Microbial compositions in geometrically shaped encapsulates

Abstract

The present disclosure relates to methods of using cold-melt extrusion during the manufacturing of a thermally sensitive product. More specifically, the present disclosure describes the use of cold-melt extrusion and hot-melt fluid-bed coating for the manufacturing of geometrically encapsulated microbes compositions (GEMs) as feed additives, comprising microorganisms selected from native microbiome of an animal such as Butyrivibrio fibrisolvens, Ruminococcus bovis, Pichia kudriavzevii, and Clostridium beijerinckii.

Description

MICROBIAL COMPOSITIONS IN GEOMETRICALLY SHAPED ENCAPSULATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application 63 / 605,357, filed on December 1, 2023, which is herein incorporated by reference in its entirety. FIELD

[0002] The present disclosure relates to dry microbial compositions, described as Geometrically Encapsulated Microbes (GEMs), composed of a geometrically shaped core comprising a microbe, a protective carrier material, and optionally, a functional ingredient, enveloped by a coating layer comprising a protective material, and optionally, a functional ingredient. The geometrically shaped cores are manufactured using a cold-melt extrusion process.

[0003] The disclosed compositions supplement beneficial microbes to any nutritious substance consumed as food by a host, thus promoting the health, performance, and or production of the host. In some embodiments, the disclosed compositions provide beneficial microbes by way of administration through a body cavity of the host for nutraceutical, prophylactic, or therapeutic purposes. In some embodiments, the host is an animal. In some embodiments, the animal is a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal, or a companion animal. In some embodiments, the host is a human. REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0004] The contents of the electronic sequence listing (ASBI_031_01WO_SeqList_ST26.xml; Size: 40,994 bytes; Date of Creation: November 19, 2024) are herein incorporated by reference in its entirety. BACKGROUND

[0005] Microorganisms are widely used in animal husbandry and agriculture to promote performance and production, promote health, prevent disease, improve milk quality and production, improve meat quality, reduce odor and pollution of excretion, and inhibit the growth of pathogens. During manufacturing, microorganisms are often exposed to multiple lethal factors required for processing the microorganisms with other ingredients to form a microbial product (such as high temperature, pressure, moisture, and pH). Specifically, the highprocessing temperatures frequently result in loss of viability leading to reduced processing yields. Therefore, maintaining a low-temperature environment during the manufacturing process is critical for thermally-sensitive microorganisms.

[0006] There is a need in the art for improved manufacturing methods for temperature- sensitive products such as feed additives comprising microorganisms. SUMMARY

[0007] The present disclosure relates to dry microbial compositions, described as Geometrically Encapsulated Microbes (GEMs), composed of a geometrically shaped core comprising a microbe, a protective carrier material, and optionally, a functional ingredient, enveloped by a coating layer comprising a protective material, and optionally, a functional ingredient.

[0008] In some embodiments, the GEMs comprise one or more microbial strains entrapped in a geometrically defined core matrix enveloped with at least one protective coating layer defined herein as the moisture-barrier layer. A high-melting protective material with a melting point above 50oC, optionally a low-melting protective material with a melting point below 50oC, or a mixture of such materials comprise at least 50% of the moisture-barrier layer.

[0009] In some embodiments, the GEMs comprise one or more microbial strains entrapped in a geometrically defined core matrix enveloped with two or more protective coating layers defined as moisture-barrier and moisture-scavenging layers. A high-melting protective coating material with a melting point above 50oC, optionally a low-melting protective coating material with a melting point below 50oC, or a mixture of such materials constitute at least 50% of the moisture-barrier layer. A mineral salt or a mixture of mineral salts consisting of the anhydrous and hydrated forms of the salts comprise at least 50% of the moisture-scavenging layer.

[0010] In some embodiments, the GEMs comprise one or more microbial strains entrapped in a geometrically defined core matrix enveloped by at least one protective coating layer consisting of a high-melting material with a melting point above 50oC as the most inner layer, or the most outer layer, optionally one protective coating layer comprising a low-melting material with a melting point below 50oC as the middle layer, and optionally, at least one functional ingredient in each layer.

[0011] In some embodiments, the GEMs comprise one or more microbial strains entrapped in a geometrically defined matrix enveloped by at least one protective coating layer comprising a high-melting material with a melting point above 50oC as the most inner layer, or the mostouter layer, optionally one protective coating layer comprising at least one mineral salt as the middle layer wherein the at least one mineral salt comprises a mixture of its anhydrous and hydrated forms in a ratio from about 0% to about 100% on a weight basis, and optionally, at least one functional ingredient in each layer. The at least one mineral salt is deposited by spraying a molten preparation of the corresponding salt hydrate onto the coated cores.

[0012] In some embodiments, the core and or the at least one protective coating layer (envelope) may optionally contain a functional ingredient selected from a group consisting of a moisture binding agent, a pH modifying agent, a release modifying agent, a disintegrating agent, an oxygen scavenging agent, or a combination thereof.

[0013] In some embodiments, the GEMs comprise one or more microbial strains entrapped in a geometrically defined core matrix enveloped by at least one protective coating layer, by at least two protective coating layers, or by at least three protective coating layers.

[0014] In some embodiments, the GEMs are manufactured in steps including: 1) encapsulation or entrapment of at least one microorganism in geometrically shaped cores; 2) coating of geometrically shaped cores with one or more protective layers to envelop the cores; and 3) optionally, drying the microbe-containing envelopes to a water activity level less than 0.4.

[0015] In some embodiments, the geometrically shaped cores are produced by any of single- screw extrusion, twin-screw extrusion, ram extrusion, granulation-extrusion-spheronization, tablet compaction, pastillation, freeze pelletization, spinning disk atomization, or spray granulation (spouted fluid-bed granulation) methods.

[0016] In some embodiments, the geometrically shaped envelopes are produced by spraying a molten preparation of protective materials onto the microbe-containing cores.

[0017] In some embodiments, the geometrically shaped envelopes are produced by compacting a powder form of protective materials over the microbe-containing cores.

[0018] In some embodiments, the geometrically shaped envelopes are produced by applying protective materials over the microbe-containing cores through a combination of hot-melt spray coating and compaction processes.

[0019] In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder prior to manufacture of cores and envelopes. In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder by freeze-drying, tray drying, vacuum drying, microwave drying, spraydrying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0020] In some embodiments, the protective carrier material has a high-melting temperature. In some embodiments, the high-melting temperature is an onset melting temperature of at least 40ºC. In some embodiments, the high-melting temperature is a peak melting temperature of at least 40ºC. In some embodiments, the protective carrier material is meltable at temperatures below 100ºC.

[0021] In some embodiments, the protective carrier material is partially or completely insoluble in aqueous media. In some embodiments, the carrier is a wax. In some embodiments, the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, monoglycerides, diglycerides, triglycerides, paraffins, alkane-based waxes, mineral- based waxes, insect-based waxes, and plant-based waxes.

[0022] In some embodiments, at least two carriers are mixed to produce a mixture exhibiting a melting temperature lower than the melting temperature of individual carriers.

[0023] In some embodiments, at least two carriers are mixed to produce a mixture exhibiting a melting temperature manifested as the eutectic point of the carrier mixture.

[0024] In some embodiments, the protective coating material has a high-melting temperature. In some embodiments, the high-melting temperature is an onset melting temperature of at least 50ºC. In some embodiments, the high-melting temperature is a peak melting temperature of at least 50ºC. In some embodiments, the protective coating material is meltable at temperatures below 100ºC.

[0025] In some embodiments, the protective coating material is partially or completely insoluble in aqueous media. In some embodiments, the protective coating material is a wax. In some embodiments, the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, monoglycerides, diglycerides, triglycerides, paraffins, alkane- based waxes, mineral-based waxes, insect-based waxes, and plant-based waxes.

[0026] In some embodiments, the fatty acid is a saturated fatty acid with an aliphatic tail of 6 to 21 carbons. In some embodiments, the saturated fatty acid is a medium-chain fatty acid (MCFA) with an aliphatic tail of 6 to 12 carbons. In some embodiments, the saturated fatty acid is a long-chain fatty acid (LCFA) with an aliphatic tail of 13 to 21 carbons. In someembodiments, the carrier is a stearic acid. In some embodiments, the stearic acid comprises at least 90% by weight of octadecanoic acid (C18:0).

[0027] In some embodiments, the plant-based wax is a hydrogenated vegetable oil selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the oil is partially hydrogenated. In some embodiments, the oil is fully hydrogenated. In some embodiments, the carrier is hydrogenated soybean oil.

[0028] In some embodiments, the mineral salt is selected from a group of salt hydrates which consist of 3 to 12 moles of hydration water per mole of salt, and exhibit a melting point from about 25oC to about 125oC.

[0029] In some embodiments, the salt hydrate is manganese nitrate hexahydrate, calcium chloride hexahydrate, sodium sulfate decahydrate, sodium carbonate decahydrate, potassium iron disulfate dodecahydrate, calcium bromide tetrahydrate, disodium hydrogen phosphate dodecahydrate, zinc nitrate hexahydrate, manganese nitrate tetrahydrate, calcium chloride tetrahydrate, calcium nitrate tetrahydrate, disodium hydrogen phosphate heptahydrate, tripotassium phosphate heptahydrate, zinc nitrate tetrahydrate, sodium silicate tetrahydrate, sodium metasilicate pentahydrate, disodium hydrogen phosphate heptahydrate, dipotassium hydrogen phosphate heptahydrate, magnesium sulfate heptahydrate, sodium thiosulfate pentahydrate, calcium nitrate trihydrate, sodium nitrate hexahydrate, zinc nitrate dihydrate, magnesium chloride tetrahydrate, manganese chloride tetrahydrate, sodium bisulfite monohydrate, sodium aluminum sulfate dodecahydrate, disodium dihydrogen pyrophosphate dodecahydrate, trisodium phosphate dodecahydrate, aluminum nitrate nonahydrate, trisodium phosphate dodecahydrate, sodium pyrophosphate decahydrate, barium hydroxide octahydrate, aluminum sulfate octadecahydrate, aluminum nitrate octahydrate, magnesium nitrate hexahydrate, ammonium aluminum sulfate dodecahydrate, aluminum sulfite dodecahydrate, magnesium chloride hexahydrate, and combination thereof.

[0030] In some embodiments, the salt hydrate is calcium chloride hexahydrate, sodium sulfate decahydrate, sodium carbonate decahydrate, sodium hydrogen phosphate dodecahydrate, sodium hydrogen phosphate heptahydrate, sodium thiosulfate pentahydrate, or magnesium sulfate heptahydrate, and combination thereof.

[0031] In some embodiments, at least two salt hydrates are mixed to produce a mixture exhibiting a melting temperature lower than the melting temperature of individual salt hydrates.

[0032] In some embodiments, at least two salt hydrates are mixed to produce a mixture exhibiting a melting temperature manifested as the eutectic point of the salt mixture.

[0033] In some embodiments, the molten salt preparation is a mixture of calcium chloride hexahydrate and sodium sulfate decahydrate, a mixture of calcium chloride hexahydrate and sodium hydrogen phosphate dodecahydrate, or a mixture of calcium chloride hexahydrate, sodium sulfate decahydrate, and sodium thiosulfate pentahydrate.

[0034] In some embodiments, the GEMs further comprise one or more functional ingredient selected from a group consisting of a moisture binding agent, a pH modifying agent, a release modifying agent, a disintegrating agent, an oxygen scavenging agent, or a combination thereof. In some embodiments, the moisture-binding agent is a zeolite, a bentonite, a diatomaceous earth, a silica, or an inorganic hydratable salt. In some embodiments, the zeolite is a natural zeolite. In some embodiments, the disintegrating agent is selected from the group consisting of a natural polysaccharide, a natural polysaccharide hydrogel, a soy polysaccharide, a psyllium husk fiber, a molecular sieve, and a mineral clay.

[0035] In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder prior to cold-melt extrusion. In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder by freeze-drying, spray drying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), tray drying, vacuum drying, microwave drying, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0036] In some embodiments, the at least one microorganism is microencapsulated with a meltable carrier. In some embodiments, the carrier has a melting temperature of at least 40oC. In some embodiments, the carrier has an onset melting temperature of at least 40ºC. In some embodiments, the carrier has a peak melting temperature of at least 40ºC. In some embodiments, the carrier is a wax. In some embodiments, the wax is selected from the group consisting of: animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, paraffins, alkane-based waxes, mineral-based waxes, insect-based waxes, and plant-based waxes. In some embodiments, the plant-based wax is a hydrogenated vegetable oil. In some embodiments, the hydrogenated vegetable oil is selected from the group consisting ofhydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the hydrogenated vegetable oil is fully hydrogenated vegetable oil. In some embodiments, the hydrogenated vegetable oil is fully hydrogenated soybean oil.

[0037] In some embodiments, the at least one microorganism is microencapsulated by freeze- drying, spray drying, fluid-bed spray granulation (spouted-bed), fluid-bed spray coating, hot- melt fluid-bed coating, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0038] In some embodiments, the composition comprises at least one microbial strain, at least two microbial strains, at least three microbial strains, at least four microbial strains, or at least five microbial strains. In some embodiments, microbial strains comprises live microorganisms.

[0039] In some embodiments, the microbial strains are selected from a family, genus, species, or functional grouping of microbes which confer improved health, enhanced performance, increased production, and nutraceutical or therapeutic benefit to the host when consumed or administered at sufficient numbers.

[0040] In some embodiments, the host is an animal. In some embodiments, the animal is a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal or a companion animal. In some embodiments, the host is a human.

[0041] In some embodiments, the total amount of the at least one microorganism is about 10% to about 90% of the composition. In some embodiments, the at least one microorganism is native to the microbiome of an animal. In some embodiments, the at least one microorganism is native to the gastrointestinal microbiome of an animal. In some embodiments, the at least one microorganism is native to the microbiome of a monogastric animal. In some embodiments, the at least one microorganism is native to the microbiome of a ruminant or of a camelid. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1- 34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequencewith at least 99% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence of any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism is alien (non-native) to the microbiome of an animal. In some embodiments, the at least one microorganism is a genetically-modified strain.

[0042] In some embodiments, the at least one microorganism is of the genus Butyrivibrio. In some embodiments, the at least one microorganism is of the genus species Butyrivibrio fibrisolvens. In some embodiments, the at least one microorganism is a Butyrivibrio fibrisolvens with a deposit accession number of NRRL B-67347. In some embodiments, the at least one microorganism is of the genus Ruminococcus. In some embodiments, the at least one microorganism is of the genus species Ruminococcus bovis. In some embodiments, the at least one microorganism is a Ruminococcus bovis with a deposit accession number of PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479.

[0043] In some embodiments, the geometrically shaped cores are manufactured by cold-melt extrusion, the method comprising the following steps: (a) feeding ingredients into an extruder wherein the ingredients comprise at least one microorganism and a protective carrier material; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the carrier to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head consisting of at least one hole with a geometrically defined shape; (e) cutting the extrudate into pellets; and (f) cooling the pellets.

[0044] In some embodiments, the temperature of the extruder is below 50ºC. In some embodiments, the temperature of the extruder is below 40ºC. In some embodiments, the temperature of the extruder is 35ºC.

[0045] In some embodiments, the extruder is a ram extruder. In some embodiments, the extruder is a single-screw extruder. In some embodiments, the extruder is a twin-screw extruder. In some embodiments, the twin-screw extruder is a co-rotating or a counter-rotating extruder.

[0046] In some embodiments, the method further comprises kneading the semi-molten or molten extrudable composition.

[0047] In some embodiments, the die head comprises a single hole or a plurality of holes with a geometrically defined shape. In some embodiments, the die hole is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a hemisphere. In some embodiments, the die hole has a round shape. In some embodiments, the die hole has a diameter of about 0.5 mm to about 50 mm. In some embodiments, the die hole has a diameter of about 1 mm to about 5 mm.

[0048] In some embodiments, the geometrically shaped core has a cubic, cylindrical, tubular, prismatic, round, or spherical shape with an aspect ratio ranging from 1 to 10. The aspect ratio for cylindrical cores is defined as the ratio of their length to diameter (L:D). The aspect ratio for tubular cores is defined as the ratio of their length to outer diameter (L:Do).The aspect ratio for cubic and prismatic cores is defined as the ratio of their length to equivalent diameter (L:De), wherein the equivalent diameter is defined as the geometric mean diameter of hypothetical circles enrobing the two end-faces of the core. The aspect ratio for round cores is defined as the ratio of their maximum Feret width to maximum Feret length (W / L). The aspect ratio for spherical cores is equivalent to 1.

[0049] In some embodiments, the cooling is cryogenic cooling. In some embodiments, the cryogenic cooling is performed using liquid nitrogen or carbon dioxide (dry ice). In some embodiments, the cryogenic cooling is an immersion freezing process. In some embodiments, the cooling is performed using a chilled dry gas. In some embodiments, the chilled dry gas is nitrogen, carbon dioxide, or dehumidified air.

[0050] In some embodiments, the cooling is taken place on a conveyor belt, wherein heat transfer is achieved by direct cooling, employing a chilled dry gas, and or indirect cooling, employing a chilled gas or liquid.

[0051] In some embodiments, the geometrically shaped core is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a sphere. In some embodiments, the core has a round shape. In some embodiments, the pellet has a diameter of about 0.5 mm to about 50 mm. In some embodiments, the core has a diameter of about 1 mm to about 4 mm.

[0052] In some embodiments, the core is enveloped with a protective layer defined herein as the moisture-barrier layer. A high-melting wax with a melting point above 50oC, a low-melting wax with a melting point below 50oC, or a mixture of such waxes constitute at least 50% of the moisture-barrier layer.

[0053] In some embodiments, the core is enveloped with two or more protective layers defined as moisture-barrier and moisture-scavenging layers. A high-melting wax with a melting point above 50oC, a low-melting wax with a melting point below 50oC, or a mixture of such waxes constitute at least 50% of the moisture-barrier layer. A mineral salt or a mixture of mineral salts consisting of the anhydrous and hydrated forms of the salts constitute at least 50% of the moisture-scavenging layer.

[0054] In some embodiments, the core is enrobed with a protective coating layer. In some embodiments, the core is coated by hot-melt fluid-bed coating. In some embodiments, the core is coated to a level of at least 5% with the additional protective material. In some embodiments, the additional protective material has a high-melting temperature. In some embodiments, the high-melting temperature is an onset melting temperature of at least 50ºC. In some embodiments, the high-melting temperature is a peak melting temperature of at least 50ºC. In some embodiments, the protective coating material is a wax. In some embodiments, the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, paraffins, alkane-based waxes, mineral-based waxes, insect-based waxes, and plant-based waxes. In some embodiments, the plant-based wax is a hydrogenated vegetable oil. In some embodiments, the hydrogenated vegetable oil is selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the hydrogenated vegetable oil is fully hydrogenated vegetable oil. In some embodiments, the protective coating material is hydrogenated soybean oil. In some embodiments, the glyceride is hydrogenated glyceride.

[0055] In some embodiments, the GEMs have a water activity below 0.4. In some embodiments, the water activity is below 0.3, 0.2, or 0.1. In some embodiments, the water activity is below 0.1.

[0056] In some embodiments, the present disclosure provides a composition produced by the cold-melt extrusion method described herein. In some embodiments, the present disclosure provides a microbial product produced by the cold-melt extrusion method described herein. In some embodiments, the present disclosure provides a feed additive or supplement produced by the cold-melt extrusion method provided herein.

[0057] In some embodiments, the present disclosure provides a composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a protective carrier, wherein the pellet is manufactured by cold-melt extrusion at a temperature of at least 5ºC below the melting temperature of the protective carrier.

[0058] In some embodiments, the present disclosure provides a composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a protective carrier, wherein the pellet is manufactured by cold-melt extrusion at a temperature at or below 50ºC.

[0059] In some embodiments, the present disclosure provides a method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: feeding ingredients into an extruder, wherein the ingredients comprise at least one biologic and a wax; conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, heating the ingredients at a temperature at or below 50ºC to form a semi- molten or molten extrudable composition; extruding the composition through a die head; cutting the composition into pellets; and cooling the pellets.

[0060] In some embodiments, the present disclosure provides a method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: feeding ingredients into an extruder, wherein the ingredients comprise at least one microorganism and a wax; conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, heating the ingredients at a temperature at or below 50ºC to form a semi-molten or molten extrudable composition; extruding the composition through a die head; cutting the composition into pellets; and cooling the pellets.

[0061] In some embodiments, the encapsulation method increases survival of the at least one microorganism. In some embodiments, the encapsulation method increases stability of the at least one microorganism. In some embodiments, the method increases the total processing yield of the at least one microorganism.

[0062] In some embodiments, the disclosed GEMs supplement beneficial microbes to any nutritious substance consumed as food by a host, thus promoting the health, performance, and or production of the host. In some embodiments of the invention, the disclosed compositions provide beneficial microbes by way of administration through a body cavity of the host for nutraceutical, prophylactic, or therapeutic purposes.

[0063] In some embodiments, the GEMs is for use in animals. In some embodiments, the animal is a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal, or a companion animal.

[0064] In some embodiments, the GEMs is for use in humans.

[0065] In some embodiments, the disclosed GEMs are developed as a probiotic composition for administration to a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal, or a companion animal.

[0066] In some embodiments, the disclosed GEMs are developed as a probiotic composition for addition to the animal’s rationed feed directly or through an aqueous-based liquid, slurry, or paste delivery system maintained at 5-30oC.

[0067] In some embodiments, less than 50% and most preferably less than 1% of entrapped microorganisms is released within 1 hour of addition of the composition to the delivery system.

[0068] In some embodiments, less than 50% and most preferably less than 1% of entrapped microorganisms is released within 0.25 hour of addition of the composition to the delivery system.

[0069] In some embodiments, at least 1% and most preferably at least 99% of entrapped microorganisms is released in vitro within 24 hours in buffered media (pH 3 - 7) at about 37oC to about 39oC.

[0070] In some embodiments, at least 1% and most preferably at least 99% of entrapped microorganisms is released in vitro within 1 hour in buffered media (pH 3 - 7) at about 37oC to about 39oC. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1A, FIG. 1B, and FIG. 1C illustrate schematic diagrams of geometrically encapsulated microbes (GEMs). FIG. 1A shows GEMs consisting of a core (101) and a protective coating layer (102) comprising a high-melting wax. FIG. 1B shows GEMs consisting of a core (201), an inner protective coating layer (202) comprising a high-melting wax, a middle protective coating layer (203) comprising a low-melting wax, and an outer protective coating layer (204) comprising a high-melting wax. FIG. 1C shows GEMs consisting of a core (301), an inner protective coating layer (302) comprising a high-melting wax, a middle protective coating layer (303) comprising a mineral salt hydrate or a mixture ofthe anhydrous and hydrated forms of a mineral salt, and an outer protective coating layer (304) comprising a high-melting wax; particle length:diameter ≥ 1.

[0072] FIG. 2A, FIG. 2B, and FIG. 2C illustrate schematic diagrams of geometrically encapsulated microbes (GEMs). FIG. 2A shows GEMs consisting of a core (401) and a protective coating layer (402) comprising a high-melting wax. FIG. 2B shows GEMs consisting of a core (501), an inner protective coating layer (502) comprising a high-melting wax, a middle protective coating layer (503) comprising a low-melting wax, and an outer protective coating layer (504) comprising a high-melting wax. FIG. 2C shows GEMs consisting of a core (601), an inner protective coating layer (602) comprising a high-melting wax, a middle protective coating layer (603) comprising a mineral salt hydrate or a mixture of the anhydrous and hydrated forms of a mineral salt, and an outer protective coating layer (604) comprising a high-melting wax; particle length:diameter ≤ 1.

[0073] FIG.3 shows Butyrivibrio fibrisolvens pellets produced with cold-melt extrusion. DETAILED DESCRIPTION Overview

[0074] Hot-melt extrusion is considered an efficient technique in developing solid molecular dispersions. However, the use of this process with temperature-sensitive drugs, probiotics, or biologics often leads to reduced stability and / or processing yields of final products. The present disclosure addresses these challenges by using a cold-melt extrusion manufacturing process. This method can be used for the manufacturing of any type of thermally sensitive composition or product, such as the manufacturing of native or genetically modified microorganisms for use in animal husbandry and agriculture.

[0075] According to some embodiments of the disclosure, methods for manufacturing microbial compositions are disclosed. Such methods can be used for, by way of non-limiting example, forming a microbial composition or supplement as detailed below. Such compositions comprise one or more microorganisms, in some embodiments, facultative or strict anaerobic microorganisms. In some embodiments, compositions comprise one or more microorganisms as disclosed in one or more of the following: U.S. Pub. Nos.2018 / 0310592, 2018 / 0333443, 2018 / 0223325, 2022 / 0174992, 2022 / 0265732, 2022 / 0386647, and PCT Pub. Nos. 2016 / 210251, 2017 / 120495, 2017 / 181203, 2018 / 201049, 2019 / 079629, 2018 / 056563, 2020 / 042148, 2020 / 227442, 2021 / 163212, 2021 / 011662, 2021 / 202804, 2022 / 226367, and 2022 / 081992 (each being herein expressly incorporated by reference for all purposes).Definitions

[0076] As used in this specification, the singular forms “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, the term “an organism type” is intended to mean a single organism type or multiple organism types. For another example, the term “an environmental parameter” can mean a single environmental parameter or multiple environmental parameters, such that the indefinite article “a” or “an” does not exclude the possibility that more than one of environmental parameters is present, unless the context clearly requires that there is one and only one environmental parameter.

[0077] Reference throughout this specification to “one embodiment”, “an embodiment”, “one aspect”, or “an aspect”, “one implementation”, or “an implementation” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0078] As used herein, in particular embodiments the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure

[0079] As used herein, “carrier”, “acceptable carrier”, or “pharmaceutical carrier” refers to a diluent, adjuvant, excipient, or vehicle with which is used with or in the microbial ensemble. Such carriers can be a liquid form carrier such as oils, including those of vegetable, animal, petroleum or synthetic origin; such as coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower oil, mineral oil, and the like. Alternatively, the carrier can be a solid form carrier, including but not limited to one or more of a binder, a glidant, a disintegrant, a release modifying agent, anencapsulating agent, a flavorant, and a colorant. The choice of carrier can be selected with regard to the intended method of feed / food / nutraceutical / pharmaceutical application, route of administration, and standard practices. See Hardee and Baggot (2021. Development and Formulation of Veterinary Dosage Forms. 2nd Ed. CRC Press. 504 pg.); Remington: The Science and Practice of Pharmacy. 23rd Ed. Elsevier); Embree et al. (U.S. Patent No. 10,448,657) and Blaser et al. (U.S. Patent No. 8,951,512), each of which is herein expressly incorporated by reference in their entirety.

[0080] The terms “microorganism” and “microbe” are used interchangeably herein and refer to any microorganism that is of the domain Bacteria, Eukarya, or Archaea. Microorganism types include without limitation, bacteria (e.g., mycoplasma, coccus, bacillus, rickettsia, spirillum), fungi (e.g., filamentous fungi, yeast), nematodes, protozoans, archaea, algae, dinoflagellates, viruses (e.g., bacteriophages), viroids and / or a combination thereof. Organism strains are subtaxons of organism types, and can be for example, a species, sub-species, subtype, genetic variant, pathovar, or serovar of a particular microorganism.

[0081] As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures. However, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.

[0082] As used herein, “microbial composition” refers to a composition comprising one or more microorganisms of the present disclosure.

[0083] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.

[0084] As used herein, “microbiome” refers to the collection of microorganisms that inhabit the digestive tract or gastrointestinal tract of an animal (including the rumen if said animal is a ruminant) and the microorganisms’ physical environment (i.e., the microbiome has a biotic andphysical component). The microbiome is fluid and may be modulated by numerous naturally occurring and artificial conditions (e.g., change in diet, disease, antimicrobial agents, influx of additional microorganisms, etc.). The modulation of the microbiome can be achieved via administration of the compositions of the disclosure, can take the form of: (a) increasing or decreasing a particular Family, Genus, Species, or functional grouping of microbe (i.e. alteration of the biotic component of the rumen microbiome); and / or (b) increasing or decreasing any other physical parameter important for gastrointestinal health (e.g., volatile fatty acids or pH).

[0085] As used herein, “probiotic” refers to a substantially pure microbe (i.e., a single isolate) or a mixture of desired microorganisms, and may also include any additional components that can be administered to a mammal or a bird for restoring microbiota. Probiotics or microbial inoculant compositions of the invention may be administered with an agent to allow the microorganisms to survive the environment of the gastrointestinal tract, i.e., to resist low pH and to grow in the gastrointestinal environment. In some embodiments, the present compositions (e.g., microbial compositions) are probiotics in some aspects.

[0086] The term “growth medium” as used herein, is any medium which is suitable to support growth of a microbe. By way of example, the media may be natural or artificial including gastrin supplemental agar, LB media, blood serum, and tissue culture gels. It should be appreciated that the media 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. The medium may be amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and / or selection of specific groups of microorganisms. For example, antibiotics (such as penicillin) or sterilants (for example, quaternary ammonium salts and oxidizing agents) could be present and / or the physical conditions (such as salinity, nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium, and micronutrients such as cobalt and magnesium), pH, and / or temperature) could be amended.

[0087] As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of interest, as compared to a control group, or as compared to a known average quantity associated with the characteristic in question. For example, “improved” milk production associated with application of a beneficial microbe, or ensemble, of the disclosure can be demonstrated by comparing the milk produced by an ungulate treated by themicroorganisms taught herein to the milk of an untreated ungulate. In the present disclosure, “improved” does not necessarily demand that the data be statistically significant (i.e. p < 0.05); rather, any quantifiable difference demonstrating that one value (e.g. the average treatment value) is different from another (e.g. the average control value) can rise to the level of “improved.”

[0088] As used herein, “inhibiting and suppressing” and like terms should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.

[0089] As used herein, the term “trait” refers to a characteristic or phenotype. Examples of desirable traits to be modulated using the compositions and methods described herein include, but are not limited to: milk production, milk quantity, milk quality, meat quality, bird digestive chemistry, mammal digestive chemistry, ruminant digestive chemistry, weight, musculature, efficiency of feed utilization and digestibility, fecal output, methane emissions, prevention of colonization of pathogenic microbes, gastrointestinal health, performance, and clearance of pathogenic microbes.

[0090] As used herein, the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar, or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar, or strain. For purposes of this disclosure homologous sequences are compared. “Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and / or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined usingsoftware programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0091] As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, and / or insertion, as is well understood in the art. For example, mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.

[0092] As used herein, the “batch size” refers to the total mass of material (e.g., the total mass of all carriers, preserved strains, etc.). The “batch amount” refers to the amount of a particular preserved strain that must be added to the batch in order to achieve a desired dose. For example, if the desired dose is 1 CFU / gram, a 5-gram batch size will require a batch amount of 1 gram of Microbe 1 if microbe material is 5 CFU / gram. In some embodiments, the batch amount for a strain is determined as a function of desired batch size and the viability / unit. In some embodiments, the batch amount of a water activity scavenger or a diluent can be determined, for example, as a function of desired batch size and the determined batch amount of each preserved strain.

[0093] As used herein “shelf-stable” refers to a functional attribute and new utility acquired by the microorganisms formulated according to the disclosure, which enable said microorganisms to exist in a useful / active state outside of their natural environment (i.e. a markedly different characteristic). Thus, shelf-stable is a functional attribute created by the formulations / compositions of the disclosure and denoting that the microbe formulated into a shelf-stable composition can exist outside the natural environment and under refrigerated and or ambient conditions for a period of time that can be determined depending upon the particular formulation utilized, but in general means that the microorganisms can be formulated to exist in a composition that is stable under refrigerated and or ambient conditions for at least a few days and generally at least one week.

[0094] The term “melting temperature” is the thermodynamic transition point of a solid into its liquid phase. For pure and homogeneous materials, the melting temperature is determinedfrom the onset temperature. In contrast, for non-homogenous materials, the peak temperature is commonly used, since such materials consist of a range of molecules of different sizes.

[0095] The term “high-melting material” as used herein refers to a product characterized with an onset melting temperature of at least 50ºC or a peak melting temperature of at least 50ºC.

[0096] The term “onset temperature” of a meltable material as used herein refers to the extrapolated onset temperature. The onset temperature is the intersection point of the extrapolated baseline and the inflectional tangent at the beginning of the melting peak. The baseline and the inflectional tangent are determined from a temperature-dependent heat flow signal. In some embodiments, the onset temperature is measured by thermoanalytical differential scanning calorimetry (DSC). In some embodiments, the onset temperature for pure and / or homogenous materials is the melting temperature.

[0097] The term “peak temperature” of a meltable material refers to the point at which the largest deviation of the heat flow signal from the virtual baseline is measured in the thermal analysis of the meltable material. In some embodiments, the peak temperature is measured by thermoanalytical differential scanning calorimetry (DSC). In some embodiments, pure homogenous materials are completely melted at the peak temperature.

[0098] The term “end temperature” of a meltable material as used herein refers to the extrapolated end temperature. The end temperature is the designed intersection point of the extrapolated baseline and the inflectional tangent at the end of the melting peak. The baseline and the inflectional tangent are determined from the temperature-dependent heat flow signal. In some embodiments, the end temperature is measured by thermoanalytical differential scanning calorimetry (DSC). In some embodiments, non-homogenous materials (e.g., polymers and waxes) are completely melted at the end temperature. In some embodiments, the high-melting carrier is a wax product.

[0099] As disclosed herein, the terms “microencapsulation”, “encapsulation”, “entrapment”, “enrobing” and “immobilization” are used interchangeably.

[0100] As used herein, the term “cold-melt extrusion” refers to a process that compounds an active ingredient and a meltable inactive ingredient by mechanical means (e.g., a series of blending, kneading, and extruding processes) in an industrial extruder at temperatures below the melting point of the meltable inactive ingredient. In some embodiments, the process temperature is maintained at least 5ºC below the onset melting point of the inactive ingredient.

[0101] In some embodiments, the term “porous material” refers to a solid material containing pores and channels which are typically filled with air but may be void, free of any fluid. The skeletal portion of a porous material is referred to as a matrix. The matrix is a continuous solid phase that surrounds, and thereby protects, some discontinuously dispersed microorganism- containing particles within the matrix. Penetration of digestive fluids such as the fluid in the rumen or the fluid in the intestine of an animal may result in at least partial dissolution or dispersion, and release of the particles and other constituents of the matrix. Microorganisms of the Present Disclosure

[0102] In some embodiments, the present disclosure provides methods of manufacturing microorganism compositions using a cold-melt extrusion process. The target population may be any microorganism suitable for the manufacturing methods described herein. In some embodiments, the microorganism is a “native” or “naturally occurring” microorganism. In some embodiments, the microorganism is genetically modified. As used herein, the term “microorganism” includes, but is not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic fungi, protists, and viruses. For example, in some embodiments, the microorganism is a species from the genera of: Ruminococcus, Butyrivibrio, Clostridium, Bacillus, Lactobacillus, Succinivibrio, Chordicoccus, Escherichia, Streptococcus, Hungatella, Atlantibacter, Enterococcus, Bacteroides, Megamonas, Fusobacterium, Collinsella, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, Sharpea, and Phyllosticta. In some embodiments, the microorganism is a species belonging to the family of Lachnospiraceae, and / or the order of Saccharomycetales.

[0103] The domain of Eukarya comprises eukaryotic organisms, which are defined by membrane-bound organelles, such as the nucleus. Protozoa are unicellular eukaryotic organisms. All multicellular organisms are eukaryotes, including animals, plants, and fungi. The eukaryotes have been classified into four kingdoms: Protista, Plantae, Fungi, and Animalia. However, several alternative classifications exist. Another classification divides Eukarya into six kingdoms: Excavata (various flagellate protozoa); amoebozoa (lobose amoeboids and slime filamentous fungi); Opisthokonta (animals, fungi, choanoflagellates); Rhizaria (Foraminifera, Radiolaria, and various other amoeboid protozoa); Chromalveolata (Stramenopiles (brown algae, diatoms), Haptophyta, Cryptophyta (or cryptomonads), andAlveolata); Archaeplastida / Primoplantae (Land plants, green algae, red algae, and glaucophytes).

[0104] Microorganisms for manufacturing by the methods described herein can also be fungi. Fungi are microorganisms that are predominant in microbial communities and include yeasts and filamentous fungi as well as the familiar mushrooms. Fungal cells have cell walls that contain glucans and chitin, a unique feature of these organisms. The fungi form a single group of related organisms, named the Eumycota that share a common ancestor. The kingdom Fungi has been estimated at 1.5 million to 5 million species, with about 5% of these having been formally classified. The cells of most fungi grow as tubular, elongated, and filamentous structures called hyphae, which may contain multiple nuclei. Some species grow as unicellular yeasts that reproduce by budding or binary fission. The major phyla (sometimes called divisions) of fungi have been classified mainly on the basis of characteristics of their sexual reproductive structures. Currently, seven phyla are proposed: Microsporidia, Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Glomeromycota, Ascomycota, and Basidiomycota.

[0105] Microorganisms for manufacturing by the methods described herein can also be viruses. A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms in the domains of Eukarya, Bacteria, and Archaea. Virus particles (known as virions) consist of two or three parts: (i) the genetic material which can be either DNA or RNA; (ii) a protein coat that protects these genes; and in some cases (iii) an envelope of lipids that surrounds the protein coat when they are outside a cell. Seven orders have been established for viruses: the Caudovirales, Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales, and Tymovirales. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). In addition, ssRNA viruses may be either sense (+) or antisense (−). This classification places viruses into seven groups: I: dsDNA viruses (such as Adenoviruses, Herpesviruses, Poxviruses); II: (+) ssDNA viruses (such as Parvoviruses); III: dsRNA viruses (such as Reoviruses); IV: (+)ssRNA viruses (such as Picornaviruses, Togaviruses); V: (−)ssRNA viruses (such as Orthomyxoviruses, Rhabdoviruses); VI: (+)ssRNA-RT viruses with DNA intermediate in life-cycle (such as Retroviruses); VII: dsDNA-RT viruses (such as Hepadnaviruses).

[0106] Microorganisms for manufacturing by the methods described herein can also be viroids. Viroids are the smallest infectious pathogens known, consisting solely of short strands of circular, single-stranded RNA without protein coats. They are mostly plant pathogens, some of which are of economic importance. Viroid genomes are extremely small in size, ranging from about 246 to about 467 nucleobases.

[0107] In some embodiments, the microorganisms of the present disclosure are obtained from animals (e.g., mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air, water (e.g., marine, freshwater, wastewater sludge), sediment, oil, plants (e.g., roots, leaves, stems), agricultural products, and extreme environments (e.g., acid mine drainage or hydrothermal systems). In some embodiments, the microorganisms are obtained from the rumen of animals. In some embodiments, the microorganisms are obtained from the intestines of animals. In some embodiments, the microorganisms are obtained from animal feces. In some embodiments, the microorganisms are obtained from marine or freshwater environments such as an ocean, river, or lake. In some embodiments, the microorganisms are obtained from the surface of the body of water, or any depth of the body of water (e.g., a deep-sea sample).

[0108] In some embodiments, the microorganisms of the present disclosure are isolated in substantially pure or mixed cultures. They may be concentrated, diluted, or provided in natural concentrations in which they are found in the source material.

[0109] In some embodiments, the microorganisms are used in a crude form, in which they are not isolated from the source material in which they naturally reside. For example, the microorganisms are provided in combination with the source material in which they reside; for example, fecal matter, cud, or other composition found in the gastrointestinal tract. In some embodiments, the source material includes one or more species of microorganisms.

[0110] In some embodiments, a mixed population of microorganisms is used in the methods of the present disclosure. In embodiments of the disclosure where the microorganisms are isolated from a source material (for example, the material in which they naturally reside), any one or a combination of standard techniques which will be readily known to skilled persons may be used. However, by way of example, these in general employ processes by which a solid or liquid culture of a single microorganism can be obtained in a substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilutive isolation into a liquid microbial growth medium. These processes may include isolation from dry material, liquid suspension, slurries, or homogenates in which the material is spreadin a thin layer over an appropriate solid gel growth medium, or serial dilutions of the material made into a sterile medium and inoculated into liquid or solid culture media.

[0111] In some embodiments, the material containing the microorganisms may be pre-treated prior to the isolation process in order to multiply or enrich all microorganisms in the material. Microorganisms can then be isolated from the enriched materials.

[0112] The microorganisms subjected to the manufacturing methods described herein can be derived from any sample type that includes a microbial community. For example, samples for use with the methods provided herein encompass without limitation, an animal sample (e.g., mammal, reptile, bird), soil, air, water (e.g., marine, freshwater, wastewater sludge), sediment, oil, plant, agricultural product, agricultural plant, agricultural soil (e.g., rhizosphere) and extreme environmental sample (e.g., acid mine drainage, hydrothermal systems). In the case of marine or freshwater samples, the sample can be from the surface of the body of water, or any depth of the body water, e.g., a deep-sea sample. The water sample, in one embodiment, is an ocean, river, or lake sample.

[0113] In some embodiments, the animal sample is a body fluid. In some embodiments, the animal sample is a tissue sample. Non-limiting animal samples include tooth, perspiration, fingernail, skin, hair, feces, urine, semen, mucus, saliva, and gastrointestinal tract. The animal sample can be, for example, a human, primate, bovine, swine, porcine, canine, feline, rodent (e.g., mouse or rat), equine, or bird sample. In some embodiments, the bird sample comprises a sample from one or more chickens. In some embodiments, the sample is a human sample. The human microbiome comprises a collection of microorganisms found on the surface and deep layers of skin, in mammary glands, saliva, oral mucosa, conjunctiva, and gastrointestinal tract. The microorganisms found in the microbiome include bacteria, fungi, protozoa, viruses, and archaea. Different parts of the body exhibit varying diversity of microorganisms. In some embodiments. the quantity and type of microorganisms signal a healthy or diseased state for an individual. The number of bacteria taxa are in the thousands, and viruses may be as abundant. The bacterial composition for a given site on a body varies from person to person, not only in type, but also in abundance or quantity.

[0114] In some embodiments, the sample is a ruminal sample. Ruminants such as cattle rely upon diverse microbial communities to digest their feed. These animals have evolved to use feed with poor nutritive value by having a modified upper digestive tract (reticulorumen or rumen) where feed is held while it is fermented by a community of anaerobic microorganisms.The rumen microbial community is very dense, with about 3 × 1010microbial cells per milliliter. Anaerobic fermenting microorganisms dominate in the rumen. The rumen microbial community includes members of all three domains of life: Bacteria, Archaea, and Eukarya. Ruminal fermentation products are required by their respective hosts for body maintenance and growth, as well as milk production (van Houtert (1993). Anim. Feed Sci. Technol.43, pp.189- 225; Bauman et al. (2011). Annu. Rev. Nutr.31, pp.299-319; each incorporated by reference in its entirety for all purposes). Moreover, milk yield and composition has been reported to be associated with ruminal microbial communities (Sandri et al. (2014). Animal 8(4), pp. 572- 579; Palmonari et al. (2010). J. Dairy Sci.93(1), pp.279-287; each incorporated by reference in its entirety for all purposes). Ruminal samples, in one embodiment, are collected via the process described in Jewell et al. (2015). Appl. Environ. Microbiol. 81(14), pp. 4697-4710, incorporated by reference herein in its entirety for all purposes.

[0115] In some embodiments, the sample is a soil sample (e.g., bulk soil or rhizosphere sample). It has been estimated that 1 gram of soil contains tens of thousands of bacterial taxa, and up to 1 billion bacteria cells as well as about 200 million fungal hyphae (Wagg et al. (2014). Proc Natl. Acad. Sci. USA 111(14), pp. 5266-5270, incorporated by reference in its entirety for all purposes). Bacteria, actinomycetes, fungi, algae, protozoa, and viruses are all found in soil. Soil microorganism community diversity has been implicated in the structure and fertility of the soil microenvironment, nutrient acquisition by plants, plant diversity and growth, as well as the cycling of resources between above- and below-ground communities. Accordingly, assessing the microbial contents of a soil sample over time and the co-occurrence of active microorganisms (as well as the number of the active microorganisms) provides insight into microorganisms associated with an environmental metadata parameter such as nutrient acquisition and / or plant diversity.

[0116] In some embodiments, the soil sample is a rhizosphere sample, i.e., the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms. The rhizosphere is a densely populated area in which elevated microbial activities have been observed and plant roots interact with soil microorganisms through the exchange of nutrients and growth factors (San Miguel et al. (2014) Appl. Microbiol. Biotechnol.98(9):4257-66. DOI 10.1007 / s00253-014-5545-6, incorporated by reference in its entirety for all purposes). As plants secrete many compounds into the rhizosphere, analysis of the organism types in the rhizosphere may be useful in determining features of the plants which grow therein.

[0117] In some embodiments, the sample is a marine or freshwater sample. Ocean water contains up to one million microorganisms per milliliter and several thousand microbial types. These numbers may be an order of magnitude higher in coastal waters with their higher productivity and higher load of organic matter and nutrients. Marine microorganisms are crucial for the functioning of marine ecosystems; maintaining the balance between produced and fixed carbon dioxide; production of more than 50% of the oxygen on Earth through marine phototrophic microorganisms such as Cyanobacteria, diatoms and pico- and nanophytoplankton; providing novel bioactive compounds and metabolic pathways; ensuring a sustainable supply of seafood products by occupying the critical bottom trophic level in marine foodwebs. Organisms found in the marine environment include viruses, bacteria, archaea, and some eukarya. Marine viruses may play a significant role in controlling populations of marine bacteria through viral lysis. Marine bacteria are important as a food source for other small microorganisms as well as being producers of organic matter. Archaea found throughout the water column in the ocean are pelagic Archaea and their abundance rivals that of marine bacteria.

[0118] In some embodiments, the sample comprises a sample from an extreme environment, i.e., an environment that harbors conditions that are detrimental to most life on Earth. Organisms that thrive in extreme environments are called extremophiles. Though the domain Archaea contains well-known examples of extremophiles, the domain bacteria can also have representatives of these microorganisms. Extremophiles include: acidophiles which grow at pH levels of 3 or below; alkaliphiles which grow at pH levels of 9 or above; anaerobes such as Spinoloricus Cinzia which does not require oxygen for growth; cryptoendoliths which live in microscopic spaces within rocks, fissures, aquifers and faults filled with groundwater in the deep subsurface; halophiles which grow in about at least 0.2M concentration of salt; hyperthermophiles which thrive at high temperatures (about 80-122°C) such as found in hydrothermal systems; hypoliths which live underneath rocks in cold deserts; lithoautotrophs such as Nitrosomonas europaea which derive energy from reduced mineral compounds like pyrites and are active in geochemical cycling; metallotolerant organisms which tolerate high levels of dissolved heavy metals such as copper, cadmium, arsenic and zinc; oligotrophs which grow in nutritionally limited environments; osmophiles which grow in environments with a high sugar concentration; piezophiles (or barophiles) which thrive at high pressures such as found deep in the ocean or underground; psychrophiles / cryophiles which survive, grow and / or reproduce at temperatures of about -15°C or lower; radioresistant organisms which are resistantto high levels of ionizing radiation; thermophiles which thrive at temperatures between 45- 122°C; xerophiles which can grow in extremely dry conditions. Polyextremophiles are organisms that qualify as extremophiles under more than one category and include thermoacidophiles (prefer temperatures of 70-80°C and pH between 2 and 3). The Crenarchaeota group of Archaea includes the thermoacidophiles.

[0119] In some embodiments, the sample includes microorganisms from one or more domains. For example, in one embodiment, the sample comprises a heterogeneous population of bacteria and / or fungi (also referred to herein as bacterial or fungal strains). For example, the one or more microorganisms can be from the domain Bacteria, Archaea, Eukarya, or a combination thereof. Bacteria and Archaea are prokaryotic, having a very simple cell structure with no internal organelles. Bacteria can be classified into gram positive / no outer membrane, gram negative / outer membrane present and ungrouped phyla. Archaea constitute a domain or kingdom of single-celled microorganisms. Although visually similar to bacteria, Archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes, notably the enzymes involved in transcription and translation. Other aspects of archaeal biochemistry are unique, such as the presence of ether lipids in their cell membranes. The Archaea are divided into four recognized phyla: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota. Microbial Strains

[0120] As used herein, “isolate”, “isolated”, “isolated microbe”, and like terms, are intended to mean that the one or more microorganisms have been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, animal tissue). Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an acceptable carrier.

[0121] In certain aspects of the disclosure, the isolated microorganisms exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microorganism, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual microbe. The culture can contain varying concentrations of said microorganism. Thepresent disclosure notes that isolated and biologically pure microorganisms often necessarily differ from less pure or impure materials. See, e.g. In re Bergstrom, 427 F.2d 1394 (CCPA 1970) (discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F.95, 103 (C.C.S.D.N.Y.1911) (Learned Hand discussing purified adrenaline), aff’d in part, rev’d in part, 196 F.496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microorganisms from those microorganisms existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microorganisms), incorporated herein by reference.

[0122] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from taxonomic families of Clostridiaceae, Ruminococcaceae, Lachnospiraceae, Acidaminococcaceae, Peptococcaceae, Porphyromonadaceae, Prevotellaceae, Neocallimastigaceae, Saccharomycetaceae, Phaeosphaeriaceae, Erysipelotrichia, Anaerolinaeceae, Atopobiaceae, Botryosphaeriaceae, Eubacteriaceae, Acholeplasmataceae, Succinivibrionaceae, Lactobacillaceae, Selenomonadaceae, Burkholderiaceae, Coprobacillaceae, and Streptococcaceae.

[0123] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Clostridiaceae, which includes but is not limited to, Acetanaerobacterium, Acetivibrio, Acidaminobacter, Alkaliphilus, Anaerobacter, Anaerostipes, Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus, Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, Candidatus Arthromitus, Clostridium, Coprobacillus, Dorea, Ethanologenbacterium, Faecalibacterium, Garciella, Guggenheimella, Hespellia, Linmingia, Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia, Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium, Thermobrachium, Thermohalobacter, and Tindallia.

[0124] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of familyRuminococcaceae, which includes but is not limited to, Ruminococcus, Acetivibrio, Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger, Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens, Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, and Candidadus Soleaferrea.

[0125] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Lachnospiraceae, which includes but is not limited to, Butyrivibrio, Roseburia, Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella, Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium, Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia, Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella, Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter, Acetatifactor, and Eisenbergiella.

[0126] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Acidaminococcaceae, which includes but is not limited to, Acidaminococcus, Phascolarctobacterium, Succiniclasticum, and Succinispira.

[0127] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Peptococcaceae, which includes but is not limited to, Desulfotomaculum, Peptococcus, Desulfitobacterium, Syntrophobotulus, Dehalobacter, Sporotomaculum, Desulfosporosinus, Desulfonispora, Pelotomaculum, Thermincola, Cryptanaerobacter, Desulfitibacter, Candidatus Desulforudis, Desulfurispora, and Desulfitospora.

[0128] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Porphyromonadaceae, which includes but is not limited to, Porphyromonas, Dysgonomonas, Tannerella, Odoribacter, Proteiniphilum, Petrimonas, Paludibacter, Parabacteroides, Barnesiella, Candidatus Vestibaculum, Butyricimonas, Macellibacteroides, and Coprobacter.

[0129] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Anaerolinaeceae which includes but is not limited to, Anaerolinea, Bellilinea, Leptolinea, Levilinea, Longilinea, Ornatilinea, and Pelolinea.

[0130] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Atopobiaceae, which includes but is not limited to, Atopbium and Olsenella.

[0131] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Eubacteriaceae, which includes but is not limited to, Acetobacterium, Alkalibacter, Alkalibaculum, Aminicella, Anaerofustis, Eubacterium, Garciella, and Pseudoramibacter.

[0132] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Acholeplasmataceae. which includes but is not limited to, Acholeplasma.

[0133] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Succinivibrionaceae. which includes but is not limited to, Anaerobiospirillum, Ruminobacter, Succinatimonas, Succinimonas, and Succinivibrio.

[0134] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Lactobacillaceae, which includes but is not limited to, Lactobacillus, Paralactobacillus, Pediococcus, and Sharpea.

[0135] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Selenomonadaceae, which includes but is not limited to, Anaerovibrio, Centipeda, Megamonas, Mitsuokella, Pectinatus, Propionispira, Schwartzia, Selenomonas, and Zymophilus.

[0136] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Burkholderiaceae, which includes but is not limited to, Burkholderia, Chitinimonas, Cupriavidus, Lautropia, Limnobacter, Pandoraea, Paraburkholderia, Paucimonas, Polynucleobacter, Ralstonia, Thermothrix, and Wautersia.

[0137] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of familyStreptococcaceae, which includes but is not limited to, Lactococcus, Lactovum, and Streptococcus.

[0138] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Anaerolinaeceae, which includes but is not limited to, Aestuariimicrobium, Arachnia, Auraticoccus, Brooklawnia, Friedmanniella, Granulicoccus, Luteococcus, Mariniluteicoccus, Microlunatus, Micropruina, Naumannella, Propionibacterium, Propionicicella, Propioniciclava, Propioniferax, Propionimicrobium, and Tessaracoccus.

[0139] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Prevotellaceae, which includes but is not limited to, Paraprevotella, Prevotella, hallella, Xylanibacter, and Alloprevotella.

[0140] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Neocallimastigaceae, which includes but is not limited to, Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces.

[0141] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Saccharomycetaceae, which includes but is not limited to, Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania (syn. Arxiozyma), Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, and Zygotorulaspora.

[0142] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Erysipelotrichaceae, which includes but is not limited to, Erysipelothrix, Solobacterium, Turicibacter, Faecalibaculum, Faecalicoccus, Faecalitalea, Holdemanella, Holdemania, Dielma, Eggerthia, Erysipelatoclostridium, Allobacterium, Breznakia, Bulleidia, Catenibacterium, Catenisphaera, and Coprobacillus.

[0143] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of familyPhaeosphaeriaceae, which includes but is not limited to, Barria, Bricookea, Carinispora, Chaetoplea, Eudarluca, Hadrospora, Isthmosporella, Katumotoa, Lautitia, Metameris, Mixtura, Neophaeosphaeria, Nodulosphaeria, Ophiosphaerella, Phaeosphaeris, Phaeosphaeriopsis, Setomelanomma, Stagonospora, Teratosphaeria, and Wilmia.

[0144] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Botryosphaeriaceae, which includes but is not limited to, Amarenomyces, Aplosporella, Auerswaldiella, Botryosphaeria, Dichomera, Diplodia, Discochora, Dothidothia, Dothiorella, Fusicoccum, Granulodiplodia, Guignardia, Lasiodiplodia, Leptodothiorella, Leptodothiorella, Leptoguignardia, Macrophoma, Macrophomina, Nattrassia, Neodeightonia, Neofusicocum, Neoscytalidium, Otthia, Phaeobotryosphaeria, Phomatosphaeropsis, Phyllosticta, Pseudofusicoccum, Saccharata, Sivanesania, and Thyrostroma.

[0145] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Candida, Vrystaatia, Orpinomyces, Neocallimastix, Sharpea, and Phyllosticta. In further embodiments, the disclosure provides compositions produced by the methods described herein comprising isolated microbial species belonging to the family of Lachnospiraceae, and / or the order of Saccharomycetales. In further embodiments, the disclosure provides compositions produced by the methods described herein comprising isolated microbial species of Candida xylopsoci, Vrystaatia aloeicola, and Phyllosticta capitalensis.

[0146] In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microorganisms selected from the genus of Clostridium, Succinivibrio, Caecomyces, Pichia, Butyrivibrio, Orpinomyces, Piromyces, Bacillus, Lactobacillus, Chordicoccus, Enterococcus, Escherichia, Streptococcus, Hungatella, Atlantibacter, Bacteroides, Fusobacterium, Collinsella, Megamonas, Prevotella, Syntrophococcus, Sharpea, or Ruminococcus. In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from the family of Lachnospiraceae. In some embodiments, the present disclosure provides compositions prepared by the methods described herein comprising isolated microbial species selected from genera of family Ruminococcaceae.

[0147] In some embodiments, the method comprises the use of cold-melt extrusion during processing of bacteria for the compositions described herein. In some embodiments, the bacteria are selected from the genus of: Clostridium, Succinivibrio, Butyrivibrio, Bacillus, Lactobacillus, Chordicoccus, Enterococcus, Escherichia, Streptococcus, Hungatella, Atlantibacter, Bacteroides, Fusobacterium, Collinsella, Megamonas, Prevotella, Syntrophococcus, Sharpea, or Ruminococcus.

[0148] In some embodiments, the method comprises the use of cold-melt extrusion during processing of fungi for the compositions described herein. In some embodiments, the fungi belong to the genus of: Caecomyces, Pichia, Orpinomyces, Piromyces, or Neocallimastix.

[0149] In some embodiments, the method comprises the use of cold-melt extrusion during the processing of genetically modified microbial strains for the compositions described herein. In some embodiments, the genetically modified or recombinant microorganisms comprise polynucleotide sequences which do not naturally occur in said microorganisms. In some embodiments, the microorganisms may comprise heterologous polynucleotides. In further embodiments, the heterologous polynucleotides may be operably linked to one or more polynucleotides native to the microorganisms.

[0150] In some embodiments, the heterologous polynucleotides may be reporter genes or selectable markers. In some embodiments, reporter genes may be selected from any of the family of fluorescence proteins (e.g., GFP, RFP, YFP, and the like), β-galactosidase, or luciferase. In some embodiments, selectable markers may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, aminoglycoside adenyltransferase, dihydrofolate reductase, acetolactase synthase, bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, and chloramphenicol acetyltransferase. In some embodiments, the heterologous polynucleotide may be operably linked to one or more promoter.

[0151] Microorganisms can be distinguished into a genus based on polyphasic taxonomy, which incorporates all available phenotypic and genotypic data into a consensus classification (Vandamme et al.1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996, 60:407-438). One accepted genotypic method for defining species is based on overall genomic relatedness, such that strains which share approximately 70% or more relatedness using DNA-DNA hybridization, with 5°C or less ΔTm (the difference in the melting temperature between homologous and heterologous hybrids), under standard conditions, are considered to be members of the same species. Thus, populations that sharegreater than the aforementioned 70% threshold can be considered to be variants of the same species. Another accepted genotypic method for defining species is to isolate marker genes of the present disclosure, sequence these genes, and align these sequenced genes from multiple isolates or variants. The microorganisms are interpreted as belonging to the same species if one or more of the sequenced genes share at least 97% sequence identity.

[0152] One approach is to observe the distribution of a large number of strains of closely related species in sequence space and to identify clusters of strains that are well resolved from other clusters. This approach has been developed by using the concatenated sequences of multiple core (housekeeping) genes to assess clustering patterns, and has been called multilocus sequence analysis (MLSA) or multilocus sequence phylogenetic analysis. MLSA has been used successfully to explore clustering patterns among large numbers of strains assigned to very closely related species by current taxonomic methods, to look at the relationships between small numbers of strains within a genus, or within a broader taxonomic grouping, and to address specific taxonomic questions. More generally, the method can be used to ask whether bacterial species exist – that is, to observe whether large populations of similar strains invariably fall into well-resolved clusters, or whether in some cases there is a genetic continuum in which clear separation into clusters is not observed.

[0153] In order to more accurately make a determination of genera, a determination of phenotypic traits, such as morphological, biochemical, and physiological characteristics can be made for comparison with a reference genus archetype. The colony morphology can include color, shape, pigmentation, production of slime, etc. Features of the cell are described as to shape, size, Gram reaction, extracellular material, presence of endospores, flagella presence and location, motility, and inclusion bodies. Biochemical and physiological features describe growth of the organism at different ranges of temperature, pH, salinity, and atmospheric conditions, growth in presence of different sole carbon and nitrogen sources. One of skill should be reasonably apprised as to the phenotypic traits that define the genera of the present disclosure.

[0154] In one embodiment, the microorganisms taught herein were identified utilizing 16S rRNA gene sequences and ITS sequences. It is known in the art that 16S rRNA contains hypervariable regions that can provide species / strain-specific signature sequences useful for bacterial identification, and that ITS sequences can also provide species / strain-specific signature sequences useful for fungal identification.

[0155] Phylogenetic analysis using the rRNA genes and / or ITS sequences are used to define “substantially similar” species belonging to common genera and also to define “substantially similar” strains of a given taxonomic species. Furthermore, physiological and / or biochemical properties of the isolates can be utilized to highlight both minor and significant differences between strains that could lead to advantageous behavior in ruminants.

[0156] Isolated microorganisms can be matched to their nearest taxonomic groups by utilizing classification tools of the Ribosomal Database Project (RDP) for 16s rRNA sequences and the User-friendly Nordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences. Examples of matching microorganisms to their nearest taxa may be found in Lan et al. (2012. PLOS one.7(3):e32491), Schloss and Westcott (2011. Appl. Environ. Microbiol.77(10):3219- 3226), and Koljalg et al. (2005. New Phytologist.166(3):1063-1068). The 16S or 18S rRNA sequences or ITS sequences are often used for making distinctions between species and strains, in that if one of the aforementioned sequences share less than a specified percent sequence identity from a reference sequence, then the two organisms from which the sequences were obtained are said to be of different species or strains. Comparisons may also be made with 23S rRNA sequences against reference sequences.

[0157] In some embodiments, the isolated microorganisms are identified by ribosomal nucleic acid sequences. Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNA genes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16S rRNA (16S rDNA) in the case of prokaryotes, have been the predominant target for the assessment of organism types and strains in a microbial community. However, the large subunit ribosomal RNA genes, 28S rDNAs, have been also targeted. rDNAs are suitable for taxonomic identification because: (i) they are ubiquitous in all known organisms; (ii) they possess both conserved and variable regions; (iii) there is an exponentially expanding database of their sequences available for comparison. In community analysis of samples, the conserved regions serve as annealing sites for the corresponding universal PCR and / or sequencing primers, whereas the variable regions can be used for phylogenetic differentiation. In addition, the high copy number of rDNA in the cells facilitates detection from environmental samples.

[0158] The internal transcribed spacer (ITS), located between the 18S rDNA and 28S rDNA, has also been targeted. The ITS is transcribed but spliced away before assembly of the ribosomes. The ITS region is composed of two highly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene. This rDNA operon occurs in multiple copies in genomes. Because theITS region does not code for ribosome components, it is highly variable. In some embodiments, the unique RNA marker can be an mRNA marker, an siRNA marker, or a ribosomal RNA marker.

[0159] The primary structure of major rRNA subunit 16S comprise a particular combination of conserved, variable, and hypervariable regions that evolve at different rates and enable the resolution of both very ancient lineages such as domains, and more modern lineages such as genera. The secondary structure of the 16S subunit include approximately 50 helices which result in base pairing of about 67% of the residues. These highly conserved secondary structural features are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analysis. Over the previous few decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

[0160] In some embodiments, a sequence identity of 94.5% or lower for two 16S rRNA genes is strong evidence for distinct genera, 86.5% or lower is strong evidence for distinct families, 82% or lower is strong evidence for distinct orders, 78.5% is strong evidence for distinct classes, and 75% or lower is strong evidence for distinct phyla. The comparative analysis of 16S rRNA gene sequences enables the establishment of taxonomic thresholds that are useful not only for the classification of cultured microorganisms but also for the classification of the many environmental sequences. Yarza et al.2014. Nature Rev. Micro.12:635-45).

[0161] Thus, one could consider microorganisms to be of the same species, if they share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence. Further, one could define microbial strains of a species, as those that share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

[0162] In one embodiment, microbial strains of the present disclosure include those that comprise polynucleotide 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 any one of SEQ ID NOs:1-34. In a further embodiment, microbial strains of the present disclosure include those that comprise polynucleotide 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 any one of SEQ ID NOs: 1-34.

[0163] Unculturable microorganisms often cannot be assigned to a definite species in the absence of a phenotype determination, the microorganisms can be given a candidatus designation within a genus provided their 16S or 18S rRNA sequences or ITS sequences subscribes to the principles of identity with known species.

[0164] Compositions of the present disclosure may include combinations of fungal spores and bacterial spores, fungal spores, and bacterial vegetative cells, fungal vegetative cells, and bacterial spores, fungal vegetative cells, and bacterial vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of spores. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria in the absence of fungi. In some embodiments, compositions of the present disclosure comprise fungi in the absence of bacteria.

[0165] Bacterial spores may include endospores and akinetes. Fungal spores may include statismospores, ballistospores, autospores, aplanospores, zoospores, mitospores, megaspores, microspores, meiospores, chlamydospores, urediniospores, teliospores, oospores, carpospores, tetraspores, sporangiospores, zygospores, ascospores, basidiospores, ascospores, and asciospores.

[0166] Non-limiting exemplary isolated microorganisms that can be cultivated and manufactured into a product according to the methods described herein are provided below in Table 1. The closest predicted taxonomy is listed in the first column, which was determined by BLAST. The strains listed below have been deposited in the labs of Native Microbials, Inc. since at least December 15, 2016. Table 1: Exemplary Microorganisms

[0167] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a bacteria and / or fungi with a sequence of at least 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% selected from any one of SEQ ID NOs: 1-34. In some embodiments, the composition comprises a bacteria and / or fungi comprising a sequence selected from any one of SEQ ID NOs: 1-34. In some embodiments, the composition comprises a bacteria and / or fungi consisting of a sequence selected from any one of SEQ ID NOs: 1-34.

[0168] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Butyrivibrio sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the composition comprises a Butyrivibrio sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 1. In some embodiments, the composition comprises Butyrivibrio sp. deposited as NRRL B-67347. In some embodiments, the Butyrivibrio sp. is Butyrivibrio fibrisolvens.

[0169] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises Butyrivibrio fibrisolvens comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the composition comprises Butyrivibrio fibrisolvens comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 1. In some embodiments, the composition comprises Butyrivibrio fibrisolvens deposited as NRRL B- 67347.

[0170] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Ruminococcus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the composition comprises a Ruminococcus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the composition comprises Ruminococcus sp. deposited as PTA-125917, NRRL B-67764, TSD-225, NCTC 14479.

[0171] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition Ruminococcus bovis comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the composition comprises Ruminococcus bovis comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 3.

[0172] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises Ruminococcus bovis comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 3. In some embodiments, the composition comprises Ruminococcus bovis comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 3. In some embodiments, the composition comprises Ruminococcus bovis deposited as PTA-125917, NRRL B-67764, TSD-225, NCTC 14479.

[0173] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 4. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 4. In some embodiments, thecomposition comprises a Clostridium sp. deposited as NRRL B-67248. In some embodiments, the Clostridium sp. is Clostridium beijerinckii.

[0174] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Pichia sp. comprises an ITS sequence comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the composition comprises a Pichia sp. comprising an ITS sequence comprising or consisting of SEQ ID NO: 5. In some embodiments, the composition comprises a Pichia sp. deposited as NRRL Y-67249. In some embodiments, the Pichia sp. is Pichia kudriavzevii.

[0175] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Bacillus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 6. In some embodiments, the composition comprises a Bacillus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 6. In some embodiments, the composition comprises a Bacillus sp. deposited as NRRL B-67266. In some embodiments, the Bacillus sp. is Bacillus subtilis. In some embodiments, the Bacillus sp. is Bacillus amyloliquefaciens.

[0176] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 7. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 7. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67691.

[0177] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Hungatella sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 7. In some embodiments, the composition comprises a Hungatella sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 7. In some embodiments, the composition comprises a Hungatella sp. deposited as NRRL B-67691. In some embodiments, the Clostridium sp. is Hungatella xylanolytica.

[0178] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNAsequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 8. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 8. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67689. In some embodiments, the Clostridium sp. is Clostridium beijerinckii. In some embodiments, the Clostridium sp. is Clostridium sensu stricto.

[0179] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67689. In some embodiments, the Clostridium sp. is Clostridium beijerinckii. In some embodiments, the Clostridium sp. is Clostridium sensu stricto.

[0180] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Prevotella sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 11. In some embodiments, the composition comprises a Prevotella sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 11. In some embodiments, the Prevotella sp. is Prevotella albensis.

[0181] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Prevotella sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 12. In some embodiments, the composition comprises a Prevotella sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 12. In some embodiments, the composition comprises a Prevotella sp. deposited as NRRL B-67552. In some embodiments, the Prevotella sp. is Prevotella albensis.

[0182] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Succinivibrio sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 13. In some embodiments, the composition comprises a Succinivibrio sp.comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 13. In some embodiments, the Succinivibrio sp. is Succinivibrio dextrinosolvens.

[0183] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Succinivibrio sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 14. In some embodiments, the composition comprises a Succinivibrio sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 14. In some embodiments, the composition comprises a Succinivibrio sp. deposited as NRRL B-67550. In some embodiments, the Succinivibrio sp. is Succinivibrio dextrinosolvens.

[0184] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Chordicoccus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 15. In some embodiments, the composition comprises a Chordicoccus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 15. In some embodiments, the Chordicoccus sp. is Chordicoccus furentiruminis.

[0185] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Chordicoccus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the composition comprises a Chordicoccus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the composition comprises a Chordicoccus sp. deposited as NRRL B-67553 or NCTC-14480. In some embodiments, the Chordicoccus sp. is Chordicoccus furentiruminis.

[0186] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 18. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 18. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67941. In some embodiments, the Clostridium sp. is Clostridium butyricum.

[0187] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Escherichia sp. comprising a 16S rRNAsequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 19. In some embodiments, the composition comprises a Escherichia sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 19. In some embodiments, the composition comprises a Escherichia sp. deposited as NRRL B-67942. In some embodiments, the Escherichia sp. is Escherichia coli.

[0188] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a bacteria comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 19. In some embodiments, the composition comprises a bacteria comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 19. In some embodiments, the composition comprises a bacteria deposited as NRRL B-67942.

[0189] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Streptococcus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 20. In some embodiments, the composition comprises a Streptococcus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 20. In some embodiments, the composition comprises a Streptococcus sp. deposited as NRRL B-67943. In some embodiments, the Streptococcus sp. is Streptococcus equinus.

[0190] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 21. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 21. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67940. In some embodiments, the Clostridium sp. is Clostridium maximum.

[0191] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Atlantibacter sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the composition comprises a Atlantibacter sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 22. In some embodiments, the composition comprises a Atlantibacter sp. deposited as NRRL B-67944. In some embodiments, the Atlantibacter sp. is Atlantibacter hermannii.

[0192] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Enterococcus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the composition comprises a Enterococcus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 23. In some embodiments, the composition comprises a Enterococcus sp. deposited as NRRL B-67972.

[0193] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 24. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 24. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67973.

[0194] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Bacteroides sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 25. In some embodiments, the composition comprises a Bacteroides sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 25. In some embodiments, the composition comprises a Bacteroides sp. deposited as NRRL B-67974. In some embodiments, the Bacteroides sp. is Bacteroides dorei.

[0195] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Megamonas sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 26. In some embodiments, the composition comprises a Megamonas sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 26. In some embodiments, the composition comprises a Megamonas sp. deposited as NRRL B-67975. In some embodiments, the Megamonas sp. is Megamonas funiformis.

[0196] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Bacteroides sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 27. In some embodiments, the composition comprises a Bacteroides sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 27. In some embodiments, the composition comprises a Bacteroides sp. deposited as NRRL B-67976.

[0197] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 28. In some embodiments, the composition comprises a Clostridium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 28. In some embodiments, the composition comprises a Clostridium sp. deposited as NRRL B-67977. In some embodiments, the Clostridium sp. is Clostridium hiranonis.

[0198] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Fusobacterium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 29. In some embodiments, the composition comprises a Fusobacterium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 29. In some embodiments, the composition comprises a Fusobacterium sp. deposited as NRRL B-67987.

[0199] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Collinsella sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 30. In some embodiments, the composition comprises a Collinsella sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 30. In some embodiments, the composition comprises a Collinsella sp. deposited as NRRL B-67986.

[0200] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Fusobacterium sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 31. In some embodiments, the composition comprises a Fusobacterium sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 31. In some embodiments, the composition comprises a Fusobacterium sp. deposited as NRRL B-67985.

[0201] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Sharpea sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 32. In some embodiments, the composition comprises a Sharpea sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 32. In some embodiments, the Sharpea sp. is Sharpea azabuensis.

[0202] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Ruminococcus sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the composition comprises a Ruminococcus sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 33. In some embodiments, the Ruminococcus sp. is Ruminococcus bromii.

[0203] In some embodiments, the disclosure provides compositions produced by the methods described herein , wherein the composition comprises a Lachnospiracea sp. comprising a 16S rRNA sequence with at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 34. In some embodiments, the composition comprises a Lachnospiracea sp. comprising a 16S rRNA sequence comprising or consisting of SEQ ID NO: 34. In some embodiments, the Lachnospiracea sp. is Lachnospiracea_incertae_sedis.

[0204] In some embodiments, the disclosure provides compositions produced by the methods described herein and comprising at least one microorganism, wherein the at least one microorganism is disclosed in one or more of the following: U.S. Pub. Nos. 2018 / 0310592, 2018 / 0333443, 2018 / 0223325, 2022 / 0174992, 2022 / 0265732, 2022 / 0386647, and PCT Pub. Nos. 2016 / 210251, 2017 / 120495, 2017 / 181203, 2018 / 201049, 2019 / 079629, 2018 / 056563, 2020 / 042148, 2020 / 227442, 2021 / 163212, 2021 / 011662, 2021 / 202804, 2022 / 226367, and 2022 / 081992 (each being herein expressly incorporated by reference for all purposes). Microbial Ensembles

[0205] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises microbial ensembles comprising a combination of at least two microorganisms. In certain embodiments, the ensembles of the present disclosure comprise two microorganisms, three microorganisms, four microorganisms, five microorganisms, six microorganisms, seven microorganisms, eight microorganisms, nine microorganisms, ten microorganisms, or more. Said microorganisms of the ensembles are different microbial species, or different strains of a microbial species.

[0206] As used herein, “microbial ensemble” refers to a composition comprising one or more active microorganisms that does not naturally exist in a naturally occurring environment and / or at ratios or amounts that do not exist in a nature. For example, a microbial ensemble (also synthetic ensemble and / or bioensemble) or aggregate could be formed from one or more isolated microbe strains, along with an appropriate medium or carrier. Microbial ensemblescan be applied or administered to a target, such as a target environment, population, individual, animal, and / or the like.

[0207] In certain embodiments of the disclosure, microbial ensembles are based on one or more isolated microorganisms that exist as isolated and biologically pure cultures.

[0208] In some embodiments, the disclosure provides compositions produced by the methods described herein and comprising microbial ensembles, wherein the microbial ensemble comprises at least two isolated microbial species selected the genera of: Clostridium, Succinivibrio, Caecomyces, Pichia, Butyrivibrio, Orpinomyces, Piromyces, Bacillus, Lactobacillus, Chordicoccus, Enterococcus, Escherichia, Streptococcus, Hungatella, Atlantibacter, Bacteroides, Fusobacterium, Collinsella, Megamonas, Prevotella, Syntrophococcus, Sharpea, or Ruminococcus.

[0209] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Butyrivibrio sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 1; a Ruminococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3; a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 4; and / or a Pichia sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO:1; a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 4 and / or a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 5.

[0210] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 4 and / or a Pichia sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 4 and / or a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 5.

[0211] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 7; and / or a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 9 and / or SEQ ID NO: 10. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 7; and / or a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0212] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Prevotella sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 12; a Succinivibrio sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 14; and / or a Chordicoccus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Prevotella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 12; a Succinivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 14; and / or a Chordicoccus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17.

[0213] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 18; a bacteria comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 19; a Streptococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 20; a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 21; and / or an Atlantibacter sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 18; a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 19; a Streptococcus sp. comprising a 16S nucleic acid sequence of SEQ IDNO: 20; a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 21; and / or an Atlantibacter sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 22.

[0214] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: an Enterococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 23; a Megamonas sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 26; and / or a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 28. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Enterococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 23; a Megamonas sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 26; and / or a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28.

[0215] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: an Enterococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 23; a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 24; a Bacteroides sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 25; a Megamonas sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 26; a Bacteroides sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 27; a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 28; a Fusobacterium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 29; a Collinsella sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 30; and / or a Fusobacterium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 31. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Enterococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 23; a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 24; a Bacteroides sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 25; a Megamonas sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 26; a Bacteroides sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 27; a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; aFusobacterium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 29; a Collinsella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 30; and / or a Fusobacterium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 31.

[0216] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Butyrivibrio sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 1; a Ruminococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3; a Clostridium sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 4; a Pichia sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 5; a Sharpea sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 32; a Ruminococcus sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 33; and / or a Lachnospiracea sp. comprising at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO: 34. In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises: a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO:1; a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 4; a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 5; a Sharpea sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; a Ruminococcus sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 33; and / or a Lachnospiracea sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 34.

[0217] In some embodiments, the disclosure provides compositions produced by the methods described herein, wherein the composition comprises a microbial ensemble comprising one or more microorganisms disclosed in one or more of the following: U.S. Pub. Nos.2018 / 0310592, 2018 / 0333443, 2018 / 0223325, 2022 / 0174992, 2022 / 0265732, 2022 / 0386647, and PCT Pub. Nos. 2016 / 210251, 2017 / 120495, 2017 / 181203, 2018 / 201049, 2019 / 079629, 2018 / 056563, 2020 / 042148, 2020 / 227442, 2021 / 163212, 2021 / 011662, 2021 / 202804, 2022 / 226367, and 2022 / 081992 (each being herein expressly incorporated by reference for all purposes). Manufacturing Methods Microbial Culture Techniques

[0218] In some embodiments, the methods comprise identifying one or more target microorganisms and / or strains, e.g., based on the discovery platform disclosed, for example in U.S. Patent No. 9,938,558. Then, once the one or more target strains have been identified, a culture of each strain is grown, and cells are of each are harvested from the cultures. Once harvested, an initial viability / unit (e.g., CFU / g or spores / g) can be determined for each strain. Thus, in some embodiments, the methods provided herein comprise identifying a target microbe and / or microbe strain, growing the target microbe and / or microbe strain to produce a population of microbial cells, preparing the population of microbial cells for preservation (for example, by combining with a preservation solution), preserving the population of microbial cells to provide a population of preserved microbial cells, harvesting viable microbial cells from the preserved population of microbial cells to provide a population of viable preserved microbial cells.

[0219] The isolation, identification, and culturing of the microorganisms of the present disclosure can be performed using standard microbiological techniques. Examples of such techniques may be found in Gerhardt, P. (ed.) Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.) Manual of Clinical Microbiology, Third Edition. American Society for Microbiology, Washington, D.C. (1980), each of which is incorporated by reference.

[0220] Isolation can be performed by streaking the specimen on a solid medium (e.g., nutrient agar plates) to obtain a single colony, which is characterized by the phenotypic traits described hereinabove (e.g., Gram positive / negative, capable of forming spores aerobically / anaerobically, cellular morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretions, etc.) and to reduce the likelihood of working with a culture which has become contaminated.

[0221] For example, for microorganisms of the disclosure, biologically pure isolates can be obtained through repeated subculture of biological samples, each subculture followed by streaking onto solid media to obtain individual colonies or colony forming units. Methods of preparing, thawing, and growing lyophilized bacteria are commonly known, for example, Gherna, R. L. and C. A. Reddy.2007. Culture Preservation, p 1019-1033. In: C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder, eds., Methods for General and Molecular Microbiology, American Society for Microbiology, Washington, D.C., 1033 pages; herein incorporated by reference. Thus, freeze dried liquid formulations andcultures stored long term at −70° C in solutions containing glycerol are contemplated for use in providing formulations of the present disclosure.

[0222] The microorganisms of the disclosure can be propagated in a liquid medium under aerobic conditions or anaerobic conditions. Medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as specially required substances such as vitamins, amino acids, nucleic acids, and the like. Examples of suitable carbon sources which can be used for growing the microorganisms include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol and glycerol and the like; oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 g / L. Preferably, glucose, starch, and / or peptone is contained in the medium as a major carbon source, at a concentration of 0.1-5% (W / V).

[0223] Examples of suitable nitrogen sources which can be used for growing the bacterial strains of the present disclosure include, but are not limited to, amino acids, yeast extract, beef extract, tryptone, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, or combinations thereof. The amount of nitrogen source varies according to the type of nitrogen source, typically between 0.1 g / L to 30 g / L.

[0224] The inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate can be used alone or in combination. The amount of inorganic acid varies according to the kind of the inorganic salt, typically between 0.001 g / L to 10 g / L. Examples of specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dried yeast, and combinations thereof.

[0225] Cultivation can be affected at a temperature, which allows the growth of the microbial strains, essentially, between 20°C and 50°C. In some aspects, a temperature range is 30°C-39°C. For optimal growth, in some embodiments, the medium can be adjusted to pH 6.0-7.4. It will be appreciated that commercially available media may also be used to culture the microbial strains, such as Nutrient Broth or Nutrient Agar available from Difco, Detroit, MI. It will be appreciated that cultivation time may differ depending on the type of culture medium used and the concentration of sugar as a major carbon source.

[0226] In some embodiments, cultivation lasts between 8-96 hours. Microbial cells are isolated using methods well known in the art. Examples include, but are not limited to, membrane filtration and centrifugal separation. The pH may be adjusted using sodium hydroxide and the like and the culture may be dried using a freeze dryer, until the water content becomes equal to 4% or less. Microbial co-cultures may be obtained by propagating each strain as described herein above. In some embodiments, microbial multi-strain cultures may be obtained by propagating two or more of the strains described hereinabove. It will be appreciated that the microbial strains may be cultured together when compatible culture conditions can be employed. Preservation Methods

[0227] In some embodiments, the microbial cells are prepared for preservation, for example, by combining with a preservation solution. An example preservation solution can include, by way of non-limiting example: an intracellular protectant (e.g., sugars, especially non-reducing sugars; sugar alcohols, such as sorbitol; and / or the like), a pH buffer (e.g., monosodium glutamate, monopotassium phosphate, dipotassium phosphate, and / or the like), a membrane protectant (e.g., non-reducing sugars such as sucrose and trehalose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone or povidone), an antioxidant (e.g., ascorbic acid) as well as components to help with the preservation (e.g., where applicable, sucrose for glass formation, etc.) and quality control (e.g., a redox indicator such as resazurin for use with anaerobic microorganisms, etc.).

[0228] In some embodiments, the intracellular protectant is selected from sorbitol, mannitol, glycerol, maltitol, xylitol, erythritol, and methyl glucoside. In some embodiments, the membrane protectant is selected from sucrose, trehalose, raffinose, maltodextrin, polyvinyl alcohol, polyethylene glycol, and polyvinylpyrrolidone. In some embodiments, the preservation solution comprises one or more buffers, e.g., phosphate salts. In some embodiments, the preservation solution comprises one or more antioxidants, e.g., ascorbic acid.

[0229] In some embodiments, the preservation solutions are tailored to the type of preservation challenges used in the serial preservation methods. One of skill in the art will be familiar withthe elements of a preservation solution (e.g., intracellular protectants, a pH buffer, membrane protectants, and the like) and the combinations applicable to each preservation method. For example, a preservation solution used for preservation by foam formation or preservation by vaporization may require higher concentrations of sugars compared to preservation solutions used for other types of preservation challenges.

[0230] Non-limiting examples of preservation solutions are provided in Tables 2 and 3 below. Additional preservation solutions are described in the art, e.g., U.S. Patent No.6,872,357. Table 2: Exemplary Preservation SolutionTable 3: Exemplary Preservation Solution

[0231] Preservation of the microbial cells can be accomplished by a variety of means known in the art, including freeze drying, lyophilization, cryopreservation, preservation by evaporation, preservation by foam formation, vitrification, stabilization by glass formation, encapsulation, preservation by vaporization, spray drying, adsorptive drying, wet extrusion- drying, or fluid bed drying. In some embodiments, the preservation is achieved by preservation by vaporization (PBV).

[0232] In some embodiments, the microbial cells are preserved by a preservation method that results in achievement of a high glass transition temperature such that the microbe is stable at ambient conditions, e.g., via PBV. According to some embodiments, there can be different preservation methods for different microorganisms and / or different strains (e.g., in an example where a Ruminococcus strain is a first strain and a Butyrivibrio strain is a second strain, each strain can be preserved via one or more different preservation methods). A. Freeze-Drying / Lyophilization

[0233] In some embodiments, a population of target microbial cells is subjected to preservation by freeze-drying (also referred to as preservation by lyophilization). Freeze-drying, or lyophilization, has been known and applied to preserve various types of proteins, cells, viruses, and microorganisms. Freeze-drying typically comprises primary drying and secondary drying. Freeze-drying can be used to produce stable bio-actives in industrial quantities. Freeze-drying can be damaging to cellular components, and can result in reduced viability. Conventionally freeze-dried products are typically only stable at or near 0°C, which can require that the bio- active material product be refrigerated from the time it is manufactured until the time it is utilized, requiring refrigeration during storage and transportation. (1) Primary Freeze-Drying

[0234] The limitations of freeze-drying, as described above, result in part from a need to utilize low pressure (or high vacuum) during a freeze-drying process. A high vacuum is required because the temperature of the material during the primary freeze-drying should be below its collapse temperature, which is approximately equal to Tg′. At such low temperatures, the primary drying takes many hours (sometimes days) because the equilibrium pressure above ice at temperatures below -25ºC. is less than 0.476 Torrs. Therefore, a new process must allow for shorter production times.

[0235] The low vacuum pressure used in freeze-drying methods limits the amount of water that can be removed from drying. Primary freeze-drying is performed by sublimation of ice from a frozen specimen at temperatures close to or below Tg′ that is a temperature at which a solution that remains not frozen between ice crystals becomes solid (vitrifies) during cooling. According to conventional beliefs, performing freeze-drying at such low temperatures is important for at least two reasons. The first reason for which freeze-drying at low temperatures (i.e., below Tg′) is important is to ensure that the cake remaining after ice removal by sublimation (primary drying) is “solid” and mechanically stable, i.e., that it does not collapse.Keeping the cake in a mechanically stable “solid” state after primary freeze-drying is important to ensure effective reconstitution of the freeze-dried material. Several methods were proposed to measure the Tg′ for a specific material. These methods rely on different interpretations of the features that can be seen in DSC (Differential Scanning Calorimeter) thermograms. The most reliable way to determine Tg′ is based on an evaluation of the temperature at which ice begins to melt and the concentration of water remaining unfrozen (Wg′) during slow cooling. The second reason typically advanced to support the importance of freeze-drying at low temperatures (i.e., below Tg′) is that the survival rate of bio-actives after freeze-drying is higher if the primary freeze-drying is performed at lower temperatures.

[0236] Freeze drying can be damaging for sensitive bio-actives. Strong freeze drying-induced injury occurs during both freezing (formation of ice crystals) and the subsequent equilibration of the frozen specimens at intermediately low temperatures during ice sublimation. Well- known factors that cause cell damage during freezing include freeze-induced dehydration, mechanical damage of cells during ice crystallization and recrystallization, phase transformation in cell membranes, increasing electrolyte concentration and others. Additionally, damages to frozen bio-actives can be caused by large pH change in the liquid phase that remains unfrozen between ice crystals. This abnormal pH change is associated with crystallization hydrolysis.

[0237] Crystallization hydrolysis occurs because ice crystals capture positive and negative ions differently. This creates a significant (about 107 V / m) electrical field inside ice crystals. Neutralization of this electrical field occurs due to electrolysis inside the ice crystals at a rate proportional to the constant of water molecule dissociation in ice. This neutralization results in a change of the pH of the liquid that remains between the ice crystals. The damaging effect of crystallization hydrolysis can be decreased by reducing the surface of ice that forms during freezing and by increasing the volume of the liquid phase that remains between the ice crystals. This remaining liquid also reduces the damaging effect of (i) the increasing electrolyte (or any other highly reactive molecules) concentration and (ii) the mechanical damage to cells between the ice crystals. The increase of the liquid between the ice crystals can be achieved by (i) increasing the initial concentration of protectants added before freezing, and (ii) by decreasing the amount of ice formed in the sample.

[0238] Avoiding freezing to temperatures equal to Tg′ or below (at which freeze-drying is typically performed) will allow to significantly reduce the amount of damage in the preservedbiological. Therefore, a new method that allows a preservation of bio-actives without subjecting the bio-actives to temperatures near or below Tg′ will significantly improve the quality of the preserved material. (2) Secondary Freeze-Drying

[0239] After the removal of ice by sublimation (primary drying) is complete, the sample may be described as a porous cake. Concentration of water in the sample at the end of primary drying is above the concentration of water that remains unfrozen in the glassy channels between ice crystals at a temperature below Tg′ (Wg′). Tg′ strongly depends on the composition of the solution, while for the majority of solutes Wg′ is about 20 wt %. At such high water concentrations, the glass transition temperature of the cake material is below the primary freeze-drying temperature, and / or significantly below -20° C. Secondary drying is performed to remove the remaining (about 20 wt %) water and increase the glass transition temperature in the cake material. As a practical matter, secondary drying cannot be performed at Tg′ or lower temperatures because diffusion of water from a material in a glass state is extremely slow. For this reason, secondary drying is performed by heating the cake to a drying temperature Td that is higher than the glass transition temperature Tg of the cake material at a given moment. If during the secondary drying step, Td is substantially higher than Tg, the cake will “collapse” and form a very viscous syrup, thereby making standard reconstitution impossible. Therefore, the collapse of the cake is highly undesirable.

[0240] The collapse phenomenon, which is kinetic by nature, has been extensively discussed in the literature. The rate of the collapse increases as the viscosity of the cake material decreases. To avoid or bring the collapse process to a negligible scale, Td is kept close to Tg during the secondary drying, thereby ensuring that the viscosity of the cake material is high and the rate of the collapse slow. B. Preservation by Vitrification (Glass Formation)

[0241] In some embodiments, a population of target microbial cells is subjected to preservation by vitrification. “Preservation by vitrification” is a transformation from a liquid into a highly immobile, noncrystalline, amorphous solid state, known as the “glass state.” Such a process may also be referred to as “preservation by glass formation”. A “glass state” is an amorphous solid state, which may be achieved by supercooling of a material that was initially in a liquid state. Diffusion in vitrified materials (e.g., “glass”) occurs at extremely low rates. Consequently, chemical and biological changes requiring the interaction of more than onemoiety are practically completely inhibited. Glass typically appears as homogeneous, transparent, brittle solids, which can be ground or milled into a powder. Above a temperature known as the glass transition temperature (Tg), the viscosity drops rapidly and the material transforms from a glass state into what is known as a deformable “rubber state.” As the temperature increases, the material transitions into a liquid state. The optimal benefits of vitrification for long-term storage may be secured only under conditions where Tg is greater than the storage temperature.

[0242] Vitrification has been broadly used to preserve biological and highly reactive chemicals. The basic premise of vitrification is that all diffusion limited physical processes and chemical reactions, including the processes responsible for the degradation of biological materials, stop in the glass state. In general terms, glasses are thermodynamically unstable, amorphous materials that are mechanically stable at their very high viscosity (1012-1014 Pa / s.). A typical liquid has a flow rate of 10 m / s compared to 10−14m / s in the glass state.

[0243] Bio-actives can be preserved at -196° C. Tg for pure water is about -145° C. If ice crystals form during cooling, the solution that remains unfrozen in the channels between the ice crystals will vitrify at Tg′, which is higher than Tg for pure water. Bio-actives that are rejected in the channels during ice growth will be stable at temperatures below Tg′. Bio-actives can be stabilized at temperatures substantially higher than -145° C provided they are placed in concentrated preservation solutions with high Tg. For example, for a solution that contains 80% sucrose, Tg is about -40° C. A solution that contains 99% sucrose is characterized by Tg of about 52° C. The presence of water in a sample results in a strong plasticizing effect, which decreases Tg. The Tg is directly dependent on the amount of water present, and may, therefore, be modified by controlling the level of hydration—the less water, the higher the Tg. Therefore, the specimens (to be vitrified at an ambient temperature) must be strongly dehydrated by drying. However, drying can be damaging to bio-actives. Therefore, to stabilize bio-actives at a room temperature and still preserve their viability and functions, they need to be dried in the presence of a protective excipient (i.e., protectant) or a combination of excipients, which have a glass transition temperature Tg higher than the room temperature. C. Preservation by Evaporation

[0244] In some embodiments, a population of target microbial cells is subjected to preservation by evaporation. “Preservation by evaporation” refers to a process comprising the removal of water by evaporative drying.

[0245] In some embodiments, activity of bio-actives dried by evaporative drying of small drops is comparable to the activity of freeze-dried samples. For example, it has been shown that labile enzymes (luciferase and isocitric dehydrogenase) can be preserved by evaporative drying for more than a year at 50° C without any detectable loss of activity during drying and subsequent storage at 50° C (Bronshtein, V., Frank, J. L., and Leopold, A. C. (1996). Protection of Desiccated Enzymes by Sugars. In: “Cryo 96 program”, Abstract 22 of a Paper Presented at the 33rd Annual Meeting of the Society for Cryobiology). Because dehydrated solutions containing protectors become viscous, it can take long periods of time to evaporate water even from small drops of a solution. D. Preservation by Foam Formation

[0246] In some embodiments, a population of target microbial cells is subjected to preservation by foam formation. During preservation by foam formation (PFF), the biological materials are first transformed into mechanically stable, dry foams by boiling them under vacuum at ambient temperatures above the freezing point (referred to as primary drying). Second the samples are subjected to stability drying at elevated temperature to increase the glass-transition temperature. Survival or activity yield after rehydration of preserved samples is achieved by proper selection of protectors (e.g., sugars) that are dissolved in the suspension before PFF and by proper selection of the vacuum and temperature protocols during PFF (See, Bronshtein, Victor. (2004). Preservation by Foam Formulation. Pharmaceutical Technology.28(4): 86-92. E. Preservation by Vaporization

[0247] In some embodiments, a population of target microbial cells is subjected to preservation by vaporization. Preservation by Vaporization (PBV) is a preservation process that comprises primary drying and stability drying. Primary drying is performed by intensive vaporization (sublimation, boiling, and evaporation) of water at temperatures significantly higher (approximately 10° C or more) than Tg′ from a partially frozen and at the same time overheated material (i.e., where the vacuum pressure is below the equilibrium pressure of water vapor).

[0248] During PBV, the boiling in the course of the primary drying does not produce a lot of splattering because the equilibrium pressure at subzero temperatures above the slush is low and ice crystals on the surface of the slush prevent or inhibit the splattering. Typically, a material (e.g., frozen solutions or suspensions) which has been subjected to PBV drying looks like a foam partly covered with a skim of a thin freeze-dried cake.

[0249] Unlike preservation by foam formation (PFF), preservation by vaporization (PBV) can be very effective for preserving bio-actives contained or incorporated within an alginate gel formulation and other gel formulations. A PBV process can be performed by drying frozen gel particles under a vacuum at small negative (on the Celsius scale) temperatures. For such hydrogel systems, vaporization comprises simultaneous sublimation of ice crystals, boiling of water inside unfrozen micro inclusions, and evaporation from the gel surface.

[0250] PBV can be different from freeze-drying because freeze-drying suggests the product processing temperature to be at or below Tg′ (which, typically, is below -25° C.) during primary drying and because freeze-drying suggests avoiding the “collapse” phenomenon during both primary and secondary drying. PBV comprises drying at temperatures substantially higher than Tg′, i.e., higher than -15° C, better higher than -10° C, and yet better higher than -5° C.

[0251] Additional details about PBV and other challenges can be found in U.S. Patent No. 9,469,835, the entirety of which is hereby expressly incorporated by reference herein for all purposes. F. Cryopreservation

[0252] In some embodiments, a population of target microbial cells is subjected to cryopreservation. Cryopreservation refers to the use of very low temperatures to preserve structurally intact living cells and tissues. The damaging effect of cryopreservation is mostly associated with freeze-induced dehydration, change in pH, increase in extracellular concentration of electrolytes, phase transformation in biological membranes and macromolecules at low temperatures, and other processes associated with ice crystallization. Potential cryodamage is a drawback in the methods that rely on freezing of bio-actives. This damage can be decreased by using cryoprotective excipients (protectants), e.g., glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), sucrose and other sugars, amino acids, synthetic, and / or biological polymers, etc. G. Spray Drying

[0253] In some embodiments, a population of target microbial cells is subjected to preservation by spray drying. Spray drying refers to a method of producing a dry powder from a liquid or slurry by rapidly drying with a hot gas. Spray-drying generally comprises spraying, in a chamber, a suspension of microorganisms in a stream of hot air, the chamber comprising an inlet for heated air, an outlet for discharging air, and an outlet for recovering the powder ofdried microorganisms. Exemplary temperatures, chamber volumes, and gases for use in spray drying methods can be found in U.S. Patent 6,010,725. H. Adsorptive drying

[0254] In some embodiments, a population of target microbial cells is subjected to preservation by adsorptive drying. Adsorptive drying refers to a method comprising the removal of water by diffusion into and adsorption onto pourous materials such as aluminas, silica gels, molecular sieves, and other chemical drying agents. I. Fluid Bed Drying

[0255] In some embodiments, a population of target microbial cells is subjected to preservation by fluid bed drying. Fluid bed drying refers to a method in which particles are fluidized in a bed and dried. A fluidized bed is formed when a quantity of solid particulates is placed under conditions that cause a solid material to behave like a fluid. In a fluid bed drying system, inlet air provides significant air flow to support the weight of the particles. J. Stability Drying

[0256] In some embodiments, a population of target microbial cells is subjected to preservation by a drying method (e.g., freeze-drying, preservation by vitrification / glass formation, preservation by evaporation, preservation by foam formation, preservation by vaporization, spray drying, adsorptive drying, or fluid bed drying) and the drying preservation method further comprises stability drying. The stability drying is performed (1) to further increase the glass transition temperature of the dry material, (2) to make it mechanically stable at ambient temperatures without vacuum, and (3) to preserve the potency and efficacy of the biological during a long-term storage at ambient temperatures.

[0257] To increase Tgof the material to for example 37°C and to thereby ensure stabilization at this temperature, the stability drying step should be performed at temperatures significantly higher than 37°C over many hours to remove water from inside of already dried material.

[0258] The process of dehydration of biological specimens at elevated temperatures may be very damaging to the subject bio-actives if the temperature used for drying is higher than the applicable protein denaturation temperature. To protect the sample from the damage that can be caused by elevated temperatures, the stability dehydration process (i.e., stability drying) may need to be performed in steps. The first step (either in air or vacuum) should be performed at a starting temperature to ensure dehydration without a significant loss of a biological’s viability and potency. After such first drying step, the process of dehydration may be continuedin subsequent steps by drying at a gradually higher temperature during each subsequent step. Each step will allow simultaneous increases in the extent of the achievable dehydration and the temperature used for drying during the following step. Extrusion

[0259] In some embodiments, a population of target microbial cells is subjected to extrusion. Extrusion refers to a method in which materials are forced through a die in order to shape them. In some embodiments, the target microbial cells are dispersed in a carrier or matrix in order to protect them from oxygen, heat, moisture, and the like. In some embodiments, the population of target microbial cells is subjected to cold-melt extrusion as described herein. Cold-Melt Extrusion

[0260] The present disclosure provides a method of manufacturing a GEMs composition by cold-melt extrusion, the method comprising the following steps: (a) feeding the ingredients into an extruder, wherein the ingredients comprise at least one biologic or bioactive and a protective carrier material; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the carrier to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head consisting of at least one hole with a geometrically defined shape; (e) cutting the composition into pellets; and (f) cooling the pellets. In some embodiments, the method further comprises kneading the semi- molten or molten extrudable composition. In some embodiments, the method further comprises processing ingredients under low pressure in the extruder by applying vacuum. In some embodiments, the method comprises pre-blending two or more ingredients before feeding to the extruder.

[0261] In some embodiments, the present disclosure provides a method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: (a) feeding the ingredients into an extruder, wherein the ingredients comprise at least one microorganism and a protective carrier material; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the carrier to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head consisting of at least one hole with a geometrically defined shape; (e) cutting the composition into pellets; and (f) cooling the pellets. In some embodiments, the method further comprises kneading thesemi-molten or molten extrudable composition. In some embodiments, the method further comprises processing ingredients under low pressure in the extruder by applying vacuum. In some embodiments, the method comprises pre-blending two or more ingredients before feeding to the extruder.

[0262] In some embodiments, the present disclosure provides a method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: (a) feeding the ingredients into an extruder, wherein the ingredients comprise at least one microorganism and a wax; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the wax to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head consisting of at least one hole with a geometrically defined shape; (e) cutting the composition into pellets; and (f) cooling the pellets. In some embodiments, the method further comprises kneading the semi-molten or molten extrudable composition. In some embodiments, the method further comprises processing ingredients under low pressure in the extruder by applying vacuum. In some embodiments, the method comprises pre-blending two or more ingredients before feeding to the extruder.

[0263] In some embodiments, the cold-melt extrusion method comprises feeding ingredients into an extruder. In some embodiment, the ingredients are dry ingredients. In some embodiments, the ingredients comprise at least one temperature-sensitive material, such as a biologic, bioactive, or microorganism. In some embodiments, the ingredients comprise at least one microorganism and a carrier. In some embodiments, the ingredients comprise at least one microorganism, a carrier, and a functional ingredient. In some embodiments, the ingredients comprise at least one microorganism, a carrier, and a stabilizing agent. In some embodiments, the ingredients comprise at least one microorganism, a carrier, a stabilizing agent, and a disintegrating agent.

[0264] In some embodiments, the cold-melt extrusion method comprises feeding ingredients into an extruder. In some embodiment, the ingredients are dry ingredients. In some embodiments, the ingredients comprise at least one temperature-sensitive material, such as a biologic, bioactive, or microorganism. In some embodiments, the ingredients comprise at least one microorganism and a carrier. In some embodiments, the said biologic, bioactive, or microorganism is encapsulated. In some embodiments, the encapsulant material is the same asthe carrier material. In some embodiments, the encapsulant material is different than the carrier material. In some embodiments, the ingredients comprise at least one encapsulated microorganism, a carrier, and a functional ingredient. In some embodiments, the ingredients comprise at least one encapsulated microorganism, a carrier, and a stabilizing agent. In some embodiments, the ingredients comprise at least one encapsulated microorganism, a carrier, a stabilizing agent, and a disintegrating agent.

[0265] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier is meltable at temperatures at or below 100ºC. In some embodiments, the carrier is meltable at temperatures at or below 100ºC, 95ºC, 90ºC, 85ºC, 80ºC, 75ºC, 70ºC, 65ºC, 60ºC, 55ºC, 50ºC, 49ºC, 48ºC, 47ºC, 46ºC, 45ºC, 44ºC, 43ºC, 42ºC, 41ºC, 40ºC, 39ºC, 38ºC, 37ºC, 36ºC, 35ºC, 34ºC, 33ºC, 32ºC, 31ºC, 30ºC, 29ºC, 28ºC, 27ºC, 26ºC, 25ºC, 24ºC, 23ºC, 22ºC, 21ºC, 20ºC, 19ºC, 18ºC, 17ºC, 16ºC, 15ºC, 14ºC, 13ºC, 12ºC, 11ºC, 10ºC, or lower.

[0266] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier has a high-melting temperature. In some embodiments, the carrier has a melting temperature of at least 25ºC. In some embodiments, the carrier has a melting temperature of at least 30ºC. In some embodiments, the carrier has a melting temperature of at least 35ºC. In some embodiments, the carrier has a melting temperature of at least 40ºC. In some embodiments, the carrier has a melting temperature of at least 45ºC. In some embodiments, the carrier has a melting temperature of at least 50ºC. In some embodiments, the carrier has a melting temperature of at least 55ºC. In some embodiments, the carrier has a melting temperature of at least 60ºC. In some embodiments, the carrier has a melting temperature of at least 65ºC. In some embodiments, the carrier has a melting temperature of at least 70ºC. In some embodiments, the carrier has a melting temperature of at least 75ºC. In some embodiments, the carrier has a melting temperature of at least 80ºC. In some embodiments, the carrier has a melting temperature of at least 85ºC. In some embodiments, the carrier has a melting temperature of at least 90ºC. In some embodiments, the carrier has a melting temperature of at least 95ºC. In some embodiments, the carrier has a melting temperature of at least 100ºC. In some embodiments, the carrier has a melting temperature of 25ºC, 26ºC, 27ºC, 28ºC, 29ºC, 30ºC, 31ºC, 32ºC, 33ºC, 34ºC, 35ºC, 36ºC, 37ºC, 38ºC, 39ºC, 40ºC, 41ºC, 42ºC, 43ºC, 44ºC, 45ºC, 46ºC, 47ºC, 48ºC, 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC, 64ºC, 65ºC, 66ºC, 67ºC,68ºC, 69ºC, 70ºC, 71ºC, 72ºC, 73ºC, 74ºC, 75ºC, 76ºC, 77ºC, 78ºC, 79ºC, 80ºC, 81ºC, 82ºC, 83ºC, 84ºC, 85ºC, 86ºC, 87ºC, 88ºC, 89ºC, 90ºC, 91ºC, 92ºC, 93ºC, 94ºC, 95ºC, 96ºC, 98ºC, 99ºC, 100ºC, or more.

[0267] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier has a high-melting temperature. In some embodiments, the carrier has an onset melting temperature of at least 50ºC. In some embodiments, the carrier has an onset melting temperature of at least 55ºC. In some embodiments, the carrier has an onset melting temperature of at least 60ºC. In some embodiments, the carrier has an onset melting temperature of at least 65ºC. In some embodiments, the carrier has an onset melting temperature of at least 70ºC. In some embodiments, the carrier has an onset melting temperature of at least 75ºC. In some embodiments, the carrier has an onset melting temperature of at least 80ºC. In some embodiments, the carrier has an onset melting temperature of at least 85ºC. In some embodiments, the carrier has an onset melting temperature of at least 90ºC. In some embodiments, the carrier has an onset melting temperature of at least 95ºC. In some embodiments, the carrier has an onset melting temperature of at least 100ºC. In some embodiments, the carrier has an onset melting temperature of 25ºC, 26ºC, 27ºC, 28ºC, 29ºC, 30ºC, 31ºC, 32ºC, 33ºC, 34ºC, 35ºC, 36ºC, 37ºC, 38ºC, 39ºC, 40ºC, 41ºC, 42ºC, 43ºC, 44ºC, 45ºC, 46ºC, 47ºC, 48ºC, 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC, 64ºC, 65ºC, 66ºC, 67ºC, 68ºC, 69ºC, 70ºC, 71ºC, 72ºC, 73ºC, 74ºC, 75ºC, 76ºC, 77ºC, 78ºC, 79ºC, 80ºC, 81ºC, 82ºC, 83ºC, 84ºC, 85ºC, 86ºC, 87ºC, 88ºC, 89ºC, 90ºC, 91ºC, 92ºC, 93ºC, 94ºC, 95ºC, 96ºC, 98ºC, 99ºC, 100ºC, or more.

[0268] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier has a high-melting temperature. In some embodiments, the carrier has a peak melting temperature of at least 50ºC. In some embodiments, the carrier has a peak melting temperature of at least 55ºC. In some embodiments, the carrier has a peak melting temperature of at least 60ºC. In some embodiments, the carrier has a peak melting temperature of at least 65ºC. In some embodiments, the carrier has a peak melting temperature of at least 70ºC. In some embodiments, the carrier has a peak melting temperature of at least 75ºC. In some embodiments, the carrier has a peak melting temperature of at least 80ºC. In some embodiments, the carrier has a peak melting temperature of at least 85ºC. In some embodiments, the carrier has a peak melting temperature of at least 90ºC. In someembodiments, the carrier has a peak melting temperature of at least 95ºC. In some embodiments, the carrier has a peak melting temperature of at least 100ºC. In some embodiments, the carrier has a peak melting temperature of 25ºC, 26ºC, 27ºC, 28ºC, 29ºC, 30ºC, 31ºC, 32ºC, 33ºC, 34ºC, 35ºC, 36ºC, 37ºC, 38ºC, 39ºC, 40ºC, 41ºC, 42ºC, 43ºC, 44ºC, 45ºC, 46ºC, 47ºC, 48ºC, 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC, 64ºC, 65ºC, 66ºC, 67ºC, 68ºC, 69ºC, 70ºC, 71ºC, 72ºC, 73ºC, 74ºC, 75ºC, 76ºC, 77ºC, 78ºC, 79ºC, 80ºC, 81ºC, 82ºC, 83ºC, 84ºC, 85ºC, 86ºC, 87ºC, 88ºC, 89ºC, 90ºC, 91ºC, 92ºC, 93ºC, 94ºC, 95ºC, 96ºC, 98ºC, 99ºC, 100ºC, or more.

[0269] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier is a wax. In some embodiments, the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, paraffins, alkanes, mineral-based waxes, insect-based waxes, and plant-based waxes. In some embodiments, the fatty acid is a saturated fatty acid with an aliphatic tail of 6 to 21 carbons. In some embodiments, the saturated fatty acid is a medium-chain fatty acid (MCFA) with an aliphatic tail of 6 to 12 carbons. In some embodiments, the saturated fatty acid is a long-chain fatty acid (LCFA) with an aliphatic tail of 13 to 21 carbons. In some embodiments, the carrier is a stearic acid. In some embodiments, the stearic acid consists of at least 90% by weight of octadecanoic acid (C18:0). In some embodiments, the glycerides are monoglycerides or triglycerides. In some embodiments, the plant-based wax is a partially hydrogenated vegetable oil. In some embodiments, the plant-based wax is a fully hydrogenated vegetable oil. In some embodiments, the plant-based wax is hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the carrier is hydrogenated rapeseed oil. In some embodiments, the carrier is hydrogenated soybean oil. In some embodiments, the carrier is partially hydrogenated soybean oil. In some embodiments, the carrier is fully hydrogenated soybean oil.

[0270] In some embodiments, the ingredients for cold-melt extrusion comprise a carrier. In some embodiments, the carrier is partially insoluble in aqueous media. In some embodiments, the carrier is completely insoluble in aqueous media.

[0271] In some embodiments, the ingredients for cold-melt extrusion comprise one or more functional ingredients. In some embodiments, the one or more functional ingredients is selectedfrom the group consisting of: a moisture-binding agent, a pH modifying agent, a release modifying agent, a disintegrating agent, and an oxygen scavenging agent.

[0272] In some embodiments, the moisture-binding agent, also referred to as a water activity scavenger (WAS), is a zeolite. In some embodiments, the zeolite is a natural zeolite. In some embodiments, the zeolite is a synthetic zeolite. In some embodiments, zeolite is selected from heulandite, analcime, chabazite, clinoptilolite, natrolite, stilbite, and phillipsite. In some embodiments, the moisture-binding agent is a bentonite clay. In some embodiments, the moisture-binding agent is a diatomaceous earth. In some embodiments, the moisture-absorbing agent is a silica (e.g., silicon dioxide and calcium silicate). In some embodiments, the moisture- absorbing agent is an inorganic salt (e.g., calcium sulfate and calcium chloride).

[0273] In some embodiments, the pH modifying agent is selected from a group consisting of a weak acid and / or its salts, a weak base and / or its salts. In some embodiments, the said acid or base is inorganic. In some embodiments, the said acid or base is organic.

[0274] In some embodiments, the release modifying agent is selected from a group consisting of salts, sugars (e.g., monosaccharides and disaccharides), sugar alcohols, short-chain fructo- oligosaccharides, and combinations thereof.

[0275] In some embodiments, the disintegrating agent is selected from the group consisting of a natural polysaccharide, a natural polysaccharide hydrogel, a soy polysaccharide, a psyllium husk fiber, and a mineral clay. In some embodiments, the mineral clay is an activated clay. In some embodiments, the mineral clay is bentonite.

[0276] In some embodiments, the oxygen-scavenging agent is selected from a group consisting of bicarbonates (e.g., sodium hydrogen carbonate), sulfites (e.g., sodium hydrogen sulfite), ferrous iron oxide, sugar alcohols (e.g., mannitol, sorbitol, xylitol) and glycols (e.g., propylene glycol, ethylene glycol), amino acids (e.g., cysteine), unsaturated fatty acids (e.g., linseed oil), ascorbic acid and its salts, enzymes (e.g., glucose oxidase), and combinations thereof.

[0277] In some embodiments, the ingredients for cold-melt extrusion comprise a material approved as an animal feed ingredient by the Association of American Feed Control Officials (AAFCO).

[0278] In some embodiments, the ingredients for cold-melt extrusion comprise a material approved as a human food ingredient by the United States Food and Drug Administration (U.S. FDA).

[0279] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the ingredients for cold-melt extrusion comprise at least two microorganisms. In some embodiments, the ingredients for cold-melt extrusion comprise at least three microorganisms. In some embodiments, the ingredients for cold-melt extrusion comprise at least four microorganisms. In some embodiments, the ingredients for cold-melt extrusion comprise at least five microorganisms. In some embodiments, the ingredients for cold-melt extrusion comprise one microorganism, two microorganisms, three microorganisms, four microorganisms, five microorganisms, six microorganisms, seven microorganisms, eight microorganisms, nine microorganisms, ten microorganisms, or more.

[0280] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is selected from any of the microorganisms described herein or incorporated by reference herein. In some embodiments, the at least one microorganism is native to the microbiome of an animal or a human. In some embodiments, the at least one microorganism is native to the gastrointestinal microbiome of an animal or a human. In some embodiments, the at least one microorganism is native to the microbiome of a monogastric animal. In some embodiments, the at least one microorganism is native to the microbiome of a ruminant or of a camelid. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria.

[0281] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence of any one of SEQ ID NOs: 1-34.

[0282] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is of the genus Butyrivibrio. In some embodiments, the at least one microorganism is of the genus species Butyrivibrio fibrisolvens. In some embodiments, the at least one microorganism is a Butyrivibrio fibrisolvens with a deposit accession number of NRRL B-67347.

[0283] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is of the genus Ruminococcus. In some embodiments, the at least one microorganism is of the genus species Ruminococcus bovis. In some embodiments, the at least one microorganism is a Ruminococcus bovis with a deposit accession number of PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479.

[0284] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is of the genus Pichia. In some embodiments, the at least one microorganism is of the genus species Pichia kudriavzevii. In some embodiments, the at least one microorganism is a Pichia kudriavzevii with a deposit accession number of NRRL B-67249.

[0285] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is of the genus Clostridium. In some embodiments, the at least one microorganism is of the genus species Clostridium beijerinckii. In some embodiments, the at least one microorganism is a Clostridium beijerinckii with a deposit accession number of NRRL B-67248.

[0286] In some embodiments, the cold-melt extrusion process is used for manufacturing of bacteria. In some embodiments, the bacteria belong to the genus of: Clostridium, Succinivibrio, Butyrivibrio, Bacillus, Lactobacillus, Chordicoccus, Enterococcus, Escherichia, Streptococcus, Hungatella, Atlantibacter, Bacteroides, Fusobacterium, Collinsella, Megamonas, Prevotella, Syntrophococcus, Sharpea, or Ruminococcus.

[0287] In some embodiments, the cold-melt extrusion process is used for manufacturing of fungi. In some embodiments, the fungi belong to the genus of: Caecomyces, Pichia, Orpinomyces, Piromyces, or Neocallimastix.

[0288] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the total amount of the at least one microorganism is about 10% to about 90% of the composition. In some embodiments, the total amount of the at least one microorganism is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the composition.

[0289] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism and a carrier. In some embodiments, the ratio of at least one microorganism to the carrier is about 50:50 (w:w) throughout the cold-extrusion process. In some embodiments, the ratio of at least one microorganism to the carrier is about 1:99, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5 (w:w) throughout the cold-extrusion process.

[0290] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder prior to the cold-melt extrusion process. In some embodiments, the at least one microorganism is formulated as a dried fine powder or dried granular powder using any of the methods described herein or incorporated by reference herein. In some embodiments, the at least one microorganism is formulated as a dried fine powder or a dried granular powder by freeze-drying, spray drying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), tray drying, vacuum drying, microwave drying, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 1000 µm. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 500 µm. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 250 µm. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 100 µm. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 50 µm. In some embodiments, the dried fine powder or dried granular powder has a mean particle size of less than 25 µm. Encapsulated Microorganisms

[0291] In some embodiments, the ingredients for cold-melt extrusion comprise at least one microorganism. In some embodiments, the at least one microorganism is encapsulated with a carrier prior to the cold-melt extrusion process. In some embodiments, the at least one microorganism is encapsulated using any of the methods described herein or incorporated by reference herein. In some embodiments, the at least one microorganism is encapsulated by freeze-drying, spray drying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), tray drying, vacuum drying, microwave drying, spray cooling (alsoreferred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0292] In some embodiments, the microorganisms of the invention are encapsulated in an encapsulating composition. An encapsulating composition protects the microorganisms from external stressors prior to entering the gastrointestinal tract of ungulates. Encapsulating compositions further create an environment that may be beneficial to the microorganisms, such as minimizing the oxidative stresses of an aerobic environment on anaerobic microorganisms. See Kalsta et al. (U.S. Patent No. 5,104,662A), Ford (U.S. Patent No. 5,733,568A), and Mosbach and Nilsson (U.S. Patent No. 4,647,536A) for encapsulation compositions of microorganisms, and methods of encapsulating microorganisms. Additional method and formulations of synthetic ensembles can include formulations and methods as disclosed in one or more of the following U.S. Patents: 5,766,520A, 6,306,345B1, 6,509,146B1, 6,534,087B2, 6,537,666B1, 6,692,695B1, 6,872,357B1, 6,884,866B2, 7,074,431B2, and / or 7,153,472B1, each of which is herein expressly incorporated by reference in its entirety.

[0293] In some embodiments, the at least one microorganism is microencapsulated with a carrier that has a melting temperature above 25oC prior to the cold-melt extrusion process. In some embodiments, the carrier has a melting temperature of at least 30ºC. In some embodiments, the carrier has a melting temperature of at least 35ºC. In some embodiments, the carrier has a melting temperature of at least 40ºC. In some embodiments, the carrier has a melting temperature of at least 45ºC. In some embodiments, the carrier has a melting temperature of at least 50ºC. In some embodiments, the carrier has a melting temperature of at least 55ºC. In some embodiments, the carrier has a melting temperature of at least 60ºC. In some embodiments, the carrier has a melting temperature of at least 65ºC. In some embodiments, the carrier has a melting temperature of at least 70ºC. In some embodiments, the carrier has a melting temperature of at least 75ºC. In some embodiments, the carrier has a melting temperature of at least 80ºC. In some embodiments, the carrier has a melting temperature of at least 85ºC. In some embodiments, the carrier has a melting temperature of at least 90ºC. In some embodiments, the carrier has a melting temperature of at least 95ºC. In some embodiments, the carrier has a melting temperature of at least 100ºC. In some embodiments, the carrier has a melting temperature of 30ºC, 31ºC, 32ºC, 33ºC, 34ºC, 35ºC, 36ºC, 37ºC, 38ºC, 39ºC, 40ºC, 41ºC, 42ºC, 43ºC, 44ºC, 45ºC, 46ºC, 47ºC, 48ºC, 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC, 64ºC, 65ºC,66ºC, 67ºC, 68ºC, 69ºC, 70ºC, 71ºC, 72ºC, 73ºC, 74ºC, 75ºC, 76ºC, 77ºC, 78ºC, 79ºC, 80ºC, 81ºC, 82ºC, 83ºC, 84ºC, 85ºC, 86ºC, 87ºC, 88ºC, 89ºC, 90ºC, 91ºC, 92ºC, 93ºC, 94ºC, 95ºC, 96ºC, 98ºC, 99ºC, 100ºC, or more. In some embodiments, the melting temperature is an onset melting temperature of at least 30ºC, at least 35ºC, at least 40ºC, at least 45ºC, at least 50ºC, at least 55ºC, at least 60ºC, at least 65ºC, at least 70ºC, at least 75ºC, at least 80ºC, at least 85ºC, at least 90ºC, at least 95ºC, at least 100ºC, or more. In some embodiments, the melting temperature is a peak melting temperature of at least 30ºC, at least 35ºC, at least 40ºC, at least 45ºC, at least 50ºC, at least 55ºC, at least 60ºC, at least 65ºC, at least 70ºC, at least 75ºC, at least 80ºC, at least 85ºC, at least 90ºC, at least 95ºC, at least 100ºC, or more.

[0294] A first category of useful meltable (fusible) encapsulants is that of normally solid fats and waxes, including fats which are already of suitable hardness and animal or vegetable fats and oils which are hydrogenated until their melting points are sufficiently high to serve the purposes of the present disclosure. Depending on the desired process and storage temperatures and the specific material selected, a particular encapsulant can be either a normally solid or normally liquid material. The terms “normally solid” and “normally liquid” as used herein refer to the state of a material at desired temperatures for storing the resulting microcapsules.

[0295] In some embodiments, the at least one microorganism is microencapsulated with a carrier that is a wax. In some embodiments, the wax is selected from the group consisting of saturated fatty acids (e.g., myristic acid, palmitic acid, stearic acid), fatty acid esters, fatty alcohols, glycerides, insect-based waxes (e.g., beeswax), paraffins (e.g., paraffin wax or petroleum wax), alkane-based waxes, mineral-based waxes (e.g., ozokerite or earthwax, montan wax), and plant-based waxes (e.g., carnauba wax, candelilla wax). In some embodiments, the glycerides are monoglycerides, diglycerides, or triglycerides. In some embodiments, the plant-based wax is a hydrogenated vegetable oil. In some embodiments, the plant-based wax is hydrogenated castor oil, hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated linseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated safflower oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the carrier is hydrogenated soybean oil. In some embodiments, the carrier is hydrogenated rapeseed oil.

[0296] In some embodiments, the at least one microorganism is microencapsulated with a carrier that is an animal fat such as beef tallow, mutton tallow, lamb tallow, lard, or pork fat.

[0297] Other specific examples of fatty acids include linoleic acid, γ-linoleic acid, dihomo-γ- linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid, elaidic acid, oleic acid, palitoleic acid, stearidonic acid, eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, margaric acid, nonadecyclic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, and octatriacontanoic acid.

[0298] In one embodiment, the encapsulating composition comprises microcapsules having a multiplicity of microbe-containing solid cores encapsulated in a solid shell material. For purposes of the disclosure, a “multiplicity” of cores is defined as two or more. In one embodiment, the encapsulating composition comprises microcapsules having a multiplicity of microbe-containing liquid cores encapsulated in a solid shell material. The said liquid is selected from a group of vegetable oils such as canola oil, castor oil, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, safflower oil, soybean oil, sunflower oil, and tung oil.

[0299] The microorganism composition (encapsulate) contemplated according to some embodiments herein constitutes from about 0.1% to about 70%, about 1% to about 60%, or about 10% to about 50% by weight of the microcapsules. In some embodiments, the microorganism composition (encapsulate) contemplated herein constitutes no more than about 30% by weight of the microcapsules. In some embodiments, the microorganism composition (encapsulate) contemplated herein constitutes about 50% by weight of the microcapsules. Depending on the implementation, the core material can be a liquid or solid at contemplated storage temperatures of the microcapsules.

[0300] In some embodiments, the encapsulating shell of the present invention can be up to 10µm, 20µm, 30µm, 40µm, 50µm, 60µm, 70µm, 80µm, 90µm, 100µm, 110µm, 120µm, 130µm, 140µm, 150µm, 160µm, 170µm, 180µm, 190µm, 200µm, 210µm, 220µm, 230µm, 240µm, 250µm, 260µm, 270µm, 280µm, 290µm, 300µm, 310µm, 320µm, 330µm, 340µm, 350µm, 360µm, 370µm, 380µm, 390µm, 400µm, 410µm, 420µm, 430µm, 440µm, 450µm, 460µm, 470µm, 480µm, 490µm, 500µm, 510µm, 520µm, 530µm, 540µm, 550µm, 560µm, 570µm, 580µm, 590µm, 600µm, 610µm, 620µm, 630µm, 640µm, 650µm, 660µm, 670µm,680µm, 690µm, 700µm, 710µm, 720µm, 730µm, 740µm, 750µm, 760µm, 770µm, 780µm, 790µm, 800µm, 810µm, 820µm, 830µm, 840µm, 850µm, 860µm, 870µm, 880µm, 890µm, 900µm, 910µm, 920µm, 930µm, 940µm, 950µm, 960µm, 970µm, 980µm, 990µm, 1000µm, 1010µm, 1020µm, 1030µm, 1040µm, 1050µm, 1060µm, 1070µm, 1080µm, 1090µm, 1100µm, 1110µm, 1120µm, 1130µm, 1140µm, 1150µm, 1160µm, 1170µm, 1180µm, 1190µm, 1200µm, 1210µm, 1220µm, 1230µm, 1240µm, 1250µm, 1260µm, 1270µm, 1280µm, 1290µm, 1300µm, 1310µm, 1320µm, 1330µm, 1340µm, 1350µm, 1360µm, 1370µm, 1380µm, 1390µm, 1400µm, 1410µm, 1420µm, 1430µm, 1440µm, 1450µm, 1460µm, 1470µm, 1480µm, 1490µm, 1500µm, 1510µm, 1520µm, 1530µm, 1540µm, 1550µm, 1560µm, 1570µm, 1580µm, 1590µm, 1600µm, 1610µm, 1620µm, 1630µm, 1640µm, 1650µm, 1660µm, 1670µm, 1680µm, 1690µm, 1700µm, 1710µm, 1720µm, 1730µm, 1740µm, 1750µm, 1760µm, 1770µm, 1780µm, 1790µm, 1800µm, 1810µm, 1820µm, 1830µm, 1840µm, 1850µm, 1860µm, 1870µm, 1880µm, 1890µm, 1900µm, 1910µm, 1920µm, 1930µm, 1940µm, 1950µm, 1960µm, 1970µm, 1980µm, 1990µm, 2000µm, 2010µm, 2020µm, 2030µm, 2040µm, 2050µm, 2060µm, 2070µm, 2080µm, 2090µm, 2100µm, 2110µm, 2120µm, 2130µm, 2140µm, 2150µm, 2160µm, 2170µm, 2180µm, 2190µm, 2200µm, 2210µm, 2220µm, 2230µm, 2240µm, 2250µm, 2260µm, 2270µm, 2280µm, 2290µm, 2300µm, 2310µm, 2320µm, 2330µm, 2340µm, 2350µm, 2360µm, 2370µm, 2380µm, 2390µm, 2400µm, 2410µm, 2420µm, 2430µm, 2440µm, 2450µm, 2460µm, 2470µm, 2480µm, 2490µm, 2500µm, 2510µm, 2520µm, 2530µm, 2540µm, 2550µm, 2560µm, 2570µm, 2580µm, 2590µm, 2600µm, 2610µm, 2620µm, 2630µm, 2640µm, 2650µm, 2660µm, 2670µm, 2680µm, 2690µm, 2700µm, 2710µm, 2720µm, 2730µm, 2740µm, 2750µm, 2760µm, 2770µm, 2780µm, 2790µm, 2800µm, 2810µm, 2820µm, 2830µm, 2840µm, 2850µm, 2860µm, 2870µm, 2880µm, 2890µm, 2900µm, 2910µm, 2920µm, 2930µm, 2940µm, 2950µm, 2960µm, 2970µm, 2980µm, 2990µm, or 3000µm thick. Extrusion Process

[0301] In some embodiments, the extruder is preset at a temperature at or below 100ºC. In some embodiments the extruder is preset at a temperature at or below 100ºC, 95ºC, 90ºC, 85ºC, 80ºC, 75ºC, 70ºC, 65ºC, 60ºC, 55ºC, 50ºC, 49ºC, 48ºC, 47ºC, 46ºC, 45ºC, 44ºC, 43ºC, 42ºC, 41ºC, 40ºC, 39ºC, 38ºC, 37ºC, 36ºC, 35ºC, 34ºC, 33ºC, 32ºC, 31ºC, 30ºC, 29ºC, 28ºC, 27ºC, 26ºC, 25ºC, 24ºC, 23ºC, 22ºC, 21ºC, 20ºC, 19ºC, 18ºC, 17ºC, 16ºC, 15ºC, 14ºC, 13ºC, 12ºC,11ºC, 10ºC, 9ºC, 8ºC, 7ºC, 6ºC, 5ºC, 4ºC, 3ºC, 2ºC, 1ºC, 0ºC, or lower. In some embodiments, the extruder is preset at a temperature at or below 50ºC. In some embodiments, the extruder is preset at a temperature at or below 40ºC. In some embodiments, the extruder is preset at a temperature of 40ºC. In some embodiments, the extruder is preset at a temperature at or below 35ºC. In some embodiments, the extruder is preset at a temperature of 35ºC. In some embodiments, the extruder is preset at a temperature of at least 5ºC below the melting temperature of the carrier. In some embodiments, the extruder is preset at a temperature of at least 5ºC below the onset melting temperature of the carrier. In some embodiments, the extruder is preset at a temperature of at least 5ºC below the peak melting temperature of the carrier.

[0302] In some embodiments, the extruder comprises one or more heating zones across the extruder barrel. In some embodiments, the one or more heating zones are preset at the same temperature across the extruder barrel for the cold-melt extrusion process. In some embodiments, the one or more heating zones are preset at different temperatures across the extruder barrel for the cold-melt extrusion process. In some embodiments, the one or more heating zones are preset at temperatures at or below 100ºC. In some embodiments, the one or more heating zones are preset at temperatures at or below 95ºC, 90ºC, 85ºC, 80ºC, 75ºC, 70ºC, 65ºC, 60ºC, 55ºC, 50ºC, 49ºC, 48ºC, 47ºC, 46ºC, 45ºC, 44ºC, 43ºC, 42ºC, 41ºC, 40ºC, 39ºC, 38ºC, 37ºC, 36ºC, 35ºC, 34ºC, 33ºC, 32ºC, 31ºC, 30ºC, 29ºC, 28ºC, 27ºC, 26ºC, 25ºC, 24ºC, 23ºC, 22ºC, 21ºC, 20ºC, 19ºC, 18ºC, 17ºC, 16ºC, 15ºC, 14ºC, 13ºC, 12ºC, 11ºC, 10ºC, 9ºC, 8ºC, 7ºC, 6ºC, 5ºC, 4ºC, 3ºC, 2ºC, 1ºC, 0ºC, or any combination thereof. In some embodiments, the one or more heating zones are preset at temperatures at or below 50ºC. In some embodiments, the one or more heating zones are preset at temperatures at or below 40ºC. In some embodiments, the one or more heating zones are preset at temperatures at or below 35ºC. In some embodiments, the one or more heating zones are preset at temperatures at or below 30ºC. In some embodiments, the one or more heating zones are preset at temperatures at or below 25ºC. In some embodiments, the one or more heating zones are preset at temperatures of at least 5ºC below the melting temperature of the carrier. In some embodiments, the one or more heating zones are preset at temperatures of at least 5ºC below the onset melting temperature of the carrier. In some embodiments, the one or more heating zones are preset at temperatures of at least 5ºC below the peak melting temperature of the carrier.

[0303] In some embodiments, the extruder comprises one or more barrels. In some embodiments, the extruder comprises one barrel, two barrels, three barrels, four barrels, fivebarrels, six barrels, seven barrels, eight barrels, nine barrels, ten barrels, or more. In some embodiments, the extruder comprises seven barrels.

[0304] In some embodiments, the pressure inside the barrels varies from about 1 bar to about 200 bar during the extrusion. In some embodiments, the pressure inside the barrels is less than 200 bar, less than 175 bar, less than 150 bar, less than 125 bar, less than 100 bar, less than 75 bar, less than 50 bar, less than 40 bar, less than 30 bar, less than 20 bar, less than 10 bar, or less than 5 bar during the extrusion.

[0305] In some embodiments, the cold-melt extrusion method comprises feeding ingredients into an extruder. In some embodiments, the ingredients are fed into the extruder at a constant feed rate. In some embodiments, the constant feed rate of the extruder is at least 100 g / min. In some embodiments, the constant feed rate of the extruder is at least 250 g / min. In some embodiments, the constant feed rate of the extruder ranges is at least 500 g / min. In some embodiments, the constant feed rate of the extruder is at least 1,000 g / min. In some embodiments, the constant feed rate of the extruder is at least 5,000 g / min. In some embodiments, the constant feed rate of the extruder is at least 10,000 g / min. In some embodiments, the constant feed rate of the extruder is about 100 g / min, about 125 g / min, about 150 g / min, about 175 g / min, about 200 g / min, 225 g / min, about 250 g / min, about 300 g / min, about 350 g / min, about 400 g / min, about 450 g / min, about 500 g / min, about 750 g / min, about 1,000 g / min, about 2,500 g / min, about 5,000 g / min, about 7,500 g / min, or about 10,000 g / min. In some embodiments, the constant feed rate of the extruder is about 500 g / min.

[0306] In some embodiments, the cold-melt extrusion process is performed at a constant screw speed. In some embodiments, the constant screw speed of the extruder is about 50 rpm to about 500 rpm. In some embodiments, the constant screw speed of the extruder is about 75 rpm to about 450 rpm. In some embodiments, the constant screw speed of the extruder is about 100 rpm to about 400 rpm. In some embodiments, the constant screw speed of the extruder is about 125 rpm to about 350 rpm. In some embodiments, the constant screw speed of the extruder is about 50 rpm, 100 rpm, about 105 rpm, about 110 rpm, about 115 rpm, about 120 rpm, about 125 rpm, about 130 rpm, about 135 rpm, about 140 rpm, about 145 rpm, about 150 rpm, about 155 rpm, about 160 rpm, about 165 rpm, about 170 rpm, about 175 rpm, about 180 rpm, about 185 rpm, about 190 rpm, about 195 rpm, about 200 rpm, about 250 prm, about 300 rpm, about 350 rpm, about 400 rpm, about 450 rpm, or about 500 rpm. In some embodiments, the constant screw speed of the extruder is about 175 rpm.

[0307] In some embodiments, the extruder used for the cold-melt extrusion process is a single- screw extruder. In some embodiments, the extruder used for the cold-melt extrusion process is a twin-screw extruder. In some embodiments, the twin-screw extruder is a co-rotating or a counter-rotating extruder.

[0308] In some embodiments, the extruder used for the cold-melt extrusion process comprises a die head, wherein the die head comprises a single hole or a plurality of holes. In some embodiments, the die head is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a hemisphere. In some embodiments, the die head has a round shape.

[0309] In some embodiments, the extruder used for the cold-melt extrusion process comprises a die head, fabricated with a single hole or a plurality of holes, wherein the die hole has a diameter of about 0.1 mm to about 50 mm. In some embodiments, the die hole has a diameter of about 0.1 to about 25 mm. In some embodiments, the die hole has a diameter of about 0.1 to about 10 mm. In some embodiments, the die hole has a diameter of about 0.1 to about 5 mm. In some embodiments, the die hole has a diameter of about 0.5 to about 2.5 mm. In some embodiments, the die hole has a diameter of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, or 5.0 mm. In some embodiments, the die hole has a diameter of 1.5 mm. In some embodiments, the die hole has a diameter of 2.0 mm.

[0310] In some embodiments, the extrudate produced as geometrically shaped cores, also referred to as pellets, are in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a hemisphere. In some embodiments, the pellet has a round shape.

[0311] In some embodiments, the extruded pellet is cooled following the cold-melt extrusion process. In some embodiments, the pellet is cooled by cryogenic cooling. In some embodiments, the cryogenic cooling is performed using liquid nitrogen or carbon dioxide (dry ice). In some embodiments, the cryogenic cooling is an immersion freezing process. In some embodiments, the pellet is cooled by using a chilled dry gas. In some embodiments, the chilled dry gas is nitrogen, carbon dioxide, or dehumidified air.

[0312] In some embodiments, the cooling is taken place on a conveyor belt, wherein heat transfer is achieved by direct cooling, employing a chilled dry gas, and or indirect cooling, employing a chilled gas or liquid.

[0313] In some embodiments, the pellets are stored at less than 25ºC. In some embodiments, the pellets are stored at less than 10ºC. In some embodiments, the pellets are stored at less than 4ºC. In some embodiments, the pellets are stored at less than 25ºC to -40ºC. In some embodiments, the pellets are stored at less than 10ºC to -40ºC. In some embodiments, the pellets are stored at less than 4ºC to -40ºC. In some embodiments, the pellets are stored at about 25ºC, about 20ºC, about 15ºC, about 10ºC, about 5ºC, about 4ºC, about 0ºC, about -5ºC, about -10ºC, about -15ºC, about -20ºC, about -25ºC, about -30ºC, about -35ºC, or about -40ºC.

[0314] In some embodiments, the pellets are stored at less than -60ºC. In some embodiments, the pellets are stored at less than -50ºC. In some embodiments, the pellets are stored at less than -40ºC. In some embodiments, the pellets are stored at about -40ºC, about -41ºC, about -42ºC, about -43ºC, about -44ºC, about -45ºC, about -46ºC, about -47ºC, about -48ºC, about -49ºC, about -50ºC, about -51ºC, about -52ºC, about -53ºC, about -54ºC, about -55ºC, about -56ºC, about -57ºC, about -58ºC, about -59ºC, about -60ºC, about -61ºC, about -62ºC, about -63ºC, about -64ºC, about -65ºC, about -66ºC, about -67ºC, about -68ºC, about -69ºC, about -70ºC about -71ºC, about -72ºC, about -73ºC, about -74ºC, about -75ºC, about -76ºC, about -77ºC, about -78ºC, about -79ºC, or about -80 ºC.

[0315] In some embodiments, the pellet extruded from the cold-melt extrusion process has a diameter of about 0.5 mm to about 50 mm. In some embodiments, the pellet has a diameter of about 0.1 to about 25 mm. In some embodiments, the pellet has a diameter of about 0.1 to about 10 mm. In some embodiments, the pellet has a diameter of about 0.1 to about 5 mm. In some embodiments, the pellet has a diameter of about 0.5 to about 2.5 mm. In some embodiments, the pellet has a diameter of about 1 mm to about 5 mm. In some embodiments, the pellet has a diameter of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, or 5.0 mm. In some embodiments, the pellet has a diameter of 1.5 mm. In some embodiments, the pellet has a diameter of 2.0 mm.

[0316] In some embodiments, the pellet extruded from the cold-melt extrusion process is coated with an additional protective material. In some embodiments, the pellet is coated by spray coating. In some embodiments, the pellet is coated by fluid-bed spray coating. In some embodiments, the pellet is coated with pan coating or drum coating.

[0317] In some embodiments, the pellet extruded from the cold-melt extrusion process is coated with an additional protective material. In some embodiments, the protective material is applied from an aqueous-based coating preparation. In some embodiments, the protective material is applied from a solvent-based coating preparation. In some embodiments, the protective material is applied from a water-organic solvent mix coating preparation. In some embodiments, the protective material is applied from a molten coating preparation. In some embodiments, the coated pellet is manufactured with hot-melt fluid-bed coating.

[0318] In some embodiments, the pellet extruded from the cold-melt extrusion process is coated with an additional protective material. In some embodiments, the pellet is coated with an additional protective material to a target coating level (i.e., the ratio of coating material to pellet). For example, in some embodiments, the ratio of the coating material to pellet is about 1:99, about 5:95, about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20, or about 90:10. In some embodiments, the pellet is coated to a level of at least 30% with the protective material. In some embodiments, the pellet is coated to a level of at least 40% with the protective material. In some embodiments, the pellet is coated to a level of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% with the protective material.

[0319] In some embodiments, the pellet extruded from the cold-melt extrusion process is coated with an additional protective material. In some embodiments, the additional carrier has a melting temperature of at least 30ºC. In some embodiments, the additional carrier has a melting temperature of at least 35ºC. In some embodiments, the additional protective material has a melting temperature of at least 40ºC. In some embodiments, the additional protective material has a melting temperature of at least 50ºC. In some embodiments, the additional protective material has a melting temperature of at least 55ºC. In some embodiments, the additional protective material has a melting temperature of at least 60ºC. In some embodiments, the additional protective material has a melting temperature of at least 65ºC. In someembodiments, the additional protective material has a melting temperature of at least 70ºC. In some embodiments, the additional protective material has a melting temperature of at least 75ºC. In some embodiments, the additional protective material has a melting temperature of at least 80ºC. In some embodiments, the additional protective material has a melting temperature of at least 85ºC. In some embodiments, the additional protective material has a melting temperature of at least 90ºC. In some embodiments, the additional protective material has a melting temperature of at least 95ºC. In some embodiments, the additional protective material has a melting temperature of at least 100ºC. In some embodiments, the additional protective material has a melting temperature of 30ºC, 31ºC, 32ºC, 33ºC, 34ºC, 35ºC, 36ºC, 37ºC, 38ºC, 39ºC, 40ºC, 41ºC, 42ºC, 43ºC, 44ºC, 45ºC, 46ºC, 47ºC, 48ºC, 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC, 64ºC, 65ºC, 66ºC, 67ºC, 68ºC, 69ºC, 70ºC, 71ºC, 72ºC, 73ºC, 74ºC, 75ºC, 76ºC, 77ºC, 78ºC, 79ºC, 80ºC, 81ºC, 82ºC, 83ºC, 84ºC, 85ºC, 86ºC, 87ºC, 88ºC, 89ºC, 90ºC, 91ºC, 92ºC, 93ºC, 94ºC, 95ºC, 96ºC, 98ºC, 99ºC, 100ºC, or more. In some embodiments, the melting temperature is an onset melting temperature of at least 30ºC, at least 35ºC, at least 40ºC, at least 45ºC, at least 50ºC, at least 55ºC, at least 60ºC, at least 65ºC, at least 70ºC, at least 75ºC, at least 80ºC, at least 85ºC, at least 90ºC, at least 95ºC, at least 100ºC, or more. In some embodiments, the melting temperature is a peak melting temperature of at least 25ºC, at least 30ºC, at least 35ºC, at least 40ºC, at least 45ºC, at least 50ºC, at least 55ºC, at least 60ºC, at least 65ºC, at least 70ºC, at least 75ºC, at least 80ºC, at least 85ºC, at least 90ºC, at least 95ºC, at least 100ºC, or more.

[0320] In some embodiments, the protective material is a wax. In some embodiments, the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, paraffins, alkane-based waxes, mineral-based waxes, insect-based waxes, and plant-based waxes. In some embodiments, the fatty acid is a saturated fatty acid with an aliphatic tail of 6 to 21 carbons. In some embodiments, the saturated fatty acid is a medium- chain fatty acid (MCFA) with an aliphatic tail of 6 to 12 carbons. In some embodiments, the saturated fatty acid is a long-chain fatty acid (LCFA) with an aliphatic tail of 13 to 21 carbons. In some embodiments, the carrier is a stearic acid. In some embodiments, the stearic acid consists of at least 90% by weight of octadecanoic acid (C18:0). In some embodiments, the glycerides are monoglycerides or triglycerides. In some embodiments, the glyceride is hydrogenated glyceride. In some embodiments, the plant-based wax is a hydrogenated vegetable oil. In some embodiments, the plant-based wax is hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenatedpalm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil. In some embodiments, the additional carrier is hydrogenated soybean oil. In some embodiments, the oil is fully hydrogenated. In some embodiments, the oil is partially hydrogenated.

[0321] In some embodiments, the composition produced using cold-melt extrusion has a water activity level below 0.8. As used herein, the term “water activity level” refers to the ratio of the water vapor pressure in a composition to the saturation water vapor pressure at the temperature of the composition. In some embodiments, the composition produced using cold-melt extrusion has a water activity level below 0.6. In some embodiments, the composition produced using cold-melt extrusion has a water activity level below 0.4. In some embodiments, the composition produced using cold-melt extrusion has a water activity level below 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, or lower.

[0322] In some embodiments, the cold-melt extrusion method increases survival of the at least one microorganism. In some embodiments, the method increases survival by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In some embodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0323] In some embodiments, the cold-melt extrusion method increases stability of the composition. As used herein, the term “stability” refers to the ability of the composition to maintain proper structure and / or activity. In some embodiments, exposure of the composition high temperatures may reduce stability of the composition. In some embodiments, the methods described herein increase stability of the composition by reducing temperature during manufacturing. In some embodiments, the method increases stability of the product by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion. In some embodiments, the composition comprises at least one microorganism described herein. In some embodiments, the at least one microorganism is afacultative or strict anaerobic bacteria. In some embodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0324] In some embodiments, the cold-melt extrusion method increases the total yield of the composition comprising at least one microorganism. In some embodiments, the method increases total yield of the product by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion.

[0325] In some embodiments, the use of cold-melt extrusion during processing increases the total yield of at least one microorganism at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E2 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E3 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E4 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E5 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E6 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E7 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E8 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E9 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E10 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E11 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E12 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E13 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E14 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E15 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In someembodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0326] In some embodiments, the composition produced using cold-melt extrusion followed by coating (e.g., hot-melt fluid-bed coating) of the composition has a water activity level below 0.8. As used herein, the term “water activity level” refers to the ratio of the water vapor pressure in a composition to the saturation water vapor pressure at the temperature of the composition. In some embodiments, the composition produced using cold-melt extrusion followed by coating has a water activity level below 0.6. In some embodiments, the composition produced using cold-melt extrusion followed by coating has a water activity level below 0.4. In some embodiments, the composition produced using cold-melt extrusion followed by coating has a water activity level below 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, or lower.

[0327] In some embodiments, the cold-melt extrusion method followed by coating (e.g., hot- melt fluid bed coating) of the composition increases survival of the at least one microorganism. In some embodiments, the method increases survival by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion followed by coating. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In some embodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0328] In some embodiments, the cold-melt extrusion method followed by coating (e.g., hot- melt fluid bed coating) of the composition increases stability of the composition. In some embodiments, the method increases stability of the product by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion followed by coating. In some embodiments, the composition comprises at least one microorganism described herein. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In some embodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0329] In some embodiments, the cold-melt extrusion method followed by coating (e.g., hot- melt fluid bed coating) of the composition increases the total yield of the composition comprising at least one microorganism. In some embodiments, the method increases total yield of the product by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% when compared to a method that does not use cold-melt extrusion followed by coating.

[0330] In some embodiments, the use of cold-melt extrusion followed by coating (e.g., hot- melt fluid bed coating) of the composition increases the total yield of at least one microorganism at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E2 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E3 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E4 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E5 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E6 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E7 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E8 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E9 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E10 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E11 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E12 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E13 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E14 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism reaches a concentration of greater than 1E15 CFU / g at the end of manufacturing. In some embodiments, the at least one microorganism is a facultative or strict anaerobic bacteria. In some embodiments, the microorganism is Ruminococcus bovis. In some embodiments, the microorganism is Butyrivibrio fibrisolvens.

[0331] In some embodiments, the present disclosure provides a product manufactured using a cold-melt extrusion process. In some embodiments, the product is a microbial product, a feed product, a food product, a nutraceutical product, a pharmaceutical product.

[0332] In some embodiments, the present disclosure provides a product manufactured using a cold-melt extrusion process. In some embodiments, the product is a microbial product such as a feed additive or supplement. In some embodiments, the product comprises materials approved as animal feed ingredient by the Association of American Feed Control Officials (AAFCO). In some embodiments, the product comprises materials approved as a human food ingredient by the United States Food and Drug Administration (U.S. FDA).

[0333] In some embodiments, the cold-melt extrusion process is used for the development of a microbial product such as a feed additive or supplement. In some embodiments, the microbial product comprises at least one microorganism. In some embodiments, the microorganism is any of the microorganisms described herein. In some embodiments, the microorganism is a bacteria or fungi. In some embodiments, the microorganism is an anaerobic, aerobic, or facultative microorganism. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism comprises a 16S or ITS nucleic acid sequence of any one of SEQ ID NOs: 1-34. In some embodiments, the at least one microorganism is of the genus Butyrivibrio. In some embodiments, the at least one microorganism is of the genus species Butyrivibrio fibrisolvens. In some embodiments, the at least one microorganism is a Butyrivibrio fibrisolvens with a deposit accession number of NRRL B-67347. In some embodiments, the at least one microorganism is of the genus Ruminococcus. In some embodiments, the at least one microorganism is of the genus species Ruminococcus bovis. In some embodiments, the at least one microorganism is a Ruminococcus bovis with a deposit accession number of PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479. Other Stabilization Methods

[0334] In some embodiments, the methods provided herein further comprise stabilization of preserved microbial cells comprising preserving a population of microbial cells to provide apopulation of preserved microbial cells; harvesting viable microbial cells from the preserved population of microbial cells to provide a population of viable preserved microbial cells; combining the population of viable preserved microbial cells with at least one water activity scavenger (WAS) to a desired homogeneity level; and packaging and sealing the mixture of the population of viable preserved microbial cells and the WAS.

[0335] In some embodiments, the present disclosure provides methods, apparatuses, and systems for stabilization of microorganisms. Such methods can be used, for example, to form a stabilized microbial composition, as detailed below. Such stabilized compositions contain and / or comprise one or more stabilized microorganisms. In some embodiments, the microorganism is a vegetative microorganism. In some embodiments, the microorganism is disclosed in one or more of the following: U.S. Pub. Nos. 2018 / 0310592, 2018 / 0333443, 2018 / 0223325, 2022 / 0174992, 2022 / 0265732, 2022 / 0386647, and PCT Pub. Nos. 2016 / 210251, 2017 / 120495, 2017 / 181203, 2018 / 201049, 2019 / 079629, 2018 / 056563, 2020 / 042148, 2020 / 227442, 2021 / 163212, 2021 / 011662, 2021 / 202804, 2022 / 226367, and 2022 / 081992 (each being herein expressly incorporated by reference for all purposes). Microbial Products or Compositions

[0336] In some embodiments, the present disclosure provides a product consisting of a Geometrically Encapsulated Microbes (GEMs) composition prepared using the manufacturing methods described herein. In some embodiments, the composition prepared by the methods described herein comprises at least one microorganism, at least one carrier, and optionally at least one protective coating material. In some embodiments, the at least one microorganism is encapsulated.

[0337] In some embodiments, the GEMs composition comprises at least one microbial strain at 102to 1015cells per gram of said composition. In some embodiments, the composition comprises at least two microbial strains at 102to 1015cells per gram of said composition. In some embodiments, the composition may be mixed with a feed composition.

[0338] In some embodiments, the GEMs compositions of the present disclosure are administered to an animal. In some embodiments, the composition is administered at least once per day. In a further embodiment, the composition is administered at least once per week. In a further embodiment, the composition is administered at least once per month. In a further embodiment, the composition is administered at least once per hour.

[0339] In some embodiments, the administration comprises injection of the composition into the rumen. In some embodiments, the composition is administered anally. In further embodiments, anal administration comprises inserting a suppository into the rectum. In some embodiments, the composition is administered orally. In some aspects, the oral administration comprises administering the composition in combination with the animal’s feed, water, medicine, or vaccine. In some embodiments, the administration occurs each time the animal is fed. In some embodiments, the oral administration comprises administering the composition in combination with the animal feed.

[0340] In some embodiments, the compositions of the present invention include feed, such as cereals (barley, maize, oats, and the like); starches (tapioca and the like); oilseed cakes; and vegetable wastes. In some embodiments, the compositions include vitamins, minerals, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickening agents, and the like.

[0341] In some embodiments, the compositions of the present invention include one or more carriers including, but not limited to: mineral earths such as clay, diatomaceous earth, dolomite, kaolin, limestone, silica, talc; calcium carbonate; calcium sulfate; calcium oxide; magnesium sulfate; magnesium oxide; products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.

[0342] In some embodiments, the compositions of the present disclosure comprise thickening agents such as mineral earths (e.g., silica and clay), starches (e.g., native starch and pregelatinized starch), polysaccharides (e.g., alginate, guar gum, locust bean gum, and xanthan gum), natural extracts of seeds or seaweed, cellulose, and polyvinyl alcohol.

[0343] In some embodiments, the compositions of the present disclosure comprise an animal- safe virucide or nematicide. In some embodiments, microbial compositions of the present disclosure comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, and the like), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts, and combinations thereof. In a further embodiment, compositions comprise polymers of agar, agarose, gellan gum, and the like.

[0344] In some embodiments, the compositions of the present disclosure comprise one or more preservatives. The preservatives may be selected from one or more of monosaccharide, disaccharide, trisaccharide, polysaccharide, acetic acid and its salts (e.g., potassium acetate),ascorbic acid and its salts (e.g., sodium ascorbate, calcium ascorbate), iso-ascorbic acid, citric acid and its salts, erythorbic acid, erythrobic acid, benzoic acid and its salts, sorbic acid and its salts (e.g., calcium sorbate, potassium sorbate, sodium sorbate), propionic acid, tartaric acid, potassium nitrate, sodium erythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite, ethyl lauroyl arginate, methyl-p-hydroxy benzoate, methyl paraben, propyl paraben, potassium benzoiate, potassium bisulphite, potassium diacetate, potassium lactate, potassium metabisulphite, propyl-p-hydroxy benzoate, sodium acetate, sodium benzoate, sodium bisulphite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite, sodium salt of methyl-p-hydroxy benzoic acid, sodium salt of propyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodium metabisulphite, sodium dithionite, calcium propionate, dimethyl dicarbonate, natamycin, potassium bisulfite, potassium metabisulfite, sodium diacetate, sodium propionate, ascorbyl palmitate, ascorbyl stearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), citric acid esters of mono- and / or diglycerides, L-cysteine, L-cysteine hydrochloride, gum guaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate, monoisopropyl citrate, propyl gallate, tertiary butyl hydroquinone, stannous chloride, thiodipropionic acid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin, sulfur dioxide, formic acid, or tocopherol(s).

[0345] In some embodiments, the GEMs compositions of the present disclosure include bacterial and / or fungal cells in spore form, vegetative cell form, and / or lysed cell form. In one embodiment, the lysed cell form acts as a mycotoxin binder, e.g. mycotoxins binding to dead cells.

[0346] In some embodiments, the compositions are shelf stable at -30-4°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the compositions are shelf stable at -30-4°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0347] In some embodiments, the compositions are shelf stable in a refrigerator (4-10°C) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the compositionsare shelf stable in a refrigerator (4-10°C) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0348] In some embodiments, the compositions are shelf stable at room temperature (20-25°C) or between 50-77°F for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the compositions are shelf stable at room temperature (20-25°C) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0349] In some embodiments, the compositions are shelf stable at 25-37°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the compositions are shelf stable at 25-37°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0350] In some embodiments, the compositions are shelf stable at 37-100°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the compositions are shelf stable at 37-100°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0351] In some embodiments, the compositions of the present disclosure are shelf stable at refrigeration temperatures (4-10°C), at room temperature (20-25°C), between 10-25°C, between -30-4°C, between 25-37°C, or between 37-100°C for a period of about 1 to 100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1 to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about 1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to 5, about 5 to 100,about 5 to 95, about 5 to 90, about 5 to 85, about 5 to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about 5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to 10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85, about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10 to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85, about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15 to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20 to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80, about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20 to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35, about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about 25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to 70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30 to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80, about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30 to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35, about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about 35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to 60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40, about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about 40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to 60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100, about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45 to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55, about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about 50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to 65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95, about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55 to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95, about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60 to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90, about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70 to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80, about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about 75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to 90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90, about 90 to 100, about 90 to 95, or 95 to 100 weeks

[0352] In some embodiments, the compositions of the present disclosure are shelf stable at refrigeration temperatures (4-10°C), at room temperature (20-25°C), between 10-25°C, between -30-4°C, between 25-37°C, or between 37-100°C for a period of 1 to 100, 1 to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95, 10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to 100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to 70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to 100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to 90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95 to 100 weeks.

[0353] In some embodiments, the compositions of the present disclosure are shelf stable at refrigeration temperatures (4-10°C), at room temperature (20-25°C), between 10-25°C, between -30-4°C, between 25-37°C, or between 37-100°C for a period of about 1 to 36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1 to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about 1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8, about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34, about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to 24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4 to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6 to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about 6 to 26, about 6 to 24, about6 to 22, about 6 to 20, about 6 to 18, about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to 8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8 to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about 8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10, about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10 to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20, about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12 to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28, about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12 to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34, about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14 to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16, about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16 to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20, about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18 to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22, about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20 to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22, about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22 to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34, about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26 to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30 to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34, or about 34 to 36 months.

[0354] In some embodiments, the compositions of the present disclosure are shelf stable at refrigeration temperatures (4-10°C), at room temperature (20-25°C), between 10-25°C, between -30-4°C, between 25-37°C, or between 37-100°C for a period of 1 to 361 to 341 to 321 to 301 to 281 to 261 to 241 to 221 to 201 to 181 to 161 to 141 to 121 to 101 to 81 to 61 one 41 to 24 to 364 to 344 to 324 to 304 to 284 to 264 to 244 to 224 to 204 to 18 4 to 164 to 144 to 124 to 104 to 84 to 66 to 366 to 346 to 326 to 306 to 286 to 266 to 246 to 226 to 206 to 186 to 166 to 146 to 126 to 106 to 88 to 368 to 348 to 328 to 308 to 288 to 268 to 248 to 228 to 208 to 188 to 168 to 148 to 128 to 1010 to 3610 to 3410 to 3210 to 3010 to 2810 to 2610 to 2410 to 2210 to 2010 to 1810 to 1610 to 1410 to 12 12 to 3612 to 3412 to 3212 to 3012 to 2812 to 2612 to 2412 to 2212 to 2012 to 1812 to 1612 to 1414 to 3614 to 3414 to 3214 to 3014 to 2814 to 2614 to 2414 to 2214 to 2014 to 1814 to 1616 to 3616 to 3416 to 3216 to 3016 to 2816 to 2616 to 2416 to 2216 to 20 16 to 1818 to 3618 to 3418 to 3218 to 3018 to 2818 to 2618 to 2418 to 2218 to 2020 to 3620 to 3420 to 3220 to 3020 to 2820 to 2620 to 2420 to 2222 to 3622 to 3422 to 3222to 3022 to 2822 to 2622 to 2424 to 3624 to 3424 to 3224 to 3024 to 2824 to 2626 to 36 26 to 3426 to 3226 to 3026 to 2828 to 3628 to 3428 to 3228 to 3030 to 3630 to 3430 to 3232 to 3632 to 34, or about 34 to 36.

[0355] In some embodiments, the compositions of the present disclosure are shelf stable at any of the disclosed temperatures and / or temperature ranges and spans of time at a relative humidity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% Animal Feed

[0356] In some embodiments, the compositions of the present invention are mixed with animal feed. In some embodiments, animal feed may be present in various forms such as pellets, capsules, granules, powder, paste, or liquid.

[0357] In some embodiments, products of the present disclosure are mixed into the premix at a feed mill, alone as a standalone premix, and / or alongside other feed additives such as trace minerals, amino acids, vitamins, etc. In one embodiment, the products of the present invention are mixed into the feed at a feed mill. In another embodiment, products of the present invention are mixed into the feed itself.

[0358] In some embodiments, the feed may be supplemented with water, premix or premixes, forage, fodder, beans (e.g., whole, cracked, or ground), grains (e.g., whole, cracked, or ground), bean- or grain-based oils, bean- or grain-based meals, bean- or grain-based haylage or silage, bean- or grain-based syrups, fatty acids, sugar alcohols (e.g., polyhydric alcohols), commercially available formula feeds, and mixtures thereof.

[0359] In some embodiments, forage encompasses hay, haylage, and silage. In some embodiments, hays include grass hays (e.g., sudangrass, orchardgrass, or the like), alfalfa hay, and clover hay. In some embodiments, haylages include grass haylages, sorghum haylage, and alfalfa haylage. In some embodiments, silages include maize, oat, wheat, alfalfa, clover, and the like.

[0360] In some embodiments, premix or premixes may be utilized in the feed. Premixes may comprise micro-ingredients such as vitamins, minerals, amino acids; chemical preservatives;pharmaceutical compositions such as antibiotics and other medicaments; fermentation products, and other ingredients. In some embodiments, premixes are blended into the feed.

[0361] In some embodiments, the feed may include feed concentrates such as soybean hulls, sugar beet pulp, molasses, high protein soybean meal, ground corn, shelled corn, wheat midds, distiller grain, cottonseed hulls, rumen-bypass protein, rumen-bypass fat, and grease. See Luhman (U.S. Publication US20150216817A1), Anderson et al. (U.S. Patent No.3,484,243) and Porter and Luhman (U.S. Patent No. 9,179,694B2) for animal feed and animal feed supplements capable of use in the present compositions and methods.

[0362] In some embodiments, feed occurs as a compound, which includes, in a mixed composition capable of meeting the basic dietary needs, the feed itself, vitamins, minerals, amino acids, and other necessary components. Compound feed may further comprise premixes.

[0363] In some embodiments, microbial compositions of the present disclosure may be mixed with animal feed, premix, and / or compound feed. Individual components of the animal feed may be mixed with the microbial compositions prior to feeding to ruminants. The microbial compositions of the present disclosure may be applied into or on a premix, into or on a feed, and / or into or on a compound feed. EXAMPLES Example 1: Thermal Analysis of Plant-based Waxes with Differential Scanning Calorimetry

[0364] Plant-based waxes including stearic acid, fractionated palm stearine, hydrogenated palm oil, hydrogenated soybean oil, hydrogenated sunflower oil, and hydrogenated rapeseed oil were analyzed by differential scanning calorimetry (DSC) to determine their melting and congealing temperatures. About 5 mg of the samples were placed in hermetically sealed pans and analyzed with the sequence of a 1st heating cycle, a cooling cycle, and a 2nd heating cycle. Each sample was first cooled to 0ºC, then heated to 100ºC at a rate of 5ºC per minute, cooled back to 0ºC at a rate of 5ºC per minute, and heated again to 100ºC at a rate of 5ºC per minute. The samples were each analyzed in duplicate. The congealing and melting (1st heating cycle) temperatures are summarized in Table 4 below. Table 4. Summary of DSC results for plant-based waxesExample 2. Production of Microbial Pellet Compositions with Cold-Melt Extrusion

[0365] Microbial compositions were produced as extruded pellets with cold-melt extrusion with the aim of development as a feed ingredient for dairy cattle. Formulation ingredients including a microorganism in freeze-dried powder form (mean particle size under 150 µm), a meltable carrier, and optionally a stabilizing agent were simultaneously fed into an extruder preset at a temperature of at least 5ºC below the peak melting point of the carrier. The barrel was segmented by design, which allowed each segment to be set at a different temperature. Feed materials were blended and heated while being moved forward by the rotary motion of the screws. The barrel temperature(s) could be varied to produce a partially molten composition with a different degree of consistency. The extrusion product in form of a soft extrudate was cut into small pellets by using a rotary knife mounted at the extruder die exit. The pellets were collected, packaged, and refrigerated until future analysis. An image of typical microbe- containing pellets is illustrated in FIG.3. Example 3. Encapsulation of Microbe-containing Pellets with Hot-Melt Fluid-Bed Coating

[0366] Encapsulation of microbe-containing pellets was achieved by spray coating of the pellets with a molten plant-based wax in a fluid-bed coater with bottom-spray setup. The spray setup consisted of a spray gun assembly and insulated feed transfer lines with electrical tracing to maintain the spray feed above the melting temperature of the wax. The fluidization process was ended once the pellets were coated to their target coating level. The coated pellets were discharged from the coater, packaged, and refrigerated until future analysis. Example 4. Stability Study Under Simulated In-Feed Conditions

[0367] Stability of microbe-containing pellets was evaluated under different simulated in-feed conditions. Pellets were filled into an open 2.5-inch by 3-inch metalized plastic bags, 4.3 mils thickness, and incubated in an environmental chamber for up to 10 days. One bag of pellets was prepared to be sampled per each time point. The bags were collected at designated time points and the pellets were analyzed for viable cell count by using a standard enumeration procedure. The environmental conditions employed for testing were as follows:

[0368] (1) Oscillating conditions: 37ºC x 75% relative humidity (RH) for 12 hours and 22ºC x 90% RH for 12 hours each day for up to 10 days;

[0369] (2) Static conditions: 37ºC x 95% RH for 24 hours; and

[0370] (3) Oscillating and Static conditions, combined: 37ºC x 75% RH for 12 hours and 22ºC x 90% RH for 12 hours per day for up to 7 days followed by 37ºC x 95% RH for 1 day. Example 5. Isolation and Cultivation of Butyrivibrio fibrisolvens

[0371] Source and Description: Butyrivibrio fibrisolvens identified as DY-19 was identified and isolated to axenicity from the rumen contents of a healthy, mid-lactation Holstein cow rumen obtained via cannula. The sample was received and isolated by Native Microbials. The isolate was deposited in the NRRL, Agricultural Research Service Culture Collection, and referenced as NRRL B-67347. B. fibrisolvens DY-19 is a prominent anaerobic, non-spore- forming, member of the ruminant gut microbiome. In the rumen, the species degrades fibrous plant material and ferments polysaccharides.

[0372] Enumeration: B. fibrisolvens pellets were assayed to determine the number of viable cells by counting colony forming units (cfu) on solid reinforced clostridial medium (RCM). Briefly, B. fibrisolvens pellets were first ground either in the presence of liquid nitrogen in a pestle and mortar set or in a centrifugal tube containing 10x 3-mm stainless steel grinding beads mounted in a SPEX® SamplePrep 2010 Geno / Grinder®. The Geno / Grinder® was operated at 1750 strokes / min for 5 minutes. About 0.10 g of ground material was transferred to a prelabeled tared 15-mL polypropylene centrifuge tube. Each sample was assayed in triplicate. The tubes were then transferred to an anaerobic chamber airlock with lids cracked to allow for exchange of air with a gas mixture of carbon dioxide, hydrogen, and nitrogen in 2-3 cycles. About 10 mL of sterile anaerobic PBSa (1x phosphate buffer saline with antioxidants, pH 7.5) was added to each sample tube and vortexed for 10-30 seconds until homogenously mixed.

[0373] The samples were plated on RCM media. Plates were then incubated in a GasPak™ EZ anaerobe container system at 37ºC ± 2ºC for 48 hours before enumeration. The number of viable cells in ‘cfu / g’ was reported as the average of 3 measurements. Example 6. Production of Butyrivibrio fibrisolvens Pellets by Cold-Melt Extrusion

[0374] Pelletized compositions of B. fibrisolvens were produced with cold-melt extrusion with the aim of development as a feed ingredient for dairy cattle.

[0375] Method: Process equipment included a co-rotating 26-mm twin-screw extruder (L / D = 28) built using a 7-barrel setup including an endplate and a pellet die (1.5 mm in diameter). Two loss-in-weight feeders were set up over the main feed in barrel 1. B. fibrisolvens (powder mean particle size was 41 µm) and food-grade hydrogenated soybean oil (HSBO, Tm68-72ºC) were fed into barrel 1. The mass ratio of B. fibrisolvens to HSBO was kept at 20:80 (w:w) throughout the process. The extrusion was performed at a constant screw speed of 175 rpm and a constant feed rate of 265 g / min (15.9 kg / h). The temperature was controlled at 35ºC across the barrel sections. Soft extrudate discharging through the die was cut into 1- to 4-mm pellets using a rotary knife mounted at the extruder die exit. The pellets were collected, packaged, and refrigerated until future analysis. Example 7. Encapsulation of Butyrivibrio fibrisolvens Pellets with Hot-Melt Fluid-Bed Coating

[0376] B. fibrisolvens pellets disclosed in Example 6 were coated with food-grade hydrogenated soybean oil (HSBO, Tm 68-72ºC) by spray coating in a laboratory fluid-bed coater equipped with a 0.15-m product chamber with bottom-spray setup. The spray setup consisted of a spray gun assembly and feed transfer lines with electrical tracing maintained at 82ºC and 92ºC, respectively. The fluidization air temperature was controlled at 21ºC while the product bed temperature varied from 28-29ºC. The fluidization process was completed once the pellets were coated to 40% coating level (HSBO:Pellet). The coated pellets were discharged from the coater, packaged, and refrigerated until future analysis. Example 8. Stability of HSBO-Coated and Uncoated Butyrivibrio fibrisolvens Pellets Under Simulated In-feed Oscillating Conditions

[0377] Stability of B. fibrisolvens pellets disclosed in Example 6 and HSBO-coated pellets disclosed in Example 7 was evaluated under simulated in-feed oscillating conditions as disclosed in Example 4 (i.e., 37ºC x 75% RH for 12 hours and 22ºC x 90% RH for 12 hourseach day). Pellets were filled into open 2.5-inch by 3-inch metalized plastic bags, 4.3 mils thickness, and incubated in an environmental chamber for 10 days. One bag of pellets was prepared to be sampled per each time point. The bags were collected after 0, 1, 2, 3, 5, 7, and 10 days of storage and the pellets were analyzed for viable cell counts by using the standard enumeration procedure outlined in Example 5. The water activity (aw) of pellets was measured using a HygroLab water activity meter (Rotronic Measurement Solutions, Hauppauge, NY, USA) equipped with water activity probes HC2-AW.

[0378] The cell enumeration and water activity measurements are provided in Table 5 below. The coated pellets exhibited improved stability under tested conditions compared to their uncoated counterparts, showing about 0.7 log cfu loss compared to 1.3 log cfu loss and 1.4 log loss compared to 1.7 log loss, respectively after 3 and 5 days of storage under oscillating conditions. The uncoated pellets exhibited no detectable live cell counts after 7 days of incubation. Table 5. Stability of HSBO-Coated and Uncoated Butyrivibrio fibrisolvens Pellets Under Oscillating Environmental ConditionsCell enumeration n=3; water activity n=2 Example 9. Stability of HSBO-Coated Butyrivibrio fibrisolvens Pellets Under Simulated In-Feed Static Conditions

[0379] Stability of HSBO-coated B. fibrisolvens pellets disclosed in Example 7 was evaluated under simulated in-feed static conditions as disclosed in Example 4 (i.e., 37ºC x 95% RH for 24 hours). Pellets were filled into open 2.5-inch by 3-inch metalized plastic bags, 4.3 mils thickness, and incubated in an environmental chamber for 24 hours. One bag of pellets was prepared to be sampled per each time point. The bags were collected after 0, 2, 4, 6, 8, 10, 18,and 24 hours of storage and the pellets were analyzed for viable cell counts by using the standard enumeration procedure outlined in Example 5. The water activity (aw) of pellets was measured using a HygroLab water activity meter (Rotronic Measurement Solutions, Hauppauge, NY, USA) equipped with water activity probes HC2-AW.

[0380] The cell enumeration and water activity measurements are provided in Table 6 below. The coated pellets exhibited excellent stability under tested conditions, showing about 1.27 log cfu loss after 24 hours of storage under static conditions. Table 6. Stability of HSBO-Coated Butyrivibrio fibrisolvens Pellets Under Static Environmental ConditionsCell enumeration n=3; water activity n=2 Example 10. Stability of HSBO-Coated Butyrivibrio fibrisolvens Pellets Under Simulated In-feed Oscillating and Static Conditions

[0381] Stability of HSBO-coated B. fibrisolvens pellets disclosed in Example 7 was evaluated under simulated in-feed oscillating and static conditions as disclosed in Example 4 (i.e., 37ºC x 75% RH for 12 hours and 22ºC x 90% RH for 12 hours per day for 3 days followed by 37ºC x 95% RH for 1 day). Pellets were filled into open 2.5-inch by 3-inch metalized plastic bags, 4.3 mils thickness, and incubated in an environmental chamber for 96 hours. One bag of pellets was prepared to be sampled per each time point. The bags were collected after 0, 24, 48, 72, 74, 76, 78, 80, 82, 84, 90, and 96 hours of storage and the pellets were analyzed for viable cell counts by using the standard enumeration procedure outlined in Example 5. The water activity (aw) of pellets was measured using a HygroLab water activity meter (Rotronic Measurement Solutions, Hauppauge, NY, USA) equipped with water activity probes HC2-AW.

[0382] The cell enumeration and water activity measurements are provided in Table 7. The coated pellets exhibited excellent stability under tested conditions, showing about 0.86 log cfuloss after 72 hours of storage under oscillating followed by 24 hours of storage under static conditions. Table 7. Stability of HSBO-coated Butyrivibrio fibrisolvens pellets Under Oscillating and Static Environmental ConditionsCell enumeration n=3; water activity n=2 Example 11. Shelf-Stability of HSBO-Coated Butyrivibrio fibrisolvens Pellets

[0383] Stability of HSBO-coated B. fibrisolvens pellets disclosed in Example 7 was evaluated under refrigerated conditions (4-8ºC). About 1.5 kg of pellets were packaged in a sealed foil laminated bag (7.5 mil). Pellet samples were collected after 0, 0.5, 1, 3, 4, and 5 months of storage post-manufacturing and analyzed by enumeration method described in Example 5. The water activity (aw) of pellets was measured using a HygroLab water activity meter (Rotronic Measurement Solutions, Hauppauge, NY, USA) equipped with water activity probes HC2- AW.

[0384] The cell enumeration results are summarized in Table 8. No loss of cell activity was observed after 4 months of storage at 4-8ºC. The initial aw of HSBO-coated pellets was 0.271 and showed no appreciable change during the storage period. Table 8. Shelf-life Stability of HSBO-Coated Butyrivibrio fibrisolvens Pellets Under Refrigerated ConditionsExample 12. Production of Butyrivibrio fibrisolvens Pellets Containing a Stabilizing Agent by Cold-Melt Extrusion

[0385] Pelletized compositions of B. fibrisolvens containing a natural zeolite (St. Cloud Mining Co., Winston, NM, USA) as the stabilizing agent were produced with cold-melt extrusion with the aim of development as a feed ingredient for dairy cattle.

[0386] Method: Process equipment included a co-rotating 26-mm twin-screw extruder (L / D = 28) built using a 7-barrel setup including an endplate and a pellet die (2.3 mm in diameter). Three loss-in-weight feeders were set up over the main feed in barrel 2. B. fibrisolvens (powder mean particle size was 41 µm), food-grade hydrogenated soybean oil (HSBO, Tm 68-72ºC), and natural zeolite powder were fed into barrel 2. The mass ratio of B. fibrisolvens to HSBO and zeolite was kept at 20:70:10 (w:w) throughout the process. The extrusion was performed at a constant screw speed of 175 rpm and a constant feed rate of 267 g / min (16.0 kg / h). The temperature was controlled at 40ºC across the barrel sections. Soft extrudate discharging through the die was cut into 1- to 4-mm pellets using a rotary knife mounted at the extruder die exit. For comparison, B. fibrisolvens:HSBO pellets at 20:80 (w:w) were produced without natural zeolite. The pellets were collected, packaged, and refrigerated until analysis for viable cell counts by using the standard enumeration procedure outlined in Example 5.

[0387] Observation: Process viability yield was calculated from the value of measured viable cell count (cfu / g) divided by expected / theoretical viable cell count (cfu / g) multiplied by 100%. The process viability yield in production of B. fibrisolvens pellets containing natural zeolite with cold-melt extrusion was about 100%, indicating no loss of cell activity, compared to 41% for the pellets formulated without zeolite. NUMBERED EMBODIMENTS

[0388] Embodiment 1. A method of manufacturing a geometrically encapsulated microbes (GEMs) composition by cold-melt extrusion, the method comprising the following steps: (a) feeding ingredients into an extruder wherein the ingredients comprise at least one microorganism and a carrier; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the carrier to form a semi-moltenor molten extrudable composition; (d) extruding the composition through a die head; (e) cutting the composition into pellets; (f) cooling the pellets to the ambient temperature; and (g) coating the pellets with a protective material.

[0389] Embodiment 2. The method of embodiment 1, wherein the temperature of the extruder is below 50ºC.

[0390] Embodiment 3. The method of embodiment 1, wherein the temperature of the extruder is below 40ºC.

[0391] Embodiment 4. The method of embodiment 1, wherein the temperature of the extruder is 35ºC.

[0392] Embodiment 5. The method of any one of embodiments 1-4, wherein the carrier is meltable at temperatures below 100ºC.

[0393] Embodiment 6. The method of any one of embodiments 1-4, wherein the melting temperature of the carrier is at least 40oC.

[0394] Embodiment 7. The method of embodiment 6, wherein the melting temperature is an onset melting temperature of at least 40ºC.

[0395] Embodiment 8. The method of embodiment 6, wherein the melting temperature is a peak melting temperature of at least 40ºC.

[0396] Embodiment 9. The method of any one of embodiments 1-8, wherein the carrier is partially or completely insoluble in aqueous media.

[0397] Embodiment 10. The method of any one of embodiments 1-9, wherein the carrier is a wax.

[0398] Embodiment 11. The method of embodiment 10, wherein the wax is selected from the group consisting of: animal fats, fatty acids, fatty acid esters, fatty alcohols, monoglycerides, diglycerides, triglycerides, insect-based waxes, and plant-based waxes.

[0399] Embodiment 12. The method of embodiment 11, wherein the fatty acid is a long-chain fatty acid and the plant-based wax is a hydrogenated vegetable oil selected from the group consisting of: hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

[0400] Embodiment 13. The method of any one of embodiments 1-12, wherein the carrier is stearic acid, hydrogenated soybean oil, or a mixture thereof.

[0401] Embodiment 14. The method of any one of embodiments 1-13, wherein the ingredients further comprise one or more functional ingredients.

[0402] Embodiment 15. The method of embodiment 14, wherein the one or more functional ingredients are selected from the group consisting of: a moisture-binding agent, a pH modifying agent, a release modifying agent, a disintegrating agent, and an oxygen scavenging agent.

[0403] Embodiment 16. The method of embodiment 15, wherein the moisture-binding agent is a zeolite, a bentonite, a diatomaceous earth, a silica, or an inorganic salt.

[0404] Embodiment 17. The method of embodiment 16, wherein the zeolite is a natural zeolite.

[0405] Embodiment 18. The method of embodiment 15, wherein the disintegrating agent is selected from the group consisting of a natural polysaccharide, a natural polysaccharide hydrogel, a soy polysaccharide, a psyllium husk fiber, and a mineral clay.

[0406] Embodiment 19. The method of any one of embodiments 1-18, wherein the at least one microorganism is formulated as a dried fine powder or a dried granular powder prior to cold- melt extrusion.

[0407] Embodiment 20. The method of embodiment 19, wherein the at least one microorganism is formulated as a dried fine powder or a dried granular powder by freeze- drying, spray drying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), tray drying, vacuum drying, microwave drying, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0408] Embodiment 21. The method of embodiment 19, wherein the at least one microorganism is microencapsulated with a carrier.

[0409] Embodiment 22. The method of embodiment 21, wherein the carrier has a melting temperature of at least 40oC.

[0410] Embodiment 23. The method of embodiment 22, wherein the melting temperature is an onset melting temperature of at least 40ºC.

[0411] Embodiment 24. The method of embodiment 22, wherein the melting temperature is a peak melting temperature of at least 40ºC.

[0412] Embodiment 25. The method of any one of embodiments 21-24, wherein the carrier is a wax.

[0413] Embodiment 26. The method of embodiment 25, wherein the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, insect- based waxes, and plant-based waxes.

[0414] Embodiment 27. The method of embodiment 26, wherein the plant-based wax is a hydrogenated vegetable oil.

[0415] Embodiment 28. The method of embodiment 27, wherein the hydrogenated vegetable oil is selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

[0416] Embodiment 29. The method of embodiment 27 or 28, wherein the hydrogenated vegetable oil is a fully hydrogenated vegetable oil.

[0417] Embodiment 30. The method of any one of embodiments 21-29, wherein the at least one microorganism is microencapsulated by freeze-drying, spray drying, fluid-bed spray granulation (spouted-bed), fluid-bed spray coating, hot-melt fluid-bed coating, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

[0418] Embodiment 31. The method of any one of embodiments 1-30, wherein the total amount of the at least one microorganism is about 10% to about 90% of the composition.

[0419] Embodiment 32. The method of any one of embodiments 1-30, wherein the at least one microorganism is native to the microbiome of an animal.

[0420] Embodiment 33. The method of any one of embodiments 1-30, wherein the at least one microorganism is native to the gastrointestinal microbiome of an animal.

[0421] Embodiment 34. The method of any one of embodiments 1-30, wherein the at least one microorganism is native to the microbiome of a monogastric animal.

[0422] Embodiment 35. The method of any one of embodiments 1-30, wherein the at least one microorganism is native to the microbiome of a ruminant or of a camelid.

[0423] Embodiment 36. The method of any one of embodiments 1-35, wherein the at least one microorganism is a facultative or strict anaerobic bacteria.

[0424] Embodiment 37. The method of any one of embodiments 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-34.

[0425] Embodiment 38. The method of any one of embodiments 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-34.

[0426] Embodiment 39. The method of any one of embodiments 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-34.

[0427] Embodiment 40. The method of any one of embodiments 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence of any one of SEQ ID NOs: 1- 34.

[0428] Embodiment 41. The method of any one of embodiments 1-40, wherein the at least one microorganism is of the genus Butyrivibrio.

[0429] Embodiment 42. The method of any one of embodiments 1-40, wherein the at least one microorganism is of the genus species Butyrivibrio fibrisolvens.

[0430] Embodiment 43. The method of any one of embodiments 1-40, wherein the at least one microorganism is a Butyrivibrio fibrisolvens with a deposit accession number of NRRL B- 67347.

[0431] Embodiment 44. The method of any one of embodiments 1-40, wherein the at least one microorganism is of the genus Ruminococcus.

[0432] Embodiment 45. The method of any one of embodiments 1-40, wherein the at least one microorganism is of the genus species Ruminococcus bovis.

[0433] Embodiment 46. The method of any one of embodiments 1-40, wherein the at least one microorganism is a Ruminococcus bovis with a deposit accession number of PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479.

[0434] Embodiment 47. The method of any one of embodiments 1-46, wherein the extruder is a single-screw extruder.

[0435] Embodiment 48. The method of any one of embodiments 1-47, wherein the extruder is a twin-screw extruder.

[0436] Embodiment 49. The method of embodiment 48, wherein the twin-screw extruder is a co-rotating or a counter-rotating extruder.

[0437] Embodiment 50. The method of any one of embodiments 1-49, wherein the method further comprises kneading the semi-molten or molten extrudable composition.

[0438] Embodiment 51. The method of any one of embodiments 1-50, wherein the die head comprises a single hole or a plurality of holes.

[0439] Embodiment 52. The method of any one of embodiments 1-51, wherein the die hole is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a hemisphere.

[0440] Embodiment 53. The method of any one of embodiments 1-51, wherein the die hole has a round shape.

[0441] Embodiment 54. The method of any one of embodiments 1-53, wherein the die hole has a diameter of about 0.5 mm to about 50 mm.

[0442] Embodiment 55. The method of embodiment 54, wherein the die hole has a diameter of about 1 mm to about 4 mm.

[0443] Embodiment 56. The method of any one of embodiments 1-55, wherein the cooling is cryogenic cooling.

[0444] Embodiment 57. The method of embodiment 56, wherein the cryogenic cooling is performed using liquid nitrogen or carbon dioxide.

[0445] Embodiment 58. The method of any one of embodiments 1-55, wherein the cooling is performed using a chilled dry gas.

[0446] Embodiment 59. The method of embodiment 58, wherein the chilled dry gas is nitrogen, carbon dioxide, or dehumidified air.

[0447] Embodiment 60. The method of any one of embodiments 1-59, wherein the pellet is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a sphere.

[0448] Embodiment 61. The method of any one of embodiments 1-59, wherein the pellet has a round shape.

[0449] Embodiment 62. The method of any one of embodiments 1-61, wherein the pellet has a diameter of about 0.5 mm to about 50 mm.

[0450] Embodiment 63. The method of embodiment 62, wherein the pellet has a diameter of about 1 mm to about 4 mm.

[0451] Embodiment 64. The method of any one of embodiments 1-63, wherein the pellet is coated with an additional protective material.

[0452] Embodiment 65. The method of embodiment 64, wherein the pellet is coated by hot- melt fluid-bed coating.

[0453] Embodiment 66. The method of embodiment 64 or 65, wherein the pellet is coated to a level of at least 5% with the additional protective material.

[0454] Embodiment 67. The method of any one of embodiments 64-66, wherein the additional protective material is an additional carrier, and wherein the additional carrier has a melting temperature of at least 40oC.

[0455] Embodiment 68. The method of embodiment 67, wherein the melting temperature is an onset melting temperature of at least 40ºC.

[0456] Embodiment 69. The method of embodiment 67, wherein the melting temperature is a peak melting temperature of at least 40ºC.

[0457] Embodiment 70. The method of any one of embodiments 64-69, wherein the additional carrier is a wax.

[0458] Embodiment 71. The method of embodiment 70, wherein the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, insect- based waxes, and plant-based waxes.

[0459] Embodiment 72. The method of embodiment 71, wherein the fatty acid is a long-chain fatty acid and the plant-based wax is a hydrogenated vegetable oil.

[0460] Embodiment 73. The method of embodiment 72, wherein the hydrogenated vegetable oil is selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenatedpalm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

[0461] Embodiment 74. The method of embodiment 72 or 73, wherein the hydrogenated vegetable oil is fully hydrogenated vegetable oil.

[0462] Embodiment 75. The method of embodiment any one of embodiments 64-69, wherein the additional protective material is stearic acid, hydrogenated soybean oil, or a mixture thereof.

[0463] Embodiment 76. The method of embodiment 71, wherein the glyceride is hydrogenated glyceride.

[0464] Embodiment 77. The method of any one of embodiments 1-76, wherein the composition has water activity below 0.4.

[0465] Embodiment 78. The method of embodiment 77, wherein the water activity is below 0.3, 0.2, or 0.1.

[0466] Embodiment 79. The method of embodiment 77, wherein the water activity is below 0.1.

[0467] Embodiment 80. The method of any one of embodiments 1-78, wherein the method increases survival of the at least one microorganism.

[0468] Embodiment 81. The method of any one of embodiments 1-80, wherein the method increases stability of the at least one microorganism.

[0469] Embodiment 82. The method of any one of embodiments 1-81, wherein the method increases the total processing yield of the at least one microorganism.

[0470] Embodiment 83. The method of any one of embodiments 1-82, wherein the composition is a probiotic for use in animals.

[0471] Embodiment 84. The method of embodiment 83, wherein the animal is a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal, or a companion animal.

[0472] Embodiment 85. A composition produced by the method of any one of embodiments 1-82.

[0473] Embodiment 86. A microbial product produced by the method of any one of embodiments 1-82.

[0474] Embodiment 87. A feed additive or supplement produced by the method of any one of embodiments 1-82.

[0475] Embodiment 88. A composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a carrier, wherein the pellet is manufactured by cold- melt extrusion at a temperature of at least 5ºC below the melting temperature of the carrier.

[0476] Embodiment 89. A composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a carrier, wherein the pellet is manufactured by cold- melt extrusion at a temperature at or below 50ºC.

[0477] Embodiment 90. A method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: (a) feeding ingredients into an extruder, wherein the ingredients comprise at least one biologic and a wax; (b) conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, (c) heating the ingredients at a temperature at or below 50ºC to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head; (e) cutting the composition into pellets; and (f) cooling the pellets.

[0478] Embodiment 91. A method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: (a) feeding ingredients into an extruder, wherein the ingredients comprise at least one microorganism and a wax; (b) conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, (c) heating the ingredients at a temperature at or below 50ºC to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head; (e) cutting the composition into pellets; and (f) cooling the pellets. INCORPORATION BY REFERENCE

[0479] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

CLAIMS 1. A method of manufacturing a geometrically encapsulated microbes (GEMs) composition by cold-melt extrusion, the method comprising the following steps: (a) feeding ingredients into an extruder wherein the ingredients comprise at least one microorganism and a carrier; (b) conveying and blending the ingredients at a temperature between the ambient temperature and the extruder temperature; (c) heating the ingredients at a temperature of at least 5ºC below the melting temperature of the carrier to form a semi-molten or molten extrudable composition; (d) extruding the composition through a die head; (e) cutting the composition into pellets; (f) cooling the pellets to the ambient temperature; and (g) coating the pellets with a protective material.

2. The method of claim 1, wherein the temperature of the extruder is below 50ºC.

3. The method of claim 1, wherein the temperature of the extruder is below 40ºC.

4. The method of claim 1, wherein the temperature of the extruder is 35ºC.

5. The method of any one of claims 1-4, wherein the carrier is meltable at temperatures below 100ºC.

6. The method of any one of claims 1-4, wherein the melting temperature of the carrier is at least 40oC.

7. The method of claim 6, wherein the melting temperature is an onset melting temperature of at least 40ºC.

8. The method of claim 6, wherein the melting temperature is a peak melting temperature of at least 40ºC.

9. The method of any one of claims 1-8, wherein the carrier is partially or completely insoluble in aqueous media.

10. The method of any one of claims 1-9, wherein the carrier is a wax.

11. The method of claim 10, wherein the wax is selected from the group consisting of: animal fats, fatty acids, fatty acid esters, fatty alcohols, monoglycerides, diglycerides, triglycerides, insect-based waxes, and plant-based waxes.

12. The method of claim 11, wherein the fatty acid is a long-chain fatty acid and the plant- based wax is a hydrogenated vegetable oil selected from the group consisting of: hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil,hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

13. The method of any one of claims 1-12, wherein the carrier is stearic acid, hydrogenated soybean oil, or a mixture thereof.

14. The method of any one of claims 1-13, wherein the ingredients further comprise one or more functional ingredients.

15. The method of claim 14, wherein the one or more functional ingredients are selected from the group consisting of: a moisture-binding agent, a pH modifying agent, a release modifying agent, a disintegrating agent, and an oxygen scavenging agent.

16. The method of claim 15, wherein the moisture-binding agent is a zeolite, a bentonite, a diatomaceous earth, a silica, or an inorganic salt.

17. The method of claim 16, wherein the zeolite is a natural zeolite.

18. The method of claim 15, wherein the disintegrating agent is selected from the group consisting of a natural polysaccharide, a natural polysaccharide hydrogel, a soy polysaccharide, a psyllium husk fiber, and a mineral clay.

19. The method of any one of claims 1-18, wherein the at least one microorganism is formulated as a dried fine powder or a dried granular powder prior to cold-melt extrusion.

20. The method of claim 19, wherein the at least one microorganism is formulated as a dried fine powder or a dried granular powder by freeze-drying, spray drying, fluid-bed drying, fluid-bed spray coating, fluid-bed spray granulation (spouted-bed), tray drying, vacuum drying, microwave drying, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

21. The method of claim 19, wherein the at least one microorganism is microencapsulated with a carrier.

22. The method of claim 21, wherein the carrier has a melting temperature of at least 40oC.

23. The method of claim 22, wherein the melting temperature is an onset melting temperature of at least 40ºC.

24. The method of claim 22, wherein the melting temperature is a peak melting temperature of at least 40ºC.

25. The method of any one of claims 21-24, wherein the carrier is a wax.

26. The method of claim 25, wherein the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, insect-based waxes, and plant-based waxes.

27. The method of claim 26, wherein the plant-based wax is a hydrogenated vegetable oil.

28. The method of claim 27, wherein the hydrogenated vegetable oil is selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

29. The method of claim 27 or 28, wherein the hydrogenated vegetable oil is a fully hydrogenated vegetable oil.

30. The method of any one of claims 21-29, wherein the at least one microorganism is microencapsulated by freeze-drying, spray drying, fluid-bed spray granulation (spouted-bed), fluid-bed spray coating, hot-melt fluid-bed coating, spray cooling (also referred to as spray chilling or spray congealing), spinning disk atomization, melt extrusion, melt granulation, roll compaction, or a combination thereof.

31. The method of any one of claims 1-30, wherein the total amount of the at least one microorganism is about 10% to about 90% of the composition.

32. The method of any one of claims 1-30, wherein the at least one microorganism is native to the microbiome of an animal.

33. The method of any one of claims 1-30, wherein the at least one microorganism is native to the gastrointestinal microbiome of an animal.

34. The method of any one of claims 1-30, wherein the at least one microorganism is native to the microbiome of a monogastric animal.

35. The method of any one of claims 1-30, wherein the at least one microorganism is native to the microbiome of a ruminant or of a camelid.

36. The method of any one of claims 1-35, wherein the at least one microorganism is a facultative or strict anaerobic bacteria.

37. The method of any one of claims 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-34.

38. The method of any one of claims 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-34.

39. The method of any one of claims 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-34.

40. The method of any one of claims 1-36, wherein the at least one microorganism comprises a 16S or ITS nucleic acid sequence of any one of SEQ ID NOs: 1-34.

41. The method of any one of claims 1-40, wherein the at least one microorganism is of the genus Butyrivibrio.

42. The method of any one of claims 1-40, wherein the at least one microorganism is of the genus species Butyrivibrio fibrisolvens.

43. The method of any one of claims 1-40, wherein the at least one microorganism is a Butyrivibrio fibrisolvens with a deposit accession number of NRRL B-67347.

44. The method of any one of claims 1-40, wherein the at least one microorganism is of the genus Ruminococcus.

45. The method of any one of claims 1-40, wherein the at least one microorganism is of the genus species Ruminococcus bovis.

46. The method of any one of claims 1-40, wherein the at least one microorganism is a Ruminococcus bovis with a deposit accession number of PTA-125917, NRRL B-67764, TSD- 225, or NCTC 14479.

47. The method of any one of claims 1-46, wherein the extruder is a single-screw extruder.

48. The method of any one of claims 1-47, wherein the extruder is a twin-screw extruder.

49. The method of claim 48, wherein the twin-screw extruder is a co-rotating or a counter- rotating extruder.

50. The method of any one of claims 1-49, wherein the method further comprises kneading the semi-molten or molten extrudable composition.

51. The method of any one of claims 1-50, wherein the die head comprises a single hole or a plurality of holes.

52. The method of any one of claims 1-51, wherein the die hole is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a hemisphere.

53. The method of any one of claims 1-51, wherein the die hole has a round shape.

54. The method of any one of claims 1-53, wherein the die hole has a diameter of about 0.5 mm to about 50 mm.

55. The method of claim 54, wherein the die hole has a diameter of about 1 mm to about 4 mm.

56. The method of any one of claims 1-55, wherein the cooling is cryogenic cooling.

57. The method of claim 56, wherein the cryogenic cooling is performed using liquid nitrogen or carbon dioxide.

58. The method of any one of claims 1-55, wherein the cooling is performed using a chilled dry gas.

59. The method of claim 58, wherein the chilled dry gas is nitrogen, carbon dioxide, or dehumidified air.

60. The method of any one of claims 1-59, wherein the pellet is in the shape of a cube, a cuboid, a cylinder, a hollow cylinder, a hexagonal prism, a pentagonal prism, a triangular prism, and a sphere.

61. The method of any one of claims 1-59, wherein the pellet has a round shape.

62. The method of any one of claims 1-61, wherein the pellet has a diameter of about 0.5 mm to about 50 mm.

63. The method of claim 62, wherein the pellet has a diameter of about 1 mm to about 4 mm.

64. The method of any one of claims 1-63, wherein the pellet is coated with an additional protective material.

65. The method of claim 64, wherein the pellet is coated by hot-melt fluid-bed coating.

66. The method of claim 64 or 65, wherein the pellet is coated to a level of at least 5% with the additional protective material.

67. The method of any one of claims 64-66, wherein the additional protective material is an additional carrier, and wherein the additional carrier has a melting temperature of at least 40oC.

68. The method of claim 67, wherein the melting temperature is an onset melting temperature of at least 40ºC.

69. The method of claim 67, wherein the melting temperature is a peak melting temperature of at least 40ºC.

70. The method of any one of claims 64-69, wherein the additional carrier is a wax.

71. The method of claim 70, wherein the wax is selected from the group consisting of animal fats, fatty acids, fatty acid esters, fatty alcohols, glycerides, insect-based waxes, and plant-based waxes.

72. The method of claim 71, wherein the fatty acid is a long-chain fatty acid and the plant- based wax is a hydrogenated vegetable oil.

73. The method of claim 72, wherein the hydrogenated vegetable oil is selected from the group consisting of hydrogenated coconut oil, hydrogenated corn oil, hydrogenated cottonseed oil, hydrogenated olive oil, hydrogenated palm oil, hydrogenated palm kernel oil, hydrogenated peanut oil, hydrogenated rapeseed oil, hydrogenated soybean oil, or hydrogenated sunflower oil.

74. The method of claim 72 or 73, wherein the hydrogenated vegetable oil is fully hydrogenated vegetable oil.

75. The method of claim any one of claims 64-69, wherein the additional protective material is stearic acid, hydrogenated soybean oil, or a mixture thereof.

76. The method of claim 71, wherein the glyceride is hydrogenated glyceride.

77. The method of any one of claims 1-76, wherein the composition has water activity below 0.

4.

78. The method of claim 77, wherein the water activity is below 0.3, 0.2, or 0.

1.

79. The method of claim 77, wherein the water activity is below 0.

1.

80. The method of any one of claims 1-78, wherein the method increases survival of the at least one microorganism.

81. The method of any one of claims 1-80, wherein the method increases stability of the at least one microorganism.

82. The method of any one of claims 1-81, wherein the method increases the total processing yield of the at least one microorganism.

83. The method of any one of claims 1-82, wherein the composition is a probiotic for use in animals.

84. The method of claim 83, wherein the animal is a livestock animal, a farm animal, a working animal including animals in sport, a zoo animal, or a companion animal.

85. A composition produced by the method of any one of claims 1-82.

86. A microbial product produced by the method of any one of claims 1-82.

87. A feed additive or supplement produced by the method of any one of claims 1-82.

88. A composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a carrier, wherein the pellet is manufactured by cold-melt extrusion at a temperature of at least 5ºC below the melting temperature of the carrier.

89. A composition comprising at least one microorganism encapsulated in a pellet, wherein the pellet comprises a carrier, wherein the pellet is manufactured by cold-melt extrusion at a temperature at or below 50ºC.

90. A method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: a) feeding ingredients into an extruder, wherein the ingredients comprise at least one biologic and a wax; b) conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, c) heating the ingredients at a temperature at or below 50ºC to form a semi-molten or molten extrudable composition; d) extruding the composition through a die head; e) cutting the composition into pellets; and f) cooling the pellets.

91. A method of manufacturing a composition by cold-melt extrusion, the method comprising the following steps: a) feeding ingredients into an extruder, wherein the ingredients comprise at least one microorganism and a wax;b) conveying and blending ingredients at a temperature between the ambient temperature and the extruder temperature, c) heating the ingredients at a temperature at or below 50ºC to form a semi-molten or molten extrudable composition; d) extruding the composition through a die head; e) cutting the composition into pellets; and f) cooling the pellets.

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