas tank at 180 kPa of H2+CO2 overpressure, and the concentrations were determined by OD 1000, as described above for the Methanosphaeria WGK6 assay in 1.1.6. 595 Record their optical density by spectrophotometry under 4 °C. Subtract the OD of BY medium control wells 595 The readings were taken as background and the % inhibition of growth of M. ruminans M1 or M. gottschalkii D5 caused by the bacteriocin extract samples was calculated relative to the positive growth control wells (which contained buffer instead of bacteriocin extract).

[0310] Table 3. Microtiter plate setup for growth inhibition assays of Methanobrevibacterium rumenicum M1 and Methanobrevibacterium gottschalkii D5.

[0311]

[0312] 1.2 Results

[0313] Bacteriocin extracts against Methanosphaeria WGK6 were screened from a total of 1712 lactic acid bacteria strains. Of these, 1,580 strains (>92%) showed less than 50% inhibition. The 1,712 lactic acid bacteria strains included 94 strains of Lactobacillus rhamnosus, of which 62 strains (~66%) showed less than 20% inhibition of WGK6, 81 strains (~86%) showed less than 50% inhibition of WGK6, and only 3 strains (~3%) showed ~80% or more inhibition of WGK6. Overall, this suggests that methanogen inhibition may be a strain-specific effect.

[0314] The bacteriocin extract of Lactobacillus rhamnosus FNZ142 showed very strong inhibition against the indicative methylotrophic methanogen Methanosphaeroides WGK6, but weak inhibition against the indicative hydrogenotrophic methanogen Methanobrevibacterium bovis JH1, and no inhibition or very weak inhibition against Methanobrevibacterium rumenicum M1 or Methanobrevibacterium gottschalkii D5, as shown in Table 4.

[0315] Table 4. Inhibition of indicator methanogen strains by bacteriocin extract JH1 of Lactobacillus rhamnosus FNZ142

[0316]

[0317]

[0318] 1.3 Discussion and Conclusion

[0319] Members of the genera Methanobacterium and Methanosphaeria are the main methanogens in the rumen across a variety of ruminant species. In particular, WGK6 is used as an indicator strain for general methylotrophic methanogens and Methanosphaeria. Methanobacterium bovis JH1, Methanobacterium rumen M1, and Methanobacterium gottschalkii D5 are used as indicator strains for Methanobacterium species. This example shows that the bacteriocin extract of Lactobacillus rhamnosus FNZ142 exhibits a strong inhibitory effect on the methylotrophic methanogen Methanosphaeria WGK6, but has a slight inhibitory effect on the hydrogenotrophic Methanobacterium bovis JH1, and has no effect on the rumen Methanobacterium M1 and Methanobacterium gottschalkii D5 methanogens.

[0320] 2. Example 2 - Effect of Lactobacillus rhamnosus FNZ142 on rumen in vitro test

[0321] 2.1 Materials and methods

[0322] 2.1.1 Preparation of bacterial cultures and supernatants for testing

[0323] Lactobacillus rhamnosus FNZ142 was used to inoculate 7 Hungate tubes, each containing 5 mL of anaerobic MRS medium (Sigma-Aldrich), and they were incubated at 39°C for 16 hours (until the culture reached stationary phase). The cultures were pooled into 250 mL CO2-flushed serum bottles. An aliquot (1 mL) of the pooled culture was added to 9 mL of sterile MRS medium to measure its OD 600 Other aliquots (0.5 mL) of the culture mixture were inoculated in triplicate into 4.5 mL of sterile anaerobic buffer, serially diluted 10-fold under CO2, and plated onto MRS plates to determine the colony forming units (CFU.mL) of the original culture. -1 ) number. Half of the remaining culture was used for a set of rumen in vitro fermentations (test culture), and the other half was filtered (Millipore 0.22 μm pore size) and the filtrate was placed in a new sterile anaerobic serum bottle (supernatant treatment, SN). Anaerobic phosphate buffer (0.46M K2HP04; 0.54M KH2PO4, pH 7) was used as a no-treatment control (buffer).

[0324] 2.1.2 Rumen fluid preparation and in vitro fermentation establishment

[0325] To inoculate the rumen in the in vitro fermentation vessel, fresh rumen contents were collected from 6 rumen-fed Friesian cows. The resulting rumen fluid (approximately 150 mL of rumen fluid) from two animals was combined after squeezing through a layer of cheesecloth to obtain 3 biological replicates. An aliquot (12.5 mL) of the mixed rumen fluid was added to 0.5 mg of hay and 36.5 mL of anaerobic phosphate buffer in a 250 mL serum bottle. Buffer, test culture, SN or bacteriocin extract treatment (1 mL) were added, and the serum bottle was then closed with a butyl rubber stopper to obtain a final fermentation volume of 50 mL containing 25% rumen fluid (v / v). Gas production and methane content were measured using an automated incubation system (Muetzel et al., 2014).

[0326] 2.1.1 VFA sample collection and analysis

[0327] Collect samples from the bottles for VFA analysis. At each time point, collect 3 mL aliquots and measure their pH. 1.8 mL samples of these aliquots are used for VFA and non-VFA analysis. The VFA samples are centrifuged at 21,000 × g for 10 min at 4 ° C, and 0.9 mL of the supernatant is removed and added to 0.1 mL of internal standard (20 mM 2-ethylbutyrate / 20% phosphoric acid), mixed and frozen at -20 ° C until analysis. After thawing and centrifuging again at 21,000 × g for 10 min at 4 ° C, 0.9 mL is collected for derivatization for non-VFA analysis, and the rest of the sample is analyzed directly by GC.

[0328] 2.2 Results

[0329] As shown in Tables 5 to 10, the effect of Lactobacillus rhamnosus FNZ142 on gas production was tested in an in vitro rumen assay. The data shown are the average of three replicates. Negative numbers indicate stimulation, rather than inhibition. Asterisks (*) are used to indicate statistical significance (***P<0.05) by Student's T test with Welch correction.

[0330] Table 5. Percent inhibition of total gas produced compared to control (ml / g substrate).

[0331]

[0332] Table 6. Percent inhibition of total methane produced compared to controls (ml / g substrate).

[0333]

[0334] Table 7. Percent inhibition of total volatile fatty acids produced compared to controls (mM).

[0335]

[0336] Table 8. Percent inhibition of acetic acid production compared to controls (mM).

[0337]

[0338] Table 9. Percent inhibition of propionic acid production compared to controls (mM).

[0339]

[0340] Table 10. Percent inhibition of butyrate production compared to controls (mM).

[0341]

[0342] In the in vitro rumen assay, total methane production by Lactobacillus rhamnosus FNZ142 was significantly reduced at 2 and 6 hours in two of the three biological replicates. This effect was also observed using culture supernatant in one biological replicate. Bacteriocin extract had no significant effect on methane production. It should be noted that the rumen in vitro assay is a closed system and may become nutritionally limited over time. Therefore, the 0-12 hour time point may more accurately reflect the in vivo situation as animals will generally ingest more food and fluid over a 24 hour period. It should be noted that the RIV replicates were performed at different times, using rumen fluid taken from different cows, based on the diet on pasture. Therefore, variability between RIV replicates may be attributed, at least in part, to seasonal changes in pasture quality.

[0343] Overall, Lactobacillus rhamnosus FNZ142 cultures resulted in a reduction in total gas produced, although this varied between RIV replicates and only reached significance at certain time points in certain replicates. In the in vitro assay, culture supernatant and bacteriocin extracts had no significant effect on total gas produced in the rumen.

[0344] There was also no significant effect on total volatile fatty acids produced or the amounts of acetate, propionate, and butyrate produced, except for an increase in acetate in one replicate of the 12 h FNZ142 culture, a decrease in propionate in one replicate of the 24 h FNZ142 culture, and an increase in propionate in the bacteriocin extract at 6 h.

[0345] 2.3 Conclusion

[0346] Rumen in vitro assays with Lactobacillus rhamnosus FNZ142 demonstrated an effect on fermentation end products, showing a significant reduction in methane production. This occurred without significantly affecting volatile fatty acid production.

[0347] 3. Example 3 - Calf Methane Emissions

[0348] 3.1 Materials and methods

[0349] 3.1.1 Calf study design, animal ethics and housing facilities

[0350] This example uses a design involving treatment with Lactobacillus rhamnosus FNZ142 and a control to test the ability of FNZ142 to reduce CH4 emissions from calves when fed during the first 14 weeks of their life. Statistical power calculations using data from previous calf CH4 emission measurements indicated that at least 20 animals per treatment group would be required to detect a 20% difference in CH4 emissions. Previous studies have experienced the exclusion of calves from the trial due to navel infections caused by navel suction through the pen cap. To mitigate these potential losses, 24 calves / group were used. This work was approved by the AgResearch Ruakura Animal Ethics Committee. The calf feeding sheds were divided into 12 m 2 Each pen houses 4 calves and is lined with wood chips, supplied with fresh water and equipped with a feeder with calf pellets and hay.

[0351] 3.1.2 Calf registration and feeding

[0352] Female Fliess calves were enrolled in the study over a 3-week period. Calf recruitment was staggered over a 3-week period to extend calf age so that three groups of animals of similar age could be measured sequentially by the bovine CH4 measuring chamber. Newborn calves were collected from their mothers twice a day and brought to the calf rearing shed. Calves were weighed upon arrival at the shed and then weighed weekly in the calf rearing facility. Calves were randomly assigned to treatment groups (FNZ142 or control) within each week of enrollment, balancing birth weight and calf stock so that 8 calves were assigned to each treatment group per week. Within the first 12 hours of entering the shed, each calf was provided with 2 feeds of 2-3L of warm colostrum, with the first morning colostrum feed containing FNZ142 or control treatment. Freeze-dried Lactobacillus rhamnosus FNZ142 was kept at -20°C until use. Each FNZ142-treated calf received 5×10 10The FNZ142 daily dose of CFU. The control treatment (3g maltrin / calf / d) is an excipient for blending the concentrated freeze-dried Lactobacillus rhamnosus FNZ142 product to the correct daily dose. After colostrum feeding, calves are fed 6L calf milk replacer (CMR) (Ancalf, NZAgBiz / Fonterra; 150g / L mixed with tap water at ~37°C) every day, divided into 3L morning and 3L afternoon feeding. FNZ142 and control treatments are added only to the 3L milk in the morning and mixed into the preheated CMR until the freeze-dried material is evenly distributed throughout the milk. Calf is also provided with solid feed in the form of granulated calf feed containing 20% ​​fiber source (alfalfa and soybean hulls) and coccidia-specific non-ionophore coccidia inhibitor (see Table 11). No hay was provided to the calves during the first 6 weeks to avoid possible variation in solid feed consumption between calves and the possible impact this would have on rumen development and possible CH4 emissions from the calves. However, after the first CH4 measurement at week 6, calves were provided with pasture hay and chopped pasture hay (chopped to 75 mm) to help stimulate salivation and stabilize rumen pH. Intake of pelleted feed and hay was measured at the pen level.

[0353] Calves were weaned from most of their milk starting at week 10, so they were receiving only 0.5L of CMR containing FNZ142 and control treatments in the morning by the end of week 11. Treatments continued to be delivered to calves in 0.5L of CMR in the morning feed until their second round of CH4 measurements were completed at week 14.

[0354] Table 11. Ingredients and nutritional composition of calf feed pellets.

[0355]

[0356] 3.1.3 Animal health

[0357] The calves are weaned, vaccinated and treated against parasites in accordance with animal health and welfare protocols.

[0358] 3.1.4 Calf methane emission measurements

[0359] CH4 measurements were performed on 20 calves per treatment group at 6 weeks of age (pre-weaning), and on the same group of calves after 14 weeks of age (post-weaning), and once at 12 months of age. Calves were measured in 4 cow respiration chambers, 4 calves per session, for 2 days each. The first pre-weaning period of CH4 measurements occurred during 34 days, and the second post-weaning period also exceeded 34 days. The third, at 12 months of age, also exceeded 34 days.

[0360] First round of CH4 measurements

[0361] The calves (4 per trip) were transported from the calf feeding shed to the methane measurement facility located at the New Zealand Animal Ruminant Methane Measurement Center. Upon arrival, the calves were housed in a fence with wood chip bedding, where they received afternoon milk (3L, before weaning) or solid feed (after weaning) supplementation, and fresh water was available. Around 8:00 am the next day, the calves were moved into individual cattle respiration chambers, where they received morning milk (CMR; 3L), and pelleted feed (plus hay after weaning) and water were available at will. The calves continued to receive their FNZ142 and control treatments and solid pelleted feed in the chamber. The calves maintained CH4 measurements for 2 days in the chamber, and the feeding and cleaning of the pens were performed twice a day. At 8:00 am on the third day, the calves were transferred from the chamber to the fence, and the next 4 calves (kept in the fence the night before) entered the chamber. At 9:00 AM, calves leaving the barn were given a morning distribution of milk and ad libitum pellets (plus weaned hay, post-weaning), and water was freely available. Two hours after feeding (~11 am), samples of rumen contents and fecal material were collected from each calf. Samples of rumen (via stomach tube) and fecal material (via digital collection) were immediately used for pH measurement and stored at -80°C for subsequent volatile fatty acid (VFA) analysis.

[0362] After the first CH4 measurement period of 6 weeks, calves were returned to the calf holding facility and provided hay ad libitum in addition to calf pellets until week 12.

[0363] Second round of CH4 measurements

[0364] Hay was replaced ad libitum with bran hay 2 weeks prior to the second round of CH4 measurements at week 14. When calves entered the respiration chamber for post-weaning measurements at 14 weeks of age, bran hay was provided at approximately 10% (500 g / d) of the daily calf pellets (5 kg) provided. Milk and solid feed intake was measured during both rounds of CH4 measurements, and feed samples were dried to estimate dry matter intake (DMI) per calf per day and used for composition analysis. Chamber measurements of CH4, hydrogen (H2), and CO2 were reported as emissions (g / d) or yields (g / kg DMI / d).

[0365] After the second round of CH4 measurements and sampling was completed at week 14, calves were transported to the farm to adjust to pasture feeding. Calves were placed on pasture with access to calf pellets at their previous daily intake, and the amount of pellets provided was reduced over a 3-week period to facilitate the transition to pasture (reducing pellet allowance by 33% per week).

[0366] The third round of CH4 measurements

[0367] At approximately 9 months of age, samples of rumen contents and fecal material were collected as previously described, however respiratory chamber measurements were not performed at this time due to facility limitations. At approximately 1 year of age, animals were returned to the respiratory chambers for measurements.

[0368] Animals were acclimated to cut grass (harvested daily) for 7 days and continued to receive cut grass in the cattle respiration chamber for 2 days. Two hours after leaving the chamber, samples of rumen contents and fecal material were collected from each animal as previously described.

[0369] Feed intake was measured during 2 days in the respiration chambers and feed samples were dried to estimate dry matter intake (DMI) per animal per day. Subsamples of the dried feed were used for compositional analysis. Respiration chamber measurements of methane, hydrogen and carbon dioxide were reported as emissions (g / d) or yields (g / kg DMI / d).

[0370] 3.1.5 Post-weaning animal growth

[0371] After the methane measurements at week 14, the calves were moved to pasture and their body weight and average daily gain were monitored monthly until 13 months of age. The treated and control animals were moved to separate pastures and pasture quality was monitored to ensure comparability of the results. Although there were seasonal variations in metabolizable energy (ME) and digestibility (in vitro DOMD%) in both pastures, there were no significant differences between pastures as expected in pasture-based farming systems. Treatment with FNZ142 was stopped after weaning at 14 weeks of age.

[0372] Statistical analyses were performed in R using the Lme4 package. Body weight models were fitted by REML and included the effects of treatment with FNZ142, time (ie month of weighing), and the interaction of the two with a random effect of animal.

[0373] ADG was calculated by averaging the daily weight gain between 4-5 months and 13-14 months for each treatment. A two sample t-test was used to explore differences in daily weight gain between treatments and controls.

[0374] 3.2 Results

[0375] A total of 48 female Friesian calves were enrolled in the study over a 3-week period from July 23 to August 12, 2021. When calves were born, they were assigned to one of 2 treatment groups (n=24 per group) - FNZ142 or control (vehicle only). 20 calves from each group were used for the following measurements.

[0376] 3.2.1 Animal growth before weaning

[0377] Calves were weighed when they first arrived at the calf raising shed. There were no statistical differences in birth weights between treatment groups (Table 12).

[0378] Table 12. Calf birth weight.

[0379]

[0380] The calves received treatment starting with the first colostrum feeding and continued to receive treatment once daily in the morning until the second CH4 measurement was completed after 14 weeks of age. The intake of pellets and hay by the calves while in the respiration chamber during the methane measurement period is shown in Table 13.

[0381] Table 13. Dietary intake of calves during CH4 measurements at 6 and 14 weeks of age*.

[0382]

[0383] * During CH4 measurements, all calves consumed 6 L CMR / day (in 2 × 3 L feeds in the AM and PM) at 6 weeks of age and 0.5 L CMR / day in the morning only at 14 weeks of age. Hay was not provided to calves until after the first CH4 measurement was completed at 6 weeks of age. NS, not significantly different from control by T-test; NA, not applicable.

[0384] A summary of live weight (LWT) and average daily gain (ADG) prior to CH4 measurements at 6 and 14 weeks is shown in Table 14. There were no significant differences in LWT or ADG between FNZ142 treated and control calves at 6 weeks of age or ADG at 14 weeks of age, but the FNZ142 treated group showed a 5% decrease in LWT relative to controls at 14 weeks of age, reaching significance (p<0.05).

[0385] It is known that CH4 measurement in respiration chambers can be stressful to animals, which may affect feed intake and growth rate, therefore, LWT and ADG of calves in respiration chambers were also examined. A few animals lost weight while passing through the chambers (mean LWT loss of 0.392kg over 4 days), but most animals maintained or gained weight (mean LWT gain of 0.774kg over 4 days, ADG 0.194kg). While in the chambers, there was no significant difference in ADG between FNZ142-treated calves and those fed the control treatment.

[0386] Table 14. Live weight and average daily gain of calves measured on CH4 at 6 and 14 weeks of age.

[0387]

[0388] SD: standard deviation; p: T-test probability of FNZ142 treatment versus control, Sig: statistical significance; NS, not significant; *p≤0.05.

[0389] 3.2.2 Methane emissions

[0390] From the calves participating in the study, 40 calves were selected for measurement of CH4 emissions. The selection was made from a weekly registration of a group of animals, and the criteria used for calf exclusion were previous health status and need for any veterinary intervention before CH4 measurement. Animals were measured in three rounds; round 1 before weaning at 6 weeks of age, round 2 after weaning at 14 weeks of age, and round 3 at approximately 1 year of age. Each round of measurements was performed in 3 batches over a 34-day period.

[0391] In round 1, calves received 2 x 3 L / d of CMR feed in the morning (including treatment) and afternoon, plus calf pellets, fed ad libitum during their time in the barn. In round 2, calves received only 0.5 L of CMR containing treatment in the morning feed plus calf pellets fed ad libitum, and 10% of their expected solid feed intake was also provided with hay with bran. In round 3, animals received cut grass.

[0392] Table 15. CH4 emissions from calves at 6 weeks (pre-weaning) and 14 weeks and 1 year (post-weaning).

[0393]

[0394] DMI: dry matter intake. For the first round of measurements at week 6, calves consumed 6L CMR / d (900g / d milk solids) plus the indicated amount of calf pellets, which represented approximately 40% of total intake. For the second round at week 14, calves consumed 0.5L CMR and were provided pellets (5kg / d), with 10% of the expected intake provided as hay with chaff (500g / d). During the 1-year measurement period, animals were fed cut pasture. NS not significant; *p<0.05; **p<0.01; ***p<0.001.

[0395] In the 6 week measurements, CH4 production of the calves was 7-10 g / d, with a CH4 yield of 12-15 g / kg DMI / d (Table 15). This is expected from pre-weaned calves on a primarily milk diet (6 L / d: 0.9 kg milk solids) and only a small amount (0.6-0.7 kg / d) of solid feed intake.

[0396] Calves treated with FNZ142 showed a significant (P<0.01) 17% reduction in CH4 production (g / d) and a significant (P<0.01) 7.5% reduction in C02 production (g / kg DMI) compared to calves receiving the control treatment (vehicle only).

[0397] The calves receiving FNZ142 had a significant (P<0.05) decrease in pellet intake of approximately 17%. However, the calves also consumed 6 L CMR / d (900 g / d milk solids), so pellet intake only accounted for approximately 40% of total intake.

[0398] After weaning at week 14, CH4 production in calves treated with FNZ142 was 47.08 g / d and in control calves was 51.55 g / d, while CH4 production in calves treated with FNZ142 was 15.39 g CH4 / kg DMI / d and in control calves was 14.88 g CH4 / kg DMI / d. These CH4 emission levels were lower than those in adult animals but within the expected range for weaned calves.

[0399] Even after the cessation of FNZ142 supplementation after the 14th week methane measurements, FNZ142-fed animals still had significantly lower CH4 production (g CH4 / day; p<0.001) and productivity (g CH4 / kg DMI; p<0.05) at one year of age (Table 15). H2 and CO2 production were also significantly reduced in animals fed FNZ142. At one year, FNZ142-fed animals also showed significantly lower total dry matter intake than control animals (p<0.001; Table 15).

[0400] Samples of rumen contents and fecal material were collected from calves after the CH4 measurement cycle at week 6, week 14, month 9, and year 1, and their pH values ​​were measured. Rumen pH did not differ significantly between the FNZ142 treated and control groups (Table 16). Fecal pH did not differ significantly between the FNZ142 treated and control groups, except for fecal pH at week 14, which was lower in the FNZ142 treated group than in the control group.

[0401] Table 16. Rumen and fecal pH measurements.

[0402]

[0403] Volatile fatty acids were measured in rumen contents from samples collected from calves after leaving the rumen at 6 weeks (pre-weaning) and 14 weeks, 9 months, and 1 year (post-weaning). Acetate was the predominant VFA detected, while propionate and butyrate made up smaller proportions (Table 17). There were no significant differences between the amounts of acetate, propionate, or total VFA found in calves fed FNZ142 compared to the control group. Butyrate was significantly reduced in FNZ142-treated animals at 1 year compared to control animals, but there were no significant differences at any other measured time points.

[0404] Table 17. Volatile fatty acids in calf rumen samples*.

[0405]

[0406] *Values ​​are expressed as mM of individual VFAs determined by the GC method. P: T-test probability; NS: not significantly different from the control; **, p<0.01.

[0407] At 14 weeks, the amount of isovalerate was significantly reduced in the treated animals compared to the control animals. However, at 9 months, the treated animals instead showed highly significant increases in the amount of isobutyrate, valerate and isovalerate present in rumen samples (Table 18).

[0408] Table 18. Proportions of trace volatile fatty acids in calf rumen samples

[0409]

[0410] Values ​​are expressed as % of individual VFA relative to total VFA. P: T-test probability; NS: no significant difference from control; *, p<0.05; **, p<0.01; ***, p<0.001.

[0411] There were no significant differences in the amounts of formate, lactate, and succinate found in treated or control animals at weeks 6 and 14 or at 9 months (Table 19). At 1 year, the amounts of formate, lactate, and succinate were significantly reduced in treated animals compared to control animals.

[0412] Table 19. Rumen non-volatile fatty acids and formate concentrations (mM).

[0413]

[0414] Values ​​are expressed as mM of individual VFAs determined by the VFA derivatization method. P: T-test probability; NS: not significantly different from control; *, p<0.05; **, p<0.01.

[0415] 3.2.3 Post-weaning animal growth

[0416] After methane measurements at week 14, calves were moved to pasture and their body weight and average daily gain were monitored monthly.

[0417] During the methane measurement period at 1 year of age, the dietary intake of FNZ142 treated animals was 5.69 kg pasture dry matter per animal per day with a standard deviation of 0.77. For control animals, the dietary intake was 7.26 kg per animal per day with a standard deviation of 0.95 (Table 15). The amount of feed consumed by FNZ142 treated animals was significantly lower (p<0.001).

[0418] The body weights of FNZ142-treated and control animals were similar from 4-5 months to 6-7 months (p>0.05) (Table 20; Figure 1 However, starting from month 7-8, the control animals gained significantly less weight and thus had a significantly lower body weight (p<0.05) than the FNZ142 treated animals (Table 20; Figure 1 ).

[0419] The average daily gain (ADG) was significantly higher in FNZ142 treated animals compared to the control group at months 4-14 (p=0.003).

[0420] Table 20. Average monthly body weights and average daily gain after weaning.

[0421]

[0422] *p<0.05; **p<0.01.

[0423] 3.3 Discussion

[0424] The first round of CH4 measurements (week 6) showed that CH4 production was reduced by about 17% in calves treated with FNZ142. Overall, CH4 production and CH4 yield of these calves were low compared to mature cattle on a pasture diet (typically about 22 g / kg DMI). This is expected when pre-weaned calves on a primarily milk diet (6 L / d: 0.9 kg milk solids) consume small amounts (0.6-0.7 kg / d) of solid (grain-based) feed.

[0425] Feeding of the FNZ142 strain did not affect CMR intake or solid feed consumption in the pen during the first CH4 measurement period. Although calves consumed all of their CMR during the first round of CH4 measurements, pellet intake in calves fed FNZ142 in the pen was approximately 100 g / d lower than in calves fed the control diet, representing approximately 17% lower solid feed intake.

[0426] The stress of CH4 measurement in the respiration chamber may have caused some animals to lose weight, but most calves in round 1 continued to gain weight, albeit at a lower rate compared to when the animals were not in the chamber. When the animals were in the chamber, there was no significant difference in ADG between calves fed FNZ142 compared to the control treatment, so the differences observed in solid feed intake while in the chamber appear to be an effect of the LAB treatment. In these pre-weaned calves, milk components accounted for the majority of dietary intake (~60%), so the lower pellet intake did not affect the overall group LWT or ADG of calves receiving the FNZ142 treatment.

[0427] After weaning at week 14, CH4 production measured from calves increased approximately 5-fold, while their CH4 yields remained similar to those measured at week 6. The increase in overall CH4 production was expected, as the post-weaning diet consisted primarily of pellets and hay with bran (mean pellet intake 3.9 kg / d, mean hay intake 0.35 kg / d) and only small amounts of CMR (mean CMR intake of solids 0.075 kg / d). CH4, H2, and CO2 production were all numerically lower in calves fed FNZ142 compared to control calves, but were no longer significantly different.

[0428] At 1 year, CH4 production and yield, H2 production, and CO2 production were significantly lower in treated animals than in untreated animals. DMI intake was also significantly lower in treated animals than in untreated animals (p<0.001). Nevertheless, treated animals showed higher body weights than untreated animals.

[0429] A potential cause for the lower DMI in calves could be related to diet-induced low rumen pH, however this did not appear to be the cause of the reduced DMI observed in calves fed FNZ 142. Although some individual rumen samples (-23%) were below 5.6 at the time of sampling (2 hours post-feeding, when rumen pH is likely at its lowest point), there were no significant differences in rumen pH between calves fed FNZ 142 compared to the control treatment, indicating that pH was not the cause of the reduced intake in calves fed FNZ 142.

[0430] VFA analysis of rumen contents of calves at weeks 6 and 14 (post-weaning) showed no significant differences between FNZ142-fed calves compared to control animals.

[0431] At 1 year old, the amount of butyrate present in rumen samples from animals treated with FNZ142 was significantly reduced. The amount of VFA present in rumen samples is a balance between rumen VFA production and VFA absorption. The reduction in the amount of VFA in rumen samples may be due to lower VFA production (e.g., because less feed is digested) and / or increased rumen absorption of VFA (meaning more VFA is available for growth and development). Together with the increased body weight observed in treated animals, the reduced rumen VFA concentration is consistent with increased absorption, resulting in increased feed efficiency.

[0432] After weaning, animals continued to show similar weight gain from 4-5 months to 6-7 months. From 7-8 to 9-10 months, FNZ142 treated animals showed significantly more weight gain, so their body weight was significantly higher than control animals (p < 0.05). From 10-11 months onwards, FNZ142 treated animals still showed numerically higher body weights than control animals, although this did not reach significance levels.

[0433] ADG in FNZ142 treated heifers during the post-weaning period (4-14 months) was also significantly higher than in control animals (p=0.003). Despite reduced dry matter intake (measured during methane chamber measurements), this suggests that FNZ142 treated animals were more productive and had greater feed efficiency than control animals.

[0434] 3.4 Conclusion

[0435] The examples show that supplementation with Lactobacillus rhamnosus FNZ142 during the first 14 weeks of life can improve feed efficiency in post-weaned animals by supporting normal or increased growth, leading to increased body weight gain despite reduced dry matter intake. This effect persists until at least 1 year of age, even when supplementation is stopped at 14 weeks of age.

[0436] The examples also show that Lactobacillus rhamnosus FNZ142 can significantly reduce methane production in pre-weaned (6-week-old) calves. Likewise, even when supplementation is stopped at week 14, this effect persists until at least 1 year of age.

[0437] Although calves fed FNZ142 produced less methane and ate less when measured before weaning, they gained live weight and average daily gain comparable to control animals. Maintaining animal growth at lower feed intake and lower methane production suggests a mechanism involving more efficient use of ingested feed for rumen metabolism and production.

[0438] The examples also show that Lactobacillus rhamnosus FNZ142 can support normal growth after weaning and lead to increased weight gain even after supplementation has been stopped.

[0439] 4. Example 4 - Effects of Lactobacillus rhamnosus FNZ142 on rumen and fecal microbiota

[0440] 4.1 Materials and methods

[0441] Rumen and fecal samples were collected at the end of the methane measurement and immediately frozen at -80 ° C until DNA extraction. DNA was extracted using the bead shaking / phenol chloroform method (Rius et al., 2012) and used in PCR reactions to generate 16S ribosomal RNA gene amplicons with bacterial, archaeal and protozoan specific barcoded sequencing primers (Kittelmann and Janssen, 2011). The amplicons were purified, standardized, merged and sequenced by an Illumina MiSeq sequencer. The sequencing results were quality controlled and filtered, and the filtered sequences were analyzed by QIIME using the Silva database with rumen-specific 16S rRNA gene sequences. Operational taxonomic units (OTUs) were selected at 99% similarity and tabulated.

[0442] 4.2 Results

[0443] Samples for rumen and fecal microbiota analysis were collected at weeks 6 and 14, while methane measurements were recorded in Example 3.

[0444] 4.2.1 Rumen microbiota (bacteria)

[0445] Overall, no significant differences in rumen bacterial microbiota were observed at the phylum level between FNZ142-treated and control animals at either week 6 or week 14 (data not shown).

[0446] At the family level, the relative abundance of Lachnospiraceae decreased significantly (p<0.05) in week 6 (Table 21). Although the difference still existed in week 14, it did not reach the significance level.

[0447] Table 21. Relative abundance of bacterial families in the rumen microbiota.

[0448]

[0449]

[0450]

[0451] *p<0.05.

[0452] At the genus level, Succinolyticus showed a significant decrease in abundance at week 14 compared to the control (Table 22). Succinolyticus is known to be involved in succinate metabolism, so this decrease may indicate a perturbation in the succinate metabolic pathway in the rumen.

[0453] Table 22. Relative abundance of bacterial genera in the rumen microbiota.

[0454]

[0455] *p<0.05.

[0456] 4.2.2 Rumen microbiota (archaea)

[0457] Overall, no significant differences in the rumen archaeal microbiota were observed between FNZ142-treated and control animals at the family, genus, or clade level at either week 6 or week 14 (data not shown).

[0458] 4.2.3 Fecal microbiota (bacteria)

[0459] At week 6, at the P level, the relative abundance of Firmicutes in feces of animals fed FNZ142 was significantly increased relative to control animals (Table 23). The effect did not persist through week 14. While Lactobacillus rhamnosus FNZ142 is a member of the Firmicutes phylum, there was no corresponding significant increase in Lactobacillus, suggesting that the increase in Firmicutes reflects an increase in other bacterial genera within the phylum, not just an increase in FNZ142.

[0460] Table 23. Relative abundance of bacteria in feces.

[0461]

[0462] *p<0.05.

[0463] At the family level, the relative abundance of Firmicutes in feces of animals fed FNZ142 was significantly increased (p<0.05) relative to control animals at week 6 (Table 24). The effect did not persist until week 14.

[0464] Table 24. Relative abundance of bacterial families in feces.

[0465]

[0466]

[0467]

[0468] *p<0.05.

[0469] At the genus level, there were no significant differences between feces of FNZ142-treated animals and controls (data not shown).

[0470] 4.2.4 Fecal microbiota (archaea)

[0471] There were no significant differences in archaeal diversity at the family, genus, or clade level in feces of FNZ142-treated animals compared with controls (data not shown).

[0472] 4.3 Conclusion

[0473] The examples show that Lactobacillus rhamnosus FNZ142 can reduce methane production in calves (as shown in Example 3) without causing major disruptions to the bacterial or archaeal microbiome. The reduction in the abundance of Lachnospiraceae in the rumen at week 6 was consistent with the reduction in methane production in sheep, but no corresponding increase in Lachnospiraceae, particularly Sharp's or Megasphaera, was observed in the rumen. At week 6, an increase in Erysipelothrix was observed in the feces.

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[0494] Industrial Applicability

[0495] The present invention relates to the use of probiotics, in particular Lactobacillus rhamnosus strain FNZ142 and / or its derivatives, in particular for improving the body weight or body composition of ruminants, increasing feed efficiency, enhancing the growth and / or productivity of ruminants and / or increasing the milk production of ruminants, inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants or reducing their abundance, reducing the ability of rumen microflora to produce methane and / or reducing methane emissions of ruminants. Also provided are methods of using Lactobacillus rhamnosus strain FNZ142 and / or its derivatives and ruminant feed compositions comprising them.

[0496]

[0497]

[0498]

Claims

1. Isolated Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or its derivatives, the strain having NMIA accession number V21 / 015448, dated August 2, 2021.

2. A food or feed composition comprising Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or its derivatives, wherein the strain has an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

3. The food or feed composition according to claim 2, wherein The composition is a ruminant feed composition.

4. A ruminant feed composition, for: a. Increase feed efficiency of ruminants, b. enhancing the growth and / or productivity of ruminants, c. improving the body weight and / or body composition of ruminants, d. increasing the yield of milk and / or milk components produced by ruminants, e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, f. reducing said ability of the rumen microflora to produce methane, g. Reduce methane emissions from ruminants, h. delivering microorganisms to ruminants, and / or i. reducing the greenhouse gas emissions footprint of ruminants, A feed composition comprising Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or a derivative thereof, wherein the strain has an NMIA accession number of V21 / 015448 and a date of August 2, 2021.

5. The ruminant feed composition according to claim 4, wherein The feed composition is a fermented yogurt type composition, wherein the fermented yogurt type composition is formed by a method of growing Lactobacillus rhamnosus FNZ142 using a milk-based carrier or a non-milk-based carrier.

6. The ruminant feed composition of claim 4, which is or comprises part or total mixed ration (TMR), corn, soybeans, forage, grains, distiller's grains, sprouted grains, legumes, fiber, forage, grass, hay, straw, silage, nuts, leaves, meal, mash feed, fruit pulp, vegetable pulp, fruit or vegetable pulp, citrus meal, wheat sorghum, corn cob meal, lick block or molasses.

7. A ruminant feed composition according to any one of claims 4 to 6, further comprising at least one microorganism of a different species or strain, a vaccine inhibiting methanogens or methanogenesis, and / or a natural or chemically synthesized inhibitor of methanogenesis and / or a methanogenic inhibitor such as bromoform.

8. A ruminant feed composition according to any one of claims 4 to 7, further comprising one or more agents selected from the group consisting of one or more prebiotics, one or more probiotics, one or more postbiotics, one or more dietary fiber sources, one or more galacto-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, one or more fructo-oligosaccharides, one or more short-chain fructo-oligosaccharides, one or more long-chain fructo-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

9. The ruminant feed composition according to any one of claims 4 to 8, wherein The derivative of the Lactobacillus rhamnosus FNZ142 is a cell lysate of the strain, a cell suspension of the strain, a metabolite of the strain, a culture supernatant of the strain or inactivated Lactobacillus rhamnosus FNZ142.

10. A method for: a. Increase feed efficiency of ruminants, b. enhancing the growth and / or productivity of ruminants, c. improving the body weight and / or body composition of ruminants, d. increasing the yield of milk and / or milk components produced by ruminants, e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, f. reducing the ability of the rumen microbiota of ruminants to produce methane, g. Reduce methane emissions from ruminants, h. delivering microorganisms to ruminants, and / or i. reducing the greenhouse gas emissions footprint of ruminants, The method comprises the steps of administering to the animal: i. The food or feed composition according to claim 2 or 3, ii. The ruminant feed composition according to any one of claims 4 to 9, or iii. An effective amount of Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142 or its derivative, the NMIA accession number of which is V21 / 015448, dated August 2, 2021.

11. The method according to claim 10, wherein: The method inhibits the growth or reduces the abundance of methylotrophic methanogens, preferably methanogens from the genus Methanosphaeria, in the forestomach of an animal.

12. The method according to claim 10 or 11, wherein: The method inhibits the growth or reduces the abundance of hydrogenotrophic methanogens, such as methanogens from the genus Methanobacterium brevis, in the forestomach of the animal.

13. The method according to claim 10 or 12, wherein: The Lactobacillus rhamnosus FNZ142 or a derivative thereof is administered in the form of a composition, which is a food, a beverage, a food additive, a beverage additive, an animal feed, an animal feed additive, an animal feed supplement, a dietary supplement, a carrier, a vitamin or mineral premix, a nutritional product, an enteral feeding product, a soluble, a supplement, a medicament, a lick block, a drench, a tablet, a capsule, a pill or an intraruminal product, such as a bolus, or wherein the Lactobacillus rhamnosus FNZ142 is encapsulated in, for example, liposomes, microbubbles, microparticles or microcapsules.

14. The method of claim 13, wherein Lactobacillus rhamnosus FNZ142 or a derivative thereof is administered in drinking water, milk, milk powder, milk replacer, milk fortifier, whey, whey powder, part or total mixed ration (TMR), corn, soy, forage, cereals, distiller's grains, sprouted cereals, pulses, vitamins, amino acids, minerals, fiber, forage, grass, hay, straw, silage, nuts, leaves, meal, solubles, supplements, mash, fruit pulp, vegetable pulp, fruit or vegetable pulp, citrus meal, wheat middlings, corn cob meal, lick block, molasses, sucrose, maltodextrin, rice husks, vermiculite, zeolite or crushed limestone.

15. The method according to any one of claims 10 to 14, wherein: The method comprises administering to the animal Lactobacillus rhamnosus FNZ142 in an amount of: a.10 4 Up to 10 13 CFU / kg dry weight of carrier feed, preferably 10 8 Up to 10 12 colony forming units / kg dry weight of carrier feed; b.10 4 Up to 10 10 Colony forming units / kg animal body weight / day, preferably 10 5 Up to 10 8 CFU / kg animal body weight / day; or c.10 4 Up to 10 13 CFU / day, preferably 10 6 Up to 10 13 Colony forming units / day.

16. The method according to any one of claims 10 to 15, wherein: The derivative of Lactobacillus rhamnosus FNZ142 is a cell lysate of the strain, a cell suspension of the strain, a metabolite of the strain, a culture supernatant of the strain or inactivated Lactobacillus rhamnosus FNZ142.

17. The method according to any one of claims 10 to 16, comprising further administering at least one microorganism of a different species or strain, a vaccine inhibiting methanogens or methanogenesis, and / or a natural or chemically synthesized inhibitor of methanogenesis and / or an inhibitor of methanogens, such as bromoform.

18. The method according to any one of claims 10 to 17, wherein: The Lactobacillus rhamnosus FNZ142 or its derivatives are administered separately, simultaneously or sequentially with one or more agents selected from one or more prebiotics, one or more probiotics, one or more postbiotics, one or more dietary fiber sources, one or more galacto-oligosaccharides, one or more short-chain galacto-oligosaccharides, one or more long-chain galacto-oligosaccharides, one or more fructo-oligosaccharides, one or more short-chain fructo-oligosaccharides, one or more long-chain fructo-oligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

19. The method according to any one of claims 10 to 18, wherein the method: a. enhancing the growth or productivity of the ruminant, b. increasing said yield of milk and / or milk components produced by said ruminant, c. increasing the yield of milk fat, milk protein or milk solids in milk produced by the ruminant, and / or d. Additionally improving the body weight and / or body composition of ruminants.

20. The method according to any one of claims 10 to 19, wherein: The ruminant is cattle, goats, sheep, bison, yaks, buffaloes, deer, camels, alpacas, llamas, wildebeests, antelopes or cows; preferably cattle or sheep; more preferably cattle.

21. The method according to any one of claims 10 to 20, wherein: a. the ruminant is a lactating animal, b. the ruminant is a pre-weaned animal, such as a calf or a lamb, c. the ruminant is a weaned animal, or d. Lactobacillus rhamnosus FNZ142 was administered to ruminants before and after weaning.

22. The method according to any one of claims 10 to 21, wherein: The administration is to a pre-weaned animal, and wherein the inhibition of the growth of methanogenic bacteria and / or archaea in the forestomach of the ruminant, the reduction in methane emissions, e.g., methane production, of the ruminant, and / or the increased feed efficiency in the ruminant persists after weaning.

23. The method according to any one of claims 10 to 22, wherein: Said inhibition of said growth of methanogenic bacteria and / or archaea in said forestomach of said ruminant, said reduction of methane emissions, e.g. methane production, of said ruminant, and / or said increase in feed efficiency of said ruminant persists for at least 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years or 7 years after the last administration of Lactobacillus rhamnosus FNZ142; preferably for the life of the ruminant.

24. A ruminant to which has been applied the method of any one of claims 10 to 23.

25. A method for producing an animal product having a reduced greenhouse gas emission footprint, the method comprising: a. Providing a ruminant according to claim 24, and b. Production of animal products from said animals.

26. Use of Lactobacillus rhamnosus strain FNZ142, or a derivative thereof, wherein the strain has NMIA accession number V21 / 015448, dated August 2, 2021, for preparing a composition for: a. Increase feed efficiency of ruminants, b. enhancing the growth and / or productivity of ruminants, c. improving the body weight and / or body composition of ruminants, d. increasing the yield of milk and / or milk components produced by ruminants, e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, f. reducing said ability of the rumen microflora to produce methane, g. Reduce methane emissions from ruminants, h. delivering microorganisms to ruminants, and / or i. Reducing the greenhouse gas emissions footprint of ruminants.

27. The use according to claim 26, wherein The composition comprises a food or feed composition according to claim 2 or 3, or a ruminant feed composition according to any one of claims 4 to 9.

28. Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ142, or a derivative thereof, having NMIA accession number V21 / 015448, dated 2 August 2021, for use in: a. Increase feed efficiency of ruminants, b. enhancing the growth and / or productivity of ruminants, c. improving the body weight and / or body composition of ruminants, d. increasing the yield of milk and / or milk components produced by ruminants, e. inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, f. reducing said ability of the rumen microflora to produce methane, g. Reduce methane emissions from ruminants, h. delivering microorganisms to ruminants, and / or i. Reducing the greenhouse gas emissions footprint of ruminants.

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