Use of Lactic Acid Bacteria to Improve Feed Efficiency

Abstract

The present invention relates to the use of a strain of probiotic bacteria or a derivative thereof to increase feed efficiency, enhance growth and / or productivity, improve body weight or body composition of a ruminant and / or increase milk production in a ruminant, inhibit the growth of methanogenic bacteria and / or archaea in the forestomach of a ruminant, reduce the ability of the ruminal microflora to produce methane, reduce methane emissions by the ruminant and / or reduce the greenhouse gas emission footprint of the ruminant. Ruminant feed compositions are also provided.

Description

[Technical field]

[0001] The present invention relates to the use of lactic acid bacteria strains to increase feed efficiency, enhance growth and / or productivity, improve body weight or body composition of ruminants and / or increase milk production in ruminants, inhibit the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, reduce the ability of the ruminal microflora to produce methane, reduce methane emissions by ruminants and / or reduce the greenhouse gas emission footprint of ruminants. Ruminant feed compositions are also provided. [Background technology]

[0002] Lactic acid bacteria (LAB) have been used as probiotics in humans for 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 explored as alternatives to antibiotics used as growth promoters.

[0003] On farms, LAB are used as direct-fed microorganisms (DFM), probiotics and silage inoculants. Their action is exerted in a strain- and host-specific manner. Some studies have reported various benefits depending on the particular strain and host used, including reduced incidence of diarrhea, enhanced ruminal development, improved feed efficiency, increased weight gain and reduced morbidity. However, their impact on performance is variable and their mode of action is unclear (Krehbiel et al., 2003).

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

[0005] The main source of methane emissions is the fermentation of organic matter by methanogenic bacteria and archaea. One of the main sources of anthropogenic methane emissions is agriculture, where methane is produced by enteric fermentation in the digestive tract of ruminants and from manure. These sources accounted for approximately 30% of global anthropogenic methane emissions in 2017 (Jackson et al., 2020). Furthermore, methane production in ruminants not only results in greenhouse gas emissions but is also energetically wasteful for animals. It has long been recognized that methane production in ruminants dramatically affects the efficiency with which these animals convert feed into metabolic energy. This reduction in efficiency is due to the fact that methane represents a caloric loss for ruminants of approximately 5–10% of their total caloric intake. However, to date, there has been limited research on the potential use of LAB to reduce methane emissions.

[0006] Thus, there remains a need for methods and compositions useful for increasing feed efficiency, increasing growth and / or productivity, improving the body weight or body composition of ruminants, and / or increasing milk production in ruminants. Methods and compositions for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of ruminants, reducing the ability of the ruminal microflora to produce methane, reducing methane emissions by ruminants, and / or reducing the greenhouse gas emission footprint of ruminants are also desirable.

[0007] It is an object of the present invention to go toward achieving one or more of these needs, or at least to provide the public with a useful choice. Summary of the Invention

[0008] In a first aspect, the present invention provides an isolated Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0009] In one embodiment, the Lacticaseibacillus rhamnosus strain FNZ118 is a biologically pure culture.

[0010] In a second aspect, the present invention provides a food or feed composition comprising Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated 2 August 2021) or a derivative thereof.

[0011] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a. Increases feed efficiency in ruminants; b. enhance growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reducing methane emissions from ruminants and increasing feed efficiency in ruminants; h. delivering microorganisms to ruminants; and / or i. reducing ruminant greenhouse gas emissions; The present invention provides a ruminant feed composition for producing a ruminant animal feed composition for producing a ruminant animal feed composition comprising Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0012] 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 nilgai feed composition.

[0013] In a further aspect, the present invention provides a method for increasing feed efficiency in a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0014] In a further aspect, the present invention provides a method for enhancing growth and / or productivity in a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0015] In a further aspect, the present invention provides a method for improving the body weight or body composition of a ruminant comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0016] In a further aspect, the present invention provides a method of increasing the yield of milk and / or milk components produced from a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0017] In a further aspect, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of a ruminant comprising administering to said animal a food or feed composition according to the second aspect or a ruminant feed composition according to the third aspect.

[0018] In a further aspect, the present invention provides a method for reducing the methane producing potential of the ruminant rumen microbiota comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0019] In a further aspect, the present invention provides a method for reducing methane emission by a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0020] In a further aspect, the present invention provides a method for delivering a microorganism to a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0021] In a further aspect, the present invention provides a method of reducing greenhouse gas emissions in a ruminant animal, said method comprising administering to said animal a food or feed composition according to the second aspect, or a ruminant feed composition according to the third aspect.

[0022] In a further aspect, the present invention provides a method for increasing feed efficiency in a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0023] In a further aspect, the present invention provides a method for enhancing growth and / or productivity in a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0024] In a further aspect, the present invention provides a method of improving body weight and / or body composition of a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0025] In a further aspect, the present invention provides a method for increasing the yield of milk and / or milk components produced from a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0026] In a further aspect, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of a ruminant comprising administering to the ruminant an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0027] In a further aspect, the present invention provides a method for reducing methane emissions by a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0028] In a further aspect, the present invention provides a method for reducing the ability of ruminal microflora to produce methane, comprising administering to an animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0029] In a further aspect, the present invention provides a method for delivering a microorganism to a ruminant, comprising administering to the ruminant an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0030] In a further aspect, the present invention provides a method for reducing greenhouse gas emissions in a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0031] In a further aspect, the present invention provides a method for improving the absorptive capacity of the forestomach, e.g., increasing the absorptive capacity of volatile fatty acids (VFAs), comprising administering to an animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0032] In a further aspect, the present invention provides a method for enhancing physical and / or functional development of the rumen in a ruminant, such as a young ruminant, such as a pre-weaned young ruminant, comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0033] In one embodiment, the method enhances rumen anatomical development. For example, the method enhances development and / or muscularization of the rumen epithelium, e.g., rumen mass growth, rumen papilla growth, increased papilla density, e.g., dorsal papilla density, and / or the total surface area of ​​the rumen wall in an animal.

[0034] In one embodiment, the method provides, for example, a reduction in rumen weight, rumen wall thickness, or cm 2 of rumen wall compared to untreated animals. 2 Increases the density of ruminal papillae around the rumen.

[0035] In one embodiment, the method increases the length, width, and / or surface area of ​​the ruminal papillae. For example, in some embodiments, the method increases the length, width, and / or surface area of ​​the ruminal papillae by 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.

[0036] In one embodiment, the method enhances ruminal functional performance, for example, the method stimulates rumination, enhances dry matter intake (DMI), enhances absorptive capacity, and / or promotes maturation to mature physiology.

[0037] In some embodiments, the method inhibits the growth of methylotrophic methanogens in the forestomach of the animal. In some embodiments, the method inhibits the growth of methanogens from the genus Methanosphaera in the forestomach of the animal.

[0038] In some embodiments, the L. rhamnosus FNZ118 or a derivative thereof is administered in a composition that is a food, beverage, food additive, beverage additive, animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral nutritional product, soluble, slurry, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet, or ruminal product, such as a bolus.

[0039] In a further aspect, the invention provides a composition comprising Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof. In some embodiments, the composition is a food, beverage, food additive, beverage additive, animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral nutritional product, soluble, slurry, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet, bolus, or ruminal product, or L. rhamnosus FNZ118 is encapsulated, for example, in a liposome, microbubble, microparticle, or microcapsule.

[0040] In some embodiments, L. rhamnosus FNZ118 or a derivative thereof is administered in drinking water, milk, milk powder, milk replacer, milk fortifier, whey, milk powder, partial or total mixed ration (TMR), corn, soybean, feed, cereals, distillers grains, germinated grains, legumes, vitamins, amino acids, minerals, fiber, forage, grass, hay, straw, silage, grains, leaves, meal, solubles, slurry, supplements, mash feed, meal, fruit pulp, vegetable pulp, fruit or vegetable pomace, citrus meal, wheat shorts, corn cob meal, molasses, sucrose, maltodextrin, rice hulls, vermiculite, zeolite, or ground limestone.

[0041] In some embodiments, the method comprises at least about 10 4 Colony forming units / kg dry weight carrier feed, e.g., at least about 10 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 The method comprises administering to the animal 10 colony forming units / kg dry weight of carrier feed of L. rhamnosus FNZ118. In some embodiments, the method comprises administering to the animal 10 colony forming units / kg dry weight of carrier feed of L. rhamnosus FNZ118. 4 ~10 13 In one embodiment, the method comprises administering 10 colony forming units of L. rhamnosus FNZ118 per kg dry weight of carrier feed to the animal. 8 ~10 12 This involves administering colony forming units of L. rhamnosu FNZ118 to the animal.

[0042] In some embodiments, the derivative of L. rhamnosus FNZ118 is a cell lysate of L. rhamnosus FNZ118, a cell suspension of L. rhamnosus FNZ118, a metabolic product of L. rhamnosus FNZ118, or a culture supernatant of L. rhamnosus FNZ118. In some embodiments, derivatives of L. rhamnosus FNZ118 are killed and / or non-replicating, for example, heat-killed, lysed, pressure-killed, irradiated, and / or UV-treated.

[0043] In some embodiments, the method further comprises administering at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methanogenesis, and / or a natural or chemically synthesized methanogenesis inhibitor and / or a methanogenesis inhibitor. An example of a useful inhibitor of methanogenesis is bromoform, which acts by inhibiting the efficiency of the methyltransferase enzyme by reacting with the reduced vitamin B12 cofactor required for the final step of methanogenesis.

[0044] In one embodiment, the method further comprises administering at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methane production, and / or a natural or chemically synthetic methanogenesis inhibitor, and / or a methanogenesis inhibitor that targets hydrogenotrophic methanogens, e.g., methanogens from the genus Methanobrevibacter.

[0045] In some embodiments, L. rhamnosus FNZ118 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 sources of dietary fiber, one or more galactooligosaccharides, one or more short chain galactooligosaccharides, one or more long chain galactooligosaccharides, one or more fructooligosaccharides, 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 a mixture of any two or more thereof.

[0046] In some embodiments, the methods further enhance the growth or productivity of the animal, for example, the methods increase the yield of milk and / or milk components produced from the ruminant animal, hi some embodiments, the methods increase the yield of milk fat, milk protein, or milk solids in the milk produced from the animal.

[0047] In some embodiments, the methods further increase body weight and / or improve body composition (eg, change muscle to fat ratio) of the ruminant.

[0048] In some embodiments, the method further increases wool growth in ruminant animals.

[0049] In some embodiments, the ruminant is a cow, goat, sheep, bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope, or nilgai. In one embodiment, the ruminant is a cow or a sheep. In one embodiment, the ruminant is a cow. In one embodiment, the ruminant is a lactating animal. In an alternative embodiment, the ruminant is a pre-weaned animal, such as a calf or lamb.

[0050] In some embodiments, the ruminant feed composition is or comprises a partial or total mixed ration (TMR), corn, soybean, forage, cereals, distillers grains, germinated grains, legumes, fiber, forage, grass, hay, straw, silage, grain, leaves, meal, mash feed, lick block, or molasses.

[0051] In some embodiments, the method further comprises administering at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methanogenesis, and / or a natural or chemically synthesized methanogenesis inhibitor and / or a methanogenesis inhibitor.

[0052] In one embodiment, the method further comprises administering at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methane production, and / or a natural or synthetic methanogenesis inhibitor, and / or a methanogenesis inhibitor that targets hydrogenotrophic methanogens, e.g., methanogens from the genus Methanobrevibacter.

[0053] In some embodiments, the ruminant feed composition further comprises 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 galactooligosaccharides, one or more short chain galactooligosaccharides, one or more long chain galactooligosaccharides, one or more fructooligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

[0054] In a further aspect, the present invention provides a ruminant to which the method of the previous aspect has been applied.

[0055] In a further aspect, the present invention provides a method for producing an animal product having a reduced greenhouse gas emission footprint, the method comprising: a. providing a ruminant animal according to the preceding aspect; b. Producing animal products from animals Includes.

[0056] In some embodiments, the animal products include dairy products, meat, or wool.

[0057] In a further aspect, the present invention relates to a method for the preparation of a Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof, a. Increases feed efficiency in ruminants; b. enhance growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reducing methane emissions from ruminants and increasing feed efficiency in ruminants; h. delivering microorganisms to ruminants; and / or i. Reducing ruminant greenhouse gas emissions The present invention provides a use for the manufacture of a composition for the treatment of a disease comprising the steps of:

[0058] In some embodiments the composition is or comprises a ruminant feed composition according to the third aspect.

[0059] In a further aspect, the present invention provides Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated 2 August 2021) or a derivative thereof, a. Increases feed efficiency in ruminants; b. enhance growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reducing methane emissions from ruminants; h. delivering microorganisms to ruminants; and / or i. Reducing ruminant greenhouse gas emissions Used for:

[0060] The invention may also be broadly described as consisting of any and all combinations of the parts, elements and features referred to or shown in the specification of this application, either individually or collectively, and any two or more of said parts, elements or features, and where a particular integer having a known equivalent in the art to which this invention pertains is referred to herein, such known equivalent is deemed to be incorporated herein as if individually set forth.

[0061] Reference to a range of numbers disclosed herein (e.g., 1-10) is intended to incorporate reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and thus all subranges of every range expressly disclosed herein are hereby expressly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly set forth in this application in a similar manner.

[0062] The term "comprise" as used in this specification means "consisting at least in part of." When interpreting each statement in this specification that contains the term "comprise," there may be other features present than the feature prefaced by that term. Related terms such as "comprise" and "comprises" should be interpreted in the same manner.

[0063] Where this specification refers to patents, other external documents, or other sources of information, it is generally for the purpose of providing a context for discussing features of the present invention. Unless specifically stated otherwise, a reference to such an external document is not to be construed as an admission that such document or such source is prior art or part of the common general knowledge in the art in any jurisdiction.

[0064] Embodiments of the present invention will now be described with reference to the drawings. [Brief description of the drawings]

[0065] [Figure 1] Figure 1 shows the body weight of heifers treated with FNZ118 (squares) or control (triangles) for the first 14 weeks of life and then transferred to pasture. * indicates a significant difference (p<0.05) between the FNZ118-treated and control groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The present invention is based on the discovery that Lacticaseibacillus rhamnosus strain FNZ118 and its derivatives increase feed efficiency in ruminants. FNZ118 and its derivatives are also shown herein to inhibit or suppress the growth of methanogens and / or archaea in the forestomach of ruminants and / or to reduce the ability of the ruminal microflora to produce methane. Inhibiting or suppressing the growth of methanogens and / or archaea can reduce methane emissions and alter the volatile fatty acid (VFA) profile, total VFA concentration, residual feed intake (RFI) and / or fermentation rate in the rumen and forestomach, which can act as an increased energy source to drive increased feed efficiency, increased weight gain, and / or increased productivity (e.g., milk, meat, or wool production), and can stimulate ruminal development, such as ruminal papilla development.

[0067] Thus, in a first aspect, the present invention provides an isolated Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0068] In a second aspect, the present invention provides a food or feed composition comprising Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated 2 August 2021) or a derivative thereof.

[0069] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a. Increases feed efficiency in ruminants; b. enhance growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reducing methane emissions from ruminants; h. delivering microorganisms to ruminants; and / or i. reducing ruminant greenhouse gas emissions; The present invention provides a ruminant feed composition for the production of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0070] In a further aspect, the present invention provides a method for increasing feed efficiency in a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0071] In a further aspect, the present invention provides a method for enhancing growth and / or productivity in a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0072] In a further aspect, the present invention provides a method of improving the body weight and / or body composition of a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0073] In a further aspect, the present invention provides a method for increasing the yield of milk and / or milk components produced from a ruminant animal, said method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0074] In a further aspect, the present invention provides a method for inhibiting the growth of methanogenic bacteria and / or archaea in the forestomach of a ruminant comprising administering to the ruminant an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0075] In a further aspect, the present invention provides a method for reducing methane emissions by a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0076] In a further aspect, the present invention provides a method for reducing the ability of ruminal microflora to produce methane, the method comprising administering to an animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0077] In a further aspect, the present invention provides a method for delivering a microorganism to a ruminant animal, the method comprising administering to the ruminant animal an effective amount of Lacticase ibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445, dated August 2, 2021), or a derivative thereof.

[0078] In a further aspect, the present invention provides a method for reducing greenhouse gas emissions in a ruminant animal, the method comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0079] In a further aspect, the present invention provides a method for improving the absorptive capacity of the forestomach, for example increasing the absorptive capacity of volatile fatty acids (VFAs), comprising administering to an animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118, NMIA accession number V21 / 015445 dated August 2, 2021, or a derivative thereof.

[0080] In a further aspect, the present invention provides a method for enhancing physical and / or functional development of the rumen in a ruminant, such as a young ruminant, such as a pre-weaned young ruminant, comprising administering to the animal an effective amount of Lacticaseibacillus rhamnosus strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

[0081] 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, e.g., rumen mass growth, rumen papilla growth, increased papilla density, e.g., dorsal papilla density, and / or the total surface area of ​​the rumen wall in an animal.

[0082] In one embodiment, the methods and compositions disclosed herein increase rumen weight, rumen wall thickness, or rumen papilla density per square rumen wall.

[0083] In one embodiment, the methods and compositions disclosed herein increase ruminal functional performance, for example, the methods stimulate rumination and / or increase dry matter intake (DMI).

[0084] In one embodiment, the methods and compositions disclosed herein alter the abundance of heterofermentative anaerobes in the ruminal microbiota.In one embodiment, the methods and compositions disclosed herein increase the abundance of heterofermentative anaerobes in the ruminal microbiota.

[0085] In one embodiment, the methods and compositions disclosed herein increase ruminal turnover rate and / or increase post-ruminal digestion. Without wishing to be bound by theory, it is hypothesized that a higher ruminal turnover rate selects for microorganisms capable of rapid heterofermentative growth on soluble sugars, which produces less hydrogen and leads to less methane formation. For example, Kamke et al. (2016) note that conversion of lactate to butyrate, but not to propionate, produces 2 moles of hydrogen per hexose, which can produce 0.5 moles of methane via the hydrogenotrophic pathway, and hypothesize that direct fermentation of hexose to butyrate and acetate, for example, by members of the Ruminococcaceae, produces 2.66 moles of hydrogen, allowing the formation of 0.66 moles of methane. Thus, lower hydrogen production via the lactate to butyrate pathway is predicted to reduce methane production.

[0086] The term "administering" refers to the act of introducing an effective amount of L. rhamnosu strain FNZ118 or a derivative thereof into the forestomach of a ruminant animal. More particularly, this administration is by the oral route. This administration can be carried out in particular by supplementing the strain in animal feed or drink and allowing the animal to ingest the supplemented feed or drink.

[0087] The term "effective amount" refers to an amount of L. rhamnosus strain FNZ118 or a derivative thereof sufficient to enable the desired effect, i.e., inhibition of the growth of methanogenic bacteria and / or archaea in the forestomach of an animal, reduction of methane emission by the animal, or increase of feed efficiency in an animal, compared to a reference. The desired effect (e.g., inhibition of the growth of methanogenic bacteria and / or archaea and / or reduction of methane production or emission) can be measured in vitro or in vivo. For example, the desired effect can be measured in vitro using the methods described herein, e.g., in the Examples below, in an artificial ruminal system, such as that described in T. Hano (1993) J. Gen. Appl. Microbiol., 39, 35-45, or by in vivo oral administration to a ruminant animal.

[0088] This effective amount can be administered to the ruminant in one or more doses.

[0089] The terms "reduced methane production" and "reduced methane emissions", e.g., "reduced methane production by animals" and "reduced methane emissions by animals", refer to the reduction of methane production or emissions by any mechanism and from any ruminant-related source. For example, the terms can refer to the reduction of methane produced in the forestomach of a ruminant animal or the reduction of methane produced or emitted by the feces of a ruminant animal.

[0090] The reduction in methane production is expected to be due to a variety of mechanisms. These may include, for example, killing methanogens (i.e., bactericidal / archaecicidal effect), inhibiting the growth of methanogens (i.e., bacteriostatic / archaecicidal effect), and / or inhibiting the methanogenic ability of the forestomach or rumen microbiota. Inhibiting the ability of the forestomach or rumen microbiota to produce methane may be through a variety of mechanisms including, for example, physical and / or chemical changes to the forestomach or rumen environment, changes to the microbiota, inhibition of one or more methanogenic pathways, and / or intermediary cross-feeding (or disruption of cross-feeding) between members of the microbiota.

[0091] It will be appreciated that reducing greenhouse gas (GHG) emissions, such as methane emissions, is desirable. GHG emissions can be reduced directly or indirectly, for example, by reducing the ability of the ruminal microflora to produce methane and / or by reducing methane emissions by ruminants. One example of an indirect reduction in GHG emissions is through land use changes or land disposal. Animals with improved feed efficiency (e.g., animals to which the methods or compositions of the present invention have been applied) require less pasture for roughage and / or less imported feed. Alternatively, or in addition, more animals can be raised on a given land area, allowing for the same production with reduced land use. In any case, a reduction in land requirements may allow unused pasture to be retired, for example by planting trees or other vegetation for carbon sequestration. Such land use changes may further reduce GHG emissions per farm, which may result in reduced GHG emissions per animal and / or per kg of animal product (milk, meat, wool, etc.).

[0092] The GHG emissions of animals and / or animal products can be determined using techniques known in the art. It will be recognized that certain GHGs produce more global warming potential than others. For example, emitting 1 kg of methane gas produces a global warming effect equivalent to 25 kg of CO2. To account for this, GHG emissions are typically reported as CO2 equivalents (CO2e), which is the amount of CO2 equivalent to global warming. GHG emission footprints can be calculated per animal or per amount of animal product (e.g., per kg of milk solids, per kg of meat, per kg of wool). As noted above, the GHG emission footprint should take into account land use changes, such as planting trees and other vegetation for carbon sequestration.

[0093] The term "animal product" refers to any product produced from or by animals, or containing animal-derived ingredients. The term is intended to include products produced directly by animals (e.g., milk, meat, and wool), as well as products that contain animal ingredients or are produced from animal ingredients, optionally with other ingredients, and optionally further processed. For example, the term is intended to include foods and beverages that contain animal ingredients, such as various dairy products (including buttermilk, cheese, cream, formula, ice cream, milk, milk powder, puddings, shakes, smoothies, and yogurt), meat products (such as chops, minced meat, hamburgers, sausages, sausage meat, and steaks), and other products that contain animal ingredients.

[0094] The term "feed efficiency" refers to the relationship between feed intake and muscle gain or milk yield. Microbial fermentation in the forestomach or rumen produces volatile fatty acids (VFAs), such as acetic acid, propionic acid, and butyric acid. These fatty acids are absorbed directly through the rumen wall and are utilized as raw materials, milk components, and other end products of digestion for the growth and development of the animal. The majority of the energy consumed by body tissues is used to produce milk and milk components, or muscle. Therefore, improved energy utilization can increase milk production, e.g., milk yield, and / or milk fat, milk protein, and / or milk solids. Improvements in body composition, such as increased muscle and / or changes in muscle / fat ratio in animals, can also be achieved.

[0095] Feed efficiency can be calculated by dividing the weight of milk produced by an animal, or the live weight of an animal by the weight of dry matter consumed by the animal. Thus, animals with higher feed efficiency will produce more milk, milk with a higher content of milk components such as (but not limited to) fat and protein, and / or show increased weight gain compared to animals with lower feed efficiency when given the same nutrient inputs. Feed efficiency can be measured by the difference in the growth of an animal by any of the following parameters: average daily gain, total gain, feed conversion ratio including both feed:gain and gain:feed, feed efficiency, mortality rate, and feed intake. That is, improved feed efficiency means that the feed intake / muscle gain ratio is reduced. Improved feed efficiency also means that the muscle weight gain / feed intake ratio 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 account for differences in protein and fat content by using energy corrected milk (ECM) yield instead of milk weight. This can be calculated using the following formula (Tyrrell and Reid, 1965): ECM = (12.82 x fat weight (lbs)) + (7.13 x protein weight (lbs)) + (0.323 x milk weight (lbs))

[0096] "Feed conversion ratio" and "residual feed intake (RFI)" are also commonly used measures of feed efficiency, and the terms are often used interchangeably. In livestock production, feed conversion ratio or feed conversion ratio is the ratio or rate measurement of efficiency with which an animal's body converts the animal's feed into a desired output. RFI is defined as the difference between an animal's actual dry matter intake (DMI) and the expected DMI required for maintenance and growth.

[0097] The primary benefit of improving feed efficiency (i.e., improving feed conversion ratio or lowering RFI) is to reduce the DMI of the animals without compromising growth performance, as feed-related costs are often the largest production expense in beef or milk production. Any reduction in DMI to produce one unit of beef or dairy results in minimizing feed costs and maximizing overall profitability for the beef or dairy industry.

[0098] In one embodiment, the feed efficiency in the ruminant animal is increased by at least about 1.01 times, such as at least about 1.02 times, 1.03 times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09 times, 1.10 times, 1.12 times, 1.14 times, 1.16 times, 1.18 times, such as at least about 1.20 times, of the feed efficiency of an untreated animal.

[0099] Increased feed efficiency may result from changes in volatile fatty acid (VFA) profile, total VFA concentration and / or fermentation rate in the rumen and forestomach.

[0100] In some embodiments, the L. rhamnosus strain FNZ118 or its derivatives promotes propionic acid production. Propionic acid has a higher ATP production efficiency than other volatile fatty acids, which promotes propionic acid production and improves feed efficiency. Propionic acid is also glucogenic, and therefore can promote lactose synthesis in the mammary gland.

[0101] In some embodiments, L. rhamnosus FNZ118 or a derivative thereof shifts hydrogen metabolism from methanogenesis to short chain / volatile fatty acid (VFA) production, such as propionic acid production. Propionic acid is primarily used as a precursor to glucose in ruminants, and higher propionic acid production appears to lead to more efficient utilization of feed energy. Maximizing metabolic hydrogen flow in the forestomach or rumen away from methane and toward VFAs (mainly propionic acid) increases the efficiency of ruminant production, reduces its environmental impact, and enhances rumen development and / or ruminal papilla development.

[0102] Acetate is the major substrate for mammary lipogenesis, along with β-hydroxybutyrate, which is produced when butyrate is taken up. As a result, a high acetate fermentation pattern provides substrates to maintain or increase milk fat.

[0103] Thus, in some embodiments, L. rhamnosus strain FNZ118 or a derivative thereof provides increased milk fat, milk protein, total milk yield and / or milk solids as a result of increased VFAs in the forestomach or rumen, which can act as an increased energy source to drive increased production.

[0104] In some embodiments, the yield of milk and / or milk components produced from the animal is increased, preferably by at least 1.5%, more preferably by at least 3.0%, at least 4.5%, or at least 6.0%.

[0105] In some embodiments, L. rhamnosus strain FNZ118 or a derivative thereof results in increased live weight, muscle mass, and / or fat deposition, and / or improved body composition (e.g., changes in muscle / fat ratio) in animals as a result of increased VFAs in the forestomach or rumen, which can act as an increased energy source to drive increased production.

[0106] In some embodiments, the live weight of the animal is preferably increased by at least 1%, more preferably at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% compared to a reference animal.

[0107] It is also expected that the present invention can be used to extend the lactation cycle of lactating ruminants, such as cows. Dairy cows direct a significant portion of their energy toward producing milk during lactation. After a long lactation period, body condition deteriorates. For this reason, lactation periods are usually shortened or shortened to prevent excessive deterioration in body condition. It is expected that the methods and ruminant feed compositions disclosed herein will increase the feed efficiency of ruminants, thus reducing the impact of milk production on body condition. As a result, it will be possible to milk dairy cows for a longer period of time.

[0108] It is also expected that the present invention can be used to reduce or improve the deterioration of body condition due to lactation. It is expected that the methods and ruminant feed compositions disclosed herein will increase the feed efficiency by ruminants, thus resulting in ruminants with improved body condition at the end of lactation. For example, as an animal enters the dry period, the animal will have a higher body condition score (BCS). As a result, ruminants will require less dry matter intake in the off-season to achieve body condition. Alternatively or additionally, the methods and ruminant feed compositions disclosed herein are useful for improving the body condition of animals prior to lactation. For example, the methods and compositions disclosed herein can improve the body composition of the mother and / or fetus or newborn. For example, the methods and compositions disclosed herein can improve the body composition and / or weight of the newborn at birth.

[0109] It is also anticipated that the present invention may be similarly useful for reducing or ameliorating deterioration in body condition during other stresses such as parturition, drought, or inadequate feed intake.

[0110] Live weight and body condition scores are commonly used in the industry as measures of animal growth and performance.

[0111] Liveweight is an objective measure used to evaluate 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 dairy heifers are growing. Significant research has focused on the relationship between liveweight and performance, particularly heifer (female cow) performance, leading to the identification of liveweight targets. Achieving liveweight targets optimizes heifer lifetime performance, extending livestock longevity and increasing return on investment for agricultural operations.

[0112] In New Zealand, liveweight targets for heifers are 30, 60 and 90% of mature liveweight at 6, 15 (pre-breeding) and 22 (pre-calving) months of age. The 22 month target includes pregnancy adjustment. Heifers grown to liveweight target are also more likely to meet body condition score (BCS) target at calving, which contributes to improved milk production in the first lactation.

[0113] Body condition score (BCS) is a subjective measure used to assess animal performance and ensure animal welfare is maintained. The industry standard dairy cattle BCS scale applies to heifers from 20 months of age onwards. The target body condition score (BCS) for heifers at 22 months of age (pre-calving) is 5.5.

[0114] There is a well-established relationship between liveweight and milk production, therefore one of the main benefits of achieving liveweight targets is increased milk production.

[0115] Pre-calving heifer liveweight has been demonstrated to have a significant impact on milk production in a variety of studies both in New Zealand (Handcock et al. 2019; McNaughton, L R and T Lopdell. 2013; MacDonald et al. 2005; van der Waaij et al. 1997) and overseas (Carson et al. 2002; Dobos et al. 2001).

[0116] Assuming a 9% milk solids trial, a response of approximately 2 kg of milk solids per lactation is expected for every 1% increase in target liveweight. For heifers with a precalving liveweight target of 500 kg, 5 kg equals 1% of liveweight. Van der Waaij et al. (1997) reported a response of 6 L of milk and 0.43 kg of milk solids per kg of liveweight at first lactation, while Dobos et al. (2001) reported a response of 5.35 L of milk and 0.42 kg of milk solids per kg of liveweight. Multiplying these values ​​by 5 gives a 5 kg benefit of 26.8 to 30 L of milk and 2.1 to 2.15 kg of milk solids per 1% increase in liveweight. A similar response was reported by McNaughton, L R and T Lopdell (2013), where in precalving heifers, every 1% increase in target liveweight achievement was associated with an increase in milk production of 23 ± 0.6 liters in the first lactation and 24 ± 0.9 liters in the second lactation (P < 0.001).

[0117] Thus, in some embodiments, the methods and compositions disclosed herein increase milk production, e.g., the yield of milk and / or milk components produced from a ruminant animal, hi some embodiments, the methods and compositions disclosed herein increase the yield of milk fat, milk protein, or milk solids in the milk produced from the animal.

[0118] In some embodiments, the methods and compositions disclosed herein increase milk production during the first lactation. In some embodiments, the methods and compositions disclosed herein increase the accumulated milk production over multiple lactations, for example, over the first two or first three lactations. In some embodiments, the methods and compositions disclosed herein increase the accumulated milk production over the entire lactation period of the animal.

[0119] In some embodiments, milk production in the animal is increased, preferably by 1% or more, more preferably 2% or more, 3% or more, 4% or more, or 5% or more, compared to an untreated or reference animal.

[0120] In some embodiments, milk production of the animal is preferably increased by at least 5 kg of milk solids per lactation, more preferably at least 6 kg, at least 7 kg, at least 8 kg, at least 9 kg, at least 10 kg, at least 11 kg, at least 12 kg, or at least 13 kg of milk solids per lactation compared to an untreated or reference animal.

[0121] In some embodiments, milk production of the animal is increased by at least 60 L of milk solids per lactation, more preferably at least 70 L, at least 80 L, at least 90 L, at least 100 L, at least 110 L, at least 120 L, or at least 130 L, compared to an untreated or reference animal.

[0122] As discussed 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 preweaned ruminants. Rumen development involves three distinct processes: (i) anatomical development (e.g., ruminal mass growth and ruminal papilla growth), (ii) functional achievement (e.g., fermentative capacity and enzyme activity), and (iii) microbial colonization (bacteria, fungi, methanogens, protozoa).

[0123] Anatomical development of the rumen is a process that occurs following three stages: non-ruminant (0–3 weeks), transitional (3–8 weeks), and ruminal (from 8 weeks). During the transitional stage, growth and development of the ruminal absorptive surface area (papillae) is essential to allow absorption and utilization of digestive end products, especially ruminal volatile fatty acids. The presence and absorption of volatile fatty acids stimulates ruminal epithelial metabolism and may be key in initiating ruminal epithelial development. Constant exposure to volatile fatty acids maintains the development, size, and function of ruminal papillae. Different volatile fatty acids stimulate such growth differently, with butyric acid being the most stimulating, followed by propionic acid. Thus, a shift in hydrogen metabolism from methanogenesis to short-chain / volatile fatty acid (VFA) production, e.g., propionic acid production, is expected to enhance ruminal epithelial growth and development.

[0124] Ruminants Ruminants are a group of herbivorous animals with a multi-compartment stomach that digests food by first microbial fermentation in the rumen to form the rumen, expels the rumen for chewing, and then swallows the chewed rumen for further digestion. This group includes, but is not limited to, the ruminants and the suborder Tylopoda, including several species of livestock. In one embodiment, the ruminant is a cow, goat, sheep, bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope, or nilgai. In a preferred embodiment, the ruminant is a cow or a sheep.

[0125] In one embodiment, the ruminant is a lactating animal, hi an alternative embodiment, the ruminant is a pre-weaned animal, such as a calf or lamb.

[0126] The rumen is divided into the non-glandular forestomach (rumen, reticulum, and omasum) and the terminal glandular stomach, the abomasum.

[0127] In some embodiments, the ruminant is a newborn, neonatal, or juvenile. For example, in some embodiments, 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 old.

[0128] In some embodiments, the L. rhamnosus FNZ118 strain or a derivative thereof is administered to the ruminant prior to weaning. In some aspects, the L. rhamnosus FNZ118 or a derivative thereof is administered to the ruminant after weaning. In some embodiments, the L. rhamnosus FNZ118 or a derivative thereof is administered to the ruminant both prior to and after weaning. For example, in some embodiments, the L. rhamnosus FNZ118 strain or a derivative thereof is administered throughout the life of the ruminant.

[0129] For example, L. rhamnosus FNZ118 or a derivative thereof is administered to a ruminant on or about day 0 of birth, e.g., on or about day 0, 1, or 2 of age. Administration may then be given at least once a day, e.g., multiple times a day, sufficient to provide a sustained effect. For example, administration may continue for 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 10 weeks, 3 months, or more from birth. In some embodiments, administration of the L. rhamnosus strain FNZ118 or a derivative thereof continues 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 for the lifetime of the ruminant.

[0130] Lacticaseibacillus rhamnosus FNZ118 A culture of Lacticaseibacillus rhamnosus FNZ118 (also known as Lactobacillus rhamnosus FNZ118) was isolated from a human source and deposited on August 2, 2021 at the National Measurement Institute of Australia (NMIA), 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207, and accorded accession number V21 / 015445, an international depository recognized under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The terms Lactobacillus rhamnosus strain FNZ118, Lactobacillus rhamnosus FNZ118, Lacticaseibacillus rhamnosus FNZ118, L. rhamnosus FNZ118, and FNZ118 are used interchangeably herein.

[0131] Whole genome sequencing using a combination of short-read (Illumina) and long-read (MinION / ONT) sequencing technologies was used to generate a hybrid genome assembly. The final hybrid assembly contained seven contigs. The total length was 2,997,051 bp (3.0 Mb). The species ID for strain FNZ118 was confirmed as Lacticaseibacillus rhamnosus using the classification sequence classification program Kraken.

[0132] All working groups and associated bioinformatics were carried out in accordance with the EFSA guidelines available at https: / / efsa.onlinelibrary.wiley.com / doi / pdf / 10.2903 / j.efsa.2018.5206 and the latest EFSA statement (July 2021) available at https: / / efsa.onlinelibrary.wiley.com / doi / full / 10.2903 / j.efsa.2021.6506.

[0133] Morphological properties The morphological characteristics of L. rhamnosus FNZ118 are described below.

[0134] When grown in MRS broth, it is a short to medium rod shaped with square ends in chains, generally 0.7 x 1.1 x 2.0-4.0 μm. It is a gram-positive, non-mobile, non-spore-forming, catalase-negative facultative anaerobic bacillus.

[0135] Further characterization Of course, there are methods widely known and available to those of skill in the art that can be used to confirm the identity of L. rhamnosus FNZ118; exemplary methods include DNA fingerprinting, genomic analysis, sequencing, and related genomic and proteomic techniques.

[0136] L. rhamnosus strain FNZ118 and its derivatives As described herein, certain embodiments of the invention utilize live L. rhamnosus strain FNZ118. In other embodiments, derivatives of L. rhamnosus strain FNZ118 are utilized.

[0137] As used herein, the term "derivatives" and its grammatical equivalents, when used in reference to bacteria (including when used in reference to specific strains of bacteria such as L. rhamnosus FNZ118), contemplates mutants and homologs of bacteria or mutants and homologs derived from bacteria, killed or attenuated bacteria such as heat-killed, lysed, fractionated, autoclaved, irradiated, and UV- or light-treated bacteria, as well as bacterial-derived materials including, but not limited to, bacterial cell wall compositions, bacterial cell lysates, lyophilized bacteria, bacterial-derived anti-methanogenic factors, bacterial metabolites, bacterial cell suspensions, bacterial culture supernatants, and the like, which derivatives retain anti-methanogenic activity. Transgenic microorganisms engineered to express one or more anti-methanogenic factors are also contemplated. Methods for producing such derivatives, such as, but not limited to, one or more mutants or one or more anti-methanogenic factors of L. rhamnosus strain FNZ118, particularly derivatives suitable for administration to ruminants (e.g., in compositions), are well known in the art.

[0138] It will be appreciated that methods suitable for identifying L. rhamnosus strain FNZ118 are equally suitable for identifying mutants or homologues of L. rhamnosus strain FNZ118, or derivatives of L. rhamnosus strain FNZ118, for example including bacterial metabolic products from L. rhamnosus strain FNZ118.

[0139] The term "anti-methanogenic factor" refers to bacterial molecules responsible for mediating anti-methanogenic activity, including but not limited to bacterial DNA motifs, RNA (including mRNA and miRNA), proteins, exosomes, bacteriocins, bacteriocin-like molecules, antimicrobial peptides, antibiotics, antimicrobial agents, small molecules, polysaccharides, or cell wall components such as lipoteichoic acid and peptidoglycan, or mixtures of any two or more thereof. As noted above, these molecules have not been definitively identified and without wishing to be bound by any theory, their presence can be inferred by the presence of anti-methanogenic activity.

[0140] The term "anti-methanogenic activity" refers to the ability of a particular microorganism to inhibit the growth of methanogenic bacteria and / or archaea and / or to reduce the production of methane by methanogenic bacteria and / or archaea. This ability can be limited to inhibiting the growth and / or methanogenic potential of a particular group of methanogenic bacteria and / or archaea, for example inhibiting the growth of hydrogenotrophic methanogens, inhibiting the methanogenic potential of hydrogenotrophic methanogens, inhibiting the growth of methylotrophic methanogens, inhibiting the methanogenic potential of methylotrophic methanogens, inhibiting the growth of a particular species of methanogens, or inhibiting the methanogenic potential of a particular species of methanogens.

[0141] Reference to retaining anti-methanogen activity is intended to mean that a derivative of the microorganism, such as a mutant or homologue of the microorganism, or an attenuated or killed microorganism, or a cell culture supernatant, still has useful anti-methanogen activity, or that a composition comprising the microorganism or a derivative thereof still has useful anti-methanogen activity. Although the bacterial molecules responsible for mediating the anti-methanogen activity have not been definitively 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. These are hypothesized to interact with components of methanogenic bacteria and / or archaea to provide a growth inhibitory effect. Preferably, the retained activity 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., about 35 to about 100%, about 50 to about 100%, about 60 to about 100%, about 70 to about 100%, about 80 to about 100%, and about 90 to about 100%).

[0142] Conventional solid substrate and liquid fermentation techniques well known in the art can be used to grow L. rhamnosus strain FNZ118 in sufficient quantities to permit use as contemplated herein. For example, L. rhamnosus strain FNZ118 can be produced in bulk for formulation using nutrient film or submerged culture growth techniques, for example under conditions described in WO99 / 10476. Briefly, growth is carried out under aerobic conditions at any temperature sufficient for growth of the organism. For example, for L. rhamnosus strain FNZ118, a temperature range of 30-40°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 for the isolate to reach stationary growth phase.

[0143] The bacterial cells may be harvested by methods well known in the art, for example, conventional filtration or sedimentation methods (e.g., centrifugation), or harvested in a dry state using a cyclone system. The bacterial cells may be used immediately or stored for as long as required using standard techniques, preferably freeze-dried or chilled at -20°C to 6°C, preferably -4°C. Cryoprotectants, cryopreservatives, and / or lyoprotectants may also be used to increase the stability and / or viability of the bacterial cells when dried and / or frozen, as known in the art.

[0144] Supernatant Further embodiments of the present invention utilize supernatants from cell cultures comprising L. rhamnosus strain FNZ118 or derivatives thereof. These embodiments include processes for preparing bacterial culture supernatants, said processes comprising culturing bacterial cells and separating the supernatant from the cultured cells, thereby obtaining a supernatant. This method also allows for further isolation of bacterial molecules responsible for mediating anti-methanogen activity, which can be obtained from the supernatant.

[0145] As will be appreciated by one of skill in the art, supernatants useful in the present invention include both supernatants from such cultures, and / or concentrates of such supernatants and / or fractions of such supernatants.

[0146] The term "supernatant" in this context refers to the medium from a bacterial culture from which the bacteria have subsequently been removed, for example by centrifugation or filtration.

[0147] The supernatants useful in the present invention can be easily obtained by a simple process for preparing bacterial culture supernatants, said process comprising: a) culturing cells of L. rhamnosus strain FNZ118; b) optionally releasing active compounds and / or extracellular components of the cells by various cell treatments, such as, but not limited to, acid or alkali modification, sonication, detergents, such as sodium dodecyl sulfate (SDS) and / or Triton X, wall-soluble enzymes, such as mutarolysin and / or lysozyme, salts and / or alcohols; and c) Separating the supernatant from cultured cells thereby obtaining the supernatant.

[0148] In a preferred embodiment of this process, the supernatant composition is further subjected to a drying step to obtain a dried culture product.

[0149] The drying step may conveniently be freeze-drying or spray-drying, but any drying process suitable for drying anti-methanogenic agents such as bacteriocins is contemplated, including vacuum drying and air drying.

[0150] Although the contents of the supernatant produced by L. rhamnosus strain FNZ118 have not yet been characterized in detail, it is known that certain bacterial strains can produce bacteriocins, which are small heat-stable proteins, and therefore, without wishing to be bound by theory, it is expected that drying methods, including spray drying, which result in moderate heating of the culture eluate product, will also result in active compositions, as demonstrated in the examples described herein.

[0151] lysate The liquid containing the contents of lysed cells is called a lysate. The lysate contains the active components of the bacterial cells and can be either crude, i.e. containing all cellular components, or partially and / or completely separated into separate fractions such as extracellular components, intracellular components, proteins, etc.

[0152] 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 sonication, enzymatic lysis, such as enzymes that degrade bacterial cell walls, chemical lysis, such as using detergents, denaturants, pressure changes, and / or osmotic shock, and combinations of the above.

[0153] Thus, a further embodiment of the present invention utilises a lysate of L. rhamnosus strain FNZ118 or a derivative thereof.

[0154] Cell suspension The present invention may also, in some embodiments, utilize a cell suspension comprising L. rhamnosus strain FNZ118 or a derivative thereof.

[0155] In the present context, the term "cell suspension" relates to a plurality of cells of L. rhamnosus strain FNZ118 or a derivative thereof dispersed or suspended in a liquid, such as a liquid nutrient medium, culture medium or saline solution.

[0156] The cells may be provided in the form of a cell suspension in a suitable solution for dispersion, which may be dispersed, for example, by spraying, dipping, or any other application process.

[0157] The cells may be viable, but the suspension may also contain inactivated or dead cells or their lysates. In one embodiment, the suspension of the present invention comprises live cells, hi another embodiment, the suspension of the present invention comprises inactivated, killed or lysed cells.

[0158] Bacteriocins Bacteriocins are antibacterial compounds produced by bacteria to inhibit other bacterial strains and species.

[0159] Lactic acid bacteria (LAB) are well 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, thus extending the shelf life and safety of foods. Bacteriocins are generally considered to be narrow-spectrum antibiotics. Moreover, bacteriocins, especially from LAB, show very low human toxicity and have been consumed in fermented foods for thousands of years.

[0160] A further aspect of the invention provides an isolated antibacterial compound obtained from L. rhamnosus strain FNZ118 or a derivative thereof. Such an antibacterial compound may be obtained, for example, from a supernatant or lysate resulting from a process described herein further comprising an isolation step.

[0161] As illustrated in the Examples disclosed herein, L. rhamnosus strain FNZ118 and / or compositions comprising L. rhamnosus strain FNZ118, and / or culture supernatants of L. rhamnosus strain FNZ118 have been found to be useful as antimicrobial compounds, particularly for inhibiting the growth of methanogenic bacteria and / or for inhibiting the ability of methanogens to produce methane.

[0162] In the present context, the term antimicrobial compound utilizes compounds that kill, impair the survival or inhibit the growth of microorganisms.

[0163] Antibacterial compounds can be classified according to the microorganisms they primarily act on: for example, antibacterial agents are used against bacteria and antifungals against fungi. They can also be classified according to their function: compounds that kill microorganisms are called bacteriocidal, those that only inhibit their growth are called bacteriostatic.

[0164] In one embodiment, the present invention relates to antimicrobial compounds that are bactericidal. In another embodiment, the present invention relates to antimicrobial compounds that are bacteriostatic. In another embodiment, the present invention relates to antimicrobial compounds that are antimicrobial.

[0165] Ruminant feed or carrier composition Ruminant feed compositions useful herein can be formulated as a food, beverage, food additive, beverage additive, animal feed, animal feed additive, animal feed supplement, nutritional supplement, carrier, vitamin or mineral premix, nutritional product, enteral nutritional product, soluble, slurry, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet or ruminal product such as a bolus. Suitable formulations can be prepared by one of ordinary skill in the art having regard to the teachings of the art and this specification.

[0166] The composition can be administered as a top dressing or mixed into standard feed ingredients such as the daily ration. Additionally, the strain can be administered in partial or total mixed feeds (TMR), pelleted feeds, mixed with liquid feeds or beverages, mixed with protein premixes, or delivered via vitamin and mineral premixes.

[0167] In one embodiment, the compositions useful herein include any edible feed product capable of carrying bacteria or bacterial derivatives. As used in this application, the term "feed" or "animal feed" refers to a substance consumed by an animal that contributes energy and / or nutrients to the animal's diet. Animal feed typically includes many different ingredients that may be present in the form of concentrates, premixes, co-products, or pellets. Examples of feeds and feed compositions include partial or complete mixed rations (TMR), corn, soybeans, forage, grains, distillers grains, germinated grains, legumes, vitamins, amino acids, minerals, fiber, forage, grass, hay, straw, silage, grains, leaves, meals, solubles, slurries, supplements, mash feed, meals, fruit pulp, vegetable pulp, fruit or vegetable pomace, citrus meal, wheat shorts, corn cob meal, and molasses. Other compositions useful as carriers include milk, milk powder, milk replacers, milk fortifiers, colostrum, whey, whey powder, sucrose, maltodextrin, rice hulls, and the like.

[0168] In certain embodiments, the feed composition is formed through a process of growing L. rhamnosus strain FNZ118 using a milk-based carrier, such as thermalized milk, or a non-milk-based carrier, to create a fermented yogurt-style composition. Methods of creating such fermented yogurt-style compositions are well known in the art and may include, for example, incubating the milk at an appropriate temperature, for example, for 12 hours or more, using a hot water bath or other heating means, until sufficient cell density is reached. In one embodiment, the temperature is 25-30°C. Optionally, the milk may contain other additives to promote bacterial growth, such as yeast extract. In certain embodiments, the method is performed in the field, such as a farm, where probiotic feed supplementation is performed. The fermented yogurt-style composition may be administered by oral application, such as drenching. In some embodiments, the fermented yogurt-style composition is administered at a dose of 1-100 ml per day, for example, 2-50, 5-30, or 10-20 ml per day.

[0169] Other suitable feed formulations for ruminants are described in EW Crumton et al., Applied Animal Nutrition, WH Freeman and Company, San Francisco, Calif., 1969, and DC Church, Livestock Feeds and Feeding, O & B Books, Corvallis, Oreg., 1977, both of which are incorporated herein by reference.

[0170] In one embodiment, compositions useful herein include any non-feed carrier that can be consumed by the animal to which the bacteria or bacterial derivatives are added, such as vermiculite, zeolite, or crushed limestone.

[0171] In a particular embodiment, the composition of the invention comprises L. rhamnosus strain FNZ 118. Methods for producing such compositions are well known in the art.

[0172] In some embodiments, the compositions of the invention comprise one or more derivatives of the L. rhamnosus FNZ118 strain. Again, methods for producing such compositions are well known in the art and can utilize standard microbiological and pharmaceutical practices. In some embodiments, the compositions comprise a dried culture product, such as a supernatant or a cell lysate, as described herein.

[0173] It is understood that a wide range of additives or carriers can be included in such compositions, for example, to improve or preserve bacterial viability or to increase the anti-methanogen activity of L. rhamnosus strain FNZ118 or its derivatives. For example, additives such as surfactants, wetting agents, humectants, stickers, dispersants, stabilizers, penetrants, and so-called stress additives (such as potassium chloride, glycerol, sodium chloride, and glucose) to improve the vitality, growth, replication, and survival of bacterial cells, as well as cryoprotectants such as maltodextrin, may be included. Additives may also include compositions that help maintain the viability of microorganisms in long-term storage, such as unrefined corn oil, or "inverse" emulsions that include a mixture of oil and wax on the outside and water, sodium alginate, and bacteria on the inside.

[0174] In some embodiments, L. rhamnosus FNZ118 or a derivative thereof is encapsulated. Methods for producing such encapsulated bacteria are well known in the art. In some embodiments, L. rhamnosus FNZ118 or a derivative thereof is encapsulated in a liposome, microbubble, microparticle, microcapsule, or the like. Such encapsulating agents include natural, semi-synthetic, or synthetic polymers, waxes, lipids, fats, fatty alcohols, fatty acids, and / or plasticizers, such as alginates, gums, kappa-carrageenan, chitosan, starch, sugar, gelatin, and the like.

[0175] In a particular embodiment, the L. rhamnosus strain FNZ118 is in a reproductively viable form and amount.

[0176] The composition may include a carbohydrate source, such as disaccharides, including, for example, sucrose, fructose, glucose, or dextrose. Preferably, the carbohydrate source is one that can be utilized by L. rhamnosus strain FNZ118 aerobically or anaerobically.

[0177] In such embodiments, the composition is capable of supporting the reproductive viability of L. rhamnosus strain FNZ118 for a period of greater than about 2 weeks, preferably greater than about 1 month, greater than about 2 months, greater than about 3 months, greater than about 4 months, greater than about 5 months, more preferably greater than about 6 months, and most preferably for at least about 2 years to about 3 years or more.

[0178] In certain embodiments, the oral composition is formulated to administer an effective amount of L. rhamnosus strain FNZ118 to allow for establishment of a population in the gastrointestinal tract of an animal upon ingestion. The established population may be a temporary or permanent population.

[0179] While a variety of routes and methods of administration are contemplated, oral administration of L. rhamnosus strain FNZ118, e.g., in a composition suitable for oral administration, is currently preferred, it being recognized, of course, that other routes and methods of administration may be utilized or may be preferred in particular circumstances.

[0180] The term "oral administration" includes oral, buccal, enteral, intraruminal, and intragastric administration.

[0181] Theoretically, one colony forming unit (cfu) should be sufficient to establish a population of L. rhamnosus strain FNZ118 in an animal, but in practical situations a minimum number of units is required to do so. Therefore, for therapeutic mechanisms that rely on a viable live population of probiotic bacteria, the number of units administered to a subject will affect efficacy.

[0182] In some embodiments, the formulation formulated for administration comprises at least about 6×10 9 cfu / day, e.g., at least about 6×10 11 In another embodiment, the formulation formulated for administration is sufficient to provide at least about 10 cfu / day of L. rhamnosus FNZ118 strain. 12This is sufficient to provide cfu of L. rhamnosu FNZ118 strain.

[0183] Methods for determining the presence of a population of intestinal and / or ruminal flora (e.g., L. rhamnosus strain FNZ118) in the gastrointestinal tract of a subject are well known in the art, and examples of such methods are provided herein. In certain embodiments, the presence of a population of L. rhamnosus strain FNZ118 can be determined directly, for example, by analyzing one or more samples obtained from the animal and determining the presence or amount of L. rhamnosus strain FNZ118 in said samples. In other aspects, the presence of a population of L. rhamnosus strain FNZ118 can be determined indirectly, for example, by observing a decrease in methane emission or methane production, a decrease in hydrogen production, or a decrease in the number of other intestinal and / or ruminal flora in a sample obtained from the animal. Combinations of such methods are also contemplated.

[0184] The efficacy of the composition useful according to the present invention can be evaluated both in vitro and in vivo. For example, see the following examples. Briefly, the composition can be tested for its ability to inhibit the growth of methanogens and / or archaea, or its ability to reduce the production of methane by methanogens and / or archaea. In in vivo studies, the composition can be fed to ruminants or injected into ruminants, and its effect on ruminal methanogens and / or archaea, as well as its effect on methane emission, can then be evaluated. Based on these results, the appropriate dosage range and administration route can be determined.

[0185] The method of calculating the appropriate dose may depend on the nature of the active agent in the composition.For example, if the composition contains live bacteria, the dose may be calculated by referring to the number of live bacteria present.For example, as described in the examples herein, the dose may be established by referring to the number of colony forming units (cfu) to be administered per day, or by referring to the number of cfu per kilogram of dry feed weight.

[0186] For example, approximately 1 × 10 of L. rhamnosus strain FNZ118 per kg of dry feed weight per day 6 cfu ~ approx. 1 × 10 12 cfu, preferably about 1 × 10 6 cfu ~ approx. 1 × 10 11 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 10 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 9 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 8 cfu / kg / day, approximately 1 × 10 6 cfu ~ approx. 5 × 10 7 cfu / kg / day, or approximately 1 x 10 6 cfu ~ approx. 1 × 10 7 A dose of about 5×10 cfu / kg / day of L. rhamnosus strain FNZ118 per day of dry feed weight is contemplated. 6 cfu ~ approx. 5 × 10 8 cfu, preferably about 5 x 10 6 cfu ~ approx. 4 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 3 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 2 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 1 × 10 8 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 9 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 8 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 7 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 6 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 5 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 4 × 10 7cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 3 × 10 7 cfu / kg / day, approximately 5 × 10 6 cfu ~ approx. 2 × 10 7 cfu / kg / day, or approximately 5 × 10 6 cfu ~ approx. 1 × 10 7 A dose of cfu / kg / day is contemplated.

[0187] In certain embodiments, the periodic dose need not vary with the subject's body weight, dry food weight, or other characteristics. In such an example, about 1×10 6 cfu ~ approx. 1 × 10 13 cfu, preferably about 1 x 10 6 cfu ~ approx. 1 × 10 12 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 11 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 10 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 9 cfu / day, approximately 1 × 10 6 cfu ~ approx. 1 × 10 8 cfu / day, approximately 1 × 10 6 cfu ~ approx. 5 × 10 7 cfu / day, or approximately 1 x 10 6 cfu ~ approx. 1 × 10 7 Dosing of cfu / day is contemplated.

[0188] In one particular embodiment, about 5×10 L. rhamnosus strain FNZ118 per kg body weight per day 7 cfu ~ approx. 5 × 10 10 cfu, preferably about 5 x 10 7 cfu ~ approx. 4 × 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 3 × 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 2 × 10 10 cfu / day, approximately 5 × 10 7 cfu ~ approx. 1 × 10 10 cfu / day, approximately 5 × 107 cfu ~ approx. 9 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 8 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 7 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 6 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 5 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 4 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 3 × 10 9 cfu / day, approximately 5 × 10 7 cfu ~ approx. 2 × 10 9 cfu / day, or approximately 5 × 10 7 cfu ~ approx. 1 × 10 9 cfu / day is contemplated. Preferably, 1×10 8 ~1×10 9 The dose is administered at cfu / kg body weight / day.

[0189] It will be understood that in certain embodiments, the dose does not need to be administered every day.For example, the composition can be formulated to be administered every 2 days, twice a week, once a week, every 2 weeks, or once a month.Alternatively, in certain embodiments, the composition can be formulated to be administered 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, with each meal, or with each mouth.

[0190] It is understood that the composition is preferably formulated to allow administration of an effective dose of L. rhamnosus FNZ118 strain and / or one or more derivatives thereof. The dose of the composition administered, the duration of administration, and the general administration regimen may vary between animals, depending on the mode of administration selected, as well as variables such as the age, sex, weight, and species of the animal. Furthermore, as mentioned above, the appropriate dose may depend on the nature of the active agent in the composition and the mode of formulation.

[0191] Furthermore, the dose of the composition may vary over time. For example, in some embodiments, an initial dosing regimen may be followed by a maintenance dosing regimen. It will be appreciated that a higher dose may be required to establish a population of L. rhamnosus FNZ118 in an animal, and a lower dose may be sufficient to maintain said population. Thus, in some embodiments, the initial dosing regimen includes administering a higher and / or more frequent dose than the maintenance dosing regimen. Preferably, the initial dosing regimen is effective to establish a population of L. rhamnosus FNZ118 in an animal, and preferably, the maintenance dosing regimen is effective to maintain a population of L. rhamnosus FNZ118 in an animal. In some embodiments, the maintenance dosing regimen includes administering a daily, every other day, twice weekly, twice weekly, every other week, or monthly dose.

[0192] In some embodiments, the effects of the methods described herein persist after administration of L. rhamnosus FNZ118. Without wishing to be bound by theory, it is expected that administration of L. rhamnosus FNZ118 as described herein may result in long-lasting or even permanent changes in the forestomach and / or rumen of a ruminant animal. In some embodiments, the effects persist for 2 days after the last administration of L. rhamnosus FNZ118, e.g., 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 L. rhamnosus FNZ118. In preferred embodiments, the effects persist for the life of the animal.

[0193] In the example where the composition comprises one or more derivatives of L. rhamnosus strain FNZ118, the dose can be calculated by reference to the amount or concentration of the derivative administered daily. For example, if the bacteria is to be inactivated, the aforementioned amount is calculated before inactivation. For a composition comprising L. rhamnosus strain FNZ118 culture supernatant, the dose 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, 1×10 9 The amount of culture supernatant equivalent to cfu / day can be calculated from the total yield of the culture and the total amount of culture supernatant.

[0194] It will be understood that the preferred compositions are formulated to provide an effective amount in a convenient form and amount. In certain embodiments, including but not limited to those in which the periodic dose does not need to vary with the weight or other characteristics of the animal, the composition can be formulated for a unit dose. Of course, administration includes a single daily administration, or multiple separate divided administrations as appropriate. For example, an effective dose of L. rhamnosus FNZ118 strain can be incorporated into feed for oral administration.

[0195] However, as a general example, we contemplate administering from about 1 mg to about 1000 mg of the compositions useful herein per day, preferably from about 50 to about 500 mg / day, alternatively from about 150 to about 410 mg / day or from about 110 to about 310 mg / day. In one embodiment, we contemplate administering from about 0.05 mg to about 250 mg / kg body weight of the compositions useful herein.

[0196] In one embodiment, the compositions useful herein comprise, consist essentially of, or consist of at least about 0.1, 0.2, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5, 99.8, or 99.9% by weight of L. rhamnosus strain FNZ118 and / or derivatives thereof, and useful ranges can be selected between any of these aforesaid values ​​(e.g., about 0.1 to about 50%, about 0.2 to about 50%, about 0.5 to about 50%, about 1 to about 50%, about 5 to about 50%, about 10 to about 50%, about 15 to about 50%, about 20 to about 50%, about 25 to about 50%, about 30 to about 50%, about 35 to about 50%, about 40 to about 50%, about 45 to about 50%, about 0.1 to about 60%, about 0.2 to about 60%, about 0.5 to about 60%, about 1 to about 60%, about 5 to about 60%, about 10 to about 60%, about 15 to about 60%, about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60%, about 45 to about 60%, about 0.1 to about 70%, about 0.2 to about About 70%, about 0.5 to about 70%, about 1 to about 70%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, about 0.1 to about 80%, about 0.2 to about 80%, about 0.5 to about 80%, about 1 to about 80%, about 5 to about 80%, about 10 to about 80%, about 15 to about 80%, about 20 to about 80%, about 25 to about 80%, about 30 to about 80%, about 35 to about 80%, about 40 to about 80%, about 45 to about 80%, about 0.1 to about 90% , about 0.2 to about 90%, about 0.5 to about 90%, about 1 to about 90%, about 5 to about 90%, about 10 to about 90%, about 15 to about 90%, about 20 to about 90%, about 25 to about 90%, about 30 to about 90%, about 35 to about 90%, about 40 to about 90%, about 45 to about 90%, about 0.1 to about 99%, about 0.2 to about 99%, about 0.5 to about 99%, about 1 to about 99%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, and about 45 to about 99%).

[0197] In one embodiment, the compositions useful herein comprise, consist essentially of, or consist of at least about 0.001, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 grams of L. rhamnosus strain FNZ118 and / or a derivative thereof. and useful ranges may be selected between any of these aforementioned values ​​(e.g., about 0.01 to about 1 gram, about 0.01 to about 10 grams, about 0.01 to about 19 grams, about 0.1 to about 1 gram, about 0.1 to about 10 grams, about 0.1 to about 19 grams, about 1 to about 5 grams, about 1 to about 10 grams, about 1 to about 19 grams, about 5 to about 10 grams, and about 5 to about 19 grams).

[0198] In certain embodiments, the compositions useful herein have at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 The present invention may comprise, consist essentially of, or consist of colony forming units (cfu) of L. rhamnosus FNZ118, and a useful range may be selected between any of these aforementioned values ​​(e.g., about 10 5 ~about 10 13 cfu, approximately 10 6 ~about 10 12 cfu, approximately 10 7 ~about 10 12 cfu, approximately 10 8 ~about 10 11 cfu, approximately 10 8 ~about 10 10 cfu, and about 10 8 ~about 10 9 cfu).

[0199] It will be apparent that the concentration of L. rhamnosus strain FNZ118 and / or one or more derivatives thereof in a composition formulated for administration may be lower than the concentration in a composition formulated for, for example, distribution or storage, and that the concentration of a composition formulated for storage and subsequently formulated into a composition suitable for administration must be sufficient to enable said composition for administration to be sufficiently concentrated so that it can be administered in an effective dose.

[0200] The 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 ingredient, beverage ingredient, dietary supplement, vitamin or mineral premix, oil, oil blend, oil-rich feed supplement, nutritional product, medical food, dietary supplement, medicine or pharmaceutical. The therapeutic agent can be a probiotic agent or factor, preferably effective in inhibiting the growth of methanogenic bacteria and / or archaea or reducing methane emission by methanogenic bacteria and / or archaea. In some embodiments, the oil, oil blend, or oil-rich feed supplement is palm kernel expeller (PKE) and / or PROLIQ.

[0201] When used in combination with another therapeutic agent, the administration of the composition useful herein and the other therapeutic agent can be simultaneous or sequential. Simultaneous administration includes administration of a single dosage form containing all components, or administration of separate dosage forms at substantially the same time. Sequential administration preferably includes administration according to different schedules, such that there is an overlap in the period during which the composition useful herein and the other therapeutic agent are provided. Examples of other therapeutic agents include at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methane production, and / or a natural or chemically synthesized methane production inhibitor and / or a methane production inhibitor such as bromoform.

[0202] Suitable agents with which the compositions useful herein 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.

[0203] Typically, the term prebiotics refers to substances that stimulate the growth and / or activity of biologically active bacteria in the digestive system of animals. Prebiotics can be selectively fermented ingredients that allow specific changes in both the composition and / or activity of the gastrointestinal microflora, conferring health benefits to the host. Probiotics generally refer to microorganisms that contribute to the balance of gut microorganisms, which play a role in maintaining health or providing other biological activities. Many species of lactic acid bacteria (LAB), such as Lacticaseibacillus and Bifidobacterium, are generally considered probiotics, although some species of Bacillus and some yeasts have also been found to be suitable candidates. Postbiotics refer to non-viable bacterial products or metabolic by-products from microorganisms, such as probiotics, that have biological activity in the host.

[0204] Useful prebiotics include galactooligosaccharides (GOS), short-chain GOS, long-chain GOS, fructooligosaccharides (FOS), short-chain FOS, long-chain FOS, inulin, galactan, fructan, 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 Used in Infant Nutrition, J.Nutr.(2008)138(9):1818S-1828S), which is incorporated herein by reference. Other useful agents may include dietary fiber, such as fully or partially insoluble or indigestible dietary fiber.

[0205] Thus, in one embodiment, L. rhamnosus strain FNZ118 and / or its derivatives may be administered separately, simultaneously or sequentially with one or more agents selected from one or more probiotics, one or more prebiotics, one or more dietary fiber sources, one or more galactooligosaccharides, one or more short chain galactooligosaccharides, one or more long chain galactooligosaccharides, one or more fructooligosaccharides, one or more short chain galactooligosaccharides, one or more long chain galactooligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or a mixture of any two or more thereof.

[0206] In certain embodiments, the composition comprises L. rhamnosus strain FNZ118 and / or a derivative thereof, and one or more prebiotics, one or more probiotics, one or more postbiotics, and one or more dietary fiber sources. In certain embodiments, the prebiotic comprises one or more fructooligosaccharides, one or more galactooligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or any mixture of any two or more thereof.

[0207] Without wishing to be bound by theory, it is believed that co-cultivation and / or simultaneous administration of two or more strains of lactic acid bacteria, such as three strains of lactic acid bacteria, can reduce the incidence of culture failure due to infection with bacteriophages. Thus, in certain embodiments, the composition comprises L. rhamnosus FNZ118 and one or more other strains of lactic acid bacteria, preferably two or more other strains of lactic acid bacteria. In other embodiments, the composition comprising L. rhamnosus FNZ118 is administered simultaneously or sequentially with one or more other compositions comprising one or more other strains of lactic acid bacteria, preferably two or more other strains of lactic acid bacteria.

[0208] It will be understood that different compositions of the present invention may be formulated with a view to administration to a particular group of ruminant subjects. For example, the formulation of a composition suitable for administration to cattle may be different from that suitable for administration to a different ruminant, such as sheep. It should also be recognized that the compositions of the present invention may be formulated differently to be suitable for administration to ruminants of different ages. For example, the formulation of a composition suitable for administration to calves or lambs may be different from that suitable for administration to adult cattle or sheep. In certain embodiments, a first composition may be formulated for administration to young animals, such as pre-weaned animals, in an initial administration regimen, and a second composition may be formulated for administration to the same animals in a maintenance administration regimen. In some embodiments, a first composition is formulated for pre-weaned animals and a second composition is formulated for post-weaned animals.

[0209] Preparation of L. rhamnosus FNZ118 strain Direct-fed microorganisms (DFM) and their use in methods for regulating ruminal function and improving ruminant performance are known in the art, as are methods for their production.

[0210] Briefly, L. rhamnosus FNZ118 strain can be cultured using conventional liquid or solid fermentation techniques. In at least one embodiment, the strain is grown in liquid nutrient broth to a level where the highest number of cells are formed. The strain is produced by fermenting a bacterial strain that can be initiated by scaling up a seed culture. This involves repeatedly and aseptically transferring the culture to increasingly larger volumes to serve as inoculum for fermentation, which can be carried out in large stainless steel fermenters in a medium containing the proteins, carbohydrates, and minerals required for optimal growth. Non-limiting exemplary media are MRS or TSB. However, other media can be used. After the inoculum is added to the fermentation vessel, the temperature and agitation are controlled to allow maximum growth. Once the culture reaches maximum population density, the culture is harvested by separating the cells from the fermentation medium. This is usually done by centrifugation.

[0211] In one embodiment, to prepare L. rhamnosus strain FNZ118, the strain is cultured at 1×10 8 CFU / ml ~ approx. 1 x 10 9 Ferment to a level of CFU / ml. The bacteria are harvested by centrifugation and the supernatant removed. The pelleted bacteria can be used to make a DFM. In at least some embodiments, the pelleted bacteria are lyophilized and then used to form a DFM. However, it is not necessary to freeze-dry the strain prior to use. The strain can be used with or without preservatives and in concentrated, non-concentrated, or diluted form.

[0212] The number of cultures can then be determined. CFU or colony forming units is the viable cell count of a sample obtained from standard microbiological plating methods. The term derives from the fact that a single cell, when plated on an appropriate medium, will grow into a viable colony in the agar medium.

[0213] The term colony forming unit is a more useful unit measurement than cell number because many cells can give rise to one visible colony. EXAMPLES

[0214] 1. Example 1 - Plate-based screening of bacteriocin extracts against indicator methanogenic strains 1.1 Materials and Methods 1.1.1 Bacteriocin extraction Bacteriocin extracts from L. rhamnosus FNZ118 culture were prepared and tested for their effect against indicator methanogen strains Methanobrevibacter boviskoreani JH1 ("JH1"), Methanosphaera sp. WGK6 ("WGK6"), Methanobrevibacter ruminantium M1 ("M1") and Methanobrevibacter gottschalkii D5 ("D5").

[0215] L. rhamnosus FNZ118 was revived from -80°C storage by plating on De Man-Rogosa-Sharpe agar (MRS, De Man et al., 1960) + lactose (2 g / L). Using a small inoculation loop, the glycerol stock was streaked onto MRS agar plates to obtain isolated colonies. The plates were incubated at 37°C for 48 h in a sealed container. After growth, a single colony was selected, picked up, restreaked onto an agar plate, and incubated at 37°C to obtain isolated colonies. After 48 h, a single colony was selected from the restreaked plate, inoculated into MRS liquid medium, and incubated at 37°C for 48 h. An inoculum (1 mL) of each revived strain was then subcultured into 16 mL of MRS + nisin liquid medium (1 ng / mL final concentration). Nisin was included in these media at a very low level to induce bacteriocin production. The cultures were incubated overnight at 37°C. An overnight grown L. rhamnosus FNZ118 culture was used for bacteriocin extraction. A drop of the culture was used to make a wet mount slide, the cells were examined using a phase contrast microscope, and a Gram stain was prepared to check the purity of the culture.

[0216] The remainder of the culture (approximately 16 mL) was transferred to a 50 mL Falcon tube and used for bacteriocin extraction according to the method of Gaspar et al. (2018) with some modifications as follows: The pH of the culture was adjusted to approximately 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, followed by incubation 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, if necessary, adjusted to pH 2 with 1 M HCl. The cells were incubated for 2 h at 4 °C with gentle 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 in a new 15 mL Falcon tube. The pH of the supernatant was adjusted to pH 6.8 with 1 M NaOH and filtered under sterile conditions through a sterile filter (Millex-GP 0.22 μm, 25 mm diameter, Millipore, Merck, Sigma-Aldrich, New Zealand) using a 10-mL syringe and needle into a sterile Hungate tube flushed with N. The filtered supernatant was frozen at −20° C. until use.

[0217] 1.1.2 Mbb.boviskoreani JH1 culture To identify potential candidate LAB strains with anti-methanogen activity, we used a microtiter plate-based methanogen growth inhibition bioassay using the model methanogen strain Methanobrevibacter boviskoreani JH1 (Li et al., 2019). Mbb. boviskoreani JH1 has the unique ability to grow using ethanol as a reducing power source to reduce CO2 to CH4, and JH1 was grown in microtiter plates and incubated under anaerobic conditions, without the need for H2 supplementation with 1 atm H2:CO2 overpressure (80:20). This allows a high-throughput JH1 screening method to identify inhibitory activity from LAB strains.

[0218] Mbb. boviskoreani JH1 cultures for inoculating plate assays were grown by syringe using anaerobic techniques in Balch tubes (anaerobic tubes, 18 × 150 mm, butyl rubber septum stoppers, aluminum crimp, 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 3–5 days and were used to inoculate the microtiter plate assays after reaching an OD600 of 0.8–1.0 against a distilled water blank. Overpressure in the JH1 culture tubes was released by inserting a needle into the butyl rubber septum and allowing accumulated gas to escape before removing the inoculum.

[0219] Freshly grown cultures were checked using wet mounts and Mbb. boviskoreani JH1 under a fluorescent microscope, where Mbb. boviskoreani JH1 appeared as short oval rods fluorescing green under ultraviolet (UV) illumination. Contamination of the cultures was checked by inoculating a sample of the culture into 9 mL of BY medium supplemented with 5 mM glucose and incubating at 39 °C for 1 day. If no turbidity was observed after 1 day, the culture was considered uncontaminated. Further culture validation was sometimes performed by extracting genomic DNA from the cultures and PCR amplifying the 16S rRNA gene using both regular bacterial 16S primers (27f-GAGTTTGATCMTGGCTCAG, 1492r-GGYTACCTTGTTACGACTT) and archaeal specific 16S primers (915af-AGGAATTGGCGGGGGAGCAC, 386r-GCGGTGTGTGCAAGGAGC). Culture purity was confirmed using the presence of bands with the archaeal primer set and the absence of bands with the bacterial primer set, as well as sequencing results from the PCR products.

[0220] 1.1.3 Cultivation of Methanosphaera sp. WGK6 Members of the genus Methanosphaera account for approximately 8% of ruminal methanogens (Henderson et al., 2015) and are generally H2-dependent methylotrophs, using H2 to reduce methanol to methane. Methanosphaera sp. WGK6 is an H2-utilizing methylotrophic methanogen isolated from the intestine of an Australian kangaroo, but can also utilize ethanol as a source of reducing power to reduce methanol to methane (Hoedt, 2017). Similar to Mbb. boviskoreani JH1, this metabolic capability theoretically allows WGK6 to grow on ethanol without the need for H2 overpressure, and thus to grow in microtiter plates. Growth of Methanosphaera sp. WGK6 was tested using BRN-RF10 medium (Balch et al., 1979; Hoedt, 2017) with H2 (H2+CO2 at 180 kPa overpressure, 80:20) or ethanol as the energy source and terminal electron acceptor, in both cases with ethanol as the terminal electron acceptor. Attempts to grow WGK6 on ethanol+methanol were unsuccessful, but WGK6 could be grown on methanol+H2 in a Hungate tube. Initial attempts to culture Methanosphaera sp. WGK6 in a pressurized gas canister with H2+CO2 atmosphere (180 kPa overpressure) in a microtiter plate format produced barely detectable growth after one week. However, good growth of Methanosphaera sp. WGK6 was obtained after increasing the concentration of cysteine ​​added to the BRN-RF10 medium. Therefore, we developed a plate quantification method using a pressurizable stainless steel gas canister (H2+CO2; 80:20).

[0221] Methanosphaera sp. WGK6 cultures for analysis were grown in Balch tubes in 9 mL of BRN-RF10 medium supplemented with 60 mM sodium formate, 1% methanol, 0.1 mL of vitamin solution (1x) and 0.1 mL of coenzyme M solution (10 μM) using anaerobic techniques and 180 kPa overpressure of H2+CO2 (80:20, BOC Gases NZ) by syringe. The tubes were incubated at 39 °C for 3-5 days without shaking until visible turbidity appeared and after reaching an OD600 of 0.8-1.0 against a distilled water blank, they were used for inoculation of the test tube assays. The overpressure in the WGK6 culture tubes was released by inserting a needle into the butyl rubber septum and allowing the accumulated gas to escape before removing the inoculum.

[0222] 1.1.4 Mbb.ruminantium M1 and Mbb.gottschalkii D5 culture The procedure for growing Methanobrevibacter ruminantium M1 and Methanobrevibacter gottschalkii D5 was identical to the WGK6 protocol described in 1.1.3 above, except that BY medium was used for growth. Cultures for the assay were grown in 9 mL of BY medium supplemented with 60 mM sodium formate, 0.1 mL of vitamin solution (1x) and 0.1 mL of coenzyme M solution (10 μM) added by syringe in Balti tubes, using anaerobic techniques and 180 kPa overpressure of H2+CO2 (80:20, BOC Gases NZ). Tubes were incubated at 39 °C without shaking until visible turbidity appeared after 3-5 days and used to inoculate the microtiter plate assays.

[0223] 1.1.5 Mbb.boviskoreani JH1 growth inhibition assay Bacteriocin extracts from L. rhamnosus FNZ118 stored frozen under anaerobic conditions in Hungate tubes were allowed to thaw at room temperature. All assay components for each assay, except the JH1 inoculum, were added to 3.75 mL of BY+formic acid medium in sterile 7.5 mL Hungate tubes via CO2-flushed syringes and needles in the proportions shown in Table 1. Each tube was then inoculated with a freshly grown JH1 culture and incubated at 39°C for 1 h before being moved into an anaerobic chamber (98% CO2-2% H2 atmosphere; Coy Laboratory Products, USA) and dispensed into the wells of a multi-well 96-well plate. The filled plate was placed into an AnaeroPack 2.5 L rectangular jar with an MCG Anaeropack-Anaero (Ngaio Diagnostics, Nelson, NZ), the lid was sealed, and the jar was removed from the anaerobic chamber and incubated at 39°C. Plates were observed daily through a transparent jar until Mbb. boviskoreani JH1 control wells showed visible turbidity (usually within 5-6 days). The optical density of each well was then recorded at 595 nm (OD595) after shaking for 5 s in a Multiscan FC microplate photometer (Thermo Scientific, Auckland, NZ). The absorbance readings of the medium control wells were subtracted as background, and the % inhibition of Mbb. boviskoreani JH1 growth caused by the bacteriocin extract samples was calculated by comparing it to the JH1 positive growth control wells (which contained buffer only).

[0224] [Table 1]

[0225] 1.1.6 Methanosphaera sp. WGK6 growth inhibition assay Each assay component of the assay, except for the WGK6 inoculum, was added via a CO2 flush syringe and needle to 3.75 mL of BRN-RF10 medium in a Hungert tube supplemented with 1% methanol (247 mM, final concentration), as described in Table 2. The tube along with the inoculation tube was then moved into the chamber. The medium containing all components except the inoculum was dispensed into the plates in the chamber, and the inoculum was then added to the appropriate wells. The plates were placed in a stainless steel gas canister laid horizontally to hold up to four microtiter plates at a time. Two anaerobic pouches (MCG Anaeropack-Anaero, Ngaio Diagnostics, Nelson, NZ) were added, the canister was sealed, and the canister was removed from the anaerobic chamber and pumped to 180 kPa with H2+CO2 (80:20, BOC Gases, NZ), then incubated at 39°C for 1 week. The canister was checked periodically to ensure that overpressure was maintained and repressurized with H2+CO2 as necessary. After 1 week of incubation, the canister was opened and the plate removed. The contents of each well were evenly resuspended by repeated pipetting with a multichannel pipettor. The optical density of each well was then immediately recorded at 595 nm (OD595) after shaking for 5 seconds in a Multiscan FC microplate photometer (Tgerni Scientific, Auckland, NZ). The absorbance readings of the medium control wells were subtracted as background to calculate the % inhibition of Methanosphaera sp. WGK6 growth caused by the bacteriocin extract samples relative to the WGK6 positive growth control wells (containing only buffer instead of the bacteriocin extract).

[0226] [Table 2]

[0227] 1.1.7 Mbb.ruminantium M1 and Mbb.gottschalkii D5 growth inhibition assay Cultures of Mbb. ruminantium M1 and Mbb. gottschalkii D5 were prepared as described above in 1.1.4. The overpressure in the tubes was released before removing the inoculum. The analytical components were added to 3.5 mL of sterile BY medium in 7.5 mL Hangert tubes via CO2 flushed syringes and needles as described in Table 3. Each tube was then inoculated with a freshly grown culture and incubated at 39°C for 1 h, then transferred into an anaerobic chamber and dispensed into wells of a 96-well multiwell plate. The plates were sealed and incubated under 180 kPa overpressure of H2+CO2 in stainless steel gas canisters and their optical densities were recorded by a spectrophotometer at OD595 as described for Methanosphaera sp. WGK6 analysis in 1.1.6 above. The OD595 readings of the BY medium control wells were subtracted as background, and the % inhibition of the increase in Mbb. ruminantium M1 or Mbb. gottschalkii D5 caused by the bacteriocin extract samples compared to the positive growth control wells (which contained buffer instead of the bacteriocin extract) was calculated.

[0228] [Table 3]

[0229] 1.2 Results A total of 1,712 bacteriocin extracts from strains of lactic acid bacteria were screened against Methanosphaera sp. WGK6. Of these, 1,580 strains (>92%) showed less than 50% inhibition. The 1,712 strains of lactic acid bacteria included 94 strains of Lacticaseibacillus rhamnosus, of which 62 strains (~66%) showed less than 20% inhibition of WGK6, 81 strains (~86%) showed less than 50% inhibition, and only 3 strains (~3%) showed more than 80% inhibition. Taken together, this indicates that inhibition of methanogens is likely a strain-specific effect.

[0230] The L. rhamnosus FNZ118 bacteriocin extract showed very strong inhibition of the indicator methylotrophic methanogen Methanosphaera sp. WGK6, but showed very weak or no inhibition of the indicator hydrogenotropic methanogens Mbb. boviskoreani JH1, Mbb. ruminantium M1 or Mbb. gottschalkii D5, as shown in Table 4.

[0231] [Table 4]

[0232] Discussion and Conclusion Members of the genera Methanobrevibacter and Methanosphaera are the predominant methanogens in the rumen of several ruminant species. WGK6 was used as an indicator strain for methylotrophic methanogens in general, and Methanosphaera species in particular. Mbb. boviskoreani JH1, Mbb. ruminantium M1, and Mbb. gottschalkii D5 were used as indicator strains for Methanobrevibacter species. This example shows that L. rhamnosus FNZ118 bacteriocin extract exhibits a strong inhibitory effect on the methylotrophic methanogen Methanosphaera species WGK6, but not on the Mbb. boviskoreani JH1, Mbb. ruminantium M1, and Mbb. gottschalkii D5 methanogens.

[0233] 2. Example 2 - Effect of L. rhamnosus FNZ118 in a ruminal in vitro assay 2.1 Materials and Methods 2.1.1 Preparation of bacterial cultures and test supernatants Seven Hungate tubes were inoculated with L. rhamnosus FNZ118. Each contained 5 ml of anaerobic MRS medium (Sigma-Aldrich) and was grown at 39 °C for 16 h (until the cultures reached stationary phase). Cultures were pooled in a 250 mL CO2-flushed serum bottle. An aliquot (1 mL) of the combined cultures was added to 9 mL of sterile MRS medium and its OD600 was measured. Further 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 on MRS plates to measure the colony forming units (CFU mL-1) of the original culture. Half of the remaining culture was used for one set of ruminal in vitro fermentations (test cultures) 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). For the untreated control (buffer), untreated phosphate buffer (0.46 M K2HPO4; 0.54 M KH2PO4, pH 7) was used.

[0234] 2.1.2 Ruminal fluid preparation and in vitro fermentation setup For inoculation of the ruminal in vitro fermentation vessels, fresh ruminal contents were collected from six rumen-fistulated Friesian cows. After squeezing through one layer of cheesecloth, the ruminal fluid obtained from two animals was combined (approximately 150 mL of ruminal fluid) to obtain three biological replicates. An aliquot (12.5 mL) of the mixed ruminal fluid was added to 0.5 mg of dried grass and 36.5 mL of anaerobic phosphate buffer in a 250 mL serum bottle. Buffer, test culture, either SN or bacteriocin extract treatment (1 mL) was added, after which the serum bottle was closed with a butyl rubber stopper to obtain a final fermentation volume of 50 mL containing 25% ruminal fluid (v / v). Gas production and methane content were measured using an automated incubation system (Muetzel et al., 2014).

[0235] 2.2.1 VFA sample collection and analysis Samples were collected from the bottles for VFA analysis. At each time point, 3 mL aliquots were collected and their pH was measured. 1.8 mL samples of these aliquots were used for VFA and non-VFA analysis. VFA samples were centrifuged at 21,000×g for 10 min at 4° C., 0.9 mL of supernatant was removed and added to 0.1 mL of internal standard (20 mM 2-ethylbutyrate in 20% phosphoric acid), mixed, and frozen at −20° C. until analysis. After thawing and recentrifuging at 21,000×g for 10 min at 4° C., 0.9 mL was collected for derivatization for non-VFA analysis, while the remainder of the sample was analyzed directly by GC.

[0236] 2.2 Results L. rhamnosus FNZ118 was tested for its effect on gas production in a ruminal in vitro assay, as shown in Tables 5-10. Data shown are the average of three replicate experiments. Negative numbers represent stimulation, not inhibition. Asterisks (*) are used to indicate statistical significance (*P<0.05; **P<0.01) by Student's T-test with Welch's correction.

[0237] [Table 5]

[0238] [Table 6]

[0239] [Table 7]

[0240] [Table 8]

[0241] [Table 9]

[0242] [Table 10]

[0243] L. rhamnosus FNZ118 produced a significant reduction in total methane produced at 2 and 6 h in the ruminal in vitro assay across three biological replicates. This effect was also seen with culture supernatant in one of the biological replicates. Bacteriocin extracts showed no significant effect. It should be noted that the ruminal in vitro assay is a closed system and nutrition may become limiting over time. Therefore, the 0-12 h time point may more accurately reflect the in vivo situation, as animals typically ingest more feed and liquid over a 24-h period. It should also be noted that the RIV replicates were performed at different times, using ruminal fluid collected from different cows on pasture-based diets. Thus, variability between RIV replicates may be at least partly due to seasonal changes in pasture quality.

[0244] Overall, L. rhamnosus FNZ118 culture, culture supernatant, and bacteriocin extract showed no significant effect on total gas produced in the ruminal in vitro assay, except in one of the biological replicates where culture supernatant was used.

[0245] There was also no significant effect on the total volatile fatty acids produced, or the amount of acetate, propionate, and butyrate produced, except in one of the biological replicates with the FNZ118 culture at 6 hours.

[0246] 2.3 Conclusion In vitro ruminal assays of L. rhamnosus FNZ118 demonstrated effects on fermentation end products, showing a significant reduction in methane production, without any significant effect on volatile fatty acid production.

[0247] 3. Example 3 – Methane emissions from calves 3.1 Materials and Methods 3.1.1 Calf study design, animal welfare and housing facilities In this example, a design including treatments with L. rhamnosus FNZ118 and a control was used to test the ability of FNZ118 to reduce CH4 emissions from calves when fed for the first 14 weeks of their lives. Statistical power using previous calf CH4 emission data required at least 20 animals per treatment group to detect a 20% difference in CH4 emissions. Previous studies have experienced calves being removed from the study due to navel infections caused by navel sucking by barn mates. To mitigate these potential losses, 24 animals / group were used. The study was approved by the AgResearch Ruakura Animal Ethics Committee. Calf rearing sheds were divided into 2000-m2 sheds, each with 12 m2 of floor space. 2 Each pen contained four calves, was fitted with wood chips, had fresh water, and was equipped with feeders for calf pellets and hay.

[0248] 3.1.2 Calf schooling and feeding Female Friesian dairy cows were enrolled in the study over a 3-week period. Calf enrollment was staggered by 3 weeks to spread the age of calves so that three groups of the same age could be measured sequentially in the cow CH4 room. Newborn calves were separated from their dams twice daily and brought into the calf-rearing facility. Calves were weighed on arrival at the barn and then weekly for the duration in the calf-rearing facility. Calves were randomly assigned to treatment groups (FNZ118 or control) within each week of enrollment, taking into account the balance of birth weight and calf sire so that eight calves were assigned to each treatment group per week. Within the first 12 hours of entering the barn, each calf was given two doses of 2–3 L of warm colostrum, with the first morning colostrum containing FNZ118 or control treatment. Freeze-dried L. rhamnosus FNZ118 was stored at −20°C until use. Calves administered FNZ118 had a 5 × 10 10CFU of FNZ118 were administered once daily. The control treatment (3 g maltrin / calf / day) was an excipient used to blend the concentrated freeze-dried L. rhamnosus FNZ118 product to the exact daily dose. After colostrum feeding, calves were fed 6 L of calf milk replacer (CMR) (Ancalf, NZAgBiz / Fonterra; 150 g / L mixed with tap water at ∼37°C) daily, divided into 3 L in the morning and 3 L in the afternoon. FNZ118 and control treatments were added to the morning 3 L milk only and mixed into the pre-warmed CMR until the freeze-dried material was evenly distributed throughout the milk. Calves were also fed solid feed in the form of pelleted calf feed containing 20% ​​fiber source (lucerne and soybean hulls) and a coccidiosis-specific non-ionophore coccidiostat (see Table 11). Calves were not offered hay for the first 6 weeks to avoid variability in solid feed intake among calves and its possible effect on ruminal development and CH4 emissions from calves. However, pasture hay and fluffed pasture hay (cut to approximately 75 mm) were offered to calves after the first CH4 measurement at week 6 to stimulate salivation and stabilise ruminal pH. Pelleted feed and hay intake were measured at pen level.

[0249] Calves were weaned from most of their milk starting at week 10 to receive only 0.5 L of CMR in the morning containing FNZ118 and control treatments by the end of week 11. Treatment continued to be given to calves with 0.5 L of CMR in the morning feed until after completion of the second round of CH4 measurements at week 14.

[0250] [Table 11]

[0251] 3.1.3 Animal Health Calves were weaned and vaccinated and treated with antiparasitic drugs in accordance with animal health and welfare protocols.

[0252] 3.1.4 Measuring methane emissions from calves CH4 measurements were made on 20 calves per treatment group at 6 weeks of age (preweaning) and after 14 weeks of age (postweaning). Four calves were measured in four cow breathing chambers, one time per 2 days. The first preweaning period of CH4 measurements lasted 34 days, and the second postweaning period lasted 34 days.

[0253] First round of CH4 measurements Calves (four per trip) were transported from the calf-rearing shed to the methane measurement facility at the New Zealand Animal Ruminant Methane Measurement Centre. On arrival, calves were housed in wood-bedding pens and were given milk (3 L, pre-weaning) or solid feed (post-weaning) in the afternoon and had access to fresh water. At approximately 8:00 the following morning, calves were moved to individual cow breathing chambers where they received their morning allowance of milk (CMR; 3 L) and had free access to pelleted feed (+chaff hay post-weaning) and water. Calves continued to receive FNZ118 and control treatments as well as solid pelleted feed while in the chambers. Calves remained in the room for two days of CH4 measurements and for twice-daily feeding and pen cleaning. At 8:00 a.m. on the third day, calves were moved from shed to shed and the next four calves (housed in the shed over the previous night) were placed in the shed. Calves emerged from the chambers at approximately 9:00 a.m. and were allotted morning milk and ad libitum pellets (plus chaffed hay after weaning), with water ad libitum. Two hours after feeding (approximately 11:00 a.m.), ruminal contents and fecal samples were collected from each calf. Ruminal (via stomach tube) and fecal (via digital collection) samples were used immediately for pH measurements and stored at -80°C for subsequent volatile fatty acid (VFA) analysis.

[0254] After the first CH4 measurements at 6 weeks, calves were returned to the calf rearing facility and fed hay ad libitum in addition to calf feed until 12 weeks.

[0255] Second round of CH4 measurements Hay was replaced with hay ad libitum 2 weeks prior to the second round of CH4 measurements at week 14. Chumpled hay was offered at approximately 10% (500 g / day) of the daily calf pellets (5 kg) offered when calves entered the respiration chamber for postweaning measurements at 14 weeks of age. Milk and solid feed intakes were measured at both rounds of CH4 measurements, and feed samples were dried to estimate dry matter intake per calf and for compositional analysis. Chamber measurements of CH4, moisture (H2), and CO2 were reported as emissions (g / d) or yield (g / kg DMI / d).

[0256] Following completion of the second round of CH4 measurements and sampling at week 14, calves were transported to the farm for adaptation to pasture feeding. Calves were placed on pasture with calf pellets available from their previous daily intake and the amount of pellets offered was reduced over a 3-week period to encourage transition to pasture (a 33% reduction in the weekly pellet allowance).

[0257] 3.1.5 Post-weaning animal development After methane measurements at 14 weeks, calves were transferred to pasture and their body weight and average daily gain were monitored monthly until 13 months of age. Treated and control animals were transferred to separate pastures and pasture quality was monitored to ensure comparability of results. As expected in pasture-based farming systems, there was seasonal variation in metabolizable energy (ME) and digestibility (in vitro DOMD%) in both pastures, but there were no significant differences between pastures. Treatment with FNZ118 was discontinued after weaning at 14 weeks of age.

[0258] Statistical analyses were performed in R using the lme4 package. A model for body weight was fitted by REML and included effects of treatment with FNZ118, time (i.e., month of weight measurement), and two interactions with a random effect for animal.

[0259] 3.2 Results 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 two treatment groups (n=24 per group): FNZ118 or control (vehicle only). Twenty calves from each group were used for the measurements described below.

[0260] 3.2.1 Preweaning animal growth Calf weights were measured when the calves first arrived at the calf house. No statistically significant differences in birth weights were observed between treatment groups (Table 12).

[0261] [Table 12]

[0262] Calves were treated from their first feeding of colostrum and continued to receive treatment once daily in the morning until completing a second CH4 measurement at 14 weeks of age. Calf pellet and hay intake while the calves were in the respiration chamber during methane measurements is shown in Table 13.

[0263] [Table 13]

[0264] Changes in live weight and daily gain before CH4 measurements at 6 and 14 weeks are shown in Table 14. No significant differences in LWT or ADG were observed between FNZ118-treated and control calves at 6 or 14 weeks of age.

[0265] Because CH4 measurements in the respiration chamber are known to be stressful for livestock and affect feed intake and growth rate, the body temperature and weight of calves in the respiration chamber were also measured. A small number of animals lost weight during their passage through the chamber (mean LWT loss of 0.392 kg over 4 days), but the majority maintained or gained weight (mean LWT gain of 0.774 kg and ADG of 0.194 kg over 4 days). However, no significant difference in ADG was observed between FNZ118-treated and control calves in the chamber.

[0266] [Table 14]

[0267] 3.2.2 Methane emissions From the calves enrolled in the study, 40 calves were selected for CH4 emission measurements. Selection was based on weekly herd registrations, and calf exclusion criteria were based on past health status and the presence or absence of veterinary treatment required prior to CH4 testing. Animals were measured in two rounds; round 1 was at 6 weeks of age, preweaning, and round 2 was at 14 weeks of age, postweaning. Each round of measurements was performed in three batches over a 34-day period.

[0268] In the first round, calves were fed 2 × 3 L / day CMR in the morning (including treatment) and afternoon, plus calf pellets ad libitum in the chambers. In the second round, calves received only 0.5 L CMR containing treatment in the morning ration and ad libitum calf pellets given as chaff hay at 10% of expected solid feed intake.

[0269] [Table 15]

[0270] At 6 weeks of age, CH4 yields ranged from 7 to 10 g / d and CH4 yields ranged from 12 to 15 g / kg DMI / d (Table 15), as would be expected from a preweaned calf on a primarily milk diet (6 L / d: 0.9 kg milk solids) with only low solid feed intake (0.6 to 0.7 kg / d).

[0271] Calves treated with FNZ118 showed a significant (P<0.001) 27% decrease in CH4 production (g / d) and a significant (P<0.05) 15% decrease in CH4 yield (g / kg DMI) compared to calves receiving the control treatment (vehicle only).

[0272] Similarly, there was a significant (P<0.01) 44% reduction in H2 production (g / d) and CO2 production (P<0.001), and a significant (P<0.05) 43% reduction in H2 yield (g / kg DMI) compared to the control. Calves receiving FNZ118 treatment had a significantly (P<0.05) lower pellet intake of approximately 15%. However, because calves also consumed 6 L CMR / day (900 g milk solids / day), pellet intake was only approximately 40% of total intake.

[0273] After 14 weeks of weaning, CH4 production was 40.86 g / day in FNZ118-treated calves and 52.28 g / day in control calves, while CH4 yield was 13.83 g / kg DMI / day in FNZ118-treated calves and 14.6 g / kg DMI / day in control calves. These C4 emissions are lower than adult cows, but are as expected for weaned calves. Calves fed FNZ118 continued to produce lower CH4 (g CH4 / day) compared with control calves (approximately 22% lower; P<0.001). CH4 yield (g CH4 / kg DMI / day) for calves fed FNZ118 was numerically lower (approximately 5%) than control calves, but was not significantly different from control calves. CO2 production was significantly lower in FNZ118-fed calves (P<0.001), but CO2 production, H2 production, and H2 production did not differ from control animals. FNZ118-fed calves also had significantly lower DMI (approximately 17%) (P<0.01).

[0274] Even though FNZ118 supplementation was discontinued after 14 weeks of methane measurements, animals fed FNZ118 continued to have significantly lower CH4 production (g CH4 / day) at 9 months and 1 year of age (P<0.001; Table 15). Animals fed FNZ118 also had lower total dry matter intake than control animals (Table 15). This did not rise to significance levels at 9 months, but the difference was highly significant at 1 year (P<0.001).

[0275] Rumen contents and faeces were collected from calves at 6 weeks, 14 weeks, 9 months, and 1 year after the CH4 measurement rounds, and pH was measured. There were no significant differences between the FNZ118-treated and control groups (Table 16).

[0276] [Table 16]

[0277] Volatile fatty acids were measured in ruminal contents from samples taken from calves after leaving the chamber at 6 weeks (preweaning) and 14 weeks (postweaning). Acetate was the predominant VFA detected, with propionate and butyrate accounting for smaller proportions (Table 17). The amounts of acetate, propionate, butyrate or total VFAs found in calves fed FNZ118 were not significantly different compared to controls at 6 or 14 weeks. At 9 months and 1 year, there was a significant reduction in acetate and butyrate, as well as total VFAs, in treated animals, although the acetate:propionate ratio remained the same compared to controls. There was also a reduction in propionate, not reaching significance at 9 months, but significant at 1 year.

[0278] [Table 17]

[0279] There was a significant increase in the amount of isobutyrate in treated animals compared to control animals at 6 weeks, 9 months, and 1 year; as well as a significant increase in the amount of isovalerate at 9 months and 1 year (Table 18).

[0280] [Table 18]

[0281] There were no significant differences in the amounts of formate, lactate, and succinate seen in treated or control animals at 6 and 14 weeks (Table 19). At 9 months and 1 year, a significant decrease in succinate was seen in treated animals compared to controls, and at 1 year, a significant decrease in lactate was also seen.

[0282] [Table 19]

[0283] 3.2.3 Post-weaning animal development After methane measurements at 14 weeks, calves were moved to pasture and their body weight and average daily gain were monitored monthly.

[0284] At 9 months, FNZ118 treated animals had numerically lower food intake, but this did not reach significance. After 1 year, treated animals had significantly lower food intake (Table 20).

[0285] [Table 20]

[0286] Body weights of FNZ118-treated and control animals were similar from 4 to 6 months (p>0.05) (Table 21; Figure 1). However, FNZ118-treated animals showed a significant increase in body weight compared to controls from 7 to 10 months, resulting in a significant increase in body weight (p<0.05) (Table 21; Figure 1). From the 11th month onwards, animals in the FNZ118-treated group showed numerically higher body weights than control animals, but this did not reach significance. Treated animals also showed significantly higher average daily gain (ADG) than controls over the entire period from 4 to 13 months (p<0.001).

[0287] [Table 21]

[0288] 3.3 Discussion During the first round of CH4 (week 6), FNZ118-treated calves had a reduction in CH4 mass by approximately 27% and CH4 mass by 15%. Overall, CH4 production and CH4 yield in these young calves were lower (typically approximately 22 g / kg DMI) compared to mature calves fed a grass diet. This was expected from preweaned calves on a primarily dairy diet (6 L / day: 0.9 kg milk solids) consuming small amounts (0.6-0.7 kg / day) of solid feed (grain-based). The corresponding strong reductions in H2 and CO2 production (P<0.001) and lower H2 production in FNZ118-fed calves were consistent with lower CH4 production and reduced yields.

[0289] Feeding the FNZ118 strain did not affect CMR intake or chow intake in the pens until the first CH4 measurements. Although the calves consumed all of the CMR during the first round of CH4 measurements, chow intake in the chambers was approximately 100 g / day lower for FNZ118-fed calves than control-fed calves, and chow intake was approximately 15% lower. The stress of CH4 measurements in the respiration chamber may have caused some animals to lose weight, but the majority of calves in the first round continued to gain weight, albeit at a lower rate than when the animals were not in the respiration chamber. No significant differences in ADG were observed in FNZ118-fed calves compared to controls while the animals were in the chambers, suggesting that the differences in chow intake in the chambers were due to the effect of LAB administration. In these preweaned calves, milk components constituted the majority of dietary intake (approximately 60%), so low pellet intake did not affect whole-group LWT or ADG in FNZ118-treated calves.

[0290] After weaning at 14 weeks, measured CH4 production from calves increased approximately five-fold, but their CH4 yields remained similar to those measured at 6 weeks. Since the postweaning diet consisted mainly of pellets and hulled hay (mean pellet intake 3.9 kg / day, mean hay intake 0.35 kg / day) with only small amounts of CMR (mean CMR intake of solids 0.075 kg / day), an increase in total CH4 production was expected. Animals fed FNZ118 showed a 22% decrease in CH4 production and a 13% decrease in CO2 production, but their H2 production and CH4, H2 and CO2 production were no longer significantly different from control animals. This was likely due mainly to a 17% decrease in DMI in calves fed FNZ118 compared to controls.

[0291] A potential cause of the reduced DMI in calves could be related to a feed-induced decrease in ruminal pH, although this is unlikely to have been the cause of the reduced DMI observed in calves fed FNZ-118. Although several individual ruminal samples (approximately 23%) were below 5.6 at the time of sampling (2 h after feeding, when ruminal pH is likely to be at its lowest point), ruminal pH was not significantly different between calves fed FNZ118 compared to control treatments, suggesting that pH was not the cause of the reduced intake in FNZ118-fed calves.

[0292] VFA analysis of ruminal contents of calves at 6 and 14 weeks (post-weaning) showed no significant differences between calves fed FNZ118 and control calves.

[0293] There was a significant reduction in the total VFAs present in ruminal samples from animals receiving FNZ118 treatment at 9 months and 1 year of age, as well as the individual amounts of acetate, butyrate, and propionate in the first year. The amount of VFAs present in a ruminal sample is a balance between VFA production and VFA absorption by the rumen. The reduction in the amount of VFAs in the ruminal samples could be due to a decrease in VFA production (e.g., due to less feed being digested) and / or an increase in VFA absorption by the rumen (meaning more VFAs are available for growth and development). Combined with the increased body weight seen in treated animals, the reduction in ruminal VFA concentrations is consistent with increased absorption leading to increased feed efficiency.

[0294] There were no significant differences in body weight or average daily gain (ADG) between FNZ118-treated and control calves at 6 or 14 weeks of age.

[0295] After weaning, animals continued to show similar weight gain at 4-6 months of age. However, between 7-10 months, the FNZ118-treated group gained significantly more weight than the control group, resulting in a significant increase in body weight (p<0.05). From the 11th month onwards, animals in the FNZ118-treated group showed numerically higher body weights than the control animals, but this did not reach significance levels.

[0296] ADG over the entire postweaning period in FNZ118 treated heifers (0.714 kg / day) was also highly significantly (p<0.001) higher than in controls (0.648 kg / day), suggesting that despite reduced dry matter intake (measured during methane chamber measurements), FNZ118 treated animals were more productive and had a higher feed efficiency than controls.

[0297] 3.4 Conclusion This example shows that supplementation with L. rhamnosus FNZ118 during the first 14 weeks of life can improve feed efficiency in postweaned animals by supporting normal or increased growth despite reduced dry matter intake, leading to increased weight gain. This effect persisted until at least 1 year of age, even though supplementation was stopped at 14 weeks of age.

[0298] This example also shows that L. rhamnosus FNZ118 can significantly reduce methane production in pre-weaned (6 weeks of age) and post-weaned (14 weeks of age) calves, again with an effect that persisted until at least 1 year of age, despite supplementation ceasing at 14 weeks.

[0299] 4. Example 4 - Effect of L. rhamnosus FNZ118 on ruminal and fecal microbiota 4.1 Materials and Methods Rumen and fecal samples were collected at the end of methane measurements and immediately frozen at -80°C until DNA extraction. DNA was extracted using the bead-beating / phenol-chloroform method (Rius et al., 2012) and used in PCR reactions to generate 16S ribosomal RNA gene amplicons using barcoded sequencing primers specific for bacteria, archaea and protozoa (Kittelmann and Janssen, 2011). Amplicons were purified, normalized, pooled and sequenced on an Illumina MiSeq sequencer. Sequencing results were quality controlled and filtered, and 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.

[0300] 4.2 Results Samples for ruminal and fecal microbiota analysis were collected at weeks 6 and 14 at the same time that methane measurements were recorded in Example 3.

[0301] 4.2.1 Rumen microflora (bacteria) Overall, no significant differences in the ruminal microbiota at the phylum level were observed between FNZ118-treated and control animals at weeks 6 or 14 (data not shown).

[0302] At the family level, a significant decrease (p<0.05) in the relative abundance of Ruminococcaceae was observed at week 6 (Table 22), a difference that did not persist at week 14. A significant increase (p<0.05) in the relative abundance of Acidaminococcaceae was also observed at week 6, but again this difference did not persist at week 14.

[0303] [Table 22-1]

[0304] [Table 22-2]

[0305] At the genus level, a significant decrease in the abundance of Ruminococcaceae group NK4A214 and Ruminococcus 2 (bromii / bovis) was observed at week 6. A significant increase in Olsenella was also observed at week 6.

[0306] Succiniclasticum showed a significant increase in abundance at week 6 compared to the control (Table 23). As succinate metabolism is known to involve Succiniclasticum, this increase may indicate a disruption of the succinate metabolic pathway in the rumen.

[0307] [Table 23]

[0308] 4.2.2 Rumen microbiota (archaea) Overall, no significant differences in the ruminal archaeal microbiota at the family or genus level were observed between FNZ118-treated and control animals at weeks 6 or 14 (data not shown).

[0309] At the clade level, there was a significant decrease in the abundance of the key methanogen, Methanobrevibacter gottschalkii, at week 14 in FNZ118-treated animals (Table 24).

[0310] [Table 24]

[0311] 4.2.3 Fecal microflora (bacteria) At the phylum level, the relative abundance of Firmicutes in the feces of animals fed FNZ118 was significantly increased (p<0.05) compared to control animals at week 6 (Table 26). This effect did not persist through week 14. Although L. rhamnosus FNZ118 is a member of the Firmicutes, there was no significant increase in the corresponding Lacticaseibacillus, suggesting that the increase in Firmicutes reflects an increase in other bacterial genera within this phylum and not simply an increase in FNZ118.

[0312] [Table 25]

[0313] At the family level, a significant increase (p<0.05) in the relative abundance of Ruminococcaceae and a significant decrease in Lachnospiraceae were observed in the faeces of animals fed FNZ118 compared to control animals at week 14 (Table 27). There was also a significant decrease in the relative abundance of Porphyromonadaceae at week 6, but this effect did not persist until week 14.

[0314] [Table 26-1]

[0315] [Table 26-2]

[0316] [Table 26-3]

[0317] At the genus level, the abundance of Ruminococcaceae UCG-005 was significantly increased and Ruminococcaceae UCG-014 was significantly decreased in the feces of animals fed FNZ118 compared to control animals at week 14 (Table 28). At week 6, the relative abundance of Parabacteroides was also significantly decreased.

[0318] [Table 27]

[0319] 4.2.4 Fecal microbiota (Archaea) No significant differences in archaeal diversity at the family, genus, or clade level were observed in feces from FNZ118-treated animals compared with control animals (data not shown).

[0320] 4.3 Conclusion This example shows that L. rhamnosus FNZ118 (as shown in Example 3) can reduce methane production in calves and significantly reduce the ruminal abundance of an important species of methanogen, Methanobrevibacter gottschalkii, directly via bacteriocidal / archaeostatic and / or bacteriostatic / archaeostatic effects and / or indirectly via inhibition or disruption of methanogenic pathway(s) and / or cross-feeding of intermediates, without causing other major disruptions to the bacterial or archaeal microbiota.

[0321] References

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[0323] [ka]

[0324] [ka]

[0325] Industrial Applicability The present invention relates to the use of probiotic bacteria, in particular L. rhamnosus strain FNZ118 and / or derivatives thereof, in particular for increasing feed efficiency, increasing growth and / or productivity, improving body weight or body composition of ruminants and / or increasing milk production in ruminants, inhibiting the growth of methanogenic bacteria in the forestomach of ruminants, reducing the ability of the ruminal microflora to produce methane and / or reducing methane emissions by ruminants. Methods of use of L. rhamnosus strain FNZ118 and / or derivatives thereof, as well as ruminant feed compositions comprising same are also provided.

[0326] [ka]

[0327] [ka]

[0328] [ka]

Claims

1. Isolated Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

2. A food or feed composition comprising Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

3. 3. The food or feed composition of claim 2, wherein the composition is a ruminant feed composition.

4. a. Increases feed efficiency in ruminants; b. enhancing growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reduce methane emissions from ruminants; h. delivering microorganisms to ruminants, and / or i. Reducing ruminant greenhouse gas emissions A ruminant feed composition for use in a ruminant feed for the treatment of malaria, comprising Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

5. 5. The ruminant feed composition of claim 4, wherein the feed composition is a fermented yogurt-style composition, the fermented yogurt-style composition being formed through a process of growing L. rhamnosus FNZ118 using a milk-based or non-dairy-based carrier.

6. 5. The ruminant feed composition of claim 4, which is or comprises a partial or complete mixed ration (TMR), corn, soybean, forage, grain, distiller's grain, germinated grain, legume, fiber, forage, grass, hay, straw, silage, grain, leaf, meal, mash feed, lick block, or molasses.

7. 5. The ruminant feed composition of claim 4, further comprising at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methane production, and / or a natural or chemically synthesized methane production inhibitor, and / or a methane production inhibitor such as bromoform.

8. 5. The ruminant feed composition of claim 4, further comprising 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 galactooligosaccharides, one or more short-chain galactooligosaccharides, one or more long-chain galactooligosaccharides, one or more fructooligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or a mixture of any two or more thereof.

9. 5. The ruminant feed composition of claim 4, wherein the derivative of L. rhamnosus FNZ118 is a cell lysate of the strain, a cell suspension of the strain, a metabolic product of the strain, a culture supernatant of the strain, or killed L. rhamnosus FNZ118.

10. A ruminant feed composition as described in claim 4, wherein the composition comprises milk, milk powder, milk replacer, milk fortifier, colostrum, whey, whey powder, sucrose, maltodextrin, and / or rice hulls.

11. a. Increases feed efficiency in ruminants; b. enhancing growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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 ruminant rumen microflora to produce methane; g. Reduce methane emissions from ruminants; h. delivering microorganisms to ruminants, and / or i. Reducing ruminant greenhouse gas emissions 1. A method for administering to an animal: i. a food or feed composition according to claim 2 or 3, ii. A ruminant feed composition according to any one of claims 4 to 10, or iii. An effective amount of Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof. Administering

12. 12. The method according to claim 11, wherein the growth of methylotrophic methanogens, preferably methanogens from the genus Methanosphaera, is inhibited in the forestomach of an animal.

13. 12. The method of claim 11, wherein L. rhamnosus FNZ118 or a derivative thereof is administered in a composition that is a food, beverage, food additive, beverage additive, animal feed, animal feed additive, animal feed supplement, dietary supplement, carrier, vitamin or mineral premix, nutritional product, enteral nutritional product, soluble, supplement, pharmaceutical, lick block, drench, tablet, capsule, pellet, bolus, or ruminal product, or wherein L. rhamnosus FNZ129 is encapsulated, for example, in a liposome, microbubble, microparticle, or microcapsule.

14. 13. The method of claim 12, wherein L. rhamnosus FNZ118 or a derivative thereof is administered in drinking water, milk, milk powder, milk replacer, milk fortifier, whey, whey powder, partial or complete mixed ration (TMR), corn, soybeans, feed, grains, distillers grains, germinated grains, legumes, vitamins, amino acids, minerals, fiber, forage, grass, hay, straw, silage, grains, leaves, meal, solubles, supplements, mash feed, meal, fruit pulp, vegetable pulp, fruit or vegetable pomace, citrus meal, wheat shorts, corn cob meal, molasses, sucrose, maltodextrin, rice hulls, vermiculite, zeolite, or ground limestone.

15. To animals, a. 10 per kilogram of dry weight carrier feed ４ ~10 １３ colony forming units per kilogram of dry weight carrier feed, preferably 10 ８ ~10 １２ colony-forming units; b. 10 per kg of animal weight ４ ~10 １０ colony forming units / day, preferably 10 per kg of animal body weight ５ ~10 ８ colony-forming units / day; or c.10 ４ ~10 １３ colony forming units / day, preferably 10 ６ ~10 １３ colony forming units / day 12. The method of claim 11, comprising administering L. rhamnosus FNZ118 in an amount of

16. 12. The method of claim 11, wherein the derivative of L. rhamnosus FNZ118 is a cell lysate of the strain, a cell suspension of the strain, a metabolic product of the strain, a culture supernatant of the strain, or killed L. rhamnosus FNZ118.

17. 12. The method of claim 11, further comprising administering at least one microorganism of a different species or strain, a methanogen or a vaccine that inhibits methane production, and / or a natural or chemically synthesized methanogenesis inhibitor and / or a methanogenesis inhibitor such as bromoform.

18. 12. The method of claim 11, wherein L. rhamnosus FNZ118 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 galactooligosaccharides, one or more short-chain galactooligosaccharides, one or more long-chain galactooligosaccharides, one or more fructooligosaccharides, inulin, one or more galactans, one or more fructans, lactulose, or a mixture of any two or more thereof.

19. a. Increase growth or productivity in ruminants; b. increasing the yield of milk and / or milk components produced by ruminant animals; c. Increase the yield of milk fat, milk protein or milk solids in milk produced by ruminants; and / or d) The method of claim 11, further improving the body weight and / or body composition of a ruminant.

20. 12. The method of claim 11, wherein the ruminant is a cow, goat, sheep, bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope, or nilgai; preferably a cow or sheep; more preferably a cow.

21. a. the ruminant is a lactating animal; b. the ruminant is a pre-weaned animal, such as a calf or lamb; c) the ruminant is a weaned animal; or d. The method of claim 11, wherein L. rhamnosus FNZ118 is administered to the ruminant both pre-weaning and post-weaning.

22. 12. The method of claim 11, wherein the administration is to a pre-weaned animal and 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, by the ruminant, and / or the increase in feed efficiency in the ruminant persists after weaning.

23. 12. The method of claim 11, wherein the inhibition of the growth of methanogenic bacteria and / or archaea in the forestomach of the ruminant, the reduction in methane emission, e.g., methane production, by the ruminant, and / or the increase in feed efficiency in the 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 from the last administration of L. rhamnosus FNZ118; preferably for the lifetime of the ruminant.

24. A ruminant to which the method according to claim 11 has been applied.

25. 1. A method for producing an animal product with a reduced greenhouse gas emissions footprint, comprising: a. providing the ruminant of claim 24, and b. Producing animal products from animals The method includes:

26. 26. The method of claim 25, wherein the animal product comprises dairy, meat, or wool.

27. Use of Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof, a. Increases feed efficiency in ruminants; b. enhancing growth and / or productivity in ruminants; c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reduce methane emissions from ruminants; h. delivering microorganisms to ruminants, and / or i. Reducing ruminant greenhouse gas emissions Use for producing a composition for

28. 28. Use according to claim 27, 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 10.

29. a. Increases feed efficiency in ruminants; b. Enhances growth and / or productivity in ruminants c. improving body weight and / or body composition in 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. Reduces the ability of the rumen microflora to produce methane; g. Reduce methane emissions from ruminants; h. delivering microorganisms to ruminants, and / or i. Used to reduce greenhouse gas emissions from ruminants Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus) strain FNZ118 (NMIA accession number V21 / 015445 dated August 2, 2021) or a derivative thereof.

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