PROBIÓTICOS PARA REDUZIR AS EMISSÕES DE GASES DE EFEITO ESTUFA EM BOVINOS

Abstract

The present invention relates to probiotic or microbiome modulator compositions and methods for reducing greenhouse gas emissions, such as methane emissions, that use such compositions. The probiotic or microbiome modulator compositions include one or more isolated bacterial strains which alter the microbiome and / or alter or kill methanogenic bacteria.

Description

ΛΓ73 Probiotics to reduce greenhouse gas emissions in cattle. XML SEQUENCE LISTING

[0001] This application contains a Sequence Listing encoded in XML format, which has been electronically archived by EFS-web and is incorporated herein by reference in its entirety. The said Sequence Listing in XML format, created on March 21, 2023, is named 2950-24_P1_ST26.XML and has a size of 18,761,302 bytes. FIELD

[0002] This disclosure relates to probiotic or microbiome-modulating compositions and methods for reducing greenhouse gas emissions, such as methane emissions. Probiotic or microbiome-modulating compositions include one or more isolated bacterial strains that reduce greenhouse gas and methane emissions, alter, disrupt or eliminate methanogenic Archaea, and modulate microbiota composition structures. BACKGROUND

[0003] Methane is produced as a byproduct of the ruminal microbial fermentation process. In particular, H2 and CO2 are byproducts of the fermentation process and are used by methanogenic Archaea (methanogens) to form methane. 40% of methane comes from natural sources, such as animals. The remaining 60% comes from human sources, including fossil fuels, landfills, and biomass burning. In the US alone, livestock production emits approximately 200 million metric tons of CO2 equivalent from methane. Much of the methane from livestock is generated in the rumen of ruminants by methanogens, with only a small amount emitted by large intestine fermentation in ruminants and non-ruminants. Methane production and emission result in a 6–12% energy loss for animals.

[0004] Methanogens, unique microbes of the Archaea domain, are responsible for the production of methane through the methanogenesis pathway, which reduces carbon dioxide, methanol, or methylamines to methane using electrons from hydrogens. They are common in Petition 870250085529, dated 09 / 22 / 2025, page 6 / 282 2 / 73 wetlands, where they are responsible for swamp gas. Examples of methane-producing genera are Methanobrevibacter, Methanobacterium, Methanosarcina, Methanococcus, and Methanospirillum. Methanogenic organisms are widely distributed in anaerobic nature and are found in mud, sewage, and sludge, and in the gastrointestinal tract of animals. Methanogens typically thrive in environments where all other electron acceptors (such as nitrate, sulfate, and trivalent iron) are limited. Methanogenic organisms, particularly methanogenic Archaea, are found in the digestive tracts of animals such as ruminants and non-ruminant animals, including humans, where they are responsible for the methane content of belches in ruminants and flatulence in humans.Ruminants are large, herbivorous, hoofed mammals that graze, capable of acquiring nutrients from plant foods by fermenting them in a specialized stomach before digestion, primarily through microbial action. In marine sediments, biological methane production is generally limited to locations where sulfates are depleted, below the surface layers and in marine sediments or lower layers, which are anaerobic. Furthermore, methanogenic Archaea populations play an indispensable role in the anaerobic treatment of wastewater and in global carbon cycles. Others are extremophiles, found in environments such as hot springs and submarine hydrothermal vents, as well as in the solid rock of the Earth's crust, kilometers below the surface.

[0005] The global population will increase by 33% in 2050 to 9.6 billion. Projected demand for meat and milk protein will increase by 73% and 58%, respectively, compared to those in 2010 (FAO). See Tackling climate change through livestock, FAO, 2013. An increase in livestock production is expected to make a significant contribution to global climate change (GHG emissions, N2O, CO2, CH4), as GHGs from livestock accounted for 18% of global emissions. Among GHGs, methane has a shorter half-life and is 28 times more potent than CO2. Enteric methane emissions account for 44.3% of GHG emissions from livestock production. A total of 100 Mton CH4 is emitted per year. Furthermore, Petition 870250085529, dated 09 / 22 / 2025, page 7 / 282 3 / 73 Methane emissions are considered a loss of energy for animals. Methane emissions can result in an energy loss of 6 to 12% for animals.

[0006] There are net-zero emissions initiatives in the livestock industry and market opportunities for reducing enteric CH4 emissions. Supply chain net-zero emissions pledges put pressure on livestock industries to meet sustainability / greenhouse gas (GHG) reduction targets. In addition, a government incentive program that provides a carbon credit can be an incentive to reduce methane emissions (e.g., the Biden administration's aggressive 2030 GHG reduction target in the US).

[0007] Directly administered microorganisms (DFMs), often called probiotics or microbiome modulators, are microorganisms that colonize an animal's gastrointestinal tract and provide some beneficial effect to it. The microorganisms can be bacterial species, for example, those of the genera Bacillus, Lactobacillus, Lactococcus, and Enterococcus. The microorganisms can also be yeasts or even molds.Microorganisms can be provided to an animal orally, such as in feed, or by other methods of administration, including, in the case of birds, administration to a fertilized egg, i.e., in ovo.

[0008] A beneficial activity provided by a DFM can be through the synthesis and secretion of vitamins or other nutritional molecules necessary for a healthy metabolism of the host animal, or by the production of agents or molecules that inhibit or affect other organisms, microbials, or bacteria. DFMs can also alter the microbiome, either in an animal or where microorganisms or bacteria are present. A DFM can protect the host animal from diseases, disorders, or clinical symptoms caused by microorganisms or other agents. For example, the DFM can naturally produce factors with inhibitory or killing activity against certain species of pathogens, such as deleterious or disease-causing bacteria. A DFM can also alter the microbiome in an animal, resulting in beneficial effects, particularly because the microbiome and its components are connected to various diseases, conditions, or activities in an animal or host. Probiotics and DFMs Petition 870250085529, dated 09 / 22 / 2025, page 8 / 282 4 / 73 provide an attractive alternative or addition to disease prevention and / or the use and application of antibiotics in animals. Antibiotics can promote resistant or less sensitive bacteria and may ultimately end up in feed products or food consumed by other animals or humans, representing a greater public health crisis. DFMs are characterized as generally safe, even termed Generally Considered Safe (GRAS), and most are not naturally resistant to antibiotics.

[0009] There is a need in the art for probiotic and microbiome-modulating compositions to provide beneficial molecules to an animal's gastrointestinal tract and thus improve animal health. There is a need to reduce GHG emissions. There is a need in the art for probiotic and microbiome-modulating compositions capable of reducing GHG emissions resulting from microorganisms, including Archaea and bacteria, both in animals and in the environment. In particular, there is a need to reduce methane emissions from animals such as livestock and, particularly, ruminant animals, or from natural sources where methanogenic Archaea reside. SUMMARY OF THE INVENTION

[0010] This disclosure provides compositions and methods for reducing greenhouse gas emissions, particularly methane emissions generated by methanogenic organisms, such as methanogenic Archaea, or by animals or other sources where methanogenic organisms, such as methanogenic Archaea, reside. Such compositions and methods include at least one bacterial strain and may include combinations of bacterial strains.

[0011] This disclosure provides compositions that include at least one selected strain of Bacillus spp. and / or Lactobacillus spp. as a probiotic or microbiome modulator, or combinations of such strains as probiotics or microbiome modulators. This disclosure provides compositions that include at least one selected strain of Bacillus spp. and / or Lactobacillus spp. as an antimethanogenic or methanogenesis modulator, or combinations of such strains as antimethanogens or methanogenesis modulators. The disclosure provides methods Petition 870250085529, dated 09 / 22 / 2025, page 9 / 282 5 / 73 to reduce greenhouse gas emissions, particularly methane emissions, using such compositions.

[0012] This disclosure provides a composition including at least one probiotic or microbiome modulator selected from a species of Bacillus and Lactobacillus spp. or combinations thereof, wherein the composition reduces methane gas emissions, such as those from or in the presence of methanogenic Archaea organisms, when an effective amount is combined with one or more methanogenic Archaea organisms or administered to an animal harboring one or more methanogenic Archaea organisms, compared with one or more methanogenic Archaea organisms in the absence of the composition or compared with an animal to which the composition has not been administered. This disclosure provides a composition that includes at least one probiotic or microbiome modulator selected from a species of Bacillus spp. and Lactobacillus spp.or combinations thereof, wherein the composition reduces methane gas emissions from a ruminant when an effective amount is administered to the ruminant, compared to a ruminant that did not receive the composition. The present disclosure provides a composition comprising one or more probiotic or microbiome-modulating bacteria, wherein the bacteria are selected from Bacillus spp. and Lactobacillus spp. bacteria, in which the composition inhibits or eliminates one or more methanogenic bacteria. The composition, therefore, serves to reduce methane gas emissions from an animal, particularly including a ruminant, when an effective amount is administered to the animal, or particularly to the ruminant, compared to an animal or ruminant to which the composition was not administered. In one embodiment, the bacteria selected from Bacillus spp. and Lactobacillus spp. bacteria...The composition alters the microbiome or modulates or alters the environment of one or more methanogenic organisms, particularly one or more methanogenic Archaea. Methane production is therefore reduced in the presence of and after the administration or addition of selected bacteria from among Bacillus spp. and Lactobacillus spp. bacteria.

[0013] In one embodiment, methanogenic organism(s) is / are organism(s) that produce(s) methane as a byproduct of their metabolism. In a Petition 870250085529, dated 09 / 22 / 2025, p. 10 / 282 6 / 73 embodiment, the methanogenic organism(s) is / are a methanogenic Archaea. In one embodiment, the methanogenic Archaea is / are of the order Archaea Methanobacteriales, Methanomicrobiales, or Methanosarcinales. In one embodiment, the methanogenic Archaea is / are one or more Methanobacterium, Methanobrevibacter, Methanococcus, Methanosarcina, Methanosphaera, Methanomassilicoccales. In one embodiment, the methanogenic Archaea is Methanobrevibacter ruminantium, Methanobrevibacter gottschalkii, Metanosfera stadtmanae, Methanomicrobium mobile, Methanomassilicoccaceae spp. Methanobacterium bryantii or Metanosarcina mazei. In one embodiment, the fermentative organism is Fibrobacter, Ruminococcus, Streptococcus bovis, or Ruminobacter.

[0014] This disclosure provides a method for reducing methane gas emissions from an animal, including administering an effective amount of a composition including at least one probiotic or microbiome modulator selected from Bacillus spp. and Lactobacillus spp. or combinations thereof, to the animal. This disclosure provides a method for reducing methane gas emissions from a ruminant, including administering an effective amount of a composition that includes at least one probiotic or microbiome modulator selected from Bacillus spp. and Lactobacillus spp. or combinations thereof, to the ruminant.

[0015] In some embodiments, methods are provided for reducing methane emission or production, in cases by methanogenic organisms, in manure, lagoons, or an aerobic digester. In some embodiments, methods are provided for reducing methane emission or production, in cases by methanogenic organisms, in manure, by combining or adding to the manure one or more compositions or fermentation products of one or more compositions, including the probiotic or microbiome-modulating compositions provided herein. In some embodiments, methods are provided for reducing methane emission or production, in cases by methanogenic organisms, in manure, by combining or adding to the manure one or more compositions or fermentation products of one or more compositions, including the anti-methanogenic or methanogenesis-modulating compositions provided herein. Petition 870250085529, dated 09 / 22 / 2025, page 11 / 282 7 / 73

[0016] Bacterial strains, particularly Bacillus and Lactobacillus strains, possess the capacity and activity to reduce methane gas production. In one embodiment, the bacterial strains possess the capacity and activity to alter the microbiome and microorganisms and to reduce methane production and / or methanogenesis. In another embodiment, the bacterial strains possess the capacity and activity to alter the microbiome and microorganism-facilitated fermentation in order to reduce methane production and / or methanogenesis. In another embodiment, the bacterial strains possess the capacity and activity to modulate or alter the colonization of methanogenic organisms, including methanogenic Archaea. In another embodiment, the bacterial strains possess the capacity and activity to reduce the growth or inhibit or alter the colonization of methanogenic organisms, including methanogenic Archaea.In one embodiment, bacterial strains possess the ability and activity to reduce methane gas production in an animal. In another embodiment, bacterial strains possess the ability and activity to reduce methane gas production in a ruminant animal. In another embodiment, bacterial strains possess the ability and activity to reduce methane gas production in a non-ruminant animal. In yet another embodiment, bacterial strains possess the ability and activity to inhibit or otherwise alter methanogenic Archaea and alter methanogenesis in an animal.

[0017] In some forms, the animal is a ruminant. In some forms, the animal is a non-ruminant.

[0018] In one embodiment, bacterial strains are provided with the capacity and activity to reduce methane gas production in a ruminant animal. In one embodiment, bacterial strains are provided with the capacity and activity to reduce methane gas production in a ruminant or non-ruminant animal. In one embodiment, bacterial strains are provided with the capacity to alter the microbiome and modulate, including inhibit, methanogenesis and methane production due to methanogenic Archaea in an animal. In one embodiment, bacterial strains are provided with the capacity to alter the microbiome and modulate, including inhibit, methanogenesis and methane production due to methanogenic Archaea in a ruminant animal. In a Petition 870250085529, dated 09 / 22 / 2025, page 12 / 282 8 / 73 modality, bacterial strains are provided with the capacity and activity to reduce the growth or inhibit the colonization of methanogenic bacteria in a ruminant animal.

[0019] In one embodiment, bacterial strains are endowed with the capacity and activity to reduce methane gas production from native methanogenic Archaea in an animal. In one embodiment, bacterial strains are endowed with the capacity and activity to reduce methane gas production from native methanogenic Archaea in the rumen of a ruminant animal. In one embodiment, bacterial strains are endowed with the capacity and activity to inhibit methanogenic Archaea and / or alter methanogenesis through the microbiome and methanogenic organisms in the rumen of a ruminant animal. In one embodiment, bacterial strains are endowed with the capacity and activity to reduce the growth or inhibit the colonization of methanogenic bacteria in the rumen of a ruminant animal.

[0020] In one embodiment, a bacterial strain of Bacillus is provided with the ability and activity to reduce methane gas production from native methanogenic Archaea or methanogenic bacteria in an animal. In one embodiment, one or more bacterial strains of Bacillus are provided with the ability and activity to reduce methane gas production from native methanogenic Archaea or bacteria in an animal. In one embodiment, a combination of one or more bacterial strains of Bacillus is provided with the ability and activity to reduce methane gas production from native methanogenic Archaea or bacteria in an animal. In one embodiment, bacterial strains of Bacillus are provided with the ability and activity to reduce methane gas production from native methanogenic Archaea or bacteria in the rumen of a ruminant animal. In one embodiment, one or more strains of Bacillus are provided with the ability and activity to inhibit methanogenic Archaea and / or bacteria in an animal.In one embodiment, a strain of Bacillus is provided with the ability and activity to reduce methanogenesis via methanogenic Archaea in an animal. In another embodiment, a strain of Bacillus is endowed with the ability and activity to reduce the growth or inhibit the colonization of Archaea and / or methanogenic bacteria in an animal. In another embodiment, bacterial strains of Bacillus are... Petition 870250085529, dated 09 / 22 / 2025, p. 13 / 282 9 / 73 possessing the capacity and activity to inhibit Archaea and / or methanogenic bacteria or to alter the microbiome comprising Archaea and / or methanogenic bacteria in the rumen of a ruminant animal. In one embodiment, bacterial strains of Bacillus possess the capacity and activity to reduce the growth or inhibit the colonization of Archaea and / or methanogenic bacteria in the rumen of a ruminant animal.

[0021] In one embodiment, a Lactobacillus bacterial strain is provided with the capacity and activity to reduce methane gas production from Archaea and / or native methanogenic bacteria in an animal. In one embodiment, one or more Lactobacillus bacterial strains are provided with the capacity and activity to reduce methane gas production from Archaea and / or native methanogenic bacteria in an animal. In one embodiment, one or more Lactobacillus bacterial strains and one or more Bacillus bacterial strains in combination are provided with the capacity and activity to reduce methane gas production from Archaea and / or native methanogenic bacteria in an animal. In one embodiment, Lactobacillus bacterial strains are provided with the capacity and activity to reduce methane gas production from Archaea and / or native methanogenic bacteria in the rumen of a ruminant animal.In one embodiment, one or more Lactobacillus strains are provided with the ability and activity to inhibit Archaea and / or methanogenic bacteria in an animal. In another embodiment, a Lactobacillus strain is provided with the ability and activity to reduce methanogenesis via Archaea and / or methanogenic bacteria in an animal. In another embodiment, a Lactobacillus strain is provided with the ability and activity to reduce the growth or inhibit the colonization of Archaea and / or methanogenic bacteria in an animal. In yet another embodiment, bacterial strains of Lactobacillus are provided with the ability and activity to inhibit Archaea and / or methanogenic bacteria or to alter the microbiome comprising Archaea and / or methanogenic bacteria in the rumen of a ruminant animal. In one embodiment, Lactobacillus bacterial strains are provided with the capacity and activity to reduce the growth or inhibit the colonization of Archaea and / or methanogenic bacteria in the rumen of a ruminant animal. Petition 870250085529, dated 09 / 22 / 2025, p. 14 / 282 10 / 73

[0022] In some embodiments, one or more strains of Bacillus or Lactobacillus are provided with the ability and activity to reduce methane gas production and / or with the ability and activity to inhibit and / or with the ability to alter the microbiome that includes methanogenic Archaea. In some embodiments, combinations of one or more strains of Bacillus or Lactobacillus have the ability and activity to reduce methane gas production and / or with the ability and activity to inhibit methanogenic Archaea and / or bacteria or to otherwise alter the environment or microbiome, including methanogenic Archaea and / or bacteria, to reduce methane production and alter methanogenesis. In some embodiments, combinations of two or more strains of Bacillus have the ability and activity to reduce methane gas production and / or with the ability and activity in relation to methanogenic Archaea and / or bacteria.In some embodiments, combinations of one or more strains of Bacillus and one strain of Lactobacillus have the capacity and activity to reduce methane gas production and / or have the capacity and activity in relation to Archaea and / or methanogenic bacteria.

[0023] In one embodiment, the BE191006 strain of Bacillus amyloliquefaciens (also referred to as ELA191006) corresponding to the deposit is provided. ATCC PTA-127065. In one embodiment, the BE202071 strain of Bacillus amyloliquefaciens (also known as ELA202071) corresponding to the deposit is provided. ATCC PTA-127064. In one embodiment, the Bacillus subtilis strain BE191105 (also referred to as ELA191105) corresponding to deposit ATCC PTA126786 is provided.

[0024] Compositions of one or more Bacillus strains are provided to reduce methane gas production and / or alter methanogenesis and / or inhibit or alter the microbiome, including methanogenic Archaea. In some embodiments, a composition includes or comprises the isolated strain of Bacillus amyloliquefaciens BE191006. In some embodiments, a composition includes or comprises the isolated strain of Bacillus amyloliquefaciens BE202071. In some embodiments, a composition includes or comprises the isolated strain of Bacillus subtilis, including BE191105. In some embodiments, a composition includes a first isolated strain BE191006 of Bacillus amyloliquefaciens and a second isolated strain BE202071 of Bacillus amyloliquefaciens. Petition 870250085529, dated 09 / 22 / 2025, page 15 / 282 11 / 73 In some embodiments, a composition includes a first isolated strain BE191006 of Bacillus amyloliquefaciens and an isolated strain BE191105 of Bacillus subtilis. In some embodiments, the composition includes a first isolated strain BE191006 of Bacillus amyloliquefaciens, a second isolated strain BE202071 of Bacillus amyloliquefaciens, and a first isolated strain BE191105 of Bacillus subtilis.

[0025] In some embodiments, the isolated strain BE191105 of Bacillus subtilis is genetically modified. In some embodiments, the isolated strain BE191105 of Bacillus subtilis is genetically modified to produce or administer an antibacterial agent, antibacterial peptide, or antimethanogenic agent or compound. In some cases, the bacterial strains are combined into a composition or are combined after administration with one or more antibacterial agents, antibacterial peptides, or antimethanogenic agents or compounds. Antibacterial peptides may include, for example, CAP 18 peptides. CAP 18 peptides are described in PCT / US2022 / 044221, filed September 21, 2022, incorporated by reference herein.

[0026] In one embodiment of the invention, a probiotic composition or microbiome modulator or direct-feeding microorganism is provided comprising at least one strain of Bacillus, wherein the Bacillus strain is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria. In another embodiment of the invention, a probiotic composition or microbiome modulator or direct-feeding microorganism is provided comprising at least one strain of Lactobacillus, wherein the Lactobacillus strain is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria.In one embodiment of the invention, a probiotic composition or microbiome modulator or direct-feeding microorganism is provided comprising at least one strain of Lactobacillus and at least one strain of Bacillus, wherein the Lactobacillus strain and the Bacillus strain are capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria. Petition 870250085529, dated 09 / 22 / 2025, p. 16 / 282 12 / 73

[0027] In one embodiment of the invention, a probiotic composition or a microbiome modulator or a methane production modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria, comprising at least one strain of Bacillus. In another embodiment of the invention, a probiotic composition or a microbiome modulator or a direct-feeding microorganism is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria, comprising a combination of at least two strains of Bacillus.In embodiments, the Bacillus strain(s) is / are selected from Bacillus strain 06 (BE191006) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity to the sequence of strain 06 (BE191006); or strain 71 (BE202071) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity to the sequence of strain 71 (BE202071). and Strain 105 (BE191105) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the Strain 105 (BE191105) sequence.

[0028] In one embodiment of the invention, a probiotic composition or a microbiome modulator or a direct-feeding microorganism capable of reducing methane gas production and / or inhibiting methanogenesis via methanogenic Archaea is provided, comprising at least one strain of Bacillus. In another embodiment of the invention, a probiotic composition or a microbiome modulator or a direct-feeding microorganism capable of reducing methane gas production and / or inhibiting methanogenesis via methanogenic Archaea is provided, comprising a combination of at least two strains of Bacillus. In embodiments, the Bacillus strain(s) is / are selected from: BE191006 or a Bacillus strain with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with sequence SEQ ID NO: 27; BE202071 or a Bacillus strain with at least 90% identity, 95% identity, 97% Petition 870250085529, dated 09 / 22 / 2025, page 17 / 282 13 / 73 identity, 98% identity, 99% genomic sequence identity with sequence SEQ ID NO: 42; and BE191105 or a Bacillus strain with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with one or more SEQ ID NO: 1, 2, 3, 4 and 5.

[0029] In some embodiments, the invention relates to related, homologous or derived Bacillus strains with significant genomic sequence identity to the genomic sequence of any of the following strains: Bacillus amyloliquefaciens BE191006 (also referred to as ELA191006) corresponding to ATCC deposit PTA-127065, Bacillus amyloliquefaciens BE202071 (also referred to as ELA202071) corresponding to ATCC deposit PTA-127064, and / or Bacillus subtilis BE191105 (also referred to as ELA191105) corresponding to ATCC deposit PTA-126786. Thus, derived, similar or nearly genetically identical strains to the Bacillus strains provided herein are contemplated by further embodiments of the invention.Bacillus strains with 80%, 85%, 90%, 95%, 97%, 98%, and 99% genomic sequence identity to a strain provided and deposited in association with this invention are contemplated and constitute embodiments of the invention. Such derived, similar, or nearly genetically identical strains should function similarly as probiotics and have activity / capacity or function in improving animal health, production, and animal performance, as detailed in the capacity and activity or function of the strains and in the examples described herein. As an example of this modality, it is observed that the BE191024 strain of Bacillus amyloliquefaciens, corresponding to ATCC deposit PTA-126784, and the BE191006 strain of Bacillus amyloliquefaciens, corresponding to ATCC deposit PTA-127065, are genetically related or similar strains, demonstrating 99% genomic sequence identity.

[0030] In embodiments, Bacillus strains are selected from BE191006 corresponding to the ATCC PTA-127065 deposit or a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the sequence of BE191006 Petition 870250085529, dated 09 / 22 / 2025, p. 18 / 282 14 / 73 corresponding to ATCC deposit PTA-127065; BE202071 corresponding to ATCC deposit PTA-127064 or a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the sequence of BE202071 corresponding to ATCC deposit PTA127064; and BE191105 corresponding to ATCC deposit PTA-126786 or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the sequence of BE191105 corresponding to ATCC deposit PTA-126786.

[0031] According to one embodiment of the invention, a probiotic composition or microbiome modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome of Archaea and / or methanogenic bacteria, comprising one or more strains of Bacillus, particularly one or more strains of Bacillus subtilis or Bacillus amyloliquefaciens.According to one embodiment of the invention, a probiotic composition or microbiome modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including methanogenic Archaea, comprising at least one of the following: a first isolated strain of Bacillus amyloliquefaciens, a second isolated strain of Bacillus amyloliquefaciens, and a first isolated strain of Bacillus subtilis; and a suitable carrier for administration to animals; wherein said composition reduces methane production and / or reduces or inhibits the colonization of an animal by a methanogenic bacterium or bacteria when an effective amount is administered to an animal, compared to an animal to which the composition was not administered.According to one embodiment of the invention, a probiotic composition or microbiome modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome, including Archaea and / or methanogenic bacteria, comprising at least one of the following: a first isolated strain of Bacillus amyloliquefaciens, a second isolated strain of Bacillus amyloliquefaciens, and a first isolated strain of Bacillus subtilis; and a suitable vehicle for animal administration; wherein said composition reduces methane production and / or reduces or inhibits colonization or methanogenesis in an animal by a methanogenic Archaea when a quantity. Petition 870250085529, dated 09 / 22 / 2025, page 19 / 282 15 / 73 effective is administered to an animal, compared to an animal to which the composition was not administered; and wherein the first isolated strain of Bacillus amyloliquefaciens comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27 and / or with nucleic acid encoding one or more proteins of SEQ ID NO: 28-41; wherein the second strain of Bacillus amyloliquefaciens comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 42 and / or with nucleic acid encoding one or more proteins of SEQ ID NO: 43-50; wherein the first Bacillus subtilis strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 1, 2, 3, 4 and / or 5,or with a nucleic acid sequence encoding one or more proteins of SEQ ID NO: 6-26.

[0032] In one embodiment, the composition comprises at least two bacterial strains. In one embodiment, the composition comprises at least two bacterial strains capable of reducing methane production in an animal and / or reducing or inhibiting colonization or the microbiome in an animal, including Archaea and / or methanogenic bacteria. In one embodiment, the composition comprises at least two bacterial strains capable of reducing methane production in a ruminant animal and / or reducing or inhibiting colonization or altering the microbiome in a ruminant animal, including methanogenic Archaea. In one embodiment, the composition comprises at least two strains of Bacillus. In one embodiment, the composition comprises one strain of Bacillus and one strain of Lactobacillus. In one embodiment, the composition comprises at least two of the following: a first isolated strain of Bacillus amyloliquefaciens,A second isolated strain of Bacillus amyloliquefaciens and an isolated strain of Bacillus subtilis. In one embodiment, the composition comprises a first isolated strain of Bacillus amyloliquefaciens, a second isolated strain of Bacillus amyloliquefaciens, and an isolated strain of Bacillus subtilis. In one embodiment, the composition comprises at least two of the following: a first isolated strain of Bacillus amyloliquefaciens, a second isolated strain of Bacillus amyloliquefaciens, Petition 870250085529, dated 09 / 22 / 2025, page 20 / 282 16 / 73 an isolated strain of Bacillus subtilis and an isolated strain of Lactobacillus reuteri. In one embodiment, the composition comprises a first isolated strain of Bacillus amyloliquefaciens or a second isolated strain of Bacillus amyloliquefaciens, an isolated strain of Bacillus subtilis and an isolated strain of Lactobacillus reuteri.

[0033] In some embodiments, the composition comprises at least two isolated strains capable of inhibiting methane production or methanogenesis, selected from one or more strains of Bacillus amyloliquefaciens, one strain of Bacillus subtilis, and one strain of Lactobacillus reuteri. In one embodiment, a composition is provided comprising at least two strains selected from the strains of Bacillus amyloliquefaciens BE191024, Bacillus amyloliquefaciens BE191006, Bacillus subtilis BE191105, and Lactobacillus reuteri 3632. Compositions comprising the strains of Bacillus amyloliquefaciens BE191024 and Bacillus amyloliquefaciens BE191006 are one embodiment. Compositions comprising the strains of Bacillus amyloliquefaciens BE191024 and Bacillus subtilis BE191105 are one embodiment. Compositions comprising Bacillus amyloliquefaciens strain BE191006 and Bacillus subtilis strain BE191105 are one embodiment.Compositions comprising strain BE191024 of Bacillus amyloliquefaciens and strain BE191105 of Bacillus subtilis are an embodiment. Compositions comprising strain BE191024 of Bacillus amyloliquefaciens and strain 3632 of Lactobacillus reuteri are an embodiment. Compositions comprising strain BE191006 of Bacillus amyloliquefaciens and strain 3632 of Lactobacillus reuteri are an embodiment. Compositions comprising strain BE191105 of Bacillus subtilis and strain 3632 of Lactobacillus reuteri are an embodiment. Compositions comprising strain BE191024 of Bacillus amyloliquefaciens or strain BE191006 of Bacillus amyloliquefaciens, strain BE191105 of Bacillus subtilis and strain 3632 of Lactobacillus reuteri are an embodiment. Compositions comprising the strain BE191024 of Bacillus amyloliquefaciens, the strain BE191006 of Bacillus amyloliquefaciens, the strain BE191105 of Bacillus subtilis and the strain 3632 of Lactobacillus reuteri are one embodiment.

[0034] In one embodiment, the composition comprises at least two strains of Bacillus amyloliquefaciens, one strain of Bacillus subtilis and one strain of Lactobacillus s. Petition 870250085529, dated 09 / 22 / 2025, p. 21 / 282 17 / 73 In one embodiment, the composition comprises at least two strains of Bacillus amyloliquefaciens, one strain of Bacillus subtilis, and one strain of Lactobacillus reuteri. In another embodiment, the isolated strain of Lactobacillus reuteri corresponds to strain 3632 of Lactobacillus reuteri, which corresponds to ATCC patent application number PTA126788, or to a strain of Lactobacillus reuteri with at least 90% identity, 95% identity, 97% identity, 98% identity, and 99% genomic sequence identity with the sequence of strain 3632 of Lactobacillus reuteri corresponding to ATCC application PTA-126788.In one embodiment, the isolated Lactobacillus reuteri strain has a nucleic acid genomic sequence including at least one of the SEQ ID NOs: 51-57, sequences having one or more nucleic acid sequence differences from the sequence of at least one of the SEQ ID NOs: 51-57, sequences having at least one nucleic acid sequence difference from the sequence of at least one of the SEQ ID NOs: 51-57 and also having at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with one or more SEQ ID NOs: 51-57.

[0035] In one embodiment, the composition includes a carrier. In one embodiment of the composition, the carrier is selected from food-grade edible material, mineral mix, gelatin, cellulose, carbohydrate, starch, glycerin, water, rice husk, glycol, molasses, calcium carbonate, whey, sucrose, dextrose, soybean oil, vegetable oil, sesame oil, and corn oil.

[0036] In one embodiment, the composition includes one or more antimethanogenic compounds. In one embodiment, the composition is administered or supplied in combination with one or more antimethanogenic compounds. A compound is determined to be antimethanogenic based on its mechanism. In one embodiment, the composition includes one or more compounds capable of reducing or inhibiting the production, release, or stability of methane (CH4). In one embodiment, the composition is administered or supplied in combination with one or more compounds capable of reducing or inhibiting the production, release, or stability of methane (CH4). Exemplary antimethanogenic compounds or methane inhibitors include inhibitors of a rate-limiting enzyme in the methanogenesis pathway, such as the enzyme methyl coenzyme. Petition 870250085529, dated 09 / 22 / 2025, page 22 / 282 18 / 73 M reductase. Examples of such inhibitors include 3-NOP, bromoethanesulfonate, bromoform, red algae / Asparagopsis taxiformis. Other exemplary antimethanogenic compounds or methane inhibitors include inhibitors of methanogen lipid biosynthesis, such as the inhibitor mevinolin. Other antimethanogenic compounds or methane inhibitors include microbiome modulators, such as monensin. Thus, compositions of one or more strains provided herein with one or more antimethanogenic compounds selected from inhibitors of a rate-limiting enzyme in the methanogenesis pathway, inhibitors of methanogen lipid biosynthesis, or microbiome modulators are contemplated and provided herein.

[0037] In one embodiment, the composition does not comprise Lactobacillus. In one embodiment, the composition does not comprise strains not belonging to Bacillus. In one embodiment, Bacillus amyloliquefaciens and / or Bacillus subtilis are the only bacterial strains in the composition. In one embodiment, Lactobacillus reuteri and a strain of Bacillus are the only bacterial strains in the composition.

[0038] The nucleic acid sequence of the genome of the Bacillus amyloliquefaciens strain BE191006 (also known as BAMY 19006 or BAMY 006) is provided in sequence SEQ ID NO: 27. The nucleic acid sequence of the genome of the Bacillus amyloliquefaciens strain BE202071 (also known as ELA202071, BAMY 202071 or BAMY 071) is provided in sequences SEQ ID NO: 42. The nucleic acid sequence of the genome of the Bacillus subtilis strain BE191105 (also known as ELA1901105 and BSUB 19105 and BSUB 105) is provided in sequences SEQ ID NOs: 1, 2, 3, 4 and / or 5. Genomically related or variant strains of Bacillus amyloliquefaciens with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity with the genome sequence of SEQ ID NO: 27 or SEQ ID NO: 42 are provided and contemplated as embodiments of the invention.Genomically related strains of Bacillus subtilis or variants with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity with the genome sequence of SEQ ID NO: 1, 2, 3, 4 and / or 5 are provided and contemplated as embodiments of the invention. Petition 870250085529, dated 09 / 22 / 2025, page 23 / 282 19 / 73

[0039] In one embodiment, the isolated Bacillus strain has a genomic nucleic acid sequence including at least one of the SEQ ID NOs: 1, 2, 3, 4 or 5, sequences having one or more nucleic acid sequence differences from the sequence of at least one of the SEQ ID NOs: 1, 2, 3, 4 or 5, sequences having at least one nucleic acid sequence difference from the sequence of at least one of the SEQ ID NOs: 1, 2, 3, 4 or 5 and yet having at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with one or more SEQ ID NOs: 1, 2, 3, 4 or 5.In one embodiment, the isolated Bacillus strain has a genomic nucleic acid sequence including at least one of the SEQ ID NO: 27 sequences, having one or more nucleic acid sequence differences from the sequence of at least one of the SEQ ID NO: 27 sequences, having at least one nucleic acid sequence difference from the sequence of at least one of the SEQ ID NO: 27 sequences, and having at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with one or more SEQ ID NO: 27 sequences.In one embodiment, the isolated Bacillus strain has a genomic nucleic acid sequence including at least one of the SEQ ID NO: 42 sequences, having one or more nucleic acid sequence differences from the sequence of at least one of the SEQ ID NO: 42 sequences, having at least one nucleic acid sequence difference from the sequence of at least one of the SEQ ID NO: 42 sequences, and having at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with one or more SEQ ID NO: 42 sequences.

[0040] Such genomically related Bacillus strains or variants are capable of reducing methane production in an animal and / or reducing or inhibiting colonization or the microbiome in an animal, including Archaea and / or methanogenic bacteria. Such genomically related Bacillus strains or variants are comparatively capable of improving animal health and animal production performance. Such genomically related Bacillus strains or variants are capable of use and application in compositions according to the invention. In one embodiment, such genomically related sequences include nucleic acid encoding one or more proteins provided herein as genes or unusual proteins of the respective strains. For example and illustratively, such proteins include SEQ ID NOs: 28-41 for the BAMY 006 strain, Petition 870250085529, dated 09 / 22 / 2025, page 24 / 282 20 / 73 include proteins SEQ ID NOs: 43-50 for the BAMY 071 strain and include proteins SEQ ID NOs: 6-26 for the BSUB 105 strain.

[0041] In some embodiments, a food additive of probiotic composition or microbiome modulator is provided. In one embodiment, the food additive comprises a combination of spore forms of at least two of the Bacillus strains presented herein. In one embodiment, the food additive comprises the spore forms of one or more Bacillus strains presented herein and also includes the Lactobacillus strain presented herein. In one embodiment, the food additive comprises one or more Bacillus strains presented herein and also includes the Lactobacillus strain presented herein.

[0042] In some embodiments, particularly when more than one Bacillus and / or Lactobacillus is included in the composition, the strain ratio is approximately 0.75-1.5:1. In some embodiments, the ratio between a first isolated strain of Bacillus amyloliquefaciens, a second isolated strain of Bacillus amyloliquefaciens and / or the isolated strain of Bacillus subtilis is approximately 0.75-1.5:1:0.75-1.5. In some embodiments, the ratio between a first and second isolated strain of Bacillus and the isolated strain of Bacillus subtilis is approximately 0.75-1.5:1:0.75-1.5. In some embodiments, the ratio between a first and second isolated strain of Bacillus and the isolated strain of Lactobacillus reuteri is approximately 0.75-1.5:1:0.75-1.5. In some embodiments, the ratio between a first strain of Bacillus and the isolated strain of Lactobacillus is approximately 0.75-1.5:1.In some embodiments, the ratio between a first strain of Bacillus and the isolated strain of Lactobacillus reuteri is approximately 0.751.5:1. In a preferred embodiment, the composition contains equal amounts of the strains disclosed herein and above. The amount or ratio can be determined or characterized by any known method. For example, the ratio or amount can be characterized by the number of viable spores per gram of dry weight of the probiotic composition.

[0043] In some embodiments, the bacterial strains of the present disclosure capable of reducing methane production in an animal and / or reducing or inhibiting the colonization of an animal by a methanogenic bacterium include those that Petition 870250085529, dated 09 / 22 / 2025, p. 25 / 282 21 / 73 comprise polypeptide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with at least one of the following: SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and 41. In a further embodiment, the bacterial strains of the present disclosure are capable of reducing methane production in an animal and / or reducing or inhibiting colonization in an animal. Sequences associated with methanogenic bacteria include those comprising polypeptide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with at least one of the following SEQ ID Nos: 43, 44, 45, 46, 47, 48, 49, and 50.In a further embodiment, the bacterial strains of the present disclosure capable of reducing methane production in an animal and / or reducing or inhibiting the colonization of an animal by a methanogenic bacterium include those comprising polynucleotide sequences encoding a polypeptide sequence that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one of the following: SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.

[0044] According to one embodiment of the invention, a probiotic composition or microbiome modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome of Archaea and / or methanogenic bacteria, comprising a strain of Lactobacillus. In one embodiment, a probiotic composition or microbiome modulator is provided that is capable of reducing methane gas production and / or inhibiting or altering the microbiome of Archaea and / or methanogenic bacteria, comprising a strain of Lactobacillus reuteri.

[0045] According to one embodiment of the invention, a probiotic or microbiome modulator composition capable of reducing methane gas production is provided, comprising at least one isolated strain of Lactobacillus; and a vehicle suitable for animal administration; wherein said composition reduces methane production. Petition 870250085529, dated 09 / 22 / 2025, p. 26 / 282 22 / 73 and / or reduces or inhibits colonization or alters the microbiome in an animal, including methanogenic Archaea, when an effective amount is administered to an animal, compared to an animal to which the composition was not administered. In one embodiment, the probiotic or microbiome modulator composition comprises at least one isolated strain of Lactobacillus reuteri; and a vehicle suitable for animal administration; wherein said composition reduces methane production and / or reduces or inhibits colonization or alters the microbiome in an animal, including methanogenic Archaea and / or bacteria, when an effective amount is administered to an animal, compared to an animal to which the composition was not administered.In one embodiment, the isolated Lactobacillus reuteri strain corresponds to Lactobacillus reuteri strain 3632, which corresponds to ATCC patent application number PTA126788, or a Lactobacillus reuteri strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity to the sequence of Lactobacillus reuteri strain 3632 corresponding to ATCC application PTA-126788.

[0046] In one embodiment, the isolated Lactobacillus reuteri strain has a nucleic acid genomic sequence including at least one of the SEQ ID NOs: 51-57, sequences having one or more nucleic acid sequence differences from the sequence of at least one of the SEQ ID NOs: 51-57, sequences having at least one nucleic acid sequence difference from the sequence of at least one of the SEQ ID NOs: 51-57 and yet having at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with one or more SEQ ID NOs: 51-57.

[0047] In one embodiment, the isolated Lactobacillus reuteri strain is Lactobacillus reuteri strain 3632, which corresponds to ATCC patent application number PTA126788. In another embodiment, the isolated Lactobacillus reuteri strain comprises or possesses a genomic nucleic acid sequence corresponding to the genomic nucleic acid sequence of ATCC strain PTA-126788, or a variant thereof, comprising or possessing a nucleic acid sequence with at least 98%, by Petition 870250085529, dated 09 / 22 / 2025, p. 27 / 282 23 / 73 minus 98.5%, at least 99%, or at least 99.5% sequence identity with the genomic nucleic acid sequence of the ATCC strain PTA-126788.

[0048] In one embodiment, a method is provided for reducing the emission or production of methane by methanogenic organisms, such as Archaea or methanogenic bacteria, in the environment, in a culture or in a process comprising the introduction or contact of the composition described herein or of a fermentation product of the composition. In one embodiment, the environment, culture or process is selected from among manure, leaf litter, lagoons, an aerobic digester and waste treatment.

[0049] In some embodiments, methods are provided for reducing methane emission or production, in the case of methanogenic organisms, in manure, by combining or adding to the manure one or more compositions of this document or fermentation product of one or more compositions of this document.

[0050] In another embodiment, a postbiotic comprising the fermentation product, culture, or supernatant of the composition of the strains described herein is provided. In one embodiment, the postbiotic reduces methane production, alters methanogenesis, and / or reduces greenhouse gas emissions. In one embodiment, the postbiotic is administered to an animal.

[0051] Although what are currently believed to be the preferred embodiments of the present invention have been described, those skilled in the art will realize that further alterations and modifications can be made without departing from the spirit of the invention, and it is intended to claim all such modifications and alterations that are within the true scope of the invention.

[0052] Other objectives and advantages will become apparent to those skilled in the art from a review of the detailed description that follows, which proceeds with reference to the following illustrative drawings and corresponding claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 represents methane emission by enteric fermentation.

[0054] FIG. 2 represents the enteric emission of methane. Petition 870250085529, dated 09 / 22 / 2025, page 28 / 282 24 / 73

[0055] FIG. 3A and B. (A) depicts methane production with an H2 consumption and H2 production pathway being shown. (B) depicts alternative H2 sinks, showing how the H2 flow can be redirected to an alternative electron acceptor (such as SO42- and NO3-) or pathway to reduce methane.

[0056] FIG. 4 represents the methanogenesis pathway (Wolwe cycle).

[0057] FIG. 5 describes the experimental design of the batch culture of the rumen and methane evaluation.

[0058] FIG. 6 illustrates a methane standard curve representing the amount of methane gas injected into the gas chromatography (GC) system.

[0059] FIG. 7 describes the results with a series of controls, the negative control being CaCO3 and the positive control CHCl3. The change in pressure (ΔP) in psig and the change in methane concentration (Concentration) in mM are plotted graphically over time in hours (h).

[0060] FIG. 8 shows results with a series of Bacillus bacteria.

[0061] FIG. 9 shows results with a series of Lactobacillus bacteria.

[0062] FIG. 10 represents a comparison result of Bacillus strains and Lactobacillus versus control.

[0063] FIG. 11 illustrates methane production (CH4 production, %) for various additives and bacterial strains tested. Methane production is defined as the increase (+ value) or decrease (- value) in methane production of a typical treatment compared to a control blank (None).

[0064] Figures 12A and 12B present the results of the evaluation of methane production in batch culture in the rumen. The results are represented in alternative graphic formats. In (A), methane production with the addition of each of the following: None (nothing added), CHCl3, vehicle, strain BE105 of B. subtilis bacteria, strain BE006 of B. amyloliquefaciens, strain BE071 of B. amyloliquefaciens, strain BE3632 of L. reuteri; another strain, designated strain A, is shown on a scale of 0 to 100. In (B), methane production with the addition of each of the following: None (nothing added), CHCl3, vehicle, strain BE105 of B. subtilis bacteria, strain BE006 of B. amyloliquefaciens, strain BE071 of B. amyloliquefaciens, strain BE3632 of L. reuteri, and Petition 870250085529, dated 09 / 22 / 2025, page 29 / 282 25 / 73 Another strain A is shown on a scale of 0 to 125. The BE006 strain of Bacillus amyloliquefaciens is observed to reduce methane production by at least 25%.

[0065] FIG. 13 illustrates the design of an in vivo study in animals to better evaluate the effect of administering bacteria capable of reducing methane production on livestock performance.

[0066] FIG. 14 illustrates the effect of adding L. reuteri BE3632 on H2, CO2, and CH4 in batch rumen culture in vitro. The left panel shows H2, the center panel shows CO2, and the right panel shows CH4. Samples of untreated medium, treated with CHCl3, medium only, and L. reuteri BE3632 bacteria are provided. Evaluations were conducted 0, 24, and 48 hours after treatment or addition.

[0067] FIG. 15 presents a diagram of the process steps for evaluating colonization by L. reuteri BE3632 in batch rumen culture in vitro. Starting from an original inoculum, which was sampled and plated as Plate 1, an inoculum was transferred serially, from the 1st transfer to the 3rd transfer, with one inoculum from each plate, as Plate 2 to Plate 4, with each transfer occurring after 2 days. The bacteria present on Plates 1 and 4 were counted.

[0068] FIG. 16 shows a sample of bacterial plate assessments from the study described in Figure 15. Bacterial colonies of L. reuteri BE3632 are evident (orange colonies).

[0069] FIG. 17 illustrates an overlapping experiment to examine strain compatibility. The evaluation of the BE105 strain of B. subtilis and the BE3632 strain of L. reuteri is illustrated. The steps and process for the other strains would be the same. DETAILED DESCRIPTION OF THE INVENTION

[0070] This disclosure provides a solution to the problem of greenhouse gas emissions, including the problem of methane emissions from various sources or under various conditions. This disclosure provides a solution to the problem of greenhouse gas emissions, including the problem of methane emissions from animals, such as ruminants, including cattle, thereby improving the sustainability of livestock production and rumen feed efficiency. Specifically, this Petition 870250085529, dated 09 / 22 / 2025, page 30 / 282 Disclosure 26 / 73 foresees the development of a feed additive to reduce enteric methane gas emissions from animals, including or particularly ruminants, including cattle, to reduce the carbon footprint of livestock production, to enable manure management and to ensure ruminal feed efficiency.

[0071] The sustainability of ruminant production in livestock farming and the improvement of feed efficiency are therefore advantages of the present invention. In particular, methane reduction leads to increased feed efficiency, which leads to sustainable livestock production. A direct effect on the reduction of enteric methane emissions is considered. A direct effect on the reduction of enteric methane emissions is considered where methane emissions or production are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% and at least 50%. A direct effect on the reduction of enteric methane emissions is considered where methane emissions or production are reduced by at least 10%. A direct effect on the reduction of enteric methane emissions is considered where methane emissions or production are reduced by at least 20%.A direct effect on reducing enteric methane emissions is considered to be one where methane emissions or production are reduced by at least 25%.

[0072] This disclosure provides a mitigation strategy for enteric methane production. Specifically, this disclosure envisions the use of probiotics or microbiome modulators to mitigate enteric methane production in animals, such as ruminants and, more particularly, cattle. Furthermore, this disclosure envisions the use of probiotics or microbiome modulators to mitigate methane production by methanogenic Archaea or in situations or locations where methanogenic Archaea is present, including in animals, in manure, at the bottom or in the depths of ponds, in anaerobic digesters, etc. Probiotics can have an effect on methanogenesis through modulation of the ruminal microbiota.Without wanting to limit ourselves to any specific theory, by modulating the structure of the microbiome, probiotics or microbiome modulators decrease the bacteria that produce H2 available for methanogenesis, thus decreasing the amount of methanogenesis with the effect of reducing methane emissions. Probiotics or microbiome modulators amplify or... Petition 870250085529, dated 09 / 22 / 2025, page 31 / 282 27 / 73 increase the H2 consumption pathway and reduce the H2 production pathway, thus reducing the H2 available to generate methane CH4 (see, for example, Figure 3A). The production of volatile fatty acids (VFA) with the fermentation process can therefore be increased as opposed to methanogenesis (see, for example, Figure 2).

[0073] This disclosure provides probiotics or microbiome modulators that alter or target methanogens in an animal's gastrointestinal tract. In particular, the probiotics in this disclosure alter the rumen methanogen fermentation process and, as such, are rumen modifiers. In particular, the probiotics in this disclosure alter the rumen methanogenic Archaea fermentation process and, as such, are rumen modifiers. In particular, the probiotics or microbiome modulators in this disclosure alter the microbiome and, therefore, the fermentation process and the production of methanogen and CH4. Methanogens that may be targeted by the probiotics in this disclosure include, without limitation, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanomassilicoccacea spp., Methanobacterium bryantii, and Methanosarcina mazei.

[0074] Methanogens are a group of microorganisms that produce methane as a byproduct of their metabolism. They play an important role in the digestive system of ruminants. The ruminant stomach contains four main parts: rumen, reticulum, omasum, and abomasum. Food mixed with saliva first passes into the rumen to be broken down into smaller particles and then moves to the reticulum, where the food is further broken down into even smaller particles. Any indigestible particles are sent back to the rumen to be chewed again. Most anaerobic microbes that aid in the breakdown of cellulose occupy the rumen and initiate the fermentation process. The animal absorbs the fatty acids, vitamins, and nutrient content as the partially digested food passes from the rumen to the omasum. This lowers the pH level and initiates the release of enzymes for further breakdown of the food, which later passes to the abomasum to absorb the remaining nutrients before excretion.This process takes about 9 to 12 hours. Petition 870250085529, dated 09 / 22 / 2025, page 32 / 282 28 / 73

[0075] Some of the microbes in the digestive system of ruminants are: Fibrobacter (Bacteroides) succinogenes is a gram-negative, cellulolytic and amylolytic methanogen that produces formates, acetates and succinates; Ruminococcus albus is a cellulolytic and xylanolytic bacterium that produces ethanol, hydrogen, carbon dioxide, formates and acetates; Ruminococcus flavefaciens is a cellulolytic and xylanolytic bacterium that produces formates, acetates, hydrogen and succinates; Butyrivibrio fibrisolvens is a proteolytic, cellulolytic and xylanolytic microbe that produces lactate, butyrate, ethanol, hydrogen, carbon dioxide, formates and acetates; Streptococcus bovis is an amylolytic microbe, a major fermenter of soluble sugar, proteolytic, resulting in lactate, acetate and formate; Ruminobacter (Bacteroides) amylophilus amylolytic, propionate, proteolytic, organism forming, formates, acetates and succinates;Prevotella (Bacteroides) ruminocola amylolytic, xylanolytic, propionate, proteolytic, microbe that creates formates, acetates, succinates and propionate; Succinimonas amylolytica amylolytic, dextrinolytic, acetate and succinate forming bacteria; Selenomonas ruminantium amylolytic, main soluble sugar fermenter, glycerol user, lactate user, proteolytic, microbe that produces acetates, lactates, hydrogen, carbon dioxide and propionates; Lachnospira propionato multiparus, proteolytic, microbe that results in the production of lactate, ethanol, hydrogen, carbon dioxide, formates and acetates; Succinivibrio propionato dextrinosolvens, dextrinolytic, formate, acetate, lactate and succinate forming bacteria; Methanobrevibacter ruminantium, a methanogenic, hydrogen-using Archaea involved in methane production; Methanosarcina barkeri, a methanogenic, hydrogen-using Archaea involved in methane production.

[0076] Ruminants (suborder Ruminantia) are herbivorous ungulate mammals that graze, capable of acquiring nutrients from plant foods by fermenting them in a specialized stomach prior to digestion, primarily through microbial action. The process, which occurs in the front part of the digestive system and is therefore called foregut fermentation, typically requires the fermented ingesta (known as rumination) to be regurgitated and chewed again. The process of re-chewing rumination further breaks down the plant matter. Petition 870250085529, dated 09 / 22 / 2025, page 33 / 282 29 / 73 and stimulating digestion is called rumination. The approximately 200 species of ruminants include domestic and wild species. Ruminant mammals include livestock, all domesticated and wild bovines such as cattle, bison, American buffalo and water buffalo, goats, sheep, giraffes, deer, gazelles, and antelopes. Ruminants play an important role in global food security and nutrition, as well as in the livelihoods of farmers and those along the agri-food chain. Ruminant animals include cattle, sheep, goats, and buffalo and constitute the largest livestock production system in the world. More than 50% of all protein provided by livestock comes from ruminants, mainly in the form of milk and meat. Globally, enteric methane emissions from ruminants and manure management practices account for more than 30% of all human-induced methane emissions.Cattle are responsible for 77% of methane emissions, buffalo for 14%, and small ruminants, such as sheep and goats, for the remainder.

[0077] Methane emissions from non-ruminant animals are also relevant and contribute to greenhouse gases (GHG). In some countries, the contribution of non-ruminant emissions is significant. For example, in East and Southeast Asia, pigs and poultry are responsible for a large part of enteric methane. Methanogens are also associated with some clinically relevant conditions in animals, including humans. Methanogens and methane production in humans are positively correlated with obesity, higher glucose levels, and higher body mass index (Mathur R et al (2013) Obesity 21(4):748-754; Mathur R et al (2013) Clin Endocrinol Metab 98(4):E698-702; Mathur R et al (2014) Res J or Endocrinol and Metab doi:10.7243 / 2053-3640-2-2).

[0078] Vertebrates lack the ability to hydrolyze the beta [1-4] glycosidic linkage of plant cellulose due to the absence of the enzyme cellulase. Thus, ruminants depend entirely on the microbial flora present in the rumen or hind intestine to digest cellulose. Food digestion in the rumen is primarily carried out by the ruminal microflora, which contains dense populations of various species of bacteria, protozoa, sometimes yeasts and other fungi – it is estimated that 1 ml of rumen Petition 870250085529, dated 09 / 22 / 2025, p. 34 / 282 30 / 73 contains 10 to 50 billion bacteria and 1 million protozoa, in addition to various yeasts and fungi.

[0079] Because the environment inside the rumen is anaerobic, most of these microbial species are obligate or facultative anaerobes, capable of decomposing complex plant materials such as cellulose, hemicellulose, starch, and proteins. The hydrolysis of cellulose results in sugars, which are subsequently fermented into acetate, lactate, propionate, butyrate, carbon dioxide, and methane.

[0080] The compositions and methods provided herein have application in non-ruminant animals, including humans. Consumption of human food leads to flatulence and the release of methane, which contributes to greenhouse gases. There are some intestinal conditions in humans associated with increased methane production. Small intestinal bacterial overgrowth (SIBO) is a condition associated with a large number of bacteria colonizing the small intestine (Rezaie A, Pimentel M, Rao SS. Curr Gastroenterol Rep. 2016;18(2):8; doi.org / 10.1007 / s11894-015-0482-9). Intestinal methanogen overgrowth (IMO) is a more recent term (possibly replacing methane-dominant SIBO) to characterize an overgrowth of Archaea throughout the intestinal tract (Pimentel M, Saad RJ, Long MD, Rao SSC. Am J Gastroenterol. 2020;115(2):165-178; (doi.org / 10.14309 / ajg.0000000000000501). IMO is an overgrowth of the Archaea group of microorganisms. Intestinal methanogen overgrowth confirmed by lactulose breath test. The standard allopathic treatment for IMO is a regimen of antibiotics to clear the system. The standard antibiotics used are rifaximin and neomycin and / or metronidazole as treatments of choice. Methanobrevibacter smithii is the predominant methanogen, and evidence suggests a strong association between the presence of methane gas and irritable bowel syndrome (IBS-C) with constipation predominance (Kim G, Deepinder F, Morales W, et al.. Dig Dis Sci. 2012;57(12):32133218; doi:10.1007 / s10620-012-2197-1). The prevalence of SIBO is higher in people with IBS when compared to healthy controls (Chen B, Kim JJ, Zhang Y, Du L, Dai N. J Gastroenterol. 2018;53(7):807-818). Petition 870250085529, dated 09 / 22 / 2025, page 35 / 282 31 / 73

[0081] Methane production in humans has been associated with obesity. A thorough examination of the gastrointestinal microbiome has demonstrated clear changes in the microbial community during obesity and, primarily, that these changes can lead to metabolic alterations (Turnbaugh PJ et al. (2009) Nature 457: 480-484; Ridaura VK et al. (2013) Science 341:124-1214). Recently, methanogenic archaea (methanogens) have been identified as a potential regulator of metabolic function. These microorganisms metabolize a variety of electron sources, including dihydrogen gas and small fatty acids in the intestinal environment to produce methane (reviewed in Pimentel M et al (2012) Am J Gastroenterol Suppl 1:28-33). Although not all humans have detectable levels of methane in their breath, studies have shown that methane production is altered in disease states.Indeed, it appears that fecal methanogen levels increase during human obesity, and the interruption of methanogens through antibiotic treatment of their bacterial syntrophs also corresponds to a reduction in insulin secretion (Mathur R et al (2016) Obesity 24:576-582). Other studies have demonstrated that intestinal methane production in obese individuals is associated with a higher body mass index, and that a higher concentration of methane detected by breathalyzer testing is a significantly greater predictor of obesity in overweight individuals (Basseri RJ et al (2012) Gastroenterology & Hepatology 8(1):22-28).

[0082] The compositions provided herein are applicable for use and in methods for relieving methane production in humans with intestinal disorders, obesity, and high body mass index (BMI). In one embodiment, the intestinal disorders are selected from SIBO, IBO, IBS, and IBS-C.

[0083] The compositions and methods described herein may be supplied as feed additives. Probiotics advantageously provide a benefit in feed efficiency.

[0084] The strains for use as probiotics or microbiome modulators in the compositions and methods described herein include Bacillus and Lactobacillus spp., which are known to alter the microbial composition or microbiome in an animal and also produce antimicrobial peptides (broad activities) and metabolites. Petition 870250085529, dated 09 / 22 / 2025, page 36 / 282 32 / 73 secondary factors that may lead to antimethanogenic activity. The antimethanogenic activities of Bacillus strains (B. amyloliquefaciens BE191006, BE202071 and B. subtilis BE191105 and Lactobacillus reuteri 3632) have been demonstrated. Without wanting to limit oneself to any specific theory, the mechanism by which probiotics or microbiome modulators act varies depending on the probiotic strain.

[0085] This disclosure provides compositions comprising Bacillus and Lactobacillus spp. to reduce methane emissions in a ruminant. Specifically, this disclosure provides compositions comprising Bacillus strains such as B. amyloliquefaciens BE191006, BE202071 and B. subtilis BE191105, as well as compositions including Lactobacillus reuteri 3632 and combinations thereof, to reduce methane emissions in an animal. This disclosure provides compositions comprising Bacillus strains such as B. amyloliquefaciens BE191006, BE202071 and B. subtilis BE191105, as well as compositions including Lactobacillus reuteri 3632 and combinations thereof, to reduce methane emissions in a ruminant animal. The present invention provides compositions comprising Bacillus strains, such as B. amyloliquefaciens BE191006, BE202071 and B. subtilis BE 191105, as well as compositions including Lactobacillus reuteri 3632 and combinations thereof, to reduce methane production in an animal.This disclosure provides compositions comprising Bacillus strains, such as B. amyloliquefaciens BE191006, BE202071 and B. subtilis BE191105, as well as compositions including Lactobacillus reuteri 3632 and combinations thereof, to reduce methane emissions in situations and locations where Archaea or methanogenic bacteria are present or located.

[0086] Bacillus Strains - Exemplary and suitable Bacillus strains may be selected from one or more strains of Bacillus amyloliquefaciens and / or one or more strains of Bacillus subtilis. In particular, exemplary strains of Bacillus amyloliquefaciens are provided in Strain 06 (also designated BE191006) and Strain 071 (also designated BE202071). In particular, an exemplary strain of Bacillus subtilis is provided in Strain 105 (also designated BE191105). The strains are described below by reference in aspects. Details about these strains, including some of their Petition 870250085529, dated 09 / 22 / 2025, page 37 / 282 33 / 73 probiotic characteristics, their metabolites, including when grown independently or in combinations, their unique genes, antibacterial peptides or enzymes, encoded proteins, and their genome nucleic acid sequence are provided and disclosed in PCT / US22 / 044211, based on U.S. Patent Application No. 63 / 083,697 filed September 25, 2020, and U.S. Patent Application No. 63 / 241,369 filed September 8, 2021, the disclosures of which are incorporated herein by reference in their entirety.

[0087] Strain 06: Bacillus amyloliquefaciens strain BE191006 (also known as ELA191006) corresponding to ATCC deposit PTA-127065. BE191006 corresponding to ATCC deposit PTA-127065 or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity to the BE191006 sequence corresponding to ATCC deposit PTA-127065. The nucleic acid genome sequence of strain BE191006 (Strain 06) is provided in SEQ ID NO: 27.

[0088] Strain 071: Bacillus amyloliquefaciens strain BE202071 (also referred to as ELA202071) corresponding to ATCC deposit PTA-127064. BE202071 corresponding to ATCC deposit PTA-127064 or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity to the BE202071 sequence corresponding to ATCC deposit PTA-127064. The nucleic acid genome sequence of strain BE202071 (strain 071) is provided in SEQ ID NO: 42.

[0089] Strain 105: Bacillus subtilis strain BE191105 (also called ELA191105) corresponding to ATCC deposit PTA-126786. BE191105 corresponding to ATCC deposit PTA-126786 or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the BE191105 sequence corresponding to ATCC deposit PTA-126786. The nucleic acid genome sequence of strain BE191105 (strain 105) is provided in SEQ ID Nos: 1, 2, 3, 4, and / or 5.

[0090] In one embodiment, the isolated strain of Bacillus amyloliquefaciens comprises strain BE191006, filed with the ATCC under patent filing number PTA. Petition 870250085529, dated 09 / 22 / 2025, p. 38 / 282 34 / 73 127065. In one embodiment, the isolated strain of Bacillus amyloliquefaciens comprises strain BE202071, filed with the ATCC under patent application number PTA127064. In another embodiment, the isolated strain of Bacillus subtilis comprises strain BE191105, filed with the ATCC under patent application number PTA-126786.

[0091] The strain BE191006 of Bacillus amyloliquefaciens (designated ELA191006) was deposited on May 11, 2021, in accordance with the Budapest Treaty, in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit was given ATCC Patent Filing Number PTA-127065.

[0092] The Bacillus amyloliquefaciens strain BE202071 (designated ELA202071) was deposited on May 11, 2021, in accordance with the Budapest Treaty, in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit was given ATCC Patent Filing Number PTA-127064.

[0093] The Bacillus subtilis strain BE191105 (designated ELA191105) was deposited on June 19, 2020, in accordance with the Budapest Treaty, in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit was given ATCC Patent Filing Number PTA-126786.

[0094] Lactobacillus - Exemplary and suitable strains of Lactobacillus may be selected from one or more strains of Lactobacillus reuteri. In particular, an exemplary strain of Lactobacillus reuteri is provided in Strain 3632. The strain is described below by reference in its aspects. Details about this strain, including some of its probiotic characteristics, metabolites, unique genes, antibacterial peptides or enzymes, encoded proteins and genome nucleic acid sequence, are provided and disclosed in document WO 2020 / 163398, published on August 13, 2020, which is incorporated herein by reference in its entirety.

[0095] Strain 3632: Patent Application Number ATCC PTA-126788. Isolated strain of Lactobacillus PTA-126788 or a Lactobacillus strain with at least 99% amino acid or nucleic acid identity with strain PTA-126788. The sequence Petition 870250085529, dated 09 / 22 / 2025, p. 39 / 282 35 / 73 of the nucleic acids from the genome of strain 3632 and ATCC PTA 126788 are provided in SEQ ID Nos: 51-57.

[0096] In one embodiment, the composition, particularly a probiotic or microbiome-modulating composition, comprises at least one isolated strain 3632 of Lactobacillus (PTA-126788) or a strain of Lactobacillus with at least 99% amino acid or nucleic acid identity with strain 3632 (PTA-126788). In one embodiment, the composition, particularly a probiotic or microbiome-modulating composition, comprises an isolated strain 3632 of Lactobacillus (PTA-126788) or a strain of Lactobacillus with at least 99% amino acid or nucleic acid identity with strain 3632 (PTA-126788) and at least one strain of Bacillus. In one embodiment, the composition, particularly a probiotic or microbiome-modulating composition, comprises either the isolated Lactobacillus strain 3632 (PTA-126788) or a Lactobacillus strain with at least 99% amino acid or nucleic acid identity to strain 3632 (PTA-126788) and a Bacillus strain.In one embodiment, the composition, particularly a probiotic or microbiome-modulating composition, comprises Lactobacillus isolate strain 3632 (PTA-126788) or a Lactobacillus strain with at least 99% amino acid or nucleic acid identity to strain 3632 (PTA-126788) and at least two Bacillus strains. In another embodiment, the composition, particularly a probiotic or microbiome-modulating composition, comprises Lactobacillus isolate strain 3632 (PTA-126788) or a Lactobacillus strain with at least 99% amino acid or nucleic acid identity to strain 3632 (PTA-126788) and a Bacillus strain selected from strain 06 (PTA-127065), strain 071 (PTA-127064), and strain 105 (PTA-126786).

[0097] In one embodiment of the invention, a probiotic composition, a microbiome modulator, or a direct-feeding microorganism comprising at least one strain of Bacillus is provided. In another embodiment of the invention, a probiotic composition or a direct-feeding microorganism comprising a combination of at least two strains of Bacillus is provided. In embodiments, the Bacillus strain(s) is / are selected from Strain 06 (BE191006) or a strain of Bacillus Petition 870250085529, dated 09 / 22 / 2025, page 40 / 282 36 / 73 with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with the sequence of Strain 06 (BE191006); Strain 71 (BE202071) or a Bacillus strain with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with the sequence of Strain 71 (BE202071); and Strain 105 (BE191105) or a Bacillus strain with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with the sequence of Strain 105 (BE191105).In some embodiments, the Bacillus strain(s) are selected from either Strain 06 (BE191006) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with sequence SEQ ID NO:27; or Strain 71 (BE202071) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with sequence SEQ ID NO:42. and Strain 105 (BE191105) or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with one or more SEQ ID NO: 1, 2, 3, 4, and / or 5.

[0098] In one embodiment of the invention, a probiotic composition, a microbiome modulator, or a direct-feeding microorganism comprising at least one Lactobacillus strain is provided. In one embodiment of the invention, a probiotic composition or a direct-feeding microorganism comprising at least one Lactobacillus reuteri strain is provided. In one embodiment of the invention, a probiotic composition or a direct-feeding microorganism comprising a combination of two Lactobacillus strains, particularly a combination of two Lactobacillus reuteri strains, is provided. In embodiments, at least one Lactobacillus strain is selected from Strain 3632 (PTA-126788) or a Lactobacillus strain with at least 90%, 95%, 97%, 98%, and 99% genomic sequence identity to the sequence of Strain 3632 (PTA-126788); and from Strain 3630 (PTA-126787).In some forms, at least one strain of Lactobacillus is present. Petition 870250085529, dated 09 / 22 / 2025, p. 41 / 282 37 / 73 selected from Strain 3632 (PTA-126788) or a Lactobacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with one or more SEQ ID NO: 51, 52, 53, 54, 55, 56, and / or 57.

[0099] Direct-feeding microorganisms (DFMs) as delivery or production systems

[0100] Suitable strains such as DFMs can provide an attractive and useful starting point for applications in the production or generation of heterologous biomolecules and proteins, including as an in vivo delivery system for the synthesis and administration of molecules or proteins with broad applications, including in therapy and animal health. These direct-feeding strains have applicability as a delivery system that can consistently deliver therapeutic molecules and useful biomolecules, such as anti-infective molecules, directly to the host, such as in the gastrointestinal tract, where bacteria, including pathogenic or methanogenic bacteria, replicate in the host. The gastrointestinal system is also frequently a point of entry for bacteria or pathogens into the host.Ideally, the delivery system is a genetically modified living microorganism, such as a bacterium, that can reproduce in—and even colonize in some cases—a host and directly deliver therapeutic molecules and biomolecules, such as anti-infective, antipathogenic, antibacterial, or antimethanogenic or methanogen-reducing agents to reduce the number or block the entry of a pathogen, bacterium, or Archaea, or the amount of methane or other molecule(s) produced. These bacterial strains provide enhanced delivery platforms and systems, including suitable vectors and nucleic acid-based systems for rapid and effective expression of heterologous proteins or genes of interest and robust generation of multiple vehicles using a single platform.

[0101] Bacillus Subtilis - Strain 105

[0102] Bacillus subtilis is a Gram-positive model bacterium widely used for the industrial production of recombinant proteins, such as alpha-amylase, protease, lipase, and other industrial enzymes. Due to the bacterium's ability to produce large Petition 870250085529, dated 09 / 22 / 2025, page 42 / 282 38 / 73 quantities of a target protein and also to secrete large quantities of a target protein into the culture medium, and to the availability of a low-cost downstream production and purification process, more than 60% of commercial industrial enzymes are produced in Bacillus subtilis and related Bacillus species (Schallmey, M.; Singh, A.; Ward, OP (2004) 50 (1): 1-17). In contrast to the frequently used recombinant protein expression host, Escherichia coli, Bacillus subtilis does not present a risk of endotoxin contamination and has been certified as a GRAS (generally considered safe) organism by the FDA, making it a choice for food-grade and pharmaceutical-grade protein production.

[0103] B. subtilis strains, particularly strain 105, provide a Bacillus subtilis expression system that can be modified and engineered to produce high levels of at least one or more heterologous biomolecules or proteins, including, in some cases, molecules displayed on the surface or secreted. The Bacillus subtilis strain BE191105, also referred to as ELA191105 and strain 105, corresponds to the ATCC deposit PTA-126786. Strain 105 is described and detailed as a genetically modified strain for administration or production in living organisms in document PCT / US2022 / 044211, filed on September 21, 2022, which is based on documents USSN 63 / 247,271 (filed on September 11, 2021), 63 / 247,273 (filed on September 22, 2021) and 63 / 247,400 (filed on September 23, 2021) (incorporated herein by reference).These applications describe native bacterial promoters, suitable signaling sequences for expression and vectors, as well as sites / genes in the bacterial genome for integration, in order to generate stable modified strains, as well as modifications to strain 105 to improve expression.

[0104] Strain 105 of B. subtilis has been described as a microorganism with beneficial effects, including in combination with one or more strains of Bacillus amyloliquefaciens. In some aspects / embodiments, strain 105 of B. subtilis can be combined with one or more isolated strains of Bacillus amyloliquefaciens, particularly selected from BE191024 (also known as ELA191024) (corresponding to ATCC deposit PTA-126784), BE191036 (also known as ELA191036) (corresponding to ATCC deposit PTA-126785), BE191006 (also Petition 870250085529, dated 09 / 22 / 2025, page 43 / 282 39 / 73 designated ELA191006) (corresponding to ATCC deposit PTA-127065) and BE202071 (also designated ELA202071) (corresponding to ATCC deposit PTA-127064). These combinations of probiotic strains, compositions, and methods for them are described and provided in document PCT / US2021 / 051973, filed on September 24, 2021, published as WO2022 / 067052 on March 31, 2022 (incorporated herein by reference). Combinations of B. subtilis 105 and one or more strains of B. amyloliquefaciens have been described for the treatment of necrotic enteritis in poultry. Bacillus strains have shown efficacy in reducing mortality in poultry and improving performance, selected based on average daily feed intake (ADF), average daily gain (ADG), and feed conversion ratio (FCR) in poultry.Strain combinations are also effective in reducing post-weaning diarrhea in pigs and improving feed intake, pen weight and / or weight gain in pigs, as well as improving performance, selected from average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) in pigs, particularly in post-weaning pigs. No effects or activities related to Archaea and / or methanogenic bacteria, methanogenesis or methane production have been described.

[0105] Lactobacillus reuteri strains 3630 and 3632 are described and detailed as novel strains suitable as DFMs, including in combination, and also as strains suitable for genetic modification and as live delivery or production strains. Lactobacillus reuteri strain 3630 was filed on June 19, 2020 in the ATCC Patent Office and received ATCC Patent Filing Number PTA126787. L. reuteri strains 3630 and 3632 are described and detailed as probiotic strains in Probiotic Compositions Comprising Lactobacillus Reuteri Strains and Methods of Use PCT / US2020 / 016668, filed on 04 / 02 / 2020, published as WO 2020 / 163398 on August 13, 2020. The corresponding publications in the US are US 2022 / 0088094, published on March 24, 2022, and US 2022 / 0125860, published on April 28, 2022. An in vivo delivery system based on L. reuteri strains 3630 or 3632...Lactobacillus reuteri is described and detailed in A Genetically Modified Lactobacillus and Uses Thereof PCT / US2020 / 016522, filed on 04 / 02 / 2020, published as WO. Petition 870250085529, dated 09 / 22 / 2025, page 44 / 282 40 / 73 2020 / 163284, August 13, 2020. This application describes native bacterial promoters, suitable signaling sequences for expression, vectors, and bacterial genome sites / genes for integration in order to generate stable modified strains. These applications are incorporated herein by reference. No effects or activities related to Archaea and / or methanogenic bacteria, methanogenesis, or methane production have been described.

[0106] In some respects, the compositions described above are used to alter the microbiome or reduce the bacterial load, of microorganisms or Archaea, particularly pathogenic bacteria, methanogenic bacteria or organisms, or clinically significant bacteria, including the number, quantity or type (genus, species) of bacteria or Archaea in the intestine, gastrointestinal tract or rumen of an animal. The bacterium may be, in particular, a methanogenic bacterium or methanogenic Archaea selected, for example, from at least one of Methanobrevibacter ruminantium, Methanobacterium formicum and Methanobacterium mobile.

[0107] In some respects, the compositions described above are used to reduce the transmission of bacteria or to alter the flora or microbiome of microorganisms or bacteria, particularly pathogenic bacteria or methanogenic organisms or bacteria, in an animal pen or in a group or herd of animals. In some respects, the compositions described above are used to reduce or alter the transmission or to alter the flora or microbiome of microorganisms or bacteria in an animal pen or in a group or herd of animals of at least one methanogenic bacterium or methanogenic Archaea, as selected from, for example, Methanobrevibacter ruminantium, Methanobacterium formicum and Methanobacterium mobile.

[0108] In embodiments of the invention, an animal may include a farm animal or livestock or a domesticated animal. Livestock or farm animal may include cattle (e.g., cows or bulls (including calves)), poultry (including broilers, chickens and turkeys), pigs (including piglets), birds, aquatic animals such as fish, gastric fish, freshwater fish such as salmon, cod, trout and carp, for example, koi carp, marine fish such as sea bass, and crustaceans such as Petition 870250085529, dated 09 / 22 / 2025, p. 45 / 282 41 / 73 shrimp, mussels, and scallops), horses (including racehorses), sheep (including lambs). As used herein, the term ruminants includes, without limitation, extensive beef cattle, intensive beef cattle, and dairy cattle. Ruminants also include sheep, buffalo, goats, bison. An animal can be a cat or a kitten. An animal can be a human.

[0109] The compositions may also include one or more components or additives. One or more components or additives may be a component or additive to facilitate administration, for example, by means of a stabilizer or vehicle, or by means of an additive to allow administration to an animal, such as by any suitable means of administration, including in aerosol or spray form, in water, in feed or in injectable form. Administration to an animal may be made by any known or standard technique. These include oral ingestion, gastric intubation or broncho-nasal spraying. The compositions disclosed herein may be administered by immersion, intranasal, intramammary, topical, mucosal or inhalation.

[0110] In some embodiments, the composition does not include antibiotics. In some embodiments, the composition includes antibiotics. Examples of antibiotics include tetracycline, bacitracin, tylosin, salinomycin, virginiamycin, and bambermycin.

[0111] In some embodiments, the Bacillus strains of this disclosure are not genetically modified or genetically engineered and do not contain heterologous genetic sequences. In some embodiments, the Lactobacillus strains of this disclosure are not genetically engineered or genetically engineered and do not contain heterologous genetic sequences.

[0112] The compositions described above may include a vehicle suitable for animal consumption or use. Examples of suitable vehicles include food-grade edible material, mineral mixture, gelatin, cellulose, carbohydrate, starch, glycerin, water, glycol, molasses, corn oil, animal feed such as cereals (barley, corn, oats and similar), starches (tapioca and similar), oilseed cakes and vegetable residues. In some embodiments, the compositions include vitamins, minerals, trace elements, emulsifiers, flavorings, binders, colorings, odorants, thickeners and the like. Petition 870250085529, dated 09 / 22 / 2025, p. 46 / 282 42 / 73

[0113] In some embodiments, the compositions include one or more biologically active molecules or therapeutic molecules. Examples of these include ionophores; vaccines; antibiotics; anthelmintics; virucides; nematicides; amino acids such as methionine, glycine and arginine; fish oil; krill oil; and enzymes.

[0114] In one embodiment, the composition includes one or more antimethanogenic compounds. In one embodiment, the composition is administered or provided in combination with one or more antimethanogenic compounds. In one embodiment, the composition includes one or more compounds capable of reducing or inhibiting the production, release, or stability of methane (CH4). In one embodiment, the composition is administered or provided in combination with one or more compounds capable of reducing or inhibiting the production, release, or stability of methane (CH4). Compositions of one or more strains provided herein with one or more antimethanogenic compounds selected from inhibitors of a rate-limiting enzyme in the methanogenesis pathway, inhibitors of methanogenic lipid biosynthesis, or microbiome modulators are contemplated and provided herein.Exemplary antimethanogenic compounds or methane inhibitors include inhibitors of the enzyme methyl coenzyme M reductase, such as 3-NOP, bromoethanesulfonate, bromoform, red algae / Asparagopsis taxiformis, inhibitors of methanogenic lipid biosynthesis, such as the inhibitor mevinolin, and / or microbiome modulators, such as monensin.

[0115] In some embodiments, the compositions or combinations may additionally include one or more prebiotics. In some embodiments, the compositions may be administered together or co-administered with one or more prebiotics. Prebiotics may include organic acids or non-digestible food ingredients that are fermented in the large intestine and may serve to select beneficial bacteria. Prebiotics may include mannan-oligosaccharides, fructooligosaccharides, galactooligosaccharides, chitooligosaccharides, isomalto-oligosaccharides, pecticoligosaccharides, xylo-oligosaccharides, and lactose-oligosaccharides.

[0116] The compositions provided herein and the products derived from them have application and use as postbiotics. Thus, the fermentation product of the strains and compositions described herein provides a useful postbiotic product and composition with application in Petition 870250085529, dated 09 / 22 / 2025, page 47 / 282 43 / 73 reduction in methane and greenhouse gas production and in altering methanogenesis. This can be applied to animals or outside the body, such as in the environment (lakes, manure, soils, etc.) or in a crop or process (anaerobic digester, waste treatment, etc.). Postbiotics refer to the residues or growth products remaining after the digestion of prebiotics and probiotics. Postbiotics can include nutrients such as vitamins, amino acids, antimicrobial peptides, fatty acids, such as short-chain fatty acids or volatile fatty acids. Fermented foods are also exemplary postbiotics, such as kefir, tempeh, and kimchi.

[0117] Probiotic or microbiome-modulating compositions have applications and uses outside of an animal's body. These applications and uses include the environment (lakes, manure, soils, etc.) or a crop or process (anaerobic digester, waste treatment, etc.). The administration or addition of a composition, as described herein, can serve to alter methanogenesis and / or reduce methane production in the environment or in a crop or process. This can include environmental GHG management, such as in manure or litter management. The compositions as prebiotics or the fermentation product of the compositions as postbiotics can be used in manure or litter management, including as additives, to reduce methane gas production.

[0118] Compositions may be formulated as animal feed, feed additive, feed ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. The composition may be formulated and suitable for use as or in one or more of the following: animal feed, feed additive, feed ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. The composition may be suitable and prepared for use as animal feed, feed additive, feed ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. When compositions are administered as a feed additive, there is a benefit in feed efficiency. Petition 870250085529, dated 09 / 22 / 2025, page 48 / 282 44 / 73

[0119] The compositions may include a vehicle in which the bacteria or any other components are suspended or dissolved. Such vehicle(s) may be any solvent or solid, or encapsulated in a material that is non-toxic to the inoculated animal and compatible with the organism. Suitable pharmaceutical vehicles include liquid vehicles, such as normal saline solution and other non-toxic salts in physiological or near-physiological concentrations, and solid vehicles, such as talc or sucrose, which may also be incorporated into farm animal feed. When used for bronchial administration, the composition is preferably presented in aerosol form. A dye may be added to the compositions described herein, including to facilitate verification or confirmation that an animal has ingested or inhaled the composition.

[0120] When administering to animals, including farm animals, administration may be oral or by injection. Oral administration may be in bolus, tablet or paste form, or as a powder or solution in feed or drinking water. The method of administration will generally depend on the species being fed or administered, the number of animals fed or administered, and other factors such as available handling facilities and risk of stress to the animal.

[0121] In one embodiment, administration comprises in ovo administration. In one embodiment, delivery comprises spray delivery. In one embodiment, administration comprises immersion, intranasal, intramammary, topical or inhalation.

[0122] The required dosages vary and must be sufficient to induce an immune response or effect an expected or desired biological or phenotypic change or response. Routine experiments will determine the required amount. Increasing amounts or multiple dosages may be implemented and used as needed.

[0123] In one embodiment of the invention, the bacterial strains are administered in doses indicated as CFU / g or colony-forming units of bacteria per gram. In one embodiment, the dose is in the range of 1x10³ to 1x10⁹ CFU / g. In another embodiment, the dose is in the range of 1x10³ to 1x10⁷. In another embodiment, the dose is in Petition 870250085529, dated 09 / 22 / 2025, page 49 / 282 45 / 73 range of 1x10⁴ to 1x10⁶. In one embodiment, the dose is in the range of 5x10⁴ to 1x10⁶. In one embodiment, the dose is in the range of 5x10⁴ to 6x10⁵. In one embodiment, the dose is in the range of 7x10⁴ to 3x10⁵. In one embodiment, the dose is approximately 50K, 75K, 100K, 125K, 150K, 200K, 300K, 400K, 500K, 600K CFU / g.

[0124] In one embodiment, where the composition, including a food additive, comprises a combination of one or more bacterial strains, the ratio between the first strain and the second or subsequent strains is, in each case, approximately 0.75-1.5:1. In one embodiment, where the composition comprises a combination of one or more bacterial strains, the ratio between the first strain and the second or subsequent strains is approximately equal, approximately 1:1, approximately the same CFU dose of each strain. In one embodiment, where the composition, including a food additive, comprises a combination of one or more bacterial strains, the amount of the CFU dose of the first strain and the second or subsequent strains is approximately equal.Thus, a composition comprising three strains, such as a combination of the first isolated strain of Bacillus amyloliquefaciens, the second isolated strain of Bacillus amyloliquefaciens, and the isolated strain of Bacillus subtilis, comprises a ratio of the first isolated strain of Bacillus amyloliquefaciens, the second isolated strain of Bacillus amyloliquefaciens, and the isolated strain of Bacillus subtilis of 0.75-1.5:1:0.75-1.5. In one embodiment, the composition comprises approximately equal quantities of the first isolated strain of Bacillus amyloliquefaciens, the second isolated strain of Bacillus amyloliquefaciens, and the isolated strain of Bacillus subtilis. Thus, a composition comprising three strains, such as a combination of two isolated strains of Bacillus and one isolated strain of Lactobacillus, comprises a ratio of the first isolated strain of Bacillus, the second isolated strain of Bacillus, and the isolated strain of Lactobacillus subtilis of 0.75-1.5:1:0.75-1.5.In one embodiment, the composition comprises approximately equal quantities of the first and second isolated strains of Bacillus and the isolated strain of Lactobacillus subtilis. In one embodiment, and in cases where two strains of Bacillus or one strain of Bacillus and one strain of Lactobacillus are included in a composition, the ratio of the first strain to the second or more strains is, in each... Petition 870250085529, dated 09 / 22 / 2025, p. 50 / 282 46 / 73 case, a ratio of about 0.75-1.5:1. In one embodiment, the ratio or quantity is characterized by the number of viable spores per gram of dry weight. In one embodiment, the composition comprises from about 10⁴ to about 10¹⁰ viable spores per gram of dry weight. In one embodiment, the composition comprises from about 10⁶ to about 10¹⁰ viable spores per gram of dry weight. In one embodiment, the composition comprises from about 10⁶ to about 10⁸ viable spores per gram of dry weight.

[0125] In one embodiment, the isolated strains are not genetically modified. In one embodiment, the isolated strains are genetically modified.

[0126] In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition according to the invention. In another embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising one or more isolated strains of Bacillus amyloliquefaciens, an isolated strain of Bacillus subtilis, or an isolated strain of Lactobacillus, such as a strain of Lactobacillus reuteri.In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising one or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, and isolated strain of Bacillus subtilis, strain 105. In another embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising one or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, isolated strain of Bacillus subtilis, strain 105, and isolated strain of Lactobacillus reuteri, strain 3632. In another embodiment, a method is provided for to reduce, inhibit, or alter the colonization of one. Petition 870250085529, dated 09 / 22 / 2025, p. 51 / 282 47 / 73 animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising two or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, isolated strain of Bacillus subtilis, strain 105, and isolated strain of Lactobacillus reuteri, strain 3632. In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization of the rumen of a ruminant animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising two or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, isolated strain of Bacillus subtilis, strain 105, and isolated strain of Lactobacillus reuteri, strain 3632. 3632.

[0127] In one embodiment, a method is provided for altering the colonization by methanogenic bacteria or methanogenic Archaea in a natural environment or anaerobic circumstance where methanogenic bacteria or methanogenic Archaea are present, the method comprising contacting or providing an effective amount of a composition comprising one or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, isolated strain of Bacillus subtilis, strain 105, and isolated strain of Lactobacillus reuteri, strain 3632.In one embodiment, a method is provided for altering the amount of methane produced by methanogenic bacteria or methanogenic Archaea in a natural environment or anaerobic circumstance where methanogenic bacteria or methanogenic Archaea are present, the method comprising contacting or providing an effective amount of a composition comprising one or more isolated strains of Bacillus amyloliquefaciens, strain 06, isolated strain of Bacillus amyloliquefaciens, strain 071, isolated strain of Bacillus subtilis, strain 105, and isolated strain of Lactobacillus reuteri, strain 3632.

[0128] In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising an isolated strain of Lactobacillus. In one embodiment, Petition 870250085529, dated 09 / 22 / 2025, p. 52 / 282 48 / 73 provides a method for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising an isolated strain of Lactobacillus reuteri. In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization of an animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to an animal an effective amount of a composition comprising an isolated strain of Lactobacillus reuteri. In another embodiment, a method is provided for reducing, inhibiting, or altering the colonization in the rumen of a ruminant animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to a ruminant animal an effective amount of a composition comprising an isolated strain of Lactobacillus.In one embodiment, a method is provided for reducing, inhibiting, or altering the colonization in the rumen of a ruminant animal by a methanogenic bacterium or methanogenic Archaea, the method comprising administering to a ruminant animal an effective amount of a composition comprising an isolated strain of Lactobacillus reuteri. In another embodiment, the method comprises administering to an animal an effective amount of strain 3632. In another embodiment, the method comprises administering to an animal an effective amount of strain 3632 and one or more strains of Bacillus. In another embodiment, the method comprises administering to an animal an effective amount of strain 3632 and one or more strains of Bacillus selected from strain 06, strain 071, and strain 105.

[0129] In some embodiments, the compositions described above are used to reduce the bacterial or microbial load, particularly methanogenic bacteria or Archaea or clinically significant bacteria, including the number or quantity of bacteria in the intestine, gastrointestinal tract, or rumen of an animal. In some embodiments, the compositions described above are used to alter the bacterial or microbial load or to alter the bacterial or microbial composition or components of the microbiome, particularly methanogenic bacteria and / or Archaea or environmentally significant bacteria, including the number, Petition 870250085529, dated 09 / 22 / 2025, p. 53 / 282 49 / 73 the quantity, types, species or relative amounts and composition of bacteria and / or Archaea in the intestine, gastrointestinal tract or rumen of an animal.

[0130] In one embodiment, the method results in the interruption or alteration of methane production or the amount of methane produced by methanogenic bacteria and / or Archaea. In one embodiment, the method results in the inhibition of the growth or the elimination of methanogenic bacteria and / or Archaea. In one embodiment, the method results in the alteration of the fermentation processes of methanogenic bacteria and / or Archaea, so that the amount of methane produced or released is controlled or reduced. In one embodiment, the method results in the reduction of methane production or the amount of methane produced by an animal. In one embodiment, the method results in the inhibition of the growth or the elimination of methanogenic bacteria and / or Archaea in an animal. In one embodiment, the method results in the alteration of the fermentation processes of methanogenic bacteria and / or Archaea, so that the amount of methane produced or released by an animal is controlled or reduced.In one embodiment, the method results in a reduction in methane production or the amount of methane produced by a ruminant animal. In another embodiment, the method results in the inhibition of the growth or elimination of methanogenic bacteria and / or Archaea in a ruminant animal. In yet another embodiment, the method results in altering the fermentation processes of methanogenic bacteria and / or Archaea, so that the amount of methane produced or released by a ruminant animal is controlled or reduced.

[0131] In one embodiment of the method(s), the composition is formulated as animal feed, feed additive, feed ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. In one embodiment, the composition comprises animal feed.

[0132] The compositions described above may include a vehicle suitable for animal consumption or use. Examples of suitable vehicles include food-grade edible material, mineral mix, gelatin, cellulose, carbohydrate, starch, glycerin, water, glycol, molasses, corn oil, animal feed such as cereals (barley, corn, oats and Petition 870250085529, dated 09 / 22 / 2025, page 54 / 282 50 / 73 similar), starches (tapioca and similar), oilseed cakes and vegetable residues. In some forms, the compositions include vitamins, minerals, trace elements, emulsifiers, flavorings, binders, colorings, odorants, thickeners and similar substances.

[0133] In some embodiments, the compositions include one or more biologically active molecules or therapeutic molecules. Examples of these include ionophore; vaccine; antibiotic; anthelmintic; virucide; nematicide; amino acids such as methionine, glycine and arginine; fish oil; krill oil; and enzymes.

[0134] In some embodiments, the compositions or combinations may additionally include one or more prebiotics. In some embodiments, the compositions may be administered together with or co-administered with one or more prebiotics. Prebiotics may include organic acids or non-digestible food ingredients that are fermented in the large intestine and may serve to select beneficial bacteria. Prebiotics may include mannan-oligosaccharides, fructooligosaccharides, galactooligosaccharides, chitooligosaccharides, isomaltooligosaccharides, pectic-oligosaccharides, xylo-oligosaccharides, and lactoseoligosaccharides.

[0135] The composition may be formulated as animal feed, feed additive, food ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. The composition may be formulated and suitable for use as or in one or more of the following: animal feed, feed additive, food ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. The composition may be suitable and prepared for use as animal feed, feed additive, food ingredient, water additive, water-mixing additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof.

[0136] In embodiments of the invention, an animal may include a farm animal or livestock or a domesticated animal. Livestock or farm animal may include livestock (by Petition 870250085529, dated 09 / 22 / 2025, page 55 / 282 51 / 73 example, cows or bulls (including calves), poultry (including broilers, chickens and turkeys), pigs (including piglets), birds, aquatic animals such as fish, gastric fish, freshwater fish such as salmon, cod, trout and carp, for example, koi carp, marine fish such as sea bass, and crustaceans such as shrimp, mussels and scallops), horses (including racehorses), sheep (including lambs). A domesticated animal may be a pet or an animal kept in a zoological setting and may include any relevant animal, including canines (e.g., dogs), felines (e.g., cats), rodents (e.g., guinea pigs, rats, mice), birds, fish (including freshwater fish and marine fish) and horses. In modalities, the animal is a ruminant. In modalities, the ruminant is a bovine. In some forms, the ruminant animal is cattle or bison.In some categories, the ruminant is a bovine, caprine, ovine, giraffe, gazelle, or antelope. In some categories, the ruminant is a bovine, caprine, or ovine animal. In some categories, the animal is a non-ruminant animal.

[0137] The animal may be a pregnant or breeding animal. The animal may be a pregnant ruminant. The animal may be a pregnant cow or bull, a pregnant sow or a pregnant sow.

[0138] As used herein, “isolated” means that the isolate in question has been separated from at least one of the materials to which it is associated in a specific environment, for example, its natural environment.

[0139] Thus, an “isolate” does not exist in its natural environment; instead, it is through the various techniques known in the art that the microbe has been removed from its natural environment and placed in a non-natural state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture in association with an acceptable carrier.

[0140] As used herein, “individual isolates” should be understood as a composition, or culture, comprising the predominance of a single species, or strain, of microorganism, following the separation of one or more other microorganisms. The expression should not be interpreted as an indication of the extent to which the Petition 870250085529, dated 09 / 22 / 2025, page 56 / 282 52 / 73 microorganisms were isolated or purified. However, “individual isolates” may include substantially only one species, or strain, of microorganism.

[0141] In certain aspects of the disclosure, the isolated strain of Bacillus and / or the isolated strain of Lactobacillus exist as isolated and biologically pure cultures. It will be understood by one skilled in the art that an isolated and biologically pure culture of a particular strain of Bacillus or Lactobacillus denotes that said culture is substantially free (within reasonable scientific limits) from other living organisms and contains only the individual strain of Bacillus and / or Lactobacillus in question. The culture may contain varying concentrations of said Bacillus and / or isolated strain of Lactobacillus. The present disclosure notes that isolated and biologically pure microbes often necessarily differ from less pure or impure materials.

[0142] In some embodiments of the present invention, the composition includes a combination of two isolated bacterial strains. In some embodiments of the present invention, the composition includes a combination of two isolated strains of Bacillus. In some embodiments of the present invention, the composition includes a combination of two or more isolated bacterial strains. In some embodiments of the present invention, the composition includes a combination of two or more isolated strains of Bacillus or Lactobacillus. In some embodiments of the present invention, the composition includes a combination of three isolated strains of Bacillus. In some embodiments of the present invention, the composition includes a combination of one isolated strain of Bacillus and one strain of Lactobacillus. In some embodiments of the present invention, the composition includes a combination of at least one isolated strain of Bacillus and one strain of Lactobacillus.

[0143] As used herein, the term bacterial consortia, bacterial consortium, microbial consortia, or microbial consortium refers to a subset of a microbial community of individual microbial species, or strains of a species, that can be described as performing a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic characteristic of interest (by Petition 870250085529, dated 09 / 22 / 2025, page 57 / 282 53 / 73 example, increased feed efficiency in poultry). The community may comprise two or more species, or strains of a species, of microbes. In some cases, microbes coexist symbiotically within the community.

[0144] As used herein, spore or spores refer to structures produced by bacteria that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, they are capable of differentiating through the process of germination. Germination is the differentiation of spores into vegetative cells capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single bacterial vegetative cell. Bacterial spores are structures adapted to survival conditions that are not normally conducive to the survival or growth of vegetative cells.

[0145] The composition may include or comprise live bacteria or bacterial spores, or a combination thereof.

[0146] As used herein, the terms “colonize” and “colonization” include “temporarily colonize” and “temporary colonization”.

[0147] As used herein, microbiome refers to the collection of microorganisms that inhabit an animal's gastrointestinal tract and the physical environment of those microorganisms (i.e., the microbiome has a biotic and a physical component). The microbiome is fluid and can be modulated by numerous natural and artificial conditions (e.g., changes in diet, disease, antimicrobial agents, influx of additional microorganisms, etc.).Modulation of the gastrointestinal microbiome can be achieved through the administration of the compositions disclosed, and may take the form of: (a) an increase or decrease in a particular Family, Genus, Species or functional grouping of a microbe (i.e., alteration of the biotic component of the gastrointestinal microbiome) and / or (b) an increase or decrease in gastrointestinal pH, an increase or decrease in volatile fatty acids in the gastrointestinal tract, an increase or decrease in any other physical parameter important for gastrointestinal health (i.e., alteration of the abiotic component of the intestinal microbiome). Petition 870250085529, dated 09 / 22 / 2025, page 58 / 282 54 / 73

[0148] As used herein, probiotic or microbial modulator refers to a substantially pure microbe (i.e., a single isolate) or a mixture of desired microbes, and may also include any additional components (e.g., carrier) that may be administered to an animal or environment to provide a beneficial health effect or to alter the microbiome of / in an animal or in / or an environment. Probiotics or microbial or microbiome modulating compositions of the invention may be administered with an agent or carrier to enable the microbes to survive in the environment, such as that of the gastrointestinal tract, i.e., to withstand low pH and grow in the gastrointestinal environment.

[0149] The term growth medium, as used herein, refers to any medium suitable for supporting the growth of a microbe. By way of example, the medium may be natural or artificial, including supplemental gastrin agar, minimal medium, rich medium, LB medium, blood serum, and tissue culture gels. It should be noted that the medium may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients.

[0150] As used herein, the term improved should be understood broadly, encompassing the improvement of a trait of interest, compared to a control group or compared to a known average quantity associated with the trait in question. For example, the improved feed efficiency associated with the application of a beneficial microbe, or microbial ensemble, of the disclosure can be demonstrated by comparing the feed efficiency of birds treated with the microbes discussed herein with the feed efficiency of untreated birds. In the present disclosure, improved does not necessarily require that the data be statistically significant (i.e., p < 0.05); instead, any quantifiable difference that demonstrates that one value (e.g., the treatment mean value) is different from another (e.g., the control mean value) can reach the level of improved.

[0151] As used herein, the term metabolite refers to an intermediate or product of metabolism. In some embodiments, a metabolite includes a small molecule. Metabolites have various functions, including fuel, structural, Petition 870250085529, dated 09 / 22 / 2025, page 59 / 282 55 / 73 of signaling, stimulatory and inhibitory effects on enzymes, as a cofactor for an enzyme, in defense, and in interactions with other organisms (such as pigments, odorants, and pheromones). A primary metabolite is directly involved in normal growth, development, and reproduction. A secondary metabolite is not directly involved in these processes but usually has an important ecological function. Examples of metabolites include, among others, antibiotics and pigments such as resins and terpenes, etc. Metabolites, as used here, include small, hydrophilic carbohydrates; large, hydrophobic lipids; and complex natural compounds.

[0152] As used herein, carrier, acceptable carrier, or pharmaceutical carrier are used interchangeably and refer to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous solutions of dextrose and glycerol are preferably employed as carriers, in some embodiments as injectable solutions. Alternatively, the carrier may be a solid pharmaceutical form carrier, including, but not limited to, one or more of: a binder (for tablets), a glidant, an encapsulating agent, a flavoring agent, and a colorant. The choice of carrier may be made taking into account the intended route of administration and standard pharmaceutical practice.See Handbook of Pharmaceutical Excipients, (Sheskey, Cook, and Cable) 2017, 8th edition, Pharmaceutical Press; Remington's Pharmaceutical Sciences, (Remington and Gennaro) 1990, 18th edition, Mack Publishing Company; Development and Formulation of Veterinary Dosage Forms (Hardee and Baggot), 1998, 2nd edition, CRC Press.

[0153] As used herein, delivery or administration means the act of providing a beneficial activity to a host. Delivery can be direct or indirect. Administration can be oral, nasal, or mucosal. For example, without limitation, an oral route can be administration via drinking water, a nasal route of administration can be via spray or vapor, and a mucosal route of administration can be via direct contact with mucosal tissue. Mucosal tissue is a Petition 870250085529, dated 09 / 22 / 2025, page 60 / 282 56 / 73 membrane rich in mucous glands, such as those lining the inner surface of the nose, mouth, esophagus, trachea, lungs, stomach, intestines, and anus. In the case of birds, administration can be in ovo, that is, administration to a fertilized egg. In ovo administration can be by means of a liquid that is sprayed onto the surface of the eggshell or injected through the shell.

[0154] As used herein, the terms “treat,” “treating,” or “treatment” include restricting, delaying, interrupting, inhibiting, reducing, mitigating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. A treatment may also be applied prophylactically to prevent or reduce the incidence, occurrence, risk, or severity of a symptom, disorder, condition, or clinical disease.

[0155] As used herein, animal includes bird, domestic fowl, a human being, or a non-human mammal. Specific examples include chickens, turkeys, dogs, cats, cattle, buffalo, bison, sheep, goats, deer, giraffes, gazelles, antelopes, salmon, fish, swine, and horses. Specific examples include farm animals such as chickens, turkeys, cattle, swine, and sheep. A chicken may be a broiler chicken, a laying hen, or an egg-producing hen.As used herein, the term domestic fowl includes domestic birds such as chickens, turkeys, ducks, and geese. The term animal includes a ruminant animal or a non-ruminant animal.

[0156] As used herein, “intestine” refers to the gastrointestinal tract, including the stomach, small intestine, and large intestine. The term “intestine” may be used interchangeably with “gastrointestinal tract.”

[0157] As used herein, a genetically modified microorganism means any microorganism that has been altered from its natural state by means of molecular biology techniques. A genetic modification may be the deletion of a part of the bacterial chromosome or of a natural plasmid. Genetic modification may also be the introduction of an artificial or exogenous nucleic acid into a part of the chromosome. The introduction may or may not disrupt or disturb the expression of a bacterial gene. Genetic modification may also be the introduction of an artificial plasmid. The genetically modified microorganism may be a bacterium, a virus, a yeast, a fungus, or a unicellular organism. Petition 870250085529, dated 09 / 22 / 2025, page 61 / 282 57 / 73

[0158] An “artificial nucleic acid” or “artificial plasmid” is any nucleic acid or plasmid that does not occur naturally but has been constructed using molecular biology techniques. Parts of the nucleic acid or plasmid may occur naturally, but these parts are in an artificial relationship or organization.

[0159] As used herein, an expression cassette is an artificial nucleic acid constructed to result in the expression of a biomolecule desired by the genetically modified microorganism. An expression cassette comprises one or more of the following: a promoter for transcriptional expression, a nucleic acid sequence encoding a signal sequence for secretion, a nucleic acid sequence encoding a cell wall anchor, at least one heterologous coding region encoding a desired biomolecule, a nucleic acid sequence encoding an expressed peptide tag for detection, and terminators for translation and transcription termination. A promoter directs the initiation of transcription of the coding regions into a messenger RNA and the translation of the mRNA into a peptide. A signal sequence for secretion, or a signal sequence for secretion, directs the peptide to be localized outside the cell membrane.The extracellular peptide can be a secreted soluble protein or it can be associated with the cell, particularly if the expression cassette contains a cell wall anchor sequence that links the extracellular peptide to a bacterial cell wall. An expressed peptide tag is any amino acid sequence that can be recognized by an antibody or other binding protein. The expressed peptide tag can also bind to an inorganic substance, such as a six-histidine tag that binds to nickel molecules. Terminators for translation can be a stop codon or an open reading frame spacer containing a stop codon.

[0160] As used herein, a “heterologous coding region” is a nucleic acid sequence containing an open reading frame that codes for a peptide. The coding region is heterologous to the associated promoter, meaning that the coding region and the promoter are not associated in their natural states.

[0161] A heterologous region of a nucleic acid construct, RNA or DNA, is an identifiable segment of RNA or DNA within a larger RNA or DNA molecule. Petition 870250085529, dated 09 / 22 / 2025, page 62 / 282 58 / 73 DNA that is not found in association with the larger molecule in nature. Thus, when the heterologous region codes for a gene, it will generally be flanked by RNA or DNA that does not flank the genomic RNA or DNA in the genome of the organism of origin.

[0162] As used herein, a “protein” is a sequence of amino acids that assumes a three-dimensional structure. A “peptide” may be used interchangeably with protein, but may also be a short linear sequence of amino acids without a defined three-dimensional structure.

[0163] As used herein, a desired biomolecule is any molecule or peptide that may be advantageous to a host when administered via a live delivery platform. The desired biomolecule may be a peptide with anti-infective activity, a probiotic factor, an immunomodulatory factor, an anti-antinutritional factor, or a growth-promoting biomolecule. The desired biomolecule may also be an enzyme that produces a substance with anti-infective activity or a probiotic factor, such as a vitamin.

[0164] As used herein, “anti-infective activity” includes any activity that prevents infection of a host with a pathogenic organism. The following molecules are examples of biomolecules possessing anti-infective activity: an antibacterial peptide; a lysine or lytic enzyme; a prophage, phage, or virus; an enzyme, for example, one that cleaves or disables a protein produced by a pathogen; and an antibody that blocks, inhibits, or eliminates a pathogenic molecule. An anti-infective may have bacteriostatic activity, which slows, reduces, or prevents the growth of a pathogenic species. A non-limiting example of an antibacterial peptide is a member of the mersacidin family or a mersacidin-like molecule, such as those described in document EP0700998. A non-limiting example of lysines are lytic molecules produced by phages.Lysins may have specificity for certain pathogenic species of bacteria and have been suggested for use as a replacement for traditional antibiotics. VA Fischetti, Viruses, vol. 10, no. 310 (2018); and R. Vazquez et al. Frontiers in Immunology, vol. 9, article 2252 (2018). Petition 870250085529, dated 09 / 22 / 2025, page 63 / 282 59 / 73

[0165] As used herein, a “probiotic factor” is a substance that, when produced by a microorganism, proves beneficial to a host. The probiotic factor may be a binding molecule or a clumping molecule that promotes colonization of the host with the microorganism and / or prolongs the period during which the microorganism colonizes the host. The longer the microorganism persists in the host, the longer the duration of colonization. The beneficial effect is provided.

[0166] As used herein, an immunomodulatory factor may be a cytokine, lymphokine, chemokine, interleukin, interferon, colony-stimulating factor, or growth factor. The immunomodulatory factor may provide a nonspecific increase in an immune response, or it may increase the number or tissue distribution of immune cells present in the host. The immunomodulatory factor may also reduce an inappropriate immune response, such as, for example, an autoimmune response.

[0167] As used herein, a “growth-promoting biomolecule” may be a growth factor, a transfer factor (such as an iron chelating molecule), a hormone, or any other factor that promotes healthy metabolic activity.

[0168] As used herein, an “antinutritional factor” may include protease inhibitors, for example, trypsin inhibitors.

[0169] As used herein, subject includes birds, domestic fowl, fish, a human being, or a non-human animal. Specific examples include chickens, turkeys, dogs, cats, cattle, buffalo, bison, sheep, goats, deer, giraffes, gazelles, antelopes, salmon, fish, pigs, and horses. Specific examples include farm animals such as chickens, turkeys, cattle, pigs, and sheep. A chicken can be a broiler hen, a laying hen, or an egg-producing hen. As used herein, the term domestic fowl includes domestic birds such as chickens, turkeys, ducks, and geese. The term subject includes a ruminant animal or a non-ruminant animal.

[0170] The term primer, as used herein, refers to an oligonucleotide, whether naturally occurring, as in a purified restriction digest, or synthetically produced, capable of acting as a starting point for synthesis when subjected to conditions in which the synthesis of a primer extension product, complementary to Petition 870250085529, dated 09 / 22 / 2025, page 64 / 282 60 / 73 a nucleic acid strand is induced, that is, in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at an appropriate temperature and pH. The primer can be single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, primer source, and method use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 25 or more nucleotides, although it may contain fewer nucleotides.

[0171] The primers described here are selected to be substantially complementary to different strands of a specific target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed in the primer, provided that the primer sequence has sufficient complementarity with the strand sequence to hybridize with it and thus form the template for the synthesis of the extension product.

[0172] As used herein, the term mutant refers to a variation in a nucleic acid sequence, DNA or RNA, or in a chromosomal structure from that which is considered a normal or wild-type sequence or a defect-free chromosome. In the context of a nucleic acid sequence, DNA or RNA, examples of mutations include point mutations, insertions, and deletions. A deletion includes the deletion of part or all of the gene. Such mutations may have functional effects such as, for example, a decrease in the function of a gene product, ablation of function in a gene product, and / or a new or altered function in a gene product.

[0173] As used herein, mutation includes any alteration in one or more nucleic acids in a genomic sequence, including one or more base alterations, deletions and / or insertions, that result in silent mutations, nonsense mutations, or any other mutations that result in a reduction Petition 870250085529, dated 09 / 22 / 2025, p. 65 / 282 61 / 73 of a gene's function or result in an inactive or non-functional protein encoded by a gene. Mutations include, but are not limited to, mutations that result in premature stop codons, aberrant splicing, altered transcription or failure, or altered translation or failure. A gene comprising a mutation may have more than one mutation. Mutations include the deletion of a gene or a significant portion of a gene, particularly such that the gene's protein is not produced or expressed and / or is inactive. Mutations include insertions, such as those in which a foreign or heterologous sequence or nucleic acid is introduced or otherwise inserted into the gene. Such an insertion may block or eliminate translation into an active or full-length protein, or may result in a significantly altered and distinct protein that is not active like the wild type.An insertion can facilitate the isolation, detection, and selection of the mutant gene, such as by introducing or inserting an antibiotic resistance gene or a detectable marker or protein. In specific embodiments of the invention and as described herein, the mutation, including one or more mutations, is an unnatural mutation and is genetically modified or recombinantly generated. In some embodiments, the mutation is genetically modified or recombinantly generated in vitro. In some embodiments, the mutation is genetically modified or recombinantly generated in a cell.

[0174] In some embodiments, a mutation is generated whereby a gene, or a large or significant portion of a gene or protein encoding a nucleic acid, is deleted. In some embodiments, one or more genes, or a large or significant portion of a gene or protein encoding a nucleic acid, are deleted, for example, by means of recombination methods. Recombination methods for targeted gene deletion are known and available to a specialist in the field. Such methods include homologous recombination, such as by means of an introduced plasmid, phage or nucleic acid, such as DNA or linear DNA fragment(s), recombination enzymes or enzyme-mediated recombination recombinase, for example, by means of recombinase recognition or save sequences, transposon-mediated recombination and gene substitution. Petition 870250085529, dated 09 / 22 / 2025, page 66 / 282 62 / 73

[0175] There are several peptides or proteins that act independently as therapeutic biomolecules. Among them are anti-infective or antibacterial peptides, which can serve to block or treat infections caused by infectious agents or bacteria.

[0176] In some embodiments, the disclosure provides for the use of any of the compositions described above in a therapy or treatment or to improve a phenotypic characteristic in an animal. In embodiments of the invention, an animal may be a ruminant or non-ruminant animal. In embodiments of the invention, an animal may include a farm animal or livestock or a domesticated animal. The animal may be a pregnant or breeding animal, such as a pregnant sow.

[0177] Examples of improvement of a phenotypic trait include improved growth or growth characteristics, improved feed efficiency, reduced methanogenesis or diversion of the fermentation pathway of methane production, increased H2 consumption, reduced H2 production, increased volatile fatty acid production, and improved gut health or characteristics (reducing permeability and inflammation). In particular, examples include reduced methanogenesis or diversion of the fermentation pathway of methane production, increased H2 consumption, reduced H2 production, increased volatile fatty acid production, and improved gut health or characteristics (reducing permeability and inflammation).In particular, examples include one or more of the following: reduction of methanogenesis or diversion of the fermentation pathway from methane production, increased H2 consumption, reduced H2 production, increased production of volatile fatty acids, and improved gut health or characteristics (reducing permeability and inflammation).

[0178] The compositions may also include one or more components or additives. One or more components or additives may be a component or additive to facilitate administration, for example, by means of a stabilizer or vehicle, or by means of an additive to allow administration to an animal, such as by any suitable administrative means, including in aerosol or spray form, in water, in feed or in an injectable form. Administration to an animal may be by any technique Petition 870250085529, dated 09 / 22 / 2025, page 67 / 282 63 / 73 known or standard. These include oral ingestion, gastric intubation, or bronchonasal spraying. The compositions disclosed here can be administered by immersion, intranasal, intramammary, topical, mucosal, or inhalation. When the animal is a bird, treatment can be administered in ovo or by inhalation spray.

[0179] Any examples or illustrations presented herein should in no way be considered as restrictions, limitations, or express definitions of any term or terms with which they are used. Instead, such examples or illustrations should be considered as being described in relation to a specific embodiment and as being merely illustrative. Those with common knowledge of the art will understand that any term or terms with which such examples or illustrations are used will encompass other embodiments that may or may not be provided with them or elsewhere in the specification, and all such embodiments should be included within the scope of such term(s). The language designating such non-limiting examples and illustrations includes, but is not limited to: for example, exemplary, e.g., and in an embodiment. In this specification, multi-parameter groups containing multiple members are described.Within a group of parameters, each member can be combined with any one or more of the other members to form additional subgroups. For example, if the members of a group are a, b, c, dee, specifically contemplated additional subgroups include any one, two, three, or four of the members, for example, aec; a, dee; b, c, dee; etc.

[0180] Throughout this specification, quantities are defined by intervals and by lower and upper interval limits. Each lower limit can be combined with each upper limit to define an interval. The lower and upper limits should be considered as a separate element. Two lower limits or two upper limits can be combined to define an interval.

[0181] This disclosure can be better understood with reference to the examples given below. The following examples are given to provide those skilled in the art with a complete description and disclosure of how the compounds, compositions and / or methods claimed herein are produced and evaluated, and are intended to be merely illustrative and not limiting of the disclosure. Petition 870250085529, dated 09 / 22 / 2025, p. 68 / 282 64 / 73 note that other embodiments and uses will be apparent to experts in the field and that the invention is not limited to these specific illustrative examples or preferred embodiments. EXAMPLE 1 Mitigation of enteric methane emissions for improved livestock production.

[0182] Methane is naturally produced as a byproduct of the ruminal microbial fermentation process (removal of H2, a thermodynamic barrier to fermentation) by methanogens. Methane emission is considered an energy loss for animals. Therefore, methane reduction should translate into greater feed efficiency. Approaches have been implemented to reduce enteric methane gas emissions in order to reduce the carbon footprint of livestock production and improve the feed efficiency of ruminants. Methane production per volume of biogas is evaluated and specific methanogenic activity is estimated from methane production.

[0183] In an in vitro batch rumen culture, which provides an ex vivo rumen assessment system, several strains of Bacillus and Lactobacillus reduced methane. In particular, the B. subtilis strain BE191006 (BE191006) demonstrated a reduction of up to 25% in methane compared to the control. Other strains reduced methane by at least 10%.

[0184] The experimental design of the batch culture in the rumen is illustrated in Figure 5. Experiments with rumen probiotics, evaluating the capacity and activity of bacterial strains in reducing methane production in an in vitro rumen batch culture, which provides an ex vivo rumen evaluation system, are conducted as follows. This system can be used to evaluate single strains or combinations of strains, one or more strains in combination with another agent, etc.

[0185] Step 1: Feed preparation 1. The feed for beef cattle was dried in an oven at 58 °C for 96 hours. 2. The dry food was ground using a 10-cup Oster food processor (pre-cleaned with 70% IPA) on high speed for 5 minutes, and the ground food was... Petition 870250085529, dated 09 / 22 / 2025, page 69 / 282 65 / 73 filtered through a Cooking Concept® mesh sieve (pre-cleaned with 70% IPA) to separate the fine feed from the pellets. 3. The fine feed was used immediately or stored at -20°C.

[0186] Step 2: Preparing the serum bottle 1. The serum bottle was cleaned using a MayTag dishwasher with a jet cleaning cycle and strong wash, high temperature wash, heated drying, extra drying and added steam sanitizing cleaning. 2. The clean serum bottle was autoclaved for 1 hour and stored at room temperature. 3. Two days before the experiment, 10% (w / v) fine feed was added to the pre-cleaned serum bottle.

[0187] Step 3: Preparation of probiotics and additives 1. One day before the experiment, all the powdered probiotics were added to the pre-filled serum bottles. CaCO3, Bacillus strains, strain BE191105, strain BE191006 and strain BE202071. 2. On the day of the experiment, all the liquid probiotic produced was added to the pre-filled serum bottles. 3. CHCl3, Lactobacillus, strain BE3632 (L. reuteri), and another strain called Strain A. 4. On the day of the experiment, serum bottles containing fine feed (10% w / v) and probiotic (1 x 108 CFU per g of feed) or enzymes were filled with 20 mL of artificial saliva, capped with a rubber stopper, and made anaerobic with N2:CO2 gas (80:20, v / v) for 3 cycles (with a vacuum time of 3-3-5 minutes). 5. The prepared mixture was filled with N2:CO2 gas (80:20, v / v) at 5 psig.

[0188] Step 4: Preparation of rumen fluid 1. Rumen contents were collected from fistulated cows in a centrifuged flask with a sealing cap. Petition 870250085529, dated 09 / 22 / 2025, page 70 / 282 66 / 73 2. The centrifuged flasks containing the rumen contents were transferred into an anaerobic chamber filled with N2:CO2:H2 (75:20:5, v / v / v) and the contents were filtered through a 4-layer cotton cloth. 3. Fresh ruminal fluid was aliquoted into a 560 mL serum bottle, stoppered with a rubber stopper, and removed from the anaerobic chamber. 4. The fresh concentrated rumen fluid was filled with N2:CO2 gas (80:20, v / v) at 10 psig. 5. The prepared probiotic mixtures were then added to 10 mL of fresh concentrated ruminal fluid using a syringe.

[0189] Stage 5: Rumen-probiotic experiment 1. Serum bottles containing rumen fluid, feed, and probiotic content were incubated at 40°C, 75 rpm. 2. Each of the bottles was then treated in the following order: a. The pressure in the bottle was measured using a gas meter in a dedicated laboratory gasification station system. b. A 100 μL gas sample was taken from the headspace of the bottle for GC analysis. c. After 3 hours, the pressure in the bottle was measured and a 100 μL gas sample from the bottle's headspace was collected for GC analysis. d. The process (step 3) was then repeated every 3 hours for a total of 6 hours, then every 6 hours for a total of 6 hours, and then every 12 hours for a total of 36 hours.

[0190] Effects of different microbial strains on ruminal methane emission

[0191] The methane-reducing effects of five microbial strains of animal origin, Table 1, were investigated in in vitro batch rumen culture. In vitro batch rumen culture is a surrogate system that simulates the microbial fermentation process in the rumen of ruminants. TABLE 1. List of probiotics / additives Probiotics / additives Final concentration Bacillus subtilis BE191105 (ELA191105) 1 x 108UFC per g of food Bacillus amyloliquefaciens BE191006 (ELA191006) 1 x 10⁸ CFU per g of feed Petition 870250085529, dated 09 / 22 / 2025, page 71 / 282 67 / 73 Bacillus amyloliquefaciens BE202071 (ELA202071) 1 x 10⁸ CFU per g of feed; Lactobacillus reuteri BE3632 (strain 3632) 1 x 10⁸ CFU per g of feed Strain A: 1.43 x 10⁹ CFU per g of feed

[0192] The composition of artificial saliva is shown in Table 2. TABLE 2. Composition of artificial saliva Compounds Concentration NaCI 8.04 mmol / L KCI 8.18 mmol / L NaH2PO4.H2O 12.25 mmol / L Na2HPO4.7H2O 13.91 mmol / L NaHCOs 95.24 mmol / L (NH4)2SO4 7.14 mmol / L CaCI2-2H2O 0.20 mmol / L MgCI2-6H2O 0.34 mmol / L NasNTA 10.00 pmol / L FeCI3.6H2O 2.96 pmol / L CoCI2.6H2O 0.42 pmol / L MnCI2.4H2O 5.05 pmol / L

[193] A summary of the cattle and feed used in the rumen experiment is as follows: Cattle breed: Holstein Friesian Type of livestock: Dairy Rumen fluid collection time: ~2 hours after morning feeding. Feed composition: mixtures of concentrate, forage, silage and hay.

[0194] The data summary is as follows: Bottle volume: ~270 mL Rumen fluid dilution factor: 1:2 (v / v) Diluted ruminal fluid used: 30 mL Petition 870250085529, dated 09 / 22 / 2025, page 72 / 282 68 / 73 Bottled feed Probiotics, powder Probiotics, liquid Negative control Positive control g (10% w / v) 0.1 g (1 x 108CFU per gram of food) - 600 μL 0.1 g of CaCO3 powder 0.2 mM CHCl3 Volume of headspace in the bottle: ~240 mL Incubation Measurement Duration 40°C, 75 rpm bottle pressure and CH4 via GC (100 μL injection volume) 36h (3h sampling for 6h, 6h sampling for 6h and 12h sampling for 36h)

[0195] A methane standard curve representing the amount of methane gas injected into the gas chromatography (GC) system is provided in Figure 6. The results with a control series are shown in Figure 7. The results with a series of Bacillus bacteria are shown in Figure 8. Each of the strains BE105 of Bacillus B. subtilis (BE191105 / ELA191105), strain BE006 of B. amyloliquefaciens (BE 191006 / ELA191006) and strain BE071 of B. amyloliquefaciens (BE 202071 / ELA202071) are compared with the blank control, where none was added. The results with a series of Lactobacillus bacteria are shown in Figure 9. The results of the comparison between the Bacillus and Lactobacillus strains and the control are presented in Figure 10. Methane production (CH4 production, %) is presented in Figure 11 for the various additives and bacterial strains tested.Methane production is defined as the increase (positive value) or decrease (negative value) in methane production of a typical treatment compared to a blank control (none). Methane production from ex vivo rumen culture 24 hours after supplementation with different probiotic strains is shown in Figures 12A and 12B.

[0196] All probiotics, except the other strain, strain A, reduced methane production after 12 hours of incubation and maintained low methane production at 30 hours. Petition 870250085529, dated 09 / 22 / 2025, page 73 / 282 69 / 73 following. The vials with added BE191006 (B. amyloliquefaciens) showed the lowest methane production compared to other probiotic treatments. The results suggested that the tested strains showed methane-reducing effects when added to rumen cultures. Most methane reductions were observed after 6 hours of strain addition and peaked at 24 hours. B. amyloliquefaciens BE191006 showed the highest level of methane reduction, ~25%, compared to the control, rumen culture without added microbial strain.

[0197] Inhibition of methanogenic bacterial strains, particularly Methanobacterium bryantii, is evaluated in vitro. The growth of methanogenic bacteria is assessed. A reduction in bacterial growth is used in a kill-type culture assay to determine the minimum inhibitory amount of one or more test strains, including one or more strains of Bacillus or Lactobacillus, alone and in combinations, to kill methanogenic bacteria. In a first set of studies, Bacillus strains 06, 071, 105, alone and in combinations of two or three strains, are tested. Bacillus spp. strains are routinely cultured in standard broth, such as Lysogeny Broth (LB), and incubated at 37 °C overnight with agitation. Methanogenic bacteria, particularly Methanobacterium bryantii, are cultured separately in an overnight culture until confluence. Bacillus are added to methanogenic bacterial cultures and their growth is evaluated.In a separate set of studies, Lactobacillus strains are evaluated individually and in combination. The cultures are observed for cell death. The assays are performed in duplicate. EXAMPLE 2

[0198] Beef cattle and dairy cattle are used in an in vivo animal study to further evaluate the effect of administering bacteria capable of reducing methane production on cattle performance. A randomized Latin square (3X3) crossover study design is shown in Figure 13. There is a 7-day baseline period, followed by a Study Phase period that includes a 24-day Treatment Adaptation Period and a 4-day sample collection period until Day 28 of the study, followed by a 14-day Flushing Period. Rumen fluid is evaluated at Petition 870250085529, dated 09 / 22 / 2025, page 74 / 282 70 / 73 throughout the study. Methane is assessed on Days 5, 6, and 7 of the baseline period and on Days 25, 26, 27, and 28 of the Study Phase Sample Collection period. Feeding interventions include: A. No additive (negative control), B. 3-NOP (positive control), and C. Microbial treatment. With feeding interventions, rumen fluid is collected 2, 4, 6, and 8 hours after morning feeding, in this case on days 25, 26, 27, and 28 of the study.

[0199] Performance parameters for beef cattle are: reduced residual feed intake, increased body weight, and reduced methane emissions. Performance parameters for dairy cattle are: increased milk volume and milk protein and lipid composition. EXAMPLE 3

[0200] An in vitro batch culture in the rumen was used to evaluate the effect of bacteria (L. reuteri strain 3632 (BE3632)) on the production of various gases in the culture. The levels and quantities of H2, CO2, and CH4 were evaluated by gas chromatography. The results are shown in Figure 14.

[0201] A significant reduction in CH4 methane production is evident with L. reuteri BE3632 at 24 hours, with methane reduction of up to 50%. The methane reduction observed with L. reuteri BE3632 is greater than the reduction provided by the addition of CHCl3 control. The L. reuteri bacterium demonstrated a reduction in H2 production in batch culture, particularly at 24 hours. CO2 production is approximately the same in the presence of L. reuteri bacteria versus untreated sample. EXAMPLE 4

[0202] Bacterial colonization in ruminal fluid was evaluated. This was done to demonstrate that bacteria remain and grow in the ruminal environment. The L. reuteri strain 3632 (BE3632), which is not native to the rumen, was evaluated for colonization.

[0203] The steps of the process to evaluate the colonization of L. reuteri BE3632 in ruminal fluid are described in Figure 15. Starting with an original inoculum, which was sampled and plated to assess the number of bacteria, an inoculum was transferred serially up to 3 times. There was a two-day period between each transfer. In each Petition 870250085529, dated 09 / 22 / 2025, page 75 / 282 71 / 73 transfer, a sample was taken for plating and the number of bacteria evaluated. A sample of plate evaluations showing L. reuteri BE3632 bacteria is provided in Figure 16. L. reuteri BE3632 bacterial colonies are visually evident by their orange color. The colonies remained with and after transfer. After the second transfer, a 3-fold reduction in L. reuteri BE3632 bacterial counts was observed (data not shown). A further reduction was observed after the third transfer (data not shown), however, some bacteria were retained. L. reuteri BE3632 bacteria were retained and survived for several days (at least 6 days or almost a week) with serial transfer in ruminal fluid mimicking the ruminal environment.

[0204] The composition of the medium for liquid cultures included ruminal fluid, which was diluted with artificial saliva in a 1:1 ratio, plus dry cattle feed (cattle feed obtained from the Virginia Tech Dairy Science Complex). The ruminal fluid was collected from dairy cattle. Ruminal fluid contains microbes (i.e., bacteria, fungi, protozoa - 1011 cells / mL), microbial metabolites (e.g., volatile fatty acids, mainly short chains, also contains medium and long-chain fatty acids, proteins, peptides), bovine saliva (the composition of saliva can be shared), micronutrients, and host proteins. pH - neutral (6.5 - 7). The composition of ruminal fluid depends on the diet, age, breed (dairy vs. cattle), and ruminal microbial population of the animal of origin. The components of ruminal fluid are described, for example, in Akula et al (Akula S et al (2023) Int J Mol Sci 24(23):16838).Cows produce saliva in large quantities to lubricate and facilitate food processing. Bovine salivary proteins include carbonic anhydrase 6, a pH-stabilizing enzyme, and carcinoma-associated protein 2A of the pulmonary, nasal, and short palate epithelium (SPLUNC2A), also called bovine salivary protein 30 kDa (BSP30) or BPIFA2B, and secretory IgA. The liquid medium was prepared by mixing 1.8 ml of diluted BiomEdit ruminal fluid with 0.2 g of dry cattle feed. The composition of the medium for the plates consisted of Difco™ Lactobacilli MRS 1X agar, Bacto agar 2.5% (w / v), and tetracycline 50 μg / ml. L. reuteri BE3632 is resistant to tetracycline. Bottles, rubber stoppers, spatulas, and beakers were used for... Petition 870250085529, dated 09 / 22 / 2025, p. 76 / 282 72 / 73 The liquid media prepared were previously autoclaved to minimize bacterial contamination. All plating and transfer were performed anaerobically. The plate was incubated anaerobically in a static state, while the anaerobic liquid medium was incubated with agitation at 100 rpm. All incubation was performed at 40 °C for 48 hours. All dilutions for plating were made with artificial saliva.

[0205] These results demonstrate that L. reuteri BE3632 is retained and survives in a ruminal environment and is able to colonize the rumen of a ruminant animal. EXAMPLE 5

[0206] Previous studies demonstrate that each of the various specific isolated strains of Bacillus and also strains of Lactobacillus reduce methane production and alter methanogenesis, including in an in vitro system that mimics a native methanogenic (ruminal) environment. In particular, all strains BE191006 of Bacillus amyloliquefaciens, strain BE202071 of Bacillus amyloliquefaciens, strain BE191105 of Bacillus subtilis and strain 3632 of Lactobacillus reuteri reduce methane. Experiments were conducted to examine the compatibility of Bacillus and Lactobacillus strains to better evaluate their use and application in combination to reduce methane emissions and alter methanogenesis.

[0207] A representation of an overlap experiment to examine strain compatibility is provided in Figure 17. An evaluation of the Bacillus subtilis strain BE191105 with the Lactobacillus reuteri strain 3632 was conducted, as described in Figure 17. The first strain (in this case, B. subtilis BE191105) is placed on a plate (such as a TSA plate) and cultured under aerobic conditions at 30 °C. The second test strain (in this case, L. reuteri 3632) is cultured separately and incubated anaerobically at 39 °C for at least 20 hours. The CFU of the second strain is quantified. After at least 16 hours of growth, the second strain (in this case, L. reuteri strain 3632) is diluted to 10⁵ CFU and the agar (such as MRS agar) is inoculated with the second strain and superimposed around the colony of the first strain (in this case, B. subtilis BE191105). The plate is incubated anaerobically at 39°C for at least 18, 24, or 48 hours, and then the growth of the first and second strains is evaluated.In particular, the inhibition of the growth of the first strain due to the presence of the second. Petition 870250085529, dated 09 / 22 / 2025, p. 77 / 282 73 / 73 strain is evaluated. In multiple technical replicates and also in multiple biological replicates, no inhibition of the growth of the first strain (in this case, B. subtilis BE191105) by the second strain (in this case, L. reuteri 3632) was observed. The experiment is conducted similarly with other alternative strain combinations, including the Bacillus amyloliquefaciens strain BE191006 or the Bacillus amyloliquefaciens strain BE202071 as the first strain and the Lactobacillus reuteri strain 3632 as the second strain.

[0208] This invention can be incorporated in other forms or carried out in other ways without departing from its spirit or essential characteristics. The present disclosure should therefore be considered illustrative in all respects and not restrictive, the scope of the invention being indicated by the appended claims, and all alterations that fall within the meaning and range of equivalence shall be encompassed by them.

[0209] Several references are cited throughout this specification, each of which is incorporated herein by reference in its entirety. Petition 870250085529, dated 09 / 22 / 2025, p. 78 / 282

Claims

1 / 5 CLAIMS 1. Probiotic or microbiome-modulating composition, characterized in that it comprises at least one selected bacterial strain of a Bacillus species and Lactobacillus species, or combinations thereof, wherein the composition reduces methane gas emissions from an animal when an effective amount is administered to the animal, compared to an animal that has not had the composition administered.

2. Composition, according to claim 1, characterized in that it comprises one or more bacterial strains selected from a strain of Bacillus amyloliquefaciens, a strain of Bacillus subtilis and a strain of Lactobacillus.

3. Composition, according to claim 1 or 2, characterized in that the Lactobacillus strain is a Lactobacillus reuteri strain.

4. Composition, according to claim 1, 2 or 3, characterized in that it comprises at least one strain 3632 of Lactobacillus reuteri or a strain of Lactobacillus having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the sequence of at least one of the SEQ ID Nos: 51-57; strain BE191006 of Bacillus subtilis or a strain of Bacillus having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the sequence SEQ ID No: 27; Bacillus strain BE202071 or a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with sequence SEQ ID NO: 42;and the BE191105 strain of Bacillus amyloliquefaciens or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with one or more SEQ ID numbers: 1, 2, 3, 4, and 5.

5. Composition, according to claim 1, 2, 3 or 4, characterized in that it comprises at least one of the following: Lactobacillus reuteri strain 3632 corresponding to patent application number ATCC PTA-126788, Bacillus amyloliquefaciens strain BE191006 (also known as ELA191006) corresponding to application ATCC PTA-127065, Bacillus amyloliquefaciens strain BE202071 (also known as ELA202071) corresponding to application ATCC PTA-127064, and Bacillus subtilis strain BE191105 (also known as ELA191105) corresponding to application ATCC PTA-126786.

6. Composition, according to claim 1, 2, 3, 4 or 5, characterized in that it comprises at least two of the following: Lactobacillus reuteri strain 3632 or a Lactobacillus strain having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with at least one of the SEQ ID Nos: 51-57; Bacillus subtilis strain BE191006 or a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the SEQ ID No: 27; strain BE202071 of Bacillus amyloliquefaciens or a strain of Bacillus with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% genomic sequence identity with sequence SEQ ID NO: 42;and the BE191105 strain of Bacillus amyloliquefaciens or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with one or more SEQ ID numbers: 1, 2, 3, 4, and 5.

7. Composition, according to any one of claims 1 to 6, characterized in that it comprises at least two strains selected from Lactobacillus reuteri strain 3632, Bacillus amyloliquefaciens strain BE191024, Bacillus amyloliquefaciens strain BE191006 and Bacillus subtilis strain BE191105.

8. Composition, according to any one of claims 1-6, characterized in that it further comprises one or more antimethanogenic compounds.

9. A method for reducing methane gas production or methane gas emissions from an animal, characterized in that it comprises administering an effective amount of a composition including at least one probiotic or microbiome modulator selected from a Bacillus species, a Lactobacillus species, or combinations thereof, to an animal. Petition 870250085529, dated 22 / 09 / 2025, page 80 / 282 3 / 5 10. Method according to claim 9, characterized in that the animal is a ruminant animal.

11. Method according to claim 10, characterized in that the ruminant animal is selected from bovine animals such as cattle, bison, American buffalo and water buffalo, goats, sheep, giraffes, deer, gazelles and antelopes.

12. Method according to claim 9, characterized in that the animal is a non-ruminant animal.

13. Method according to claim 12, characterized in that the non-ruminant animal is selected from among poultry, pigs, birds, aquatic animals such as fish and crustaceans, horses, dogs, cats and humans.

14. Method according to claim 9, characterized in that it comprises one or more bacterial strains selected from a strain of Bacillus amyloliquefaciens, a strain of Bacillus subtilis and a strain of Lactobacillus.

15. Method according to claim 9, characterized in that the composition or a fermentation product of the composition is administered.

16. Method, according to any one of claims 9 to 15, characterized in that the Lactobacillus strain is a Lactobacillus reuteri strain.

17. Method, according to any one of claims 9 to 16, characterized in that the composition comprises at least one Lactobacillus reuteri strain 3632 or a Lactobacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with at least one of the SEQ ID Nos: 51 to 57; or a Bacillus subtilis strain BE191006 or a Bacillus strain with at least 90%, 95%, 97%, 98%, or 99% genomic sequence identity with the SEQ ID No: 27; strain BE202071 of Bacillus amyloliquefaciens or a strain of Bacillus with at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity of the genomic sequence with the sequence SEQ ID NO: 42; and strain BE191105 of Bacillus amyloliquefaciens or a strain of Bacillus with at least 90% of Petition 870250085529, dated 09 / 22 / 2025, p.81 / 282 4 / 5 identity, 95% identity, 97% identity, 98% identity, 99% identity of the genomic sequence with one or more SEQ ID NO: 1, 2, 3, 4 and 5.

18. Method, according to any one of claims 9 to 17, characterized in that it comprises at least one strain BE191006 of Bacillus amyloliquefaciens (also known as ELA191006) corresponding to ATCC filing PTA-127065, strain BE202071 of Bacillus amyloliquefaciens (also known as ELA202071) corresponding to ATCC filing PTA-127064, strain BE191105 of Bacillus subtilis (also known as ELA191105) corresponding to ATCC filing PTA-126786 and strain 3632 of Lactobacillus reuteri corresponding to ATCC patent filing number PTA-126788.

19. Method, according to any one of claims 9 to 18, characterized in that the composition comprises at least two isolated strains selected from Bacillus amyloliquefaciens strain BE191024, Bacillus amyloliquefaciens strain BE191006, Bacillus subtilis strain BE191105 and Lactobacillus reuteri strain 3632.

20. A method according to any one of claims 9 to 19, characterized in that it further comprises the administration of one or more antimethanogenic compounds.

21. Method according to claim 20, characterized in that the antimethanogenic compound is selected from among an inhibitor of a rate-limiting enzyme in the methanogenesis pathway, an inhibitor of methanogen lipid biosynthesis, or a microbiome modulator.

22. A method, according to any one of claims 9 to 20, characterized in that the animal is a human.

23. A method, according to claim 22, characterized in that the human being has an intestinal disorder, obesity, or a high body mass index (BMI).

24. Method for reducing methane emission or production by selected methanogenic organisms of Archaea or methanogenic bacteria in the environment, in a culture or in a process, characterized in that it comprises the administration of the composition according to any of claims 1 to 8 or a fermentation product of the composition.

25. Method according to claim 24, characterized in that the environment, culture or process is selected from among manure, leaf litter, lagoons, an aerobic digester and waste treatment.

26. Method, according to claim 24, for reducing methane emission or production in manure, characterized in that it combines or adds to the manure the composition according to any of claims 1 to 8 or a fermentation product of the composition.

27. Postbiotic, characterized in that it comprises a fermentation product of the composition according to any one of claims 1 to 8, wherein the postbiotic reduces methane production, alters methanogenesis and / or reduces greenhouse gas emissions.

28. Postbiotic, according to claim 27, characterized in that it is formulated as a feed additive, a food or feed additive, or otherwise for an animal.

29. Postbiotic, according to claim 28, characterized in that the animal is a ruminant animal.

30. Postbiotic, according to claim 28, characterized in that the animal is selected from bovine animals, such as cattle, bison, American buffalo and water buffalo, goats, sheep, giraffes, deer, gazelles, antelopes, poultry, pigs, birds, aquatic animals such as fish and crustaceans, horses, dogs, cats and humans.

31. Composition, according to any one of claims 1 to 8, or the postbiotic, according to claim 27, characterized in that it is formulated as a food additive. Petition 870250085529, dated 09 / 22 / 2025, pp. 83 / 282

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