Microbial cells, method for producing the same, and use thereof

Abstract

The present invention relates to microbial cells, including but not limited to aerobic and anaerobic bacterial cells, and yeast cells, as well as methods for producing these cells, feed additives and compositions containing the cells, and uses including administering the cells to animals. The microbial cells are preferably cultured under anaerobic conditions in a culture medium containing at least two carbon sources and can be harvested. After harvesting, the microbial cells can be concentrated, frozen, or freeze-dried as needed.

Description

Technical Field

[0001] Background of the Disclosure Field of the Invention The present invention relates to Megasphaera elsdenii cells, methods for producing M. elsdenii cells, feed additives and compositions comprising the cells, and uses comprising administering the cells to animals, for example, for improving growth performance and / or health. The present invention also relates to microbial cells including, but not limited to, aerobic bacteria, anaerobic bacteria and yeast cells, methods for producing microbial cells, feed additives and compositions comprising the microbial cells, and uses comprising administering the microbial cells to animals, for example, for improving growth performance and / or health.

Background Art

[0002] Background Megasphaera elsdenii (i.e., M. elsdenii) is a non-motile Gram-negative diplococcus that utilizes lactate as a preferred carbon source and can help prevent acidosis, which is a common digestive disorder affecting millions of beef and dairy cattle every year.

[0003] When cattle and other ruminants consume large amounts of starch-based foods (e.g., grains) or monosaccharides, opportunistic microorganisms in the stomach may rapidly ferment these compounds into lactic acid. Lactic acid is a strong organic acid that can cause lactic acidosis, which can disrupt normal digestive activity in ruminants and cause extensive damage to the inner lining of the digestive tract. Affected animals have poor performance. Furthermore, the most acute form of lactic acidosis can cause irreversible damage to the animal's digestive and respiratory systems, as well as an increased mortality rate.

[0004] M. elsdenii can contribute to controlling lactic acidosis by its ability to convert lactic acid into volatile fatty acids (VFA), which are harmless organic compounds (e.g., butyrate, propionate, and acetate). However, the population of M. elsdenii in the gastrointestinal tract of ruminants is often too low to prevent the risk of acidosis. Therefore, a liquid culture of live cells derived from a strain of M. elsdenii, namely Lactipro®, has been developed to increase the colonization rate of M. elsdenii in the gastrointestinal tract of ruminants. See, for example, U.S. Patent No. 7,550,139. However, there are practical limitations that restrict the use of products containing M. elsdenii, including the difficulty of maintaining M. elsdenii products under anaerobic conditions required by the organism, and the difficulty of transporting M. elsdenii products from the production facility to the end - use location within 14 days (beyond which the survival rate of M. elsdenii in the product significantly decreases). U.S. Patent No. 4,138,498 generally discusses the possibility of freeze - drying M. elsdenii, but does not provide any method for producing freeze - dried M. elsdenii that can be used on a commercial scale to overcome existing commercial limitations. Furthermore, at least one group has recently reported that freeze - drying microorganisms, including anaerobic bacteria such as M. elsdenii, is not commercially practical. See, for example, WO2017 / 015022.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Means for Solving the Problem

[0006] Therefore, there is a need for Megasphaera cells, such as Megasphaera elsdenii cells, that overcome existing limitations, including methods for generating them. There is also a need for other microbial cells, such as Bifidobacterium such as B. breve, Lactobacillus such as L. plantarum, Bifidobacterium such as B. animalis subsp. lactis, Pediococcus such as P. acidilactici, Lactobacillus such as L. casei, Bacillus such as B. subtilis, Saccharomyces such as S. boulardii and S. cerevisiae, including methods for generating them, that overcome existing limitations.

[0007] Brief Summary of the Disclosure The present disclosure is a method for generating freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture comprising a growth medium containing M. elsdenii cells and at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, sorbitol, mannitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) freezing the cells; and (d) freeze-drying the cells, wherein about 1×10 3 ~ about 1×10 12 CFU / g of freeze-dried M. elsdenii cells are produced.

[0008] In certain embodiments, at least two carbon sources consist of about 50-90% of a first carbon source and about 10-50% of a second carbon source, the second carbon source being different from the first carbon source, and 100% of the at least two carbon sources consists of the first carbon source and the second carbon source.

[0009] The present disclosure is directed to a method for producing freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture comprising M. elsdenii cells and a growth medium; (b) harvesting the cells under anaerobic conditions within 12 hours after the culture has ended its logarithmic growth phase and before the culture begins its stationary growth phase; (c) freezing the cells; and (d) freeze-drying the cells, whereby freeze-dried M. elsdenii cells are produced.

[0010] In certain embodiments, the harvesting step comprises at least one technique selected from the group consisting of centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. In certain embodiments, the filtration comprises tangential flow filtration.

[0011] In certain embodiments, the culture comprises a liquid, and the harvesting step comprises removing from about 60% to about 100% of the liquid.

[0012] In certain embodiments, the freezing step is at a temperature of about -80°C to about -210°C.

[0013] The present disclosure is directed to a method for producing freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture comprising M. elsdenii cells and a growth medium; (b) harvesting the cells; (c) freezing the cells at a temperature of about -80°C to about -210°C within 5 hours of harvesting; and (d) freeze-drying the cells, whereby freeze-dried M. elsdenii cells are produced.

[0014] In certain embodiments, the freezing step comprises contacting a container containing M. elsdenii cells with liquid nitrogen.

[0015] In certain embodiments, the freezing step comprises contacting the cells with liquid nitrogen.

[0016] In certain embodiments, the freezing step is at a temperature of about -196 °C, producing a frozen pellet containing the cells, and the diameter of the frozen pellet is from about 0.001 to about 0.5 inches.

[0017] In certain embodiments, the pH of the M. elsdenii culture before harvesting is between about 4.5 and about 7.0.

[0018] In certain embodiments, about 1×10 3 ~ about 1×10 12 CFU / g of freeze-dried M. elsdenii cells are viable after storage at a temperature of about 25 °C for at least two weeks.

[0019] In certain embodiments, about 1×10 3 ~ about 1×10 12 CFU / g of freeze-dried M. elsdenii cells are viable after storage at about 4 °C for at least one month.

[0020] In certain embodiments, the culture further comprises at least one cryoprotective agent.

[0021] In certain embodiments, the at least one cryoprotective agent is selected from the group consisting of fructose, glucose, sucrose, powdered milk, infant formula, nonfat dry milk, trehalose, maltodextrin, betaine, and combinations thereof.

[0022] In certain embodiments, the at least one cryoprotective agent is present in an amount of about 1% to about 20% (w / v) of the culture.

[0023] In certain embodiments, freeze-dried M. elsdenii is produced on a commercial scale.

[0024] In certain embodiments, the volume of the culture is at least about 50 liters.

[0025] In certain embodiments, about 1×10 3 ~1×10 12 CFU / g of M. elsdenii cells are viable after freeze-drying.

[0026] In certain embodiments, the cells in the culture consist of M. elsdenii cells.

[0027] The present disclosure is directed to solid feed additives comprising freeze-dried M. elsdenii cells produced by any of the above methods.

[0028] In certain embodiments, the solid feed additive further comprises another microorganism.

[0029] In certain embodiments, the solid feed additive is selected from the group consisting of powders, granules, microparticles, pellets, cakes, or combinations thereof.

[0030] In certain embodiments, the solid feed additive is a probiotic.

[0031] The present disclosure is directed to compositions comprising freeze-dried M. elsdenii cells produced by any of the above methods, or any of the above solid feed additives.

[0032] In certain embodiments, the composition is a capsule.

[0033] The present disclosure is directed to kits comprising freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions.

[0034] The present disclosure is directed to a method of administering M. elsdenii cells to an animal, the method comprising administering to the animal freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions.

[0035] The present disclosure is directed to a method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions.

[0036] In certain embodiments, the condition or disorder is acidosis.

[0037] In certain embodiments, the condition or disorder is rumen acidosis.

[0038] In certain embodiments, the condition or disorder is a respiratory disease.

[0039] In certain embodiments, the condition or disorder is laminitis.

[0040] In certain embodiments, the condition or disorder is an infectious disease.

[0041] In certain embodiments, the infectious disease is caused by Salmonella or Campylobacter.

[0042] The present disclosure is directed to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions.

[0043] In certain embodiments, the opportunistic microorganism is pathogenic.

[0044] In certain embodiments, the opportunistic microorganism is Salmonella or Campylobacter.

[0045] The present disclosure is directed to a method for improving the bioavailability of plant-derived phosphorus in an animal's diet, the method comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions.

[0046] The present disclosure is directed to a method for improving the growth performance of an animal, the method comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by any of the above methods, any of the above solid feed additives, or any of the above compositions, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, carcass weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0047] In certain embodiments, the freeze-dried M. elsdenii cells, solid feed additive, or composition is administered to the animal before, simultaneously with, or after providing food to the animal.

[0048] In certain embodiments, the method further comprises mixing the freeze-dried M. elsdenii cells or solid feed additive with a liquid prior to administration.

[0049] In certain embodiments, the liquid is administered orally or by spraying the liquid onto the animal.

[0050] In certain embodiments, the step of administering comprises a single administration of the M. elsdenii cells, feed additive, or composition.

[0051] In certain embodiments, the administering step comprises daily administration of M. elsdenii cells, a feed additive or a composition.

[0052] In certain embodiments, the administering step comprises more than one administration of M. elsdenii cells, a feed additive or a composition in a day.

[0053] In certain embodiments, the animal is a ruminant.

[0054] In certain embodiments, the ruminant is selected from the group consisting of cows, sheep, goats, deer, buffaloes and reindeer.

[0055] In certain embodiments, the animal is a non-ruminant.

[0056] In certain embodiments, the non-ruminant is selected from the group consisting of equines, poultry and pigs.

[0057] In certain embodiments, the animal is a poultry animal.

[0058] In certain embodiments, the poultry animal is selected from the group consisting of chickens, geese, ducks, turkeys, guinea fowls or pigeons.

[0059] In certain embodiments, the poultry animal is selected from the group consisting of broilers, broiler-breeder chickens and laying hens.

[0060] In certain embodiments, the poultry animal is a chicken.

[0061] In certain embodiments, the animal is an equine.

[0062] In certain embodiments, the equine is a horse, a pony, a donkey or a mule.

[0063] The present disclosure is directed to a method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells.

[0064] In certain embodiments, the condition or disorder is acidosis.

[0065] In certain embodiments, the condition or disorder is a respiratory disease.

[0066] In certain embodiments, the condition or disorder is an infectious disease.

[0067] In certain embodiments, the infectious disease is caused by Salmonella or Campylobacter.

[0068] The present disclosure is directed to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells.

[0069] In certain embodiments, the opportunistic microorganisms are pathogenic.

[0070] In certain embodiments, the opportunistic microorganisms are Salmonella or Campylobacter.

[0071] The present disclosure is directed to a method for improving the bioavailability of plant-derived phosphorus in the diet of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells.

[0072] The present disclosure is directed to a method for improving the growth performance of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, egg production, bone mineralization, or a combination thereof.

[0073] In certain embodiments, the poultry animal is selected from the group consisting of chickens, turkeys, ducks, quails, guinea fowl or pigeons.

[0074] In certain embodiments, the poultry animal is selected from the group consisting of broilers, broiler-breeder chickens and laying hens.

[0075] In certain embodiments, the poultry animal is a chicken.

[0076] The present disclosure is directed to a method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of equids, the method comprising administering to the equid an effective amount of M. elsdenii cells.

[0077] In certain embodiments, the condition or disorder is acidosis.

[0078] In certain embodiments, the condition or disorder is a respiratory disease.

[0079] In certain embodiments, the condition or disorder is laminitis.

[0080] In certain embodiments, the condition or disorder is an infectious disease.

[0081] In certain embodiments, the infectious disease is caused by Salmonella or Campylobacter.

[0082] The present disclosure is directed to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of equids, the method comprising administering to the equid an effective amount of M. elsdenii cells.

[0083] In certain embodiments, the opportunistic microorganisms are pathogenic.

[0084] In certain embodiments, the opportunistic microorganism is Salmonella or Campylobacter.

[0085] The present disclosure is directed to a method for improving the bioavailability of plant-derived phosphorus in equine diets, the method comprising administering to the equine an effective amount of M. elsdenii cells.

[0086] The present disclosure is directed to a method for improving the growth performance of equines, the method comprising administering to the equine an effective amount of M. elsdenii cells, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, loin muscle weight gain, milk production, bone mineralization, or a combination thereof.

[0087] In certain embodiments, the equine is a horse, pony, donkey, or mule.

[0088] In certain embodiments, the feed additive comprises M. elsdenii cells.

[0089] In certain embodiments, the feed additive is a powder, granule, microparticle, pellet, cake, liquid, gel, or a combination thereof.

[0090] In certain embodiments, the composition comprises M. elsdenii cells, or a feed additive comprising the cells.

[0091] In certain embodiments, the composition is a capsule.

[0092] In certain embodiments, the M. elsdenii cells are freeze-dried cells.

[0093] In certain embodiments, the M. elsdenii cells are administered in liquid form.

[0094] In certain embodiments, the method further comprises rehydrating the feed additive or the freeze-dried cells to produce a liquid.

[0095] In certain embodiments, the liquid is administered by oral gavage or by spraying the liquid onto the animal.

[0096] In certain embodiments, the M. elsdenii cells are administered before, simultaneously with, or after feeding the animal.

[0097] In certain embodiments, the administering step comprises a single administration of M. elsdenii cells.

[0098] In certain embodiments, the administering step comprises daily administration of M. elsdenii cells.

[0099] In certain embodiments, the administering step comprises more than one administration of M. elsdenii cells in a day.

[0100] The present disclosure is a method for producing encapsulated freeze-dried Megasphaera cells, comprising: (a) preparing a culture comprising Megasphaera cells and a growth medium comprising at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; and (e) encapsulating the cells, wherein about 1×10 3 ~ about 1×10 12 CFU / g of encapsulated freeze-dried Megasphaera cells are produced.

[0101] In certain embodiments, the method comprises administering to the animal freeze-dried Megasphaera cells encapsulated in a capsule.

[0102] In certain embodiments, a method of improving the growth performance of an animal comprises administering to the animal an effective amount of freeze-dried Megasphaera cells encapsulated in a capsule.

[0103] In certain embodiments, the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of the carcass, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0104] In certain embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal comprises administering to the animal an effective amount of freeze-dried Megasphaera cells encapsulated in a capsule.

[0105] In certain embodiments, the composition comprises freeze-dried Megasphaera cells encapsulated in a capsule.

[0106] The present disclosure is a method for producing freeze-dried anaerobic bacterial cells, comprising: (a) preparing a culture comprising an anaerobic bacterial cell and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under anaerobic conditions or partially anaerobic conditions; (b) harvesting the cells under anaerobic conditions or partially anaerobic conditions; (c) freezing the cells; and (d) freeze-drying the cells, wherein about 1×10 3 ~ about 1×10 12 CFU / g of freeze-dried anaerobic bacterial cells are produced.

[0107] In certain embodiments, the method comprises administering the freeze-dried anaerobic bacterial cells to an animal.

[0108] In certain embodiments, a method for improving the growth performance of an animal comprises administering an effective amount of the freeze-dried anaerobic bacterial cells to the animal.

[0109] In certain embodiments, the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of breast meat, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0110] In certain embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal comprises administering an effective amount of the freeze-dried anaerobic bacterial cells to the animal.

[0111] In certain embodiments, the composition comprises the freeze-dried anaerobic bacterial cells.

[0112] The present disclosure is a method for producing encapsulated freeze-dried anaerobic bacterial cells, comprising: (a) preparing a culture comprising an anaerobic bacterial cell and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under anaerobic conditions or semi-anaerobic conditions; (b) harvesting the cells under anaerobic conditions or semi-anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; and (e) encapsulating the cells, wherein about 1×10 3 ~ about 1×10 12 CFU / g of encapsulated freeze-dried anaerobic bacterial cells are produced.

[0113] In certain embodiments, the method comprises administering the encapsulated freeze-dried anaerobic bacterial cells to an animal.

[0114] In certain embodiments, a method for improving the growth performance of an animal comprises administering an effective amount of the encapsulated freeze-dried anaerobic bacterial cells to the animal.

[0115] In certain embodiments, the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, increase in carcass weight, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0116] In certain embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal comprises administering to the animal an effective amount of encapsulated freeze-dried anaerobic bacterial cells.

[0117] In certain embodiments, the composition comprises encapsulated freeze-dried anaerobic bacterial cells.

[0118] The present disclosure is a method for producing freeze-dried aerobic bacteria and / or yeast cells, comprising: (a) preparing a culture comprising a growth medium containing at least two carbon sources selected from the group consisting of aerobic bacterial cells and / or yeast cells, and casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under aerobic conditions; (b) harvesting the cells; (c) freezing the cells; and (d) freeze-drying the cells, wherein about 1×10 3 ~about 1×10 12 CFU / g of freeze-dried aerobic bacteria and / or yeast cells are produced.

[0119] In certain embodiments, the method comprises administering to the animal freeze-dried aerobic bacterial cells and / or yeast cells.

[0120] In certain embodiments, a method for improving the growth performance of an animal comprises administering to the animal an effective amount of freeze-dried aerobic bacterial cells and / or yeast cells.

[0121] In certain embodiments, the improvement in the growth performance of an animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0122] In certain embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal comprises administering to the animal an effective amount of freeze-dried aerobic bacterial cells and / or yeast cells.

[0123] In certain embodiments, the composition comprises freeze-dried aerobic bacterial cells and / or yeast cells.

[0124] The present disclosure is a method for producing encapsulated freeze-dried aerobic bacterial cells and / or yeast cells, comprising: (a) preparing a culture comprising a growth medium containing at least two carbon sources selected from the group consisting of aerobic bacterial cells and / or yeast cells, and casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under aerobic conditions; (b) harvesting the cells under aerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; and (e) encapsulating the cells, wherein encapsulated freeze-dried aerobic bacterial cells and / or yeast cells of about 1×10 3 ~about 1×10 12 CFU / g are produced.

[0125] In certain embodiments, the method comprises administering to the animal encapsulated freeze-dried aerobic bacterial cells and / or yeast cells.

[0126] In certain embodiments, a method of improving the growth performance of an animal comprises administering to the animal an effective amount of encapsulated freeze-dried aerobic bacterial cells and / or yeast cells.

[0127] In certain embodiments, the improvement in the growth performance of an animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of carcass meat, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0128] In certain embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal comprises administering to the animal an effective amount of encapsulated freeze-dried aerobic bacterial cells and / or yeast cells.

[0129] In certain embodiments, the composition comprises encapsulated freeze-dried aerobic bacterial cells and / or yeast cells. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for producing freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture comprising M. elsdenii cells and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, and combinations thereof, under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; comprising about 1×10 3 to about 1×10 12 CFU / g of freeze-dried M. elsdenii cells are produced. Method. (Item 2) The method according to item 1, wherein the at least two carbon sources consist of about 50 to 90% of a first carbon source and about 10 to 50% of a second carbon source, the second carbon source is different from the first carbon source, and 100% of the at least two carbon sources consists of the first carbon source and the second carbon source. (Item 3) A method for producing freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture comprising M. elsdenii cells and a growth medium; and (b) harvesting the cells under anaerobic conditions within 12 hours after the culture has ended its logarithmic growth phase and before the culture starts its stationary growth phase; (c) freezing the cells; (d) freeze-drying the cells comprising freeze-dried M. elsdenii cells are produced. (Item 4) The method according to any one of items 1 to 3, wherein the harvesting step comprises at least one technique selected from the group consisting of centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. (Item 5) The method according to item 4, wherein the filtration comprises tangential flow filtration. (Item 6) The method according to any one of items 1 to 5, wherein the culture contains a liquid, and the harvesting step includes removing about 60% to about 100% of the liquid. (Item 7) The method according to any one of items 1 to 6, wherein the freezing step is at a temperature of about -80°C to about -210°C. (Item 8) A method for producing freeze-dried Megasphaera elsdenii cells, comprising: (a) preparing a culture containing M. elsdenii cells and a growth medium; (b) harvesting the cells; (c) freezing the cells at a temperature of about -80°C to about -210°C within 5 hours of harvesting; (d) freeze-drying the cells and a method by which freeze-dried M. elsdenii cells are produced. (Item 9) The method according to any one of items 1 to 8, wherein the freezing step includes contacting the container containing the M. elsdenii cells with liquid nitrogen. (Item 10) The method according to any one of items 1 to 9, wherein the freezing step includes contacting the cells with liquid nitrogen. (Item 11) The method according to any one of items 1 to 10, wherein the freezing step is at a temperature of about -196°C, generating a frozen pellet containing the cells, and the diameter of the frozen pellet is about 0.001 to about 0.5 inches. (Item 12) The method according to any one of items 1 to 11, wherein the pH of the M. elsdenii culture before harvesting is between about 4.5 and about 7.0. (Item 13) About 1×10 3 ~about 1×10 12 CFU / g of the freeze-dried M. elsdenii cells are viable after storage at a temperature of about 25°C for at least 2 weeks. The method according to any one of items 1 to 12. (Item 14) About 1×10 3 ~about 1×10 12 CFU / g of the freeze-dried M. elsdenii cells are viable after storage at about 4°C for at least 1 month. The method according to any one of items 1 to 12. (Item 15) The method according to any one of items 1 to 14, wherein the culture further contains at least one cryoprotectant. (Item 16) The method according to item 15, wherein the at least one cryoprotective substance is selected from the group consisting of fructose, glucose, sucrose, powdered milk, infant formula milk powder, skim milk powder, trehalose, maltodextrin, betaine, and combinations thereof. (Item 17) The method according to item 15 or 16, wherein the at least one cryoprotective substance is present in an amount of about 1% to about 20% (w / v) of the culture. (Item 18) The method according to any one of items 1 to 17, wherein the freeze-dried M. elsdenii is produced on a commercial scale. (Item 19) The method according to any one of items 1 to 18, wherein the volume of the culture is at least about 50 liters. (Item 20) About 1×10 3 ~1×10 12 CFU / g of M. elsdenii cells are viable after freeze-drying, according to the method of any one of items 3 to 19. (Item 21) The method according to any one of items 1 to 20, wherein the cells in the culture consist of M. elsdenii cells. (Item 22) A solid feed additive comprising freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21. (Item 23) The solid feed additive according to item 22, further comprising another microorganism. (Item 24) The solid feed additive according to item 22 or 23, selected from the group consisting of powders, granules, microparticles, pellets, cakes, or combinations thereof. (Item 25) The solid feed additive according to any one of items 22 to 24, which is a probiotic. (Item 26) A composition comprising freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, or the solid feed additive according to any one of items 22 to 25. (Item 27) The composition according to item 26, which is in the form of a capsule. (Item 28) A kit comprising freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, the solid feed additive according to any one of items 22 to 25, or the composition according to item 26 or 27. (Item 29) A method of administering M. elsdenii cells to an animal, comprising the step of administering to the animal freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, a solid feed additive according to any one of items 22 to 25, or a composition according to item 26 or 27. (Item 30) A method for treating or preventing a condition or disorder related to lactic acid production in the gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, a solid feed additive according to any one of items 22 to 25, or a composition according to item 26 or 27. (Item 31) The method according to item 30, wherein the condition or disorder is acidosis. (Item 32) The method according to item 30 or 31, wherein the condition or disorder is rumen acidosis. (Item 33) The method according to item 30, wherein the condition or disorder is a respiratory disease. (Item 34) The method according to item 30, wherein the condition or disorder is laminitis. (Item 35) The method according to item 30, wherein the condition or disorder is an infectious disease. (Item 36) The method according to item 35, wherein the infectious disease is caused by Salmonella or Campylobacter. (Item 37) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, a solid feed additive according to any one of items 22 to 25, or a composition according to item 26 or 27. (Item 38) The method according to item 37, wherein the opportunistic microorganism is pathogenic. (Item 39) The method according to item 37 or 38, wherein the opportunistic microorganism is Salmonella or Campylobacter. (Item 40) A method for improving the bioavailability of plant-derived phosphorus in the diet of an animal, comprising the step of administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by the method according to any one of items 1 to 21, a solid feed additive according to any one of items 22 to 25, or a composition according to item 26 or 27. (Item 41) A method for improving growth performance in animals, comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by the method described in any one of items 1 to 21, a solid feed additive described in any one of items 22 to 25, or a composition described in item 26 or 27, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, increase in carcass weight, milk production in milk-producing animals, number of eggs laid in poultry, bone mineralization, or a combination thereof. (Item 42) The method according to any one of items 29 to 41, wherein the freeze-dried M. elsdenii cells, the solid feed additive or the composition are administered to the animal before, simultaneously with, or after feeding the animal food. (Item 43) The method according to any one of items 29 to 41, further comprising the step of mixing the freeze-dried M. elsdenii cells or the solid feed additive with a liquid before administration. (Item 44) The method according to item 43, wherein the liquid is administered orally or by spraying the liquid onto the animal. (Item 45) The method according to any one of items 29 to 44, comprising a single administration of the M. elsdenii cells, feed additive or composition. (Item 46) The method according to any one of items 29 to 45, comprising daily administration of the M. elsdenii cells, feed additive or composition. (Item 47) The method according to any one of items 29 to 46, comprising more than one administration of the M. elsdenii cells, feed additive or composition per day. (Item 48) The method according to any one of items 29 to 47, wherein the animal is a ruminant. (Item 49) The method according to item 48, wherein the ruminant is selected from the group consisting of cows, sheep, goats, deer, buffaloes and reindeer. (Item 50) The method according to any one of items 29 to 47, wherein the animal is a non-ruminant. (Item 51) The method according to item 50, wherein the non-ruminant is selected from the group consisting of equids, poultry and pigs. (Item 52) The method according to any one of items 29 to 33, 35 to 47, 50 or 51, wherein the animal is a poultry animal. (Item 53) The method according to item 52, wherein the poultry animal is selected from the group consisting of chicken, goose, duck, quail, turkey or pigeon. (Item 54) The method according to item 52 or 53, wherein the poultry animal is selected from the group consisting of broiler, broiler-breed chicken and laying hen. (Item 55) The method according to any one of items 52 to 54, wherein the poultry animal is a chicken. (Item 56) The method according to any one of items 29 to 47, 50 or 51, wherein the animal is a member of the family Equidae. (Item 57) The method according to item 56, wherein the member of the family Equidae is a horse, pony, donkey or mule. (Item 58) A method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells. (Item 59) The method according to item 58, wherein the condition or disorder is acidosis. (Item 60) The method according to item 58, wherein the condition or disorder is a respiratory disease. (Item 61) The method according to item 58, wherein the condition or disorder is an infectious disease. (Item 62) The method according to item 61, wherein the infectious disease is caused by Salmonella or Campylobacter. (Item 63) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells. Method. (Item 64) The method according to item 63, wherein the opportunistic microorganism is pathogenic. (Item 65) The method according to item 63 or 64, wherein the opportunistic microorganism is Salmonella or Campylobacter. (Item 66) A method for improving the bioavailability of plant-derived phosphorus in the diet of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells. (Item 67) A method for improving the growth performance of a poultry animal, the method comprising administering to the poultry animal an effective amount of M. elsdenii cells, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, egg production, bone mineralization or a combination thereof. (Item 68) The method according to any one of items 58 to 67, wherein the poultry animal is selected from the group consisting of chicken, goose, duck, quail, turkey or pigeon. (Item 69) The method according to any one of items 58 to 68, wherein the poultry animal is selected from the group consisting of broiler, broiler-breeder chicken and laying hen. (Item 70) The method according to any one of items 58 to 69, wherein the poultry animal is chicken. (Item 71) A method for treating or preventing a condition or disorder related to lactic acid production in the gastrointestinal tract of Equidae, the method comprising administering to the Equidae an effective amount of M. elsdenii cells. (Item 72) The method according to item 71, wherein the condition or disorder is acidosis. (Item 73) The method according to item 71, wherein the condition or disorder is respiratory disease. (Item 74) The method according to item 71, wherein the condition or disorder is laminitis. (Item 75) The method according to item 71, wherein the condition or disorder is an infectious disease. (Item 76) The method according to item 75, wherein the infectious disease is caused by Salmonella or Campylobacter. (Item 77) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of Equidae, the method comprising administering to the Equidae an effective amount of M. elsdenii cells. (Item 78) The method according to item 77, wherein the opportunistic microorganism is pathogenic. (Item 79) The method according to item 77 or 78, wherein the opportunistic microorganism is Salmonella or Campylobacter. (Item 80) A method for improving the bioavailability of plant-derived phosphorus in the diet of Equidae, the method comprising administering to the Equidae an effective amount of M. elsdenii cells. (Item 81) A method for improving the growth performance of Equidae, the method comprising administering to the Equidae an effective amount of M. elsdenii cells, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, carcass weight gain, milk production, bone mineralization or a combination thereof. (Item 82) The method according to any one of items 71 to 81, wherein the Equidae is horse, pony, donkey or mule. (Item 83) The method according to any one of items 58 to 82, wherein the feed additive contains the M. elsdenii cells. (Item 84) The method according to item 83, wherein the feed additive is in the form of powder, granules, microparticles, pellets, cakes, liquid, gel or a combination thereof. (Item 85) The method according to any one of items 58 to 84, wherein the composition comprises the M. elsdenii cells or a feed additive containing the cells. (Item 86) The composition according to item 85, wherein the composition is in the form of capsules. (Item 87) The method according to any one of items 58 to 86, wherein the M. elsdenii cells are freeze-dried cells. (Item 88) The method according to any one of items 58 to 85, wherein the M. elsdenii cells are administered in liquid form. (Item 89) The method according to item 88, further comprising the step of rehydrating the feed additive or the freeze-dried cells to produce the liquid. (Item 90) The method according to item 88 or 89, wherein the liquid is administered by oral gavage or by spraying the liquid onto the animal. (Item 91) The method according to any one of items 58 to 90, wherein the M. elsdenii cells are administered before, simultaneously with, or after feeding the animal. (Item 92) The method according to any one of items 58 to 91, comprising a single administration of the M. elsdenii cells. (Item 93) The method according to any one of items 58 to 91, comprising daily administration of the M. elsdenii cells. (Item 94) The method according to any one of items 58 to 93, comprising more than one administration of the M. elsdenii cells in a day. (Item 95) A method for producing encapsulated freeze-dried Megasphaera cells, comprising: (a) preparing a culture comprising Megasphaera cells and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract and combinations thereof, under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; (e) encapsulating the cells and about 1 × 10 3 to about 1 × 10 12 CFU / g of encapsulated freeze-dried Megasphaera cells are produced, method. (Item 96) A method of administering Megasphaera cells to an animal, comprising the step of administering to the animal the encapsulated freeze-dried Megasphaera cells produced by the method according to Item 95. (Item 97) A method of improving the growth performance of an animal, comprising the step of administering to the animal an effective amount of the encapsulated freeze-dried Megasphaera cells produced by the method according to Item 95, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of breast meat, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof. (Item 98) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of the encapsulated freeze-dried Megasphaera cells produced by the method according to Item 95. (Item 99) A composition comprising encapsulated freeze-dried Megasphaera cells produced by the method according to Item 95. (Item 100) A method for producing freeze-dried anaerobic bacterial cells, (a) preparing a culture comprising an anaerobic bacterial cell and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof under anaerobic conditions or semi-anaerobic conditions; (b) harvesting the cells under anaerobic conditions or semi-anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells and about 1 × 10 3 ~about 1×10 12 CFU / g of freeze-dried anaerobic bacterial cells are produced, method. (Item 101) A method of administering anaerobic bacterial cells to an animal, comprising the step of administering to the animal the freeze-dried anaerobic bacterial cells produced by the method according to Item 100. (Item 102) A method of improving the growth performance of an animal, comprising the step of administering to the animal an effective amount of the freeze-dried anaerobic bacterial cells produced by the method according to Item 100, wherein the improvement of the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of limb meat, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof. (Item 103) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of the freeze-dried anaerobic bacterial cells produced by the method according to Item 100. (Item 104) A composition comprising freeze-dried anaerobic bacterial cells produced by the method according to Item 100. (Item 105) A method for producing encapsulated freeze-dried anaerobic bacterial cells, comprising: (a) preparing a culture comprising an anaerobic bacterial cell and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof under anaerobic or partially anaerobic conditions; (b) harvesting the cells under anaerobic or partially anaerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; (e) encapsulating the cells and about 1×10 3 ~about 1×10 12 CFU / g of encapsulated freeze-dried anaerobic bacterial cells are produced, method. (Item 106) A method of administering anaerobic bacterial cells to an animal, comprising the step of administering to the animal the encapsulated freeze-dried anaerobic bacterial cells produced by the method according to item 105. (Item 107) A method of improving the growth performance of an animal, comprising the step of administering to the animal an effective amount of the encapsulated freeze-dried anaerobic bacterial cells produced by the method according to item 105, wherein the improvement of the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof. (Item 108) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of the encapsulated freeze-dried anaerobic bacterial cells produced by the method according to item 105. (Item 109) A composition comprising the encapsulated freeze-dried anaerobic bacterial cells produced by the method according to item 105. (Item 110) A method for producing freeze-dried aerobic bacteria and / or yeast cells, (a) preparing a culture comprising a growth medium containing at least two carbon sources selected from the group consisting of aerobic bacterial cells and / or yeast cells, and casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under aerobic conditions; (b) harvesting the cells; (c) freezing the cells; (d) freeze-drying the cells and producing from about 1×10 3 to about 1×10 12 CFU / g of freeze-dried aerobic bacteria and / or yeast cells. Method. (Item 111) A method of administering aerobic bacteria and / or yeast cells to an animal, comprising the step of administering to the animal the freeze-dried aerobic bacterial cells and / or yeast cells produced by the method according to item 110. (Item 112) A method for improving the growth performance of an animal, comprising the step of administering to the animal a freeze-dried aerobic bacterial cell and / or yeast cell produced by the method described in item 110 in an effective amount, wherein the improvement of the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof. (Item 113) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to the animal a freeze-dried aerobic bacterial cell and / or yeast cell produced by the method described in item 110 in an effective amount. (Item 114) A composition comprising a freeze-dried aerobic bacterial cell and / or yeast cell produced by the method described in item 110. (Item 115) A method for producing encapsulated freeze-dried aerobic bacterial cells and / or yeast cells, comprising: (a) preparing a culture comprising a growth medium containing at least two carbon sources selected from the group consisting of aerobic bacterial cells and / or yeast cells, and casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof, under aerobic conditions; (b) harvesting the cells under aerobic conditions; (c) freezing the cells; (d) freeze-drying the cells; (e) encapsulating the cells and producing encapsulated freeze-dried aerobic bacterial cells and / or yeast cells of about 1×10 3 to about 1×10 12 CFU / g. Method. (Item 116) A method for administering aerobic bacteria and / or yeast cells to an animal, comprising the step of administering to the animal the encapsulated freeze-dried aerobic bacterial cells and / or yeast cells described in item 115. (Item 117) A method for improving the growth performance of an animal, comprising administering to the animal an effective amount of encapsulated freeze-dried aerobic bacterial cells and / or yeast cells produced by the method described in item 115, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof. (Item 118) A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising administering to the animal an effective amount of encapsulated freeze-dried aerobic bacterial cells and / or yeast cells produced by the method described in item 115. (Item 119) A composition comprising encapsulated freeze-dried aerobic bacterial cells and / or yeast cells produced by the method described in item 115.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0158] Detailed Description of the Invention The present invention relates to Megasphaera elsdenii cells, methods for producing M. elsdenii cells, feed additives and compositions containing the cells, and uses including administering the cells to animals for, for example, improving growth performance and / or health. The present invention also relates to, without limitation, aerobic bacteria, anaerobic bacteria and yeasts, and microbial cells including Bifidobacterium such as B. breve, Lactobacillus such as L. plantarum, Bifidobacterium such as B. animalis subsp. lactis, Pediococcus such as P. acidilactici, Lactobacillus such as L. casei, Bacillus such as B. subtilis, Saccharomyces such as S. boulardii and S. cerevisiae, and methods for producing microbial cells, feed additives and compositions containing the microbial cells, and uses including administering the microbial cells to animals for, for example, improving growth performance and / or health.

[0159] All publications, patents and other references specified herein are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Further, any citation or identification of a reference in this application is not to be construed as an admission that such reference is available as prior art to the present invention. Terminology

[0160] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. Unless specifically required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0161] To the extent that section headings are used, these headings should not necessarily be construed as limitations.

[0162] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. The terms "a", "an", "the", "one or more", and "at least one" can be used interchangeably herein, for example.

[0163] As used herein, the term "about" when used to modify an amount associated with the present invention, for example, by standard testing and handling; by accidental error in such testing and handling; refers to variations in quantities that may occur due to differences in the manufacture, source, or purity of the components used in the present invention. Whether or not modified by the term "about", the claims include equivalents of the recited amounts. In some embodiments, the term "about" means plus or minus 10% of the reported numerical value.

[0164] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0165] The terms "comprises", "comprising", "includes", "including", "having" and their cognates are interchangeable and mean "including but not limited to". When an aspect is described herein with the language "comprising", it is to be understood that other similar aspects are also provided with respect to "consisting of" and / or "consisting essentially of".

[0166] The term "consisting of" means "including and limited to".

[0167] The term "consisting essentially of" means the specified materials of a composition, or the specified steps of a method, and additional materials or steps that do not substantially affect the basic characteristics of the materials or method.

[0168] The term "and / or", as used herein, is considered to specifically disclose each of two specified features or components, including or excluding others. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0169] As used herein, the terms "culturing", "culturing cells", and "cultured cells" mean incubating cells or maintaining cells in a viable state under in vitro conditions that permit cell growth or division. The term "culture" can also be used herein to refer to cells incubated under in vitro conditions (e.g., cells incubated in a liquid growth medium).

[0170] As used herein, the term "probiotic" refers to one or more viable microorganisms (bacteria and / or yeast), which may or may not contain other components, and when administered in an appropriate amount, can provide health, digestive, and / or performance benefits to an animal or subject.

[0171] As used herein, the term "direct-fed microbial product" refers to a product that contains one or more viable microorganisms (bacteria and / or yeast), which may or may not contain other components, and can be administered to an animal or subject in a feed mixture, bolus, and / or oral paste, and when administered in an appropriate amount, can provide health benefits to the animal or subject.

[0172] As used herein, the term "feed additive" refers to one or more components, products, or substances (e.g., cells) that are used alone or together in a nutrient (e.g., to improve the quality of food (e.g., animal feed), to improve the performance and health of animals, and / or to enhance the digestibility of food or materials in food). A feed additive can be, for example, a probiotic.

[0173] As used herein, the terms "growth medium" and "culture medium" refer to solid (e.g., agar), semi-solid (e.g., agar), or liquid (e.g., broth) compositions that contain components that support cell growth.

[0174] As used herein, the terms "harvest" and "harvesting" refer to collecting cells from a culture, e.g., collecting cells from a culture into a growth medium, collecting cells by removing a quantity of growth medium from the cells (e.g., concentrating cells in a liquid culture or separating cells from the growth medium), or stopping culturing the cells. The terms include collecting or removing a large quantity of liquid containing cells from a liquid culture, including a concentrated amount of cells.

[0175] As used herein, the term "isolated" indicates isolation or separation from a natural form or natural environment, although it does not necessarily reflect the degree to which an isolate is purified. An isolate can include, but is not limited to, an isolated microorganism, an isolated biomass, or an isolated culture.

[0176] As used herein, the term "effective amount" refers to an amount that achieves a desired result.

[0177] As used herein, "therapeutically effective amount" or "clinically effective amount" refers to an amount that achieves a desired therapeutic result, including, for example, an amount used alone or in combination with other components. Therapeutic results can include, for example, alleviation of symptoms, extension of survival, improvement of motility, etc. The therapeutic result need not be "cure".

[0178] As used herein, "excipient" refers to a component or mixture of components used to impart desirable characteristics to a feed additive, food, or composition disclosed herein. Excipients of the present invention, when added to a pharmaceutical composition, can be described as "pharmaceutically acceptable" excipients, which are compounds, materials, compositions, salts, and / or dosage forms suitable for contact with the tissues of animals (i.e., humans and non-human animals) within the scope of sound medical judgment, with a reasonable benefit / risk ratio, without undue toxicity, irritation, allergic response, or other problematic complications over a desired contact period.

[0179] The terms "treating", "treatment" and "treat" refer to both therapeutic treatment and prophylactic or preventive measures, with the purpose of preventing or decelerating (mitigating) an undesirable physiological condition, disease or disorder, or obtaining a beneficial or desired physiological outcome (e.g., clinical, medical and / or veterinary outcomes). For purposes of the present invention, beneficial or desired outcomes include, but are not limited to, alleviation or elimination of symptoms or precursors associated with a condition, disease or disorder; reduction in the extent of a condition, disease or disorder; stabilization of a condition, disease or disorder (i.e., the condition, disease or disorder does not deteriorate); delay in the onset or progression of a condition, disease or disorder; recovery from a condition, disease or disorder; remission of a condition, disease or disorder (whether partial or total and whether detectable or undetectable); or enhancement or improvement of a condition, disease or disorder. Treatment includes inducing a physiologically significant response without undue side effects. Treatment also includes prolonging survival as compared to expected survival in the absence of receiving treatment.

[0180] As used herein, the terms "prevent" and "prevention" refer to partially or completely delaying the onset of an infectious disease, disease, disorder and / or condition; partially or completely delaying the onset of one or more precursors, symptoms, features or signs (e.g., clinical or physiological precursors, symptoms, features or signs) of a particular infectious disease, disease, disorder and / or condition; partially or completely delaying the progression from an infectious disease, particular disease, disorder and / or condition; and / or reducing the risk of developing a pathology associated with an infectious disease, disease, disorder and / or condition.

[0181] As used herein, the term "yield" refers to the amount of viable or live cells, including amounts at a specific volume (e.g., colony forming units per milliliter ("CFU / mL")) or a specific weight (e.g., CFU per gram ("CFU / g")).

[0182] As used herein, the term "viable" refers to a living organism or organisms (e.g., a living microbial cell or cells). "Viability" refers, inter alia, to the ability to survive under certain specific conditions.

[0183] As used herein, "purify", "purified" and "purification" mean to make substantially pure or to eliminate unnecessary components, substance contamination, mixtures or incompleteness.

[0184] The terms "invention" and "disclosure", when described, can be used interchangeably or, for example, in the phrasing "the present invention" or "the present disclosure".

[0185] The term "animal" or "subject" refers to any organism belonging to the animal kingdom, and includes, without limitation, aquatic animals such as fish and terrestrial animals; commercial fish; ornamental fish; fry; bivalves; mollusks; crustaceans; shellfish; shrimp; juvenile shrimp; Artemia; rotifers; brine shrimp; filter feeders; amphibians; reptiles; mammals; humans; non-human animals; domestic animals; farm animals; zoo animals; sports animals; breeding stock; race animals; fair animals; heirloom animals; rare or endangered animals; companion animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice or horses; primates such as monkeys (e.g., macaques, rhesus monkeys, African green monkeys, patas monkeys, cynomolgus monkeys and cercopithecus), apes, orangutans, baboons, langurs and chimpanzees; canids such as dogs and wolves; felids such as cats, lions and tigers; equids such as horses, ponies, donkeys, mules and zebras; food animals such as cows, buffalo, cattle, pigs, poultry and sheep; ungulates such as deer and giraffes; birds (i.e., fowl); poultry such as chickens, ducks, geese, turkeys, pheasants, pigeons, emus, ostriches, and any other fowl used as food or farm animals including broilers, broiler-breeder chickens and egg-laying chickens; rodents such as mice, rats, hamsters and guinea pigs, etc. Animal feeds include, but are not limited to, aquaculture feeds, feeds for domestic animals including pet feeds, feeds for zoo animals, feeds for working animals, feeds for livestock and combinations thereof. Food includes animal feeds and human food.

[0186] For clarity, it is understood that certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention described in the context of a single embodiment may also be provided separately, or in any suitable sub-combination, or as suitable in any other described embodiment of the invention. A particular feature described in the context of various embodiments is not considered an essential feature of those embodiments unless the embodiment is inoperative without those elements.

[0187] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims. Megasphaera elsdenii

[0188] Megasphaera elsdenii cells derived from any strain, or any combination of strains, can be used in the invention described herein.

[0189] The M. elsdenii strain(s) can be selected from a stock culture collection (e.g., American Type Culture Collection (“ATCC®”), National Collection of Industrial, Food and Marine Bacteria (“NCIMB”), National Collection of Type Cultures (“NCTC”), American Research Service (“ARC”) culture collection (i.e., “NRRL”), National Institute of Animal Health (NIAH) culture collection), or can be a strain isolated from a natural source (e.g., from the gastrointestinal tract of a ruminant).

[0190] Examples of M. elsdenii strains that can be selected from a culture collection include, but are not limited to, the strains listed by the deposit numbers in Table 1. Alternative designations for the deposit numbers are also shown. [Table 1]

[0191] In some embodiments, the M. elsdenii cells are derived from a strain having a deposit number selected from the group consisting of ATCC® 25940, ATCC® 17752, ATCC® 17753, NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331, NCIMB 702409, NCIMB 702410, NCIMB 41125, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102, and combinations thereof, including any of the alternative designations in Table 1.

[0192] In some embodiments, the M. elsdenii cells are derived from a strain isolated from a ruminant (e.g., a cow). See, e.g., U.S. Patent No. 7,550,139.

[0193] In some embodiments, the M. elsdenii cells are derived from strains isolated from non-ruminant animals (e.g., humans).

[0194] In some embodiments, the M. elsdenii cells are derived from strains selected for lactate utilization (e.g., strains that utilize lactate in the presence of sugars), resistance to ionophore antibiotics, relatively high growth rates, the ability to produce mainly acetate, the ability to grow at low pH values less than 5.0 and 4.5, volatile fatty acid (VFA) production, phytase activity, and combinations thereof. See, e.g., U.S. Patent No. 7,550,139.

[0195] In some embodiments, strains selected for lactate utilization utilize lactate as a preferred carbon source in the presence of soluble carbohydrates (e.g., glucose and / or maltose). Lactate utilization can be determined, for example, based on growth in a medium containing lactate and lacking soluble carbohydrates compared to the same medium supplemented with soluble carbohydrates.

[0196] In some embodiments, the M. elsdenii cells are derived from strains that have a high growth rate compared to other strains. The growth rates of various strains can be determined, for example, by culturing the cells in liquid medium and monitoring the increase in optical density over time.

[0197] In some embodiments, the M. elsdenii cells are derived from a strain capable of producing VFAs, which can be determined, for example, by gas chromatography. In some embodiments, the VFAs are six-carbon fatty acids capable of inhibiting the growth of Salmonella and / or Campylobacter. In some embodiments, the VFAs are caproic acid. In some embodiments, Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella Typhimurium and / or Entiritidis. In some embodiments, Campylobacter is Campylobacter jejuni or Campylobacter coli.

[0198] In some embodiments, the M. elsdenii cells are derived from a strain having phytase activity.

[0199] In some embodiments, the M. elsdenii cells are derived from NCIMB41125, which is a Megasphaera elsdenii strain. This strain of Megasphaera elsdenii has a high specific growth rate (0.94 generations / hour), can grow in a pH range of 4.5 - 6.5 or higher, uses D- and L-lactate as its preferred substrate, but also has the ability to utilize glucose and other carbohydrates and is tolerant to ionophores.

[0200] In some embodiments, the M. elsdenii cells are derived from NCIMB41787, which is a Megasphaera elsdenii strain. In some embodiments, the M. elsdenii cells are derived from NCIMB41788, which is a Megasphaera elsdenii strain.

[0201] In some embodiments, the M. elsdenii cells are derived from ATCC® 25940, which is a Megasphaera elsdenii strain.

[0202] In some embodiments, the M. elsdenii cells are derived from a strain selected from a stock culture collection or isolated from a natural source. Cells “derived from” a strain can be natural or artificial derivatives, such as, for example, sub isolates, mutants, variants or recombinant strains. Preparation of cultures containing anaerobic, aerobic and / or yeast cells

[0203] M. elsdenii is an anaerobic bacterium that must be cultured under strict anaerobic conditions to obtain maximum yields and viability.

[0204] In some embodiments, the culture comprises M. elsdenii cells and a growth medium.

[0205] In some embodiments, the culture comprises one or more strains of M. elsdenii cells. In some embodiments, the culture comprises a single strain of M. elsdenii cells. In some embodiments, the culture consists of one or more strains of M. elsdenii cells (i.e., the cells in the culture consist of M. elsdenii cells, such as, for example, one or more strains of M. elsdenii cells). In some embodiments, the culture consists of a single strain of M. elsdenii cells.

[0206] In some embodiments, the culture comprises Megasphaera cells and a growth medium.

[0207] In some embodiments, the culture comprises anaerobic bacterial cells and a growth medium. In some embodiments, the culture comprises Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, Lactobacillus cells such as L. casei, and a growth medium.

[0208] In some embodiments, the culture comprises aerobic bacterial cells and a growth medium. In some embodiments, the culture comprises Bacillus cells such as B. subtilis and a growth medium.

[0209] In some embodiments, the culture comprises yeast cells and a growth medium. In some embodiments, the culture comprises Saccharomyces cells such as S. boulardii and S. cerevisiae.

[0210] A variety of fermentation parameters related to the inoculation, growth, and harvesting of microbial cells can be used, including continuous fermentation (i.e., continuous culture) or batch fermentation (i.e., batch culture). See, e.g., U.S. Patent No. 7,550,139.

[0211] The growth medium for the microbial cells can be solid, semi-solid, or liquid. The medium can contain nutrients that provide the essential elements and specific factors that allow for growth. A variety of microbiological media and variations are well known in the art. The medium can be added to the culture at any time, including at the start of the culture, during the culture, or intermittently / continuously.

[0212] Examples of growth media include, but are not limited to, (1) a semi-defined medium containing peptone, 3 g / L; yeast, 3 g / L; vitamin solution, 2 mL / L; inorganic solution, 25 mL / L; indigo carmine (0.5%), 1 g / L; 12.5% L-cysteine, 2 g / L; 12.5% sodium sulfide, 2 g / L, supplemented with either sodium lactate (semi-defined lactate, SDL), glucose (semi-defined glucose, SDG), or maltose (semi-defined maltose, SDM); (2) a modified reinforced Clostridium agar / broth medium (pre-reduced type) containing peptone, 10 g / L; beef extract, 10 g / L; yeast extract, 3 g / L; dextrose 5 g / L; NaCl, 5 g / L; soluble starch, 1 g / L; L-cysteine HCl, 0.5 g / L; sodium acetate, 3 g / L; and resazurin (0.025%), 4 mL / L; (3) trypticase soy agar / broth containing defibrinated sheep blood; (4) sodium lactate (70%), 10 g / l; peptone, 2 g / l; KH2PO4 1 g / l; (NH4)2SO4 3 g / l; MgSO4 7H2O 0.2 g / l; CaCl2.2H2O 0.06 g / l; vitamins (pyridoxol hydrochloride, 4 mg / l; pyridoxamine, 4 mg / l; riboflavin, 4 mg / l; thiamine chloride, 4 mg / l; nicotinamide, 4 mg / l; calcium D-pantothenate, 4 mg / l; 4-aminobenzoic acid, 0.2 mg / l, biotin, 0.2 mg / l, folic acid, 0.1 mg / l, and cyanocobalamin, 0.02 mg / 1); Na2S.9H2O, 0.25 g / l; cysteine, 0.25 g / l; antifoaming agent, 0.Containing 07 ml / l and monensin, 10 mg / l; adding sodium lactate and an inorganic solution to a reservoir bottle and autoclaving for 60 minutes; dissolving peptone in 300 ml of distilled H2O and autoclaving separately; filter-sterilizing a vitamin solution and two reducing agents in advance; after autoclaving, gassing the reservoir bottle with an anaerobic gas overnight; adding other constituent substances separately after cooling; and a semi-defined first gastric juice-free medium prepared by adjusting the pH to the desired value with 5N HCl; and (5) 400 ml of clarified first gastric juice incubated from lucerne-fed sheep, 371 ml of distilled water, 2 g of peptone, 15 g of agar, 100 ml of 10% (w / v) D, L-sodium lactate solution, 100 ml of 0.04% (w / v) bromocresol purple solution and 40 g / l KH2PO4; 120 g / l (NH4)2SO4; 8 g / l MgSO4.7H2O and 2.4 g / l CaCl2.2H2O, an incubated first gastric juice acetate ("IRFL") medium containing 25 ml of an inorganic solution, after adjusting the pH to 5.5 using lactic acid (90% w / v), autoclaving at 121°C for 25 minutes, and then cooling in a 50°C water bath while aerating with an anaerobic gas mixture, adding 2 milliliters each of Na2S.9H2O (12.5% w / v) and cysteine.HCl.H2O (12.5% w / v).

[0213] In some embodiments, the culture comprises a growth medium comprising at least one carbon source. In some embodiments, the at least one carbon source is selected from the group consisting of casein, starch (e.g., gelatinized starch and / or soluble starch), lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof.

[0214] In some embodiments, the culture comprises a growth medium containing at least two carbon sources. In some embodiments, the at least two carbon sources are selected from the group consisting of casein, starch (e.g., gelatinized starch and / or soluble starch), lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fats, oils, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof.

[0215] In some embodiments, the at least two carbon sources consist of from about 1 to 99% of a first carbon source (e.g., any carbon source described herein) and from about 1 to 99% of a second carbon source (e.g., any carbon source described herein that is different from the first carbon source), and 100% of the at least two carbon sources consists of the first and second carbon sources. In some embodiments, the at least two carbon sources consist of from about 50 to 60% of the first carbon source and from about 40 to 50% of the second carbon source, from about 50 to 70% of the first carbon source and from about 30 to 50% of the second carbon source, from about 50 to 80% of the first carbon source and from about 20 to 50% of the second carbon source, or from about 50 to 90% of the first carbon source and from about 10 to 50% of the second carbon source. In another embodiment, the at least two carbon sources consist of from about 65 to 75% of the first carbon source and from about 25 to 35% of the second carbon source. In some embodiments, the first carbon source is lactate.

[0216] In some embodiments, M. elsdenii cells are grown at about 39°C to about 40°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0217] In some embodiments, the microbial cells are grown at about 15°C to about 45°C, about 20°C to 40°C, 25°C to 35°C, 30°C to 39°C. In some embodiments, the microbial cells are grown at about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0218] In some embodiments, the microbial cells are cooled to about 18°C to about 25°C for storage.

[0219] In some embodiments, the pH of a culture containing M. elsdenii cells (e.g., during culture and / or at harvest) is about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, about 4.5 to about 5.0, about 4.6 to about 6.9, about 4.7 to about 6.8, about 4.8 to about 6.7, about 4.9 to about 6.6, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.0 to about 5.5, about 5.1 to about 6.9, about 5.2 to about 6.8, about 5.3 to about 6.7, about 5.4 to about 6.6, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.1 to about 6.4, about 5.2 to about 6.3, about 5.3 to about 6.2, about 5.4 to about 6.1, about 5.5 to about 6.0, about 5.0 to about 6.1, about 5.0 to about 6.2, about 5.0 to about 6.3, about 5.0 to about 6.4, about 5.1 to about 6.5, about 5.2 to about 6.5, about 5.3 to about 6.5, or about 5.4 to about 6.5.

[0220] In some embodiments, the pH of a culture containing microbial cells (e.g., during culture and / or at harvest) is between about 4.0 and about 9.0, between about 4.5 and about 8.5, between about 5.0 and about 8.0, between about 5.5 and about 7.5, between about 6.0 and about 7.0.

[0221] To culture microbial cells, fermenters of different sizes and designs can be used to maintain anaerobic conditions. The fermenter may be capable of fermenting a culture volume sufficient for commercial production of, for example, M. elsdenii cells. In some embodiments, the culture volume is about 2 liters, about 10 liters, about 50 liters, about 100 liters, about 150 liters, about 200 liters, about 250 liters, about 300 liters, about 350 liters, about 400 liters, about 450 liters, about 500 liters, about 600 liters, about 800 liters, about 1,000 liters, about 1,200 liters, about 1,500 liters, about 1,800 liters, about 2,000 liters, about 2,200 liters, about 2,500 liters, about 2,750 liters, about 3,000 liters, about 4,000 liters, about 5,000 liters, about 6,000 liters, about 7,000 liters, about 8,000 liters, about 9,000 liters, about 10,000 liters, at least about 20,000 liters, at least about 50,000 liters, or at least about 75,000 liters.In some embodiments, the fermentation volume is from about 2 liters to about 75,000 liters, from about 250 liters to about 750 liters, from about 300 liters to about 800 liters, from about 350 liters to about 850 liters, from about 400 liters to about 900 liters, from about 450 liters to about 950 liters, from about 500 liters to about 1,000 liters, from about 750 liters to about 1,250 liters, from about 1,000 liters to about 2,000 liters, from about 2,000 liters to about 4,000 liters, from about 4,000 liters to about 8,000 liters, from about 5,000 liters to about 10,000 liters, from about 50 liters to about 75,000 liters, from about 50 liters to about 50,000 liters, from about 50 liters to about 25,000 liters, from about 50 liters to about 20,000 liters, from about 50 liters to about 15,000 liters, from about 50 liters to about 10,000 liters, from about 100 liters to about 10,000 liters, from about 100 liters to about 5,000 liters, from about 100 liters to about 4,000 liters, from about 100 liters to about 3,000 liters, from about 100 liters to about 2,900 liters, from about 100 liters to about 2,850 liters, from about 100 liters to about 2,800 liters, from about 100 liters to about 2,750 liters, about 2 liters, about 10 liters, about 50 liters, about 100 liters, about 200 liters, about 500 liters, about 1,000 liters, about 1,500 liters, about 10,000 liters, about 20,000 liters, about 50,000 liters, or about 75,000 liters.

[0222] In some embodiments, a culture containing M. elsdenii cells also contains another microorganism (i.e., a microbial cell that is not an M. elsdenii cell). In some embodiments, the culture contains M. elsdenii cells and another microorganism that is an obligate anaerobe. In some embodiments, the culture contains M. elsdenii cells and another microorganism selected from the group consisting of Lactobacillus, Megasphaera, Bifidobacterium, Escherichia, Enterococcus, Bacillus, Propionibacterium, Streptococcus, Candida, Clostridium, Pediococcus, Aspergillus, and Saccharomyces. Freeze-dried aerobic, anaerobic, and yeast cells

[0223] In one aspect, the present invention is directed to a method for producing freeze-dried Megasphaera elsdenii cells.

[0224] In another aspect, the present invention is directed to freeze-dried M. elsdenii cells.

[0225] In another aspect, the present invention is directed to freeze-dried M. elsdenii cells produced by the methods disclosed herein.

[0226] In another aspect, the present invention is directed to a method for producing freeze-dried Megasphaera cells.

[0227] In another aspect, the present invention is directed to freeze-dried Megasphaera cells.

[0228] In another aspect, the present invention is directed to freeze-dried Megasphaera cells produced by the methods disclosed herein.

[0229] In another aspect, the present invention is directed to a method for producing freeze-dried anaerobic bacterial cells.

[0230] In another aspect, the present invention is directed to a method for producing freeze-dried Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, and Lactobacillus cells such as L. casei.

[0231] In another aspect, the present invention is directed to freeze-dried anaerobic bacterial cells.

[0232] In another aspect, the present invention is directed to freeze-dried Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, and Lactobacillus cells such as L. casei.

[0233] In another aspect, the present invention is directed to freeze-dried anaerobic bacteria produced by the methods disclosed herein.

[0234] In another aspect, the present invention is directed to freeze-dried Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, and Lactobacillus cells such as L. casei produced by the methods disclosed herein.

[0235] In another aspect, the present invention is directed to a method for producing freeze-dried aerobic bacterial cells.

[0236] In another aspect, the present invention is directed to a method for producing freeze-dried Bacillus cells such as B. subtilis.

[0237] In another aspect, the present invention is directed to freeze-dried aerobic bacterial cells.

[0238] In another aspect, the present invention is directed to freeze-dried Bacillus cells such as B. subtilis.

[0239] In another aspect, the present invention is directed to freeze-dried aerobic bacterial cells produced by the methods disclosed herein.

[0240] In another aspect, the present invention is directed to freeze-dried Bacillus cells such as B. subtilis produced by the methods disclosed herein.

[0241] In another aspect, the present invention is directed to a method for producing freeze-dried yeast cells.

[0242] In another aspect, the present invention is directed to a method for producing freeze-dried Saccharomyces cells such as S. boulardii and S. cerevisiae.

[0243] In another aspect, the present invention is directed to freeze-dried yeast cells.

[0244] In another aspect, the present invention is directed to freeze-dried Saccharomyces such as S. boulardii and S. cerevisiae.

[0245] In another aspect, the present invention is directed to freeze-dried yeast cells produced by the methods disclosed herein.

[0246] In another aspect, the present invention is directed to freeze-dried Saccharomyces cells, such as S. boulardii and S. cerevisiae, produced by the methods disclosed herein.

[0247] In another aspect, a method for producing freeze-dried M. elsdenii cells includes preparing a culture comprising M. elsdenii cells and a growth medium, harvesting the cells, freezing the cells, and freeze-drying the cells, whereby freeze-dried M. elsdenii cells are produced. In some embodiments, the method is performed in the order of preparing the culture, then harvesting the cells (i.e., harvesting the cultured cells), then freezing the cells (i.e., freezing the harvested cells), then freeze-drying the cells (i.e., freeze-drying the frozen cells).

[0248] In some embodiments, the method is performed under anaerobic conditions. In some embodiments, the method includes, under anaerobic conditions, preparing the culture, harvesting the cells, freezing the cells, freeze-drying the cells, and combinations thereof.

[0249] The method can include any of the methods for preparing a culture described herein. The culture of the method can also include any of the characteristics of the cultures described herein.

[0250] In some embodiments, the cells in the culture comprise M. elsdenii cells. In some embodiments, the cells in the culture consist of M. elsdenii cells.

[0251] In some embodiments, the growth medium comprises at least two carbon sources selected from the group consisting of casein, lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fats, oils, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, and combinations thereof.

[0252] In some embodiments, the method comprises harvesting the cells within 12 hours after the culture has ended its logarithmic growth phase and before the culture begins its stationary growth phase. The culture can be cooled to room temperature to stop growth upon harvesting.

[0253] In some embodiments, the culture comprises a liquid and the method comprises harvesting M. elsdenii cells (e.g., concentrated M. elsdenii cells) in terms of the percentage of the liquid. In some embodiments, the method comprises recovering the cells with about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of the liquid. In some embodiments, the method comprises recovering the cells with less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the liquid.

[0254] In some embodiments, the culture contains a liquid and the method includes harvesting M. elsdenii cells (e.g., concentrated M. elsdenii cells) by removing a portion of the liquid. In some embodiments, harvesting the cells includes removing about 50% to about 100% of the liquid, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the liquid. In some embodiments, harvesting the cells includes removing at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the liquid.

[0255] In some embodiments, the method includes harvesting M. elsdenii cells by concentrating the cells. In some embodiments, the step of harvesting the cells includes concentrating the cells by at least one technique selected from the group consisting of centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. In some embodiments, the filtration includes clay filtration. In some embodiments, the filtration includes tangential flow filtration, also known as crossflow filtration.

[0256] In some embodiments, the pH of the culture containing M. elsdenii cells at the time of harvest is from about 4.5 to about 7.0, from about 4.5 to about 6.5, from about 4.5 to about 6.0, from about 4.5 to about 5.5, from about 4.5 to about 5.0, from about 4.6 to about 6.9, from about 4.7 to about 6.8, from about 4.8 to about 6.7, from about 4.9 to about 6.6, from about 5.0 to about 7.0, from about 5.0 to about 6.5, from about 5.0 to about 6.0, from about 5.0 to about 5.5, from about 5.1 to about 6.9, from about 5.2 to about 6.8, from about 5.3 to about 6.7, from about 5.4 to about 6.6, from about 5.5 to about 7.0, from about 5.5 to about 6.5, from about 5.1 to about 6.4, from about 5.2 to about 6.3, from about 5.3 to about 6.2, from about 5.4 to about 6.1, from about 5.5 to about 6.0, from about 5.0 to about 6.1, from about 5.0 to about 6.2, from about 5.0 to about 6.3, from about 5.0 to about 6.4, from about 5.1 to about 6.5, from about 5.2 to about 6.5, from about 5.3 to about 6.5, or from about 5.4 to about 6.5.

[0257] In some embodiments, the method comprises inoculating a growth medium in a fermenter with an inoculum containing M. elsdenii cells to prepare a culture, and incubating the culture at a temperature of about 39 °C until the pH of the culture reaches about 6.0. In some embodiments, the inoculum containing M. elsdenii cells is a flask culture of M. elsdenii cells or a portion thereof. In some embodiments, the method comprises inoculating the growth medium in the fermenter at a ratio of inoculum to medium of 1 / 50 to 1 / 4,000. In some embodiments, the ratio of inoculum to medium is 1 / 100.

[0258] In some embodiments, the culture further comprises at least one cryoprotective substance. In some embodiments, the at least one cryoprotective substance is selected from the group consisting of fructose, glucose, sucrose, powdered milk, infant formula, nonfat dry milk, trehalose, maltodextrin, betaine, and combinations thereof. In some embodiments, the at least one cryoprotective substance is present in an amount of about 1% to about 50% (w / v) of the culture, about 1% to about 40% (w / v) of the culture, about 1% to about 30% (w / v) of the culture, about 1% to about 20% (w / v) of the culture, about 1% to about 10% (w / v) of the culture, about 1% to about 5% (w / v) of the culture, about 10% to about 20% (w / v) of the culture, about 15% to about 25% (w / v) of the culture, about 20% to about 30% (w / v) of the culture, about 30% to about 40% (w / v) of the culture, about 40% to about 50% (w / v) of the culture, about 60% to about 70% (w / v) of the culture, about 70% to about 80% (w / v) of the culture. In some embodiments, the cryoprotective substance is added by directly adding the powdered cryoprotective substance to the concentrated M. elsdenii cells. In some embodiments, the cryoprotective substance is added by directly adding a solution of the cryoprotective substance to the concentrated M. elsdenii cells at a ratio of 1 / 1, 1 / 5, or 1 / 10.

[0259] In some embodiments, the step of freezing the cells comprises placing the cells in a freezer or contacting the cells with dry ice, liquid nitrogen, or a combination thereof. The step of freezing the cells comprises freezing the cells while the cells are present within a container. The step of contacting the cells comprises contacting a container containing the cells with a medium for freezing the cells. The medium for freezing the cells includes, but is not limited to, a freezer, an acetone-dry ice bath, liquid nitrogen, or a combination thereof.

[0260] In some embodiments, the method includes freezing the cells at a temperature of about -20°C to about -210°C. In some embodiments, the method includes freezing the cells at a temperature of about -20°C to about -80°C. In some embodiments, the method includes freezing the cells at a temperature of about -80°C to about -210°C. In some embodiments, the method includes freezing the cells at a temperature of about -20°C to about -196°C. In some embodiments, the method includes freezing the cells at a temperature of about -80°C to about -196°C. In some embodiments, the method includes freezing the cells at a temperature of about -20°C. In some embodiments, the method includes freezing the cells at a temperature of about -80°C. In some embodiments, the method includes freezing the cells at a temperature of about -196°C. In some embodiments, the method includes the step of freezing the cells by contacting the cells with liquid nitrogen.

[0261] In some embodiments, the method includes the step of freezing the cells under anaerobic conditions.

[0262] In some embodiments, the freezing step produces a frozen pellet containing the cells. For example, the freezing step can be achieved using a flash freezer (e.g., model 250-S01 Crygran, IFQ Inc.).

[0263] In some embodiments, the diameter of the frozen pellets is from about 0.001 to about 1.0 inch, from about 0.01 to about 1.0 inch, from about 0.1 to about 1.0 inch, from about 0.2 to about 1.0 inch, from about 0.3 to about 1.0 inch, from about 0.4 to about 1.0 inch, from about 0.5 to about 1.0 inch, from about 0.6 to about 1.0 inch, from about 0.7 to about 1.0 inch, from about 0.8 to about 1.0 inch, from about 0.9 to about 1.0 inch, from about 0.001 to about 0.9 inch, from about 0.01 to about 0.9 inch, from about 0.1 to about 0.9 inch, from about 0.2 to about 0.9 inch, from about 0.3 to about 0.9 inch, from about 0.4 to about 0.9 inch, from about 0.5 to about 0.9 inch, from about 0.6 to about 0.9 inch, from about 0.7 to about 0.9 inch, from about 0.8 to about 0.9 inch, from about 0.001 to about 0.8 inch, from about 0.01 to about 0.8 inch, from about 0.1 to about 0.8 inch, from about 0.2 to about 0.8 inch, from about 0.3 to about 0.8 inch, from about 0.4 to about 0.8 inch, from about 0.5 to about 0.8 inch, from about 0.6 to about 0.8 inch, from about 0.7 to about 0.8 inch, from about 0.001 to about 0.7 inch, from about 0.01 to about 0.7 inch, from about 0.1 to about 0.7 inch, from about 0.2 to about 0.7 inch, from about 0.3 to about 0.7 inch, from about 0.4 to about 0.7 inch, from about 0.5 to about 0.7 inch, from about 0.6 to about 0.7 inch, from about 0.001 to about 0.6 inch, from about 0.01 to about 0.6 inch, from about 0.1 to about 0.6 inch, from about 0.2 to about 0.6 inch, from about 0.3 to about 0.6 inch, from about 0.4 to about 0.6 inch, from about 0.5 to about 0.6 inch, from about 0.001 to about 0.5 inch, from about 0.01 to about 0.5 inch, from about 0.05 to about 0.5 inch, from about 0.1 to about 0.5 inch, from about 0.15 to about 0.5 inch, from about 0.2 to about 0.5 inch, from about 0.3 to about 0.5 inch, or from about 0.4 to about 0.5 inch.

[0264] Frozen M. elsdenii cells can be freeze-dried after cryopreservation (e.g., below 0 °C) or can be freeze-dried immediately. In some embodiments, the frozen M. elsdenii cells are stored at a temperature of less than about 0 °C, less than about -10 °C, less than about -20 °C, less than about -50 °C, less than about -80 °C or less than about -196 °C. In some embodiments, the frozen M. elsdenii cells are stored at a temperature of about -20 °C, about -30 °C, about -40 °C, about -50 °C, about -60 °C, about -70 °C, about -80 °C, about -90 °C, about -100 °C, about -150 °C, about -196 °C or about -210 °C.

[0265] In some embodiments, the frozen M. elsdenii cells are lyophilized. In some embodiments, the frozen M. elsdenii cells are freeze-dried. The freeze-drying step includes, for example, the removal of liquid from the frozen cells.

[0266] In some embodiments, the step of freeze-drying M. elsdenii cells includes placing the frozen cells in a freeze-dryer. In some embodiments, the freeze-drying step includes subjecting the frozen cells to reduced pressure and gradually warming the cells to room temperature.

[0267] In some embodiments, the method includes the step of freeze-drying the cells under anaerobic conditions.

[0268] In some embodiments, freeze-dried M. elsdenii is produced on a commercial scale.

[0269] In some embodiments, about 1×10 3 ~1×10 12 CFU / g of freeze-dried M. elsdenii cells are produced by the methods disclosed herein. In some embodiments, about 1×10 3 ~1×10 12 CFU / g of M. elsdenii cells are viable after freeze-drying.

[0270] In one aspect, a method for generating freeze-dried Megasphaera elsdenii cells according to the present disclosure comprises: (a) preparing a culture comprising M. elsdenii cells and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate (e.g., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) freezing the cells; and (d) freeze-drying the cells, wherein freeze-dried M. elsdenii cells are produced at about 1×10 3 ~ about 1×10 12 CFU / g of freeze-dried M. elsdenii cells are produced.

[0271] In another aspect, a method for generating freeze-dried Megasphaera elsdenii cells according to the present disclosure comprises: (a) preparing a culture comprising M. elsdenii cells and a growth medium; (b) harvesting the cells under anaerobic conditions within 12 hours after the culture has ended its logarithmic growth phase and before the culture starts its stationary growth phase; (c) freezing the cells; and (d) freeze-drying the cells, wherein freeze-dried M. elsdenii cells are produced.

[0272] In another aspect, a method for generating freeze-dried Megasphaera elsdenii cells according to the present disclosure comprises: (a) preparing a culture comprising M. elsdenii cells and a growth medium; (b) harvesting the cells; (c) freezing the cells at a temperature of about -80°C to about -210°C within 5 hours of harvesting; and (d) freeze-drying the cells, wherein freeze-dried M. elsdenii cells are produced.

[0273] In some embodiments, the amount of freeze-dried M. elsdenii cells produced by the methods disclosed herein and / or the amount of viable M. elsdenii cells after freeze-drying is from about 1x10 3 CFU / g to about 1x10 12 CFU / g, about 1x10 3 CFU / g to about 1x10 11 CFU / g, about 1x10 3 CFU / g to about 1x10 10 CFU / g, about 1x10 3 CFU / g to about 1x10 9 CFU / g, about 1x10 3 CFU / g to about 1x10 8 CFU / g, about 1x10 3 CFU / g to about 1x10 7 CFU / g, about 1x10 3 CFU / g to about 1x10 6 CFU / g, about 1x10 3 CFU / g to about 1x10 5 CFU / g, about 1x10 4 CFU / g to about 1x10 12 CFU / g, about 1x10 5 CFU / g to about 1x10 12 CFU / g, about 1x10 6 CFU / g to about 1x10 12 CFU / g, about 1x10 7 CFU / g to about 1x10 12 CFU / g, about 1x10 8 CFU / g to about 1x10 12 CFU / g, about 1x10 9 CFU / g to about 1x10 12 CFU / g, about 1x10 10 CFU / g to about 1x10 12 CFU / g, about 1x10 3 CFU / g to about 1x10 5 CFU / g, about 1x10 4 CFU / g to about 1x10 6 CFU / g, about 1x10 5 CFU / g to about 1x10 7 CFU / g, about 1x10 6 CFU / g to about 1x108 CFU / g, about 1x10 7 CFU / g to about 1x10 9 CFU / g, about 1x10 8 CFU / g to about 1x10 10 CFU / g, about 1x10 9 CFU / g to about 1x10 11 CFU / g, or about 1x10 10 CFU / g to about 1x10 12 CFU / g.

[0274] In some embodiments, the freeze-dried M. elsdenii cells are viable at temperatures of about -80°C, about -20°C, about 4°C, about 25°C, or combinations thereof for about 14 days to about 24 months. In some embodiments, the freeze-dried M. elsdenii cells are viable at temperatures of about -80°C, about -20°C, about 4°C, about 25°C, or combinations thereof for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months.

[0275] In some embodiments, about 1x10 3 CFU / g to about 1x10 12 CFU / g about 1x10 3 CFU / g to about 1x10 11 CFU / g, about 1x10 3 CFU / mL to about 1x10 10 CFU / g, about 1x10 3 CFU / g to about 1x10 9 CFU / g, about 1x10 3 CFU / g to about 1x10 8 CFU / g, about 1x10 3 CFU / g to about 1x10 7 CFU / g, about 1x10 3 CFU / g to about 1x10 6 CFU / g, about 1x10 3 CFU / g to about 1x10 5 CFU / g, about 1x10 4 CFU / g to about 1x10 12 CFU / g, about 1x105 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 6 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 7 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 8 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 9 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 10 CFU / g to approximately 1 x 10 12 CFU / g, approximately 1 x 10 3 CFU / g to approximately 1 x 10 5 CFU / g, approximately 1 x 10 4 CFU / g to approximately 1 x 10 6 CFU / g, approximately 1 x 10 5 CFU / g to approximately 1 x 10 7 CFU / g, approximately 1 x 10 6 CFU / g to approximately 1 x 10 8 CFU / g, approximately 1 x 10 7 CFU / g to approximately 1 x 10 9 CFU / g, approximately 1 x 10 8 CFU / g to approximately 1 x 10 10 CFU / g, approximately 1 x 10 9 CFU / g to approximately 1 x 10 11 CFU / g, or approximately 1 x 10 10 CFU / g to approximately 1 x 10 12 CFU / g of freeze-dried M. elsdenii cells are viable after storage at a temperature of about -80°C, about -20°C, about 4°C, or a combination thereof for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months.

[0276] In some embodiments, about 1 x 10 3 CFU / g to approximately 1 x 10 12 CFU / g approximately 1 x 10 3 CFU / g to approximately 1 x 10 11 CFU / g, approximately 1 x 10 3CFU / g to approximately 1x10 10 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 9 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 8 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 7 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 6 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 5 CFU / g, approximately 1x10 4 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 5 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 6 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 7 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 8 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 9 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 10 CFU / g to approximately 1x10 12 CFU / g, approximately 1x10 3 CFU / g to approximately 1x10 5 CFU / g, approximately 1x10 4 CFU / g to approximately 1x10 6 CFU / g, approximately 1x10 5 CFU / g to approximately 1x10 7 CFU / g, approximately 1x10 6 CFU / g to approximately 1x10 8 CFU / g, approximately 1x10 7 CFU / g to approximately 1x10 9 CFU / g, approximately 1x10 8 CFU / g to approximately 1x10 10 CFU / g, approximately 1x10 9 CFU / g to approximately 1x10 11 CFU / g, or approximately 1x10 10 CFU / g to approximately 1x10 12CFU / g of freeze-dried M. elsdenii cells are viable after storage at a temperature of about 25 °C for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months. Feed Additives, Compositions, and Kits

[0277] In one aspect, the feed additive comprises M. elsdenii cells disclosed herein.

[0278] In some embodiments, the feed additive comprises freeze-dried M. elsdenii cells disclosed herein. In some embodiments, the feed additive comprises freeze-dried M. elsdenii cells produced by the methods disclosed herein.

[0279] In one aspect, the feed additive comprises Megasphaera cells disclosed herein.

[0280] In some embodiments, the feed additive comprises freeze-dried Megasphaera cells disclosed herein. In some embodiments, the feed additive comprises freeze-dried Megasphaera cells produced by the methods disclosed herein.

[0281] In one aspect, the feed additive comprises anaerobic bacterial cells disclosed herein. In another aspect, the feed additive comprises Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, Lactobacillus cells such as L. casei.

[0282] In some embodiments, the feed additive comprises the freeze-dried anaerobic bacterial cells disclosed herein. In some embodiments, the feed additive comprises the freeze-dried anaerobic bacterial cells produced by the methods disclosed herein.

[0283] In some embodiments, the feed additive comprises freeze-dried Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, and Lactobacillus cells such as L. casei. In some embodiments, the feed additive comprises freeze-dried Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, and Lactobacillus cells such as L. casei produced by the methods disclosed herein.

[0284] In one aspect, the feed additive comprises the aerobic bacterial cells disclosed herein. In another aspect, the feed additive comprises Bacillus cells such as B. subtilis.

[0285] In some embodiments, the feed additive comprises the freeze-dried aerobic bacterial cells disclosed herein. In some embodiments, the feed additive comprises the freeze-dried aerobic bacterial cells produced by the methods disclosed herein.

[0286] In some embodiments, the feed additive comprises freeze-dried Bacillus cells such as B. subtilis. In some embodiments, the feed additive comprises freeze-dried Bacillus cells such as B. subtilis produced by the methods disclosed herein.

[0287] In one aspect, the feed additive comprises the yeast cells disclosed herein. In another aspect, the feed additive comprises Saccharomyces cells such as S. boulardii and S. cerevisiae.

[0288] In some embodiments, the feed additive comprises the freeze-dried yeast cells disclosed herein. In some embodiments, the feed additive comprises the freeze-dried yeast cells produced by the methods disclosed herein.

[0289] In some embodiments, the feed additive comprises freeze-dried Saccharomyces cells such as S. boulardii and S. cerevisiae. In some embodiments, the feed additive comprises freeze-dried Saccharomyces cells such as S. boulardii and S. cerevisiae produced by the methods disclosed herein.

[0290] In some embodiments, the feed additive is solid (i.e., "solid feed additive") or liquid (i.e., "liquid feed additive"). In some embodiments, the feed additive is semi-solid or gel-like (i.e., "semi-solid or gel-like feed additive"). The gel-like feed additive can contain an oxygen scavenger (e.g., ascorbic acid).

[0291] In some embodiments, the solid feed additive is a powder (e.g., a free-flowing powder), fine granules (i.e., pellets), particles (i.e., microparticles), pellets, cakes, water-soluble concentrates, pastes, boluses, tablets, fine powders, their components, or combinations thereof.

[0292] In some embodiments, the liquid feed additive is a solution (e.g., an aqueous, organic, or aqueous-organic solution), suspension, emulsion, drencher, spray, injection, beverage (e.g., a milk replacer), their components, or combinations thereof.

[0293] In some embodiments, the gelled feed additive is an organic gel. In some embodiments, the gelled feed additive is an oral gel (i.e., a gel for oral administration).

[0294] In some embodiments, the feed additive is for use as a top dress (i.e., for adding to the surface of food or mixing with food (e.g., animal feed)). In some embodiments, the feed additive is for administration as a liquid.

[0295] In some embodiments, the freeze-dried M. elsdenii cells disclosed herein can be used as a liquid feed additive by rehydrating, dissolving, solubilizing, and / or suspending the freeze-dried cells in a liquid.

[0296] In some embodiments, the feed additive comprises a carrier (i.e., one or more carriers).

[0297] Examples of suitable carriers include, but are not limited to, vegetable materials (i.e., the whole plant or part of the plant, including dried or processed plants or parts of plants, such as seeds, stems, leaves, flowers and / or roots), dried grains (e.g., dried distillers grains), alfalfa, corn meal, citrus meal, fermentation residues, ground oyster shells, attapulgus clay, wheat short, molasses soluble, corn cob meal, edible vegetable substances, bran, soybean mill feed, antibiotic mycelium, vermiculite, soybean grits, whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice hulls, yeast cultures, dried starch, sodium silicoaluminate, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, essential oils, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc., as well as combinations thereof.

[0298] In some embodiments, the feed additive includes excipients (i.e., one or more excipients), including, but not limited to, crystalline cellulose; lactose; sodium citrate; calcium carbonate; calcium hydrogen phosphate and glycine; disintegrants such as starch, sodium starch glycolate, croscarmellose sodium and certain complex silicates; granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; bulking agents such as maltodextrin; moisture scavengers such as silicon dioxide; oxygen scavengers such as ascorbic acid; and / or lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc.

[0299] In some embodiments, the feed additive is a fine granule comprising a core containing M. elsdenii cells and / or the feed additive, and a coating over the core. In some embodiments, the coating is a hydrated barrier salt. The salt coating can provide improved heat resistance, improved storage stability, and protection from other components in the fine granule that can have a detrimental effect (e.g., on stability) on the M. elsdenii cells and / or the feed additive in the absence thereof.

[0300] In some embodiments, the freeze-dried M. elsdenii is admixed with a dry formulation of additives including, but not limited to, a growth substrate, an enzyme, a sugar, a carbohydrate, an extract, and a growth-promoting micronutrient. The sugar can include, but is not limited to, lactose, maltose, dextrose, maltodextrin, glucose, fructose, mannose, tagatose, sorbose, raffinose, amylose, starch, and galactose. The sugar can be in the range of 50 - 95% either individually or in combination. The extract can include, but is not limited to, yeast or dried yeast fermentation solubles in the range of 5 - 50%. The growth substrate can include, but is not limited to, trypticase in the range of 5 - 25%; sodium lactate in the range of 5 - 30%; and Tween 80 in the range of 1 - 5%. The carbohydrate can include, but is not limited to, mannitol, sorbitol, adonitol, and arabitol. The carbohydrate can be in the range of 5 - 50% either individually or in combination. The micronutrients can include, but are not limited to, calcium carbonate in the range of 0.5 - 5.0%; calcium chloride in the range of 0.5 - 5.0%; dipotassium phosphate in the range of 0.5 - 5.0%; calcium phosphate in the range of 0.5 - 5.0%; manganese protein in the range of 0.25 - 1.00%; and manganese in the range of 0.25 - 1.00%.

[0301] In some embodiments, the M. elsdenii feed additive is prepared by mixing M. elsdenii cells, including a culture containing cells and / or freeze-dried cells, with any additional components of the feed additive (e.g., carriers and / or excipients) (e.g., by a mixer). In some embodiments, the components are mixed to obtain a homogeneous mixture.

[0302] In some embodiments, the feed additive is a top-dress animal feed additive (e.g., freeze-dried cells) containing the M. elsdenii cells and carriers disclosed herein. In some embodiments, the carrier is selected from the group consisting of whey, maltodextrin, sucrose, dextrose, limestone (i.e., calcium carbonate), rice hulls, yeast culture, dried starch, and sodium silicoaluminate, milk, water, and combinations thereof.

[0303] In some embodiments, the animal feed additive is a replacer milk drench, spray, or supplement containing the M. elsdenii cells (e.g., freeze-dried cells) and a water-soluble carrier disclosed herein. In some embodiments, the carrier is selected from the group consisting of whey, maltodextrin, sucrose, dextrose, dried starch, sodium silicoaluminate, milk, water, and combinations thereof.

[0304] In one aspect, the present invention is directed to foods (e.g., animal feeds) comprising M. elsdenii cells (e.g., freeze-dried cells as disclosed herein, e.g., freeze-dried cells produced by the methods disclosed herein) and / or feed additives as disclosed herein. The food product is any food for animal consumption (i.e., non-human or human), and includes both solid and liquid compositions. The food includes, but is not limited to, common foods; liquid products including water, milk, beverages, therapeutic beverages, and nutritional beverages; functional foods; dietary supplements; nutritional supplements; infant formulas (i.e., including non-human and human infants) including prepared powdered milk for premature infants; foods for pregnant or lactating animals; foods for adult animals; and foods for the elderly. In some embodiments, the food includes a liquid (e.g., a beverage, e.g., water, milk, or substitute milk) comprising a feed additive.

[0305] In another aspect, the present invention is directed to compositions comprising M. elsdenii cells (e.g., freeze-dried cells) and / or feed additives as disclosed herein. In some embodiments, the composition comprises freeze-dried M. elsdenii cells produced by the methods disclosed herein.

[0306] In some embodiments, the M. elsdenii cells (e.g., freeze-dried cells) and / or feed additives as disclosed herein can be further chemically or physically modified or processed by any known technique, based on the requirements of the composition.

[0307] The composition of the present invention can include one or more excipients. In some embodiments, the excipients can be, but are not limited to, alkalizing agents, stabilizers, antioxidants, adhesives, separating agents, coating agents, exterior phase components, controlled release components, solvents, surfactants, humectants, buffering agents, fillers, emollients, or combinations thereof. The excipients can include, in addition to those discussed herein, excipients listed in Remington: The Science and Practice of Pharmacy, 21st Edition (2005), but are not limited thereto. Inclusion of an excipient in a particular classification herein (e.g., "solvent") is not intended to limit the role of the excipient, but rather to illustrate. A particular excipient can fit into multiple classifications.

[0308] In some embodiments, the composition is a pharmaceutical composition (e.g., for the treatment of non-human animals or humans). In some embodiments, the composition is a medical food (e.g., veterinary food). A medical food is a food intended for a specific dietary management of a condition in which unique nutritional requirements, based on recognized scientific principles, that are present in and recognized in the composition consumed or administered externally under the supervision of a physician (e.g., veterinarian) are established by medical evaluation. In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients. In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized international pharmacopeia for use in animals, more particularly humans.

[0309] In the case of oral administration of the composition, M. elsdenii cells (e.g., freeze-dried cells) or feed additives can be combined with excipients well-known in the art. Such carriers can enable the M. elsdenii cells or feed additives of the present invention to be formulated, for example, as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject to be treated. In some embodiments, the composition is a tablet, pill, caplet or capsule. Suitable excipients include, but are not limited to, sugars such as lactose, sucrose, mannitol and sorbitol; fillers such as cellulose preparations such as, but not limited to, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone (PVP), etc., but are not limited thereto. If desired, disintegrants such as, but not limited to, cross-linked polyvinylpyrrolidone, agar or alginic acid, or salts thereof such as sodium alginate can be added. Compositions that can be used orally include, but are not limited to, capsules made of gelatin, as well as soft-sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. In some embodiments, the dosage form is a vegetarian dosage form, in which case the dosage form is not formed from and does not contain any components derived from animal sources. In some embodiments, the vegetarian dosage form is a vegetarian capsule.

[0310] In one aspect, the present invention is directed to a capsule comprising M. elsdenii cells (e.g., freeze-dried cells) and / or a feed additive disclosed herein. In some embodiments, the capsule is a gelatin capsule. In some embodiments, the capsule comprises freeze-dried M. elsdenii cells produced by the methods disclosed herein or a feed additive disclosed herein. In some embodiments, the capsule is degradable. In some embodiments, the capsule is a degradable gelatin capsule.

[0311] In another aspect, the present invention is directed to an encapsulated freeze-dried composition comprising anaerobic bacterial cells, wherein the freeze-dried powder is encapsulated by dispensing the freeze-dried powder into heated oil. In another aspect, the present invention is directed to an encapsulated freeze-dried composition comprising M. elsdenii cells, wherein the freeze-dried powder is encapsulated by dispensing the freeze-dried powder into heated oil.

[0312] In another aspect, the present invention is directed to a kit or package comprising M. elsdenii cells, a feed additive, food, and / or a composition disclosed herein. The kit or package can include units (e.g., one or more units) of a feed additive, food, composition, or combinations thereof. In some embodiments, the kit comprises freeze-dried cells produced by the methods disclosed herein, a feed additive disclosed herein, or a capsule disclosed herein. Method of administering anaerobic bacterial cells, aerobic bacterial cells, and / or yeast cells to an animal

[0313] In one aspect, the present invention is directed to a method of administering M. elsdenii cells to an animal.

[0314] In one aspect, the present invention is directed to a method of administering Megasphaera cells to an animal.

[0315] In one aspect, the present invention is directed to a method of administering anaerobic bacterial cells to an animal. In another aspect, the present invention is directed to a method of administering Bifidobacterium cells such as B. breve, Lactobacillus cells such as L. plantarum, Bifidobacterium cells such as B. animalis subsp. lactis, Pediococcus cells such as P. acidilactici, Lactobacillus cells such as L. casei to an animal.

[0316] In one aspect, the present invention is directed to a method of administering aerobic bacterial cells to an animal. In another aspect, the present invention is directed to a method of administering Bacillus cells such as B. subtilis cells to an animal.

[0317] In one aspect, the present invention is directed to a method of administering yeast cells to an animal. In another aspect, the present invention is directed to a method of administering Saccharomyces cells such as S. boulardii and S. cerevisiae cells to an animal.

[0318] In some embodiments, the method comprises administering to the animal M. elsdenii cells, a feed additive, food, or a composition (e.g., a capsule) described herein.

[0319] Administration can be by any suitable route including, for example, oral (i.e., ingestible liquid or solid, oral drench, feed additive, food, composition or capsule), or spraying onto the body (i.e., by misting) and / or by injection.

[0320] In some embodiments, the method comprises administering a solid, liquid or gel comprising M. elsdenii cells.

[0321] In some embodiments, the method includes administering a solid feed additive comprising cells. In some embodiments, the solid feed additive is a powder (e.g., a free-flowing powder), fine granules (i.e., pellets), particles (i.e., microparticles), pellets, cakes, water-soluble concentrates, pastes, boluses, tablets, fine powders, their components, or combinations thereof.

[0322] In some embodiments, the method includes administering a liquid feed additive comprising cells. In some embodiments, the method includes administering cells in a liquid. In some embodiments, the liquid is a solution (e.g., an aqueous, organic, or aqueous-organic solution), a suspension, an emulsion, a drencher, an aerosol, an injectable, a beverage (e.g., a milk replacer), their components, or combinations thereof. In some embodiments, the liquid is administered orally or by spraying the liquid onto the animal.

[0323] In some embodiments, the method includes combining M. elsdenii cells, or a feed additive comprising cells, with another animal feed additive to form a supplement or premix for addition to animal feed. In some embodiments, the other feed additive comprises cells other than M. elsdenii.

[0324] In some embodiments, M. elsdenii cells (e.g., freeze-dried cells) can be added to the feed additive as a liquid (e.g., in broth or a broth equivalent such as, for example, rehydrated freeze-dried cells), as a reconstituted cell paste, or as freeze-dried (e.g., lyophilized) cells. Microorganisms comprising freeze-dried M. elsdenii can also be encapsulated prior to addition to the feed additive. Dosage forms (e.g., a predetermined volume of drencher or capsule) can also be formed, and if desired, the microorganisms can be added directly to the feed by sprinkling a liquid broth and / or freeze-dried cells over the animal feed or by mixing into the feed.

[0325] In some embodiments, the method includes rehydrating a solid feed additive (e.g., powder, granule, microparticle, pellet, cake, freeze-dried cell, or a combination thereof) to produce a liquid for administration.

[0326] In some embodiments, the method includes applying M. elsdenii cells to an animal feed by a delivery system that rehydrates the solid feed additive or freeze-dried cells, including batch by batch. For example, the freeze-dried powder can be predicted from a polyvinyl hopper into a flushing system that dilutes the powder and sprays it onto the feed to be mixed.

[0327] In some embodiments, the method includes applying M. elsdenii cells to an animal feed using a volumetric metering device with a storage bin. For example, the freeze-dried cells (e.g., powder containing the cells) can be stored in the storage bin and introduced into water or a water bath immediately before being sprayed onto the animal feed.

[0328] In one aspect, the present invention is a method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of M. elsdenii cells (e.g., freeze-dried cells) disclosed herein, freeze-dried M. elsdenii cells produced by the methods disclosed herein, feed additives disclosed herein, or compositions (e.g., capsules) disclosed herein.

[0329] In some embodiments, the condition or disorder is acidosis. In some embodiments, the condition or disorder is rumen acidosis. In some embodiments, the condition or disorder is a respiratory disease. In some embodiments, the condition or disorder is laminitis. In some embodiments, the condition or disorder is an infectious disease. In some embodiments, the infectious disease is caused by Salmonella or Campylobacter. In some embodiments, Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella Typhimurium and / or Entiritidis. In some embodiments, Campylobacter is Campylobacter jejuni or Campylobacter coli.

[0330] In another aspect, the present invention is a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of M. elsdenii cells (e.g., freeze-dried cells) disclosed herein, freeze-dried M. elsdenii cells produced by the methods disclosed herein, feed additives disclosed herein, or compositions disclosed herein.

[0331] In some embodiments, the opportunistic microorganisms are pathogenic. In some embodiments, the opportunistic microorganisms are Salmonella or Campylobacter. In some embodiments, Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella Typhimurium and / or Entiritidis. In some embodiments, Campylobacter is Campylobacter jejuni or Campylobacter coli.

[0332] In another aspect, the present invention is a method for improving the bioavailability of plant-derived phosphorus in the diet of an animal, the method comprising administering to the animal an effective amount of M. elsdenii cells (e.g., freeze-dried cells) disclosed herein, freeze-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In some embodiments, the M. elsdenii cells comprise phytase activity. In some embodiments, the method reduces environmental phosphorus waste resulting from the administration of the animal diet in the absence of M. elsdenii cells.

[0333] In another aspect, the present invention is a method for improving the growth performance of an animal, the method comprising administering to the animal an effective amount of M. elsdenii cells (e.g., freeze-dried cells) disclosed herein, freeze-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In some embodiments, the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, breast meat weight gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0334] In another aspect, the present invention is a method for acidifying the lower gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of M. elsdenii cells (e.g., freeze-dried cells) disclosed herein, freeze-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In some embodiments, the lower gastrointestinal tract is the cecum of a poultry animal.

[0335] In some embodiments, the M. elsdenii cells disclosed herein (e.g., freeze-dried cells), the freeze-dried M. elsdenii cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein are administered before, simultaneously with, or after feeding an animal.

[0336] In some embodiments, the method further comprises mixing, prior to administration, the freeze-dried M. elsdenii cells disclosed herein, the freeze-dried cells produced by the methods disclosed herein, or the solid feed additives disclosed herein with a liquid.

[0337] In some embodiments, the liquid is administered orally (e.g., via an oral drench) or by spraying the liquid onto the animal (e.g., by mist spraying).

[0338] In some embodiments, the method comprises a single administration of the M. elsdenii cells disclosed herein (e.g., freeze-dried cells), the freeze-dried M. elsdenii cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein.

[0339] In some embodiments, the method comprises daily administration of the M. elsdenii cells disclosed herein (e.g., freeze-dried cells), the freeze-dried M. elsdenii cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the administration is at least once daily, at least twice daily, at least three times daily, or more than three times daily. In some embodiments, the administration is voluntary (e.g., self-administration by drinking an available liquid or eating available food comprising M. elsdenii cells (e.g., freeze-dried cells), the freeze-dried M. elsdenii cells produced by the methods disclosed herein, feed additives or compositions).

[0340] In some embodiments, the method comprises more than one administration in a day of the M. elsdenii cells disclosed herein (e.g., freeze-dried cells), the freeze-dried M. elsdenii cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the administration is 2, 3, 4, 5, 6 or more administrations in a day. In some embodiments, the method comprises one or more days without administration after more than one administration in a day. In some embodiments, one or more days without administration are 1, 2, 3, 4, 5 or 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months without administration.

[0341] In some embodiments, the animal is a ruminant. In some embodiments, the ruminant can be, but is not limited to, cows, buffalo, sheep, goats, deer, reindeer, elk, giraffe, yaks and serows. In some embodiments, the ruminant is selected from the group consisting of cows, buffalo, sheep, goats, deer and reindeer.

[0342] In some embodiments, the animal is a non-ruminant. In some embodiments, the non-ruminant can be, but is not limited to, Equidae, poultry, pigs, dogs, and cats. In some embodiments, the non-ruminant is selected from the group consisting of Equidae, poultry, and pigs.

[0343] In some embodiments, the animal is a zoo animal.

[0344] In some embodiments, the animal is a poultry animal. In some embodiments, the poultry animal is a bird (i.e., fowl) used as a food animal, including, but not limited to, chickens, geese, ducks, quails, turkeys, pigeons, emus, or ostriches. In some embodiments, the poultry animal is selected from the group consisting of chickens, geese, ducks, quails, turkeys, or pigeons. In some embodiments, the poultry animal is selected from the group consisting of broilers, broiler-breeder chickens, and laying hens. In some embodiments, the poultry animal is a chicken.

[0345] In some embodiments, the animal is Equidae. In some embodiments, Equidae is a horse, pony, donkey, or mule.

Examples

[0346] Reference is now made to the following examples, which, together with the above description, illustrate some embodiments of the invention in a non-limiting manner. (Example 1) Effect of temperature on the yield of liquid culture of M. elsdenii

[0347] At 0, 7, 14, 21 and 28 days, storage temperatures of 4 °C, 20 °C and 39 °C were tested to evaluate the viability of cells in a liquid culture of M. elsdenii NCIMB41125 (Lactipro®). The results revealed that storing the product at 4 °C led to a substantial improvement in the viability of the culture (P < 0.001) compared to storage at 20 °C or 39 °C, regardless of the sampling day. After 28 days, the product stored at 4 °C had 3.98×10 6 and 6.3×10 5 colony-forming units (CFU / mL), compared to 1.26×10 6 CFU / mL and 6.3×10CFU / mL for the products stored at 20 °C and 39 °C, respectively (P < 0.01; Figure 1). Thus, the results indicate that decreasing the storage temperature improves the viability of the liquid culture of M. elsdenii NCIMB41125.

[0348] Additional data from separate studies indicate that the yield of Megasphaera elsdenii NCIMB41125 (Lactipro advance®) in liquid culture decreases after storage at room temperature for 0, 7, 14, 21 and 28 days (Figure 2). (Example 2) Use of tangential flow filtration to concentrate cultures of M. elsdenii

[0349] Tangential flow filtration (TFF) was considered as a method for high-throughput concentration of large volumes of culture.

[0350] A pilot-scale TFF system (see Figure 3) was integrated into the production line of M. elsdenii, tested for 500 liter (L) production runs, and the pilot-scale system was evaluated at volume reduction levels of 70%, 80%, and 90%. A TFF module with one RC100 kDa membrane (channel height of 0.875 mm) was used. For each reduction level, three production runs were performed for a total of nine runs. Samples were collected for each run and analyzed for pH, optical density (OD), presence of aerobic contaminants, osmolality, volatile fatty acid profile, M. elsdenii concentration, and growth characteristics. Samples were collected from nine M. elsdenii cultures ("non-freeze-dried") before the start of filtration, from the permeate (the "permeate"), which is the volume removed by the system, and from the retentate (the "retentate"), which is the concentrated M. elsdenii culture volume containing the cells remaining after volume reduction.

[0351] Permeate samples collected at the beginning, middle, and end of the concentration process had consistent OD readings across all volume reduction levels, all of which were less than 0.045 (data not shown). The amount of M. elsdenii recovered in the permeate increased over the concentration process (P = 0.0006), but was still negligible compared to the amount of cells collected in the retentate (< 3 × 10 4 CFU / mL (< 0.002%). The volume reduction level had no effect on the M. elsdenii concentrate recovered in the permeate (P > 0.9; Table 2).

Table 2

[0352] The yield of M. elsdenii in the holding solution did not differ among the bags collected at the beginning, middle, or end of each bagging process (P = 0.6088; data not shown). The yield of the holding solution was affected by the volume reduction level (P < 0.0001; Table 2 and Figure 4). The holding solution with 90% volume reduction had a higher yield than the holding solutions with 70% and 80% volume reduction (P < 0.0001). However, the 70% and 80% holding solutions did not differ from each other (P > 0.09).

[0353] The filtration process did not affect the ability of M. elsdenii cells to grow when inoculated back into SDL-20 medium (Table 3). The slope of the logarithmic phase was not affected by the volume reduction level (P > 0.3), but was affected by the sample type: "holding solution" vs. "non-freeze-dried" (P < 0.001). The time shift was affected by both the volume reduction level and the sample type (P < 0.001). The time shift for the holding solution decreased with increasing volume reduction, which represented a higher cell concentration.

Table 3

[0354] The holding solution obtained by TFF was used to test the freezing protocol, cryoprotectant content, and various freeze-drying parameters.

[0355] The holding solution was transferred to a sterilized and degassed serum bottle and aseptically mixed with various cryoprotectant formulations (w / v): no cryoprotectant (Ctrl), skim milk powder (SM), trehalose (T), and betaine. The holding solution mixed with the appropriate cryoprotectant was sampled to determine the concentration of M. elsdenii (i.e., viable count) before freeze-drying. The mixture was transferred to 10 mL vials (4 mL / vial) and snap-frozen in liquid nitrogen or slowly frozen at -80 °C overnight. Using either a slow or rapid cycle, the vials were transferred to a freeze-dryer to be freeze-dried. When freeze-drying was complete, the freeze-dried product in the anaerobic chamber was resuspended in an anaerobic diluent, rehydrated at room temperature for 40 minutes, and then plated on SDL20 agar to determine bacterial viability.

[0356] Cell loss was calculated by subtracting the concentration of M. elsdenii recovered after freeze-drying from the initial concentration of M. elsdenii measured in the corresponding holding solution with or without the cryoprotectant mixed. Using SAS® software, the cell loss data was calculated by analyzing the interaction among volume reduction levels (70%, 80%, or 90%), freeze-drying cycles (slow vs. rapid), freezing methods (-80 °C vs. liquid nitrogen), and cryoprotectants (none, betaine, trehalose, skim milk powder, maltodextrin, trehalose / skim milk powder (T / SM), and maltodextrin / skim milk powder (M / SM)).

[0357] Regardless of other criteria, the cell loss observed in the control treatment (no cryoprotectant) was 5 Log (CFU / mL) or higher. Similarly, regardless of other criteria, the cell loss observed in the betaine treatment was 3.96 Log CFU / mL or higher. The acceptable cell loss limit was set at 1.6 log CFU / mL. The holding solutions mixed with T / SM or M / SM, except for T / SM freeze-dried after freezing at -80 °C using either a slow or rapid cycle, were all below that threshold regardless of the freeze-drying cycle or freezing method used (Figure 5). B. Effect of freeze-drying conditions on storage

[0358] Megasphaera elsdenii NCIMB41125 was concentrated 10-fold using a filtration device and subjected to various characteristics: rapid or slow initial freezing (liquid nitrogen vs. -20 °C), with or without trehalose (0%, 4%, 7.5% and 10%), and gentle or rapid freeze-drying cycles (2 × 10 -6 Torr for 38 h vs. 135 × 10 -6 Torr for 16.5 h). A freeze-drying assay was performed. All the freeze-drying processes tested were able to maintain the viability of the product sufficient to initiate culture growth even after long-term storage at room temperature for 4 - 12 months after rehydration. Nevertheless, differences in bacterial viability depending on the freeze-drying characteristics used were observed (Figure 6). Slow freezing, 7.5% trehalose formulation and gentle freeze-drying steps that maintained the final water activity above 0.04 were associated with higher survival of Megasphaera elsdenii. C. Effect of cryoprotective substances on the viability of freeze-dried M. elsdenii

[0359] When centrifuged M. elsdenii NCIMB41125 cell concentrate was resuspended in infant formula powder before freeze-drying and the cells were subsequently rehydrated, the cell viability decreased by only 1 log. After testing the addition of 4% trehalose and 7.5% skim milk powder to the M. elsdenii concentrate, slow freezing at -80 °C or rapid freezing in liquid nitrogen were performed to determine the cell loss encountered during the initial freezing process. As shown in Figure 7, rapid freezing with 4% trehalose or 7.5% skim milk powder gave the highest recovery of viable cells (a decrease in viable cell count of 0.79 log). The product without cryoprotective substances lost Megasphaera elsdenii between 2.34 - 1.95 log CFU / mL regardless of the freezing technique used. (Example 4) Effect of storage conditions on the yield and stability of freeze-dried M. elsdenii

[0360] To determine the effect of freeze-drying protocols and storage conditions on the growth characteristics and shelf life of Megasphaera elsdenii NCIMB41125, the holding solution obtained from 90% volume reduction was mixed with 8% trehalose / 15% skim milk powder (T / SM) or 8% maltodextrin / 15% skim milk powder (M / SM) using rapid cycling, frozen at -80 °C or liquid nitrogen (LiqN), and freeze-dried. Next, using growth curve analysis and spread plate culture techniques, holding solution samples were tested for bacterial growth characteristics and cell survival during storage at 4 °C or 25 °C under aerobic or anaerobic conditions for 0, 2, 4, 8, 12, 16, 20, and 24 weeks. Briefly, the stored product was sampled, serially diluted, and plate cultured on SDL agar plates. Additionally, the rehydrated freeze-dried product was inoculated (1:100) into growth medium (SDL), and the optical density (OD, 600 nm) was recorded until the culture reached the stationary phase. The experiment was repeated on three different days, and all treatments were performed in triplicate. Undiluted, the absorbance exceeded the limit due to the presence of skim milk powder, so the rehydrated freeze-dried samples were diluted to generate growth curves. The same dilution was performed on the non-freeze-dried samples used as controls to facilitate comparison of the growth curves.

[0361] Results: Samples were freeze-dried and stored at room temperature or 4 °C under aerobic or anaerobic conditions for 6 months. Regardless of treatment, samples stored under aerobic conditions rapidly disintegrated with further cell loss after only 2 weeks of storage, compared to their anaerobic equivalents in the range of 0.4 - 3.2 Log. Based on these results, to improve the clarity of the drawings, only the cell loss observed in the freeze-dried products stored anaerobically is presented in Fig. 8. During storage under anaerobic conditions, samples stored at room temperature disintegrated faster than their equivalents stored at 4 °C, except for the T / SM samples frozen in liquid nitrogen. The T / SM samples frozen in liquid nitrogen and stored at room temperature after freeze-drying did not lose statistically more cells than their equivalents stored at 4 °C over a 16-week storage period (P > 0.1). However, after 20 weeks of storage, the difference between samples stored at room temperature and 4 °C became significant (P = 0.0002), with a difference of 0.84 log after 20 weeks and 0.89 log after 24 weeks of storage. All M / SM samples disintegrated faster than their T / SM equivalents. After 24 weeks of storage (Fig. 9), the cell loss of the T / SM samples frozen in liquid nitrogen and stored at 4 °C under anaerobic conditions was significantly lower than any of the other treatments (P < 0.02). The T / SM samples frozen in liquid nitrogen and stored at 4 °C under anaerobic conditions had a loss of 2.16 log compared to the M. elsdenii concentration observed before freeze-drying, with 0.82 log loss arising from the 24-week storage period. Growth curve experiments were performed to compare the growth characteristics of freeze-dried and non-freeze-dried products on each sampling day. Fig. 10 shows the growth curves prepared for samples frozen in liquid nitrogen, freeze-dried by rapid cycling (18.5 h at 250 mTorr), and stored anaerobically at 4 °C or room temperature. The non-freeze-dried samples used for each growth curve were "new" (less than 2 days old). The freeze-dried products stored at 4 °C had a shorter lag time than those stored at room temperature, which is consistent with the difference in M. elsdenii concentration observed in these samples (Table 4). The T / SM samples had a shorter lag time than the M / SM samples after 16 weeks of storage, regardless of the storage temperature.

Table 4

[0362] The slopes of the non-freeze-dried and MSM-treated samples stored at 25°C for 12 weeks and the MSM-treated samples stored at 20°C for 16 weeks were abnormally low (Table 5). After 20 and 24 weeks of storage, the slopes of the logarithmic phases of the freeze-dried samples were numerically not different from those of the non-freeze-dried controls.

Table 5

[0363] Fifty-six days before the start of the experiment, vials to be used in this study as pre-rehydrated M. elsdenii cultures (for rehydration and top-dress treatments) were prepared and stored in a refrigerator at 4°C while awaiting use. Each vial contained the equivalent of 5 mL of freeze-dried product. The vials were freeze-dried in six different freeze-drying runs. For each run, three vials were rehydrated after freeze-drying and three additional vials were rehydrated on the day of the study to determine the M. elsdenii concentration per vial (Table 6). The contents of a vial equivalent to 1.84×10 10 CFU of M. elsdenii were administered to each animal in the rehydration and top-dress groups by drench.

Table 6

[0364] After the start of the experiment, the extra vials from Runs #5 and #6 were maintained at 4°C and three vials from each of these runs were rehydrated monthly (Figure 11).

[0365] The M. elsdenii concentration in the vials was 4.96×10 during storage at 4°C for 11 months10 Changed from CFU / g to 4.17×10 10 CFU / g, showing a yield loss of only 0.07 log.

[0366] Approximately 30 days before the start of the experiment, the product (top-dress treatment) used in this study as the daily top-dress M. elsdenii culture was prepared and stored in a refrigerator in a polyfoil bag. The top-dress product was freeze-dried in six different freeze-drying runs, packaged in 15 individual polyfoil bags (1 bag / day for the feed), flushed with nitrogen, sealed, and stored at -80 °C until use. For each freeze-drying run, three packages were rehydrated after freeze-drying. The average M. elsdenii concentration was 4×10 10 CFU / package. Three additional packages were rehydrated at the start of each week and used to determine the M. elsdenii concentration given to the animals daily by adding the rehydration solution to the M. elsdenii cells, leaving the cells for 40 minutes, diluting the cells, and plating the cells (Figure 12). Statistical analysis showed an increase in cell concentration over time. All products were prepared 30 days before the start of the study, and the product used as the top-dress was approximately 80 days old at the end of the study. The animals received an average of 2.19×10 8 CFU of M. elsdenii daily (2.45×10 10 CFU / package). Study in B. bovis

[0367] Castrated cattle (n = 462; initial weight 900 lbs) were blocked by weight into a control group that did not receive Megasphaera elsdenii (17 pens; 7 animals / pen); a non-freeze-dried group that received 50 mL of an M. elsdenii product containing 2×10 8 CFU / mL (10 10 CFU of Megasphaera elsdenii NCIMB41125) (16 pens; 7 animals / pen); a freeze-dried culture that was rehydrated immediately before administration (10 10The rehydrated group (16 barns; 7 animals / barn) received Megasphaera elsdenii NCIMB41125) in CFU; and the lyophilized culture rehydrated prior to and immediately before administration (10 10 CFU of Megasphaera elsdenii NCIMB41125), and the lyophilized product (10 8 CFU of Megasphaera elsdenii NCIMB41125, daily; 17 barns; 7 animals / barn) were randomly assigned to one of four treatments consisting of the top-dress group. The non-freeze-dried group, rehydrated group and top-dress group of castrated cattle were put into a 10-day step-up program after receiving a drencher of Megasphaera elsdenii in advance, while the control group was put into a 21-day step-up program. Twenty-six hours after the first ration was given, rumen fluid was extracted by rumen puncture. Samples of rumen fluid were analyzed for pH, volatile fatty acid concentration, endotoxin level and the availability of lactic acid in the in vitro disappearance assay. For the lactate disappearance assay, 0.1 mL of rumen fluid was inoculated into 10 10-mL tubes containing semi-defined lactate medium. Optical density was measured in duplicate every 6 hours for 24 hours to evaluate the ability to grow with lactic acid as the primary substrate. To continuously determine the lactic acid concentration, the tubes were placed in the freezer immediately after the optical density was observed. One castrated cattle per barn was fitted with a continuous high-frequency bolus to track the pH measurement over a 56-day study. The castrated cattle were weighed on days 0, 28 and 56 to determine the average daily weight gain, dry matter intake and feed efficiency.

[0368] To evaluate viable M. elsdenii ingested by animals, freeze-dried product was top-dressed onto sterilized ground corn in an aluminum pot, and samples were collected after 0, 1, 2, and 4 hours of exposure to the ambient atmosphere in the laboratory or outdoors in the sun. This experiment was repeated four times from July to August 2016 (Figure 13). Statistical analysis revealed an interaction between exposure time and storage conditions (P = 0.0007), an effect of exposure time (P < 0.0001), and an effect of storage conditions (P < 0.0001). The concentration of M. elsdenii in the freeze-dried product mixed with ground corn and exposed to the atmosphere in the laboratory decreased numerically during the 4-hour exposure, but there was no significant difference from its initial concentration. The concentration of M. elsdenii in the samples exposed to outdoor conditions decreased much faster. Only 6.4% of the initially present M. elsdenii was recovered after 1 hour of exposure. The concentration of M. elsdenii in the samples exposed to outdoor conditions was significantly different from the initial concentration and their counterparts maintained at room temperature after 2 and 4 hours of exposure. The concentration of M. elsdenii in these samples varied greatly from experiment to experiment, as indicated by a large standard deviation. This could be due to differences in outdoor conditions (heat and humidity), but could also be due to differences in the freeze-dried products used, which may have some tolerance to heat and humidity. During the animal study, immediately before feeding, the freeze-dried product was mixed with ground corn and then top-dressed promptly when the samples were placed in the feed trough. The animals pounced on the ground corn, and as a result, the freeze-dried product was likely consumed within less than 1 hour after mixing. Assuming the product was consumed within 1 hour, the animals should have received approximately 1.4×10 7 CFU / head / day of viable cells (average concentration of 6.4% in the top-dressed product of 2.2×10 8 CFU / head / day).

[0369] Accelerated step - up of castrated cattle treated with M. elsdenii resulted in similar 28 - day body weights (P = 0.53; Table 7), average daily weight gain (P = 0.71), and feed efficiency (P = 0.69). The top - dress treatment had lower dry - matter intake than the control (P = 0.05). Feedlot performance over the first 56 days of feeding resulted in similar outcomes among treatments (P>0.10). For the control receiving a conservative 21 - day step - up period, these similarities among treatments indicate that an accelerated step - up program using any of the three forms of M. elsdenii treatment can be implemented without adversely affecting cattle health or feedlot performance.

[0370] The pH of the rumen fluid (extracted 26 hours after the first diet was given) was similar among treatments (P>0.10; Table 8). There was a difference in total VFA concentration, and the top - dress treatment was significantly higher than the other treatments (P<0.01). The top - dress treatment had a higher acetate concentration than the re - hydration treatment (P = 0.02). Although there were significant differences in acetate concentration, the acetate:propionate ratio was similar among treatments (P = 0.96). The other VFAs were similar among treatments (P>0.18).

[0371] The endotoxin concentration in the rumen fluid (extracted 26 hours after the first diet was given) was similar among treatments (P = 0.3462; Table 14). Endotoxin levels were measured as an indicator of bacterial lysis in the rumen. Animals suffering from acidosis have been reported to have endotoxin levels exceeding 150,000 EU / mL. All endotoxin levels measured in this study were below that threshold, regardless of treatment.

[0372] One castrated bull in each of 32 barns was equipped with an indwelling rumen pH probe (8 castrated bulls / treatment) that reported rumen pH measurements every hour over a 56-day study (Figure 15). Mean rumen pH measurements for each treatment are reported in Figure 15 at 1-hour intervals. An interaction of treatment by day (P<0.01) was detected for rumen pH. There were also effects of treatment (P<0.01) and feeding day (P<0.01) on rumen pH. With the accelerated step-up of the M. elsdenii treatment, the mean rumen pH did not decline to a state of clinical acidosis. As the number of feeding days increased, the freeze-dried treatment (rehydrate and top dress) maintained a higher rumen pH than the control and non-freeze-dried treatments.

Table 7

Table 8

[0373] Optical density measurements were observed at 6-hour intervals over a 24-hour period to determine the growth curves of mixed rumen microorganisms inoculated into semi-defined lactate medium, and these data are presented in Figure 16. There was an interaction between treatment and detection time (P<0.02). Individual effects of treatment (P<0.01) and time (P<0.01) were also found. Differences between treatments were not detected until 12 hours when rehydrated freeze-dried was higher than daily freeze-dried (P<0.02) and control (P = 0.007). At 24 hours, there was no difference between M. elsdenii treatments (P>0.10), but the control treatment had significantly lower microbial growth than all of the M. elsdenii treatments (P<0.01).

[0374] Figure 17 illustrates the disappearance of L-lactate in semi-defined lactate medium inoculated with rumen microbes and incubated for 0, 6, 12, 18 or 24 hours. An interaction between treatment and time was detected (P = 0.007) along with the effects of treatment (P = 0.01) and incubation time (P < 0.0001). The non-freeze-dried treatment contained lower L-lactate at time 0 than the other treatments (P = 0.04). The concentration of L-lactate was similar between treatments (P > 0.10) across the 6, 12 and 18 hour time points. Similar to the optical density results above, rumen microbes from control castrated cattle utilized less lactate in total during 24 hours of incubation compared to the M. elsdenii treatment (P < 0.003), while no differences were detected between M. elsdenii treatments (P > 0.10). These data illustrate that rumen microbes from castrated cattle treated with M. elsdenii grew more efficiently in semi-defined lactate medium than microbes from control castrated cattle, suggesting higher lactate utilization ability in these treatments. Furthermore, the Megasphaera treatment was similar with respect to L-lactate utilization ability.

[0375] For the samples used in the optical density and lactate disappearance assays, VFA concentrations were measured and presented in Table 9. An interaction of treatment by time was detected for total VFA (P = 0.002), acetate (P = 0.0002), isobutyrate (P = 0.04), butyrate (P < 0.0001), isovalerate (P = 0.0007) and valerate (P < 0.0001) concentrations, and for the acetate:propionate ratio (P = 0.02). An effect of time was found for total VFA and all individual VFAs (P < 0.0001). Differences between treatments were found for isobutyrate (P = 0.02), butyrate (P = 0.0004), and valerate (P = 0.001). The concentration of isobutyrate at 18 h was lower in the non-freeze-dried and top-dress treatment groups (P < 0.005 and P < 0.004, respectively) compared to the control and rehydration. At the 24 h time point, top-dress had less isobutyrate than the non-freeze-dried and rehydration treatments (P = 0.002), but was similar to the control (P > 0.10). The butyrate concentration of the rehydrated samples was higher than that of the top-dress after 18 h of incubation (P = 0.02). The control treatment produced less butyrate at 24 h than the M. elsdenii treatments (P < 0.0001). The difference in butyrate was also detected between the Megasphaera treatments at this time point for non-freeze-dried samples containing higher concentrations than the rehydration treatment (P = 0.01). By 18 h, it was revealed that the valerate concentration was lower for rehydration compared to the other treatments (P = 0.01). Similar observations were made for the concentrations of valerate and butyrate at 24 h of incubation. The control contained less valerate than all M. elsdenii treatments (P < 0.0001), while the concentration was higher in non-freeze-dried samples than during rehydration (P = 0.03). For the other VFAs, no main effect of treatment was detected (P > 0.05). Trace concentrations of isocaproate, caproate and heptanoate were measured.A treatment×time interaction (P = 0.02) was found for the acetate:propionate ratio and the effect of time (P>0.0001), although it was similar between treatments (P = 0.57).

Table 9-1

Table 9-2

[0376] The in vitro phytase activity of M. elsdenii cells was evaluated.

[0377] Megasphaera elsdenii NCIMB41125 cells were cultured under anaerobic conditions as described herein in semi-defined lactate medium containing either inorganic phosphate (KH2PO4) or phytate as a phosphorus source. Samples of 1 milliliter (mL) were removed from the culture at 2-hour intervals from the start of the culture (0 hours) to 8 hours and centrifuged to form a cell pellet. Phytase activity in the cell pellet was then determined using the molybdenum blue method. See Yanke et al., Microbiol. 144: 1565-1573 (1998). Briefly, inorganic phosphate released by enzymatic cleavage in an incubation at pH 5.0, 37 °C for up to 30 minutes was quantified by spectrophotometry at 700 nanometers (nm) and compared to a standard curve. Phytase activity was determined as the amount of inorganic phosphorus released from the cell pellet per minute under the test conditions.

[0378] Figure 18 shows that the growth of Megasphaera elsdenii cells results in significant phytase activity either in the presence or absence of phytate, which supports that (1) Megasphaera elsdenii NCIMB41125 cells produce phytase, (2) phytase production is not inhibited by the presence of phosphorus in the medium, and (3) phytase production by Megasphaera elsdenii appears to be higher in the presence of phytate in the medium. (Example 7) Effect of Megasphaera elsdenii on Salmonella concentration and morbidity

[0379] Day-old broiler chicks (n = 384) were randomly assigned to four different treatment groups: 1) a control group not receiving Megasphaera, 2) a free group with free access to a bottle feeder containing liquid Megasphaera elsdenii NCIMB41125, 3) a freeze-dried group receiving freeze-dried Megasphaera elsdenii NCIMB41125 daily in the feed, and 4) an oral gavage group receiving liquid Megasphaera elsdenii NCIMB41125 on day 0.

[0380] Each of the four treatments was represented by 16 cages containing six birds each. Animal weight, feed consumption, and feed conversion were recorded over a 15-day test period. After a 15-day feeding period, two animals per cage were randomly selected, sacrificed, and the ceca were recovered to determine Salmonella prevalence. Briefly, the ceca were removed, placed in Ziploc bags, and stored on ice. The ceca were then washed with 70% ethanol, kneaded by hand, and the contents were extracted. One milliliter of the recovered contents was serially diluted in phosphate-buffered saline (PBS) and plated onto brilliant green agar (BGA). The BGA plates were incubated at 37 °C for 24 h. Presumptive Salmonella colonies (pink colonies) were counted and confirmed as Salmonella using the Oxoid Salmonella latex test FT0203 (Oxoid-Thermo Scientific, Hampshire, UK). Additionally, 1 milliliter of the cecal content sample was added to 9 mL of Rappaport-Vassiliadis (RV) for selective enrichment. If no detectable Salmonella growth was observed on the BGA plates, the RV enrichment was plated onto BGA plates and incubated at 37 °C for 24 h to assess the presence of Salmonella. Samples that showed no growth when plated directly but positive growth when RV enriched were counted as arbitrarily 9 (less than 1 is the theoretical detection limit), and samples that showed no growth in either direct plating or RV enrichment were counted as 0.

[0381] The results showed that birds receiving the freeze-dried material had a lower cecal Salmonella concentration in colony-forming units per milliliter (CFU / mL) with a -1 Log difference compared to the control group. See Figure 19. The Salmonella prevalence in the samples of the freeze-dried group also decreased by 13% when compared to the control group. See Figure 20. (Example 8) Effect of Megasphaera elsdenii on the growth performance and cecal characteristics of broiler chickens A. Experimental design and treatment

[0382] Male chicks at the age of one day were used, and 24 replicates of three treatments in a randomized complete block design were conducted using Cobb 500 broiler chicks that were one day old at the start of the treatment (Cobb-Vantress, Siloam Springs, Arkansas). The treatments were either oral force-feeding or M. elsdenii strain NCIMB41125 (MS-Biotec, Wamego, Kansas) administered as an aerosolized mist applied to the body surface of the chicks and the control (without M. elsdenii). The chicks were housed in 72 pens, and each pen contained 35 chicks at the start of the experiment (2,520 chicks in total).

[0383] Before the administration of M. elsdenii, a 5L foil bag of the new culture was shaken vigorously to homogenize the contents. A manually operated dosing device was connected to the bag using a Tygon tube. The reservoir of the dosing device was filled and dispensed several times and air was exhausted. When the culture containing the oxygen indicator maintained its normal color tone, the contents were considered to be free of ambient air.

[0384] The chicks were assigned to 35 groups and the weight of each group was recorded. The groups of chicks were treated block by block, and the experimental treatments were randomly assigned within each block.

[0385] Using a Scorex Classic 173.05005 automatic filling syringe (Ecublens, Switzerland), 0.2 mL of a new culture containing 1.97×10 9 CFU / mL of M. elsdenii strain NCIMB41125 was administered by oral gavage to 24 pens (35 chicks / pen; 840 chicks in total). The technician held the chicks down using the thumb and index finger and directly inserted the contents of the syringe into the oral cavity of the chicks while keeping the beak open.

[0386] Applied by an air pressure type drain cleaner equipped with a spray tip, aerosolized mist of a new culture containing 1.97×10 9 CFU / mL of M. elsdenii strain NCIMB41125 was administered to 24 poultry houses (35 birds / poultry house; a total of 840 birds). The birds were placed in plastic barrels (50 cm × 35 cm × 40 cm), and the culture was applied to the body surface of the birds as an atomized mist at a rate of 60 mL per poultry house (about 1.7 mL / bird).

[0387] Twenty-four poultry houses (35 birds / poultry house; a total of 840 birds) were not exposed to M. elsdenii and served as controls. To prevent cross-contamination with the treated birds, the control birds were handled only by a designated manager who did not come into contact with the treated birds, placed on a designated loading platform, weighed, and transferred to the poultry house. In one instance, there was a miscount of the birds, and poultry house 51 contained 33 birds instead of 35 due to an error by the technician. B. Feeding and watering

[0388] Fresh water was provided ad libitum through straws (6 straws / poultry house) suspended from the water supply line. The height of the straws was adjusted throughout the study to accommodate the growth of the birds. Table 10 below shows the diets used in the experiment. All diets were provided using gravity feeders suspended in the center of the poultry house. Feed was added as needed to ensure free access throughout the study period. Before placing the birds in the poultry house, 5 kg of starter diet was placed in the gravity feeder.

Table 10

[0389] The starter diet was removed from the poultry house on day 16 of the study. The remaining feed was weighed, removed from each feeder, and placed in bins numbered corresponding to the poultry house number. The feeders were replenished with grower diet. This process was repeated on day 30 of the study, at which point the grower diet was replaced with the finisher diet. On day 36, the experiment was terminated, and the remaining finisher feed was weighed and recorded for each poultry house.

[0390] The total feed consumption per barn for each period (starter, grower, and finishing) was calculated as the amount of feed added - the amount of feed recovered.

[0391] The intake per bird per day was calculated as the total amount of feed consumed ÷ [number of birds per day in the barn × total number of feeding days]. C. Bird weight

[0392] The barn weight was recorded at the end of each feeding period (starter, grower, finishing). At the end of the starter period (day 16), all the birds in each barn were placed in buckets (50 cm × 35 cm × 40 cm) and weighed. The weight of the buckets was subtracted from the total weight to determine the weight of the birds in the barn. At the end of the grower period (day 30), all the birds in each barn were placed in two equally weighted buckets (each 103 cm × 55 cm × 41 cm), weighed, and the weights were added together. The weight of each bucket (weighed before placing the birds in the bucket) was subtracted from the total weight to determine the weight of the birds in the barn. At the end of the finishing period (day 36), all the birds in each barn were placed in two equally weighted buckets (each 103 cm × 55 cm × 41 cm) and weighed. The tare was measured on the scale at an appropriate location for this machine. Then, the weights of the birds in each bucket were added together to determine the total barn weight. To compensate for the accumulation of manure, the tare between the barns was measured again on the scale. At each weighing period, when the birds were placed in the buckets, the number of birds was confirmed. D. Sampling procedure

[0393] At each week (days 7, 14, 21, 28, and 35), 1 - 3 birds were randomly selected from each pen and euthanized by cervical dislocation. Cecal contents (0.5 g) were collected and mixed with deionized water (2 mL) in a 20 mL HDPE scintillation vial (Fisher Sci.; 03 - 337 - 23B) using a vortex mixer. The pH was determined using a portable pH meter (Thermo Scientific Orion 3 - star portable pH meter, Waltham, MA). Four parts of the cecal mixture were added to one part of 25% w / v metaphosphoric acid solution and homogenized using a vortex mixer. The sample was then transferred in 1 mL aliquots to two microcentrifuge tubes and frozen at - 18 °C pending analysis of volatile fatty acids (VFA).

[0394] On days 7 and 21, the cecal contents were divided into two aliquots. One aliquot was used for VFA analysis and prepared as described above. The other (0.5 g) was placed directly into individual 20 mL HDPE scintillation vials (Fisher Sci.; 03 - 337 - 23B) and frozen (-80 °C) for quantification of bacterial counts using quantitative real - time PCR. E. Analysis of cecal pH and volatile fatty acids

[0395] Previously diluted and acidified cecal samples were thawed, homogenized using a vortex mixer, and centrifuged at 24 x g for 18 minutes. The aqueous supernatant was transferred to a vial for gas chromatography. Volatile fatty acids were measured using an Agilent 7890 gas chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a DB-WAX capillary column (30 m x 0.53 mm x 0.5 mm film thickness; Sigma Aldrich, St. Louis, MO) and a flame ionization detector. Helium was used as the carrier gas at a flow rate of 22 cm / s, with a 1 μL split injection and a split flow of 50:1. The initial oven temperature was 80 °C, and the temperature was raised to 220 °C at a rate of 10 °C / min. The inlet and detector temperatures were 250 °C. Volatile fatty acids were quantified by comparison with a known standard (Supelco Volatile Fatty Acid Standard Mix; Sigma-Aldrich, St. Louis, MO) containing acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, and heptanoate. F. Breast meat measurement

[0396] Chickens were slaughtered at 5 weeks of age to determine breast meat measurements. Feeding was stopped approximately 4 hours before slaughter. Five average-sized chickens were selected from each poultry house and placed in capture boxes for transportation to the processing area. The five chickens were weighed per poultry house to determine the live weight, and immediately thereafter, they were slaughtered by cervical dislocation followed by whole blood collection. The chickens were bled for 2 minutes and then placed in a rotary boiling water bath at 63 °C for approximately 30 seconds. To remove the feathers, the chickens were transferred to a rotary drum mechanical plucker for 30 seconds. The legs, head, and backbone were removed, and the breast meat viscera were removed from the incision around the anus. The breast meat was then weighed per poultry house to determine the warm breast meat yield. G. Statistical analysis

[0397] Data were analyzed using the complex procedure of SAS (registered trademark) software version 9.4. The model included fixed effects of treatment, variable effects of block, and the poultry house as the experimental unit. Significance was indicated by P < 0.05. Differences between least squares means were determined using the PDiff option of SAS (registered trademark) software. H. Results

[0398] Broilers demonstrated similar feed intakes, feed efficiencies, and average daily weight gains among treatments. Live weight and mortality were also not affected by treatment. However, broilers receiving M. elsdenii via oral gavage had less breast meat than those receiving M. elsdenii as a control or as an aerosolized mist.

[0399] Cecal pH was lower in broilers receiving M. elsdenii either by mist or oral administration compared to controls (P < 0.01; Table 11).

Table 11-1

Table 11-2

[0400] The mean cecal pH for the treatments of control, aerosolized mist, and oral gavage were 6.76, 6.63, and 6.60, respectively. Treatment-by-day interactions were detected for cecal acetate (P<0.01), propionate (P=0.03), butyrate (P<0.01), acetate:propionate ratio (P=0.01; A:P ratio), caproate (P=0.002), and total VFA (P<0.01) concentrations (Table 4). Acetate increased from day 7 to day 14 and peaked on day 14. Cecal contents of birds receiving M. elsdenii as oral gavage contained higher acetate, butyrate, and caproate concentrations than those of control birds on day 14 (P<0.01). By day 21, acetate concentration decreased across all treatments, but the concentration of acetate in the cecum was higher in control birds compared to those treated by either aerosolized mist or oral gavage (P<0.01). Propionate and butyrate concentrations were also higher in the cecal contents of control birds than those of birds treated with M. elsdenii on day 21 (P<0.01). Propionate concentration peaked on day 21 across all treatments and continued to increase until day 35, but there were no differences across treatments from day 28 to day 35 (P>0.05). The A:P ratio was greater in the cecal contents of birds treated with M. elsdenii compared to control on day 7, and the A:P ratios were 31.96, 41.33, and 42.03 for control, aerosolized mist, and oral gavage, respectively. The A:P ratio of the cecum of birds treated with aerosolized mist of M. elsdenii (40.44 mM) on day 14 was greater than that of control birds (29.80 mM) or birds receiving oral gavage of M. elsdenii (33.69; P<0.03). There were no differences in the A:P ratio of the cecum across treatments from day 21 to day 35 (P>0.05). Concentrations of isobutyrate, valerate, isovalerate, isocaproate, and heptanoate in the cecal contents were not affected by treatment (P>0.10).The total VFA concentration in the ceca was greater in the force-fed chickens than in the control chickens on day 14 (P<0.001). However, the total VFA concentration was lower in the cecal contents of chickens receiving M. elsdenii as an aerosol mist or by force-feeding (64.90 mM and 64.82 mM, respectively) than in the control (87.57 mM) on day 21 (P<0.05). The total VFA concentration in the ceca was similar over the 7-day, 28-day, and 35-day treatments (P>0.30). (Example 9) Effect of Megasphaera elsdenii on the growth performance of broiler chickens A. Experimental design and treatment - Study 1

[0401] Six treatments with 18 replicates each were conducted using 1-day-old Cobb 500 broiler chicks (Cobb-Vantress, Siloam Springs, Arkansas) at the start of treatment, blocked by battery and by pen. Treatments were control (no probiotic), Lactipro Advance® (non-freeze-dried liquid culture of Megasphaera elsdenii strain NCIMB 41125, MS Biotec, Wamego, Kansas) administered by force-feeding, Megasphaera elsdenii strain KS249 culture administered by force-feeding, Megasphaera elsdenii strain ATCC® 25940 administered by force-feeding, Lactipro Advance® (non-freeze-dried liquid culture of Megasphaera elsdenii strain NCIMB 41125, MS Biotec, Wamego, Kansas) applied to the body surface of the chickens as an aerosol, and freeze-dried Megasphaera elsdenii strain NCIMB 41125 (MS Biotec, Wamego, Kansas). The chickens were housed in 108 pens, each pen containing 8 chickens at the start of the experiment (1,152 chickens total).

[0402] Eight groups of chickens were counted and the weight of each group was recorded. The groups of chickens were processed by block, and the experimental treatments were randomly assigned to pens within each block.

[0403] Eighteen pens (8 chickens / pen; 144 chickens total) were orally gavaged (force-fed) with 0.2 mL of Lactipro Advance® containing 1.97×10 9 CFU / mL of the non-freeze-dried liquid culture of Megasphaera elsdenii strain NCIMB41125. Eighteen pens (8 chickens / pen; 144 chickens total) were orally gavaged (force-fed) with 0.2 mL of a new culture containing an unknown concentration of Megasphaera elsdenii strain KS249. Attempts to evaluate the CFU / mL for this strain were unsuccessful. Eighteen pens (8 chickens / pen; 144 chickens total) were orally gavaged (force-fed) with 0.2 mL of a new culture containing 1.06×10 9 CFU / mL of Megasphaera elsdenii strain ATCC® 25940. All orally treated chickens were held in the palm of the technician's hand, and the beak was held open using the thumb and index finger, and the culture was directly introduced into the oral cavity using an Eppendorf® Reference repeater pipette (Hamburg, Germany).

[0404] Eighteen pens (8 chickens / pen; 144 chickens total) were misted with 15 mL per pen of Lactipro Advance® containing 1.97×10 9 CFU / mL of Megasphaera elsdenii strain NCIMB41125 (at approximately 1.88 mL / chicken). The chickens were placed in plastic tubs (50 cm long × 35 cm wide × 40 cm deep), and the culture was applied to the body surface of the chickens as an atomized mist using a pneumatic drenching device equipped with a spray tip. To minimize cross-contamination, the misted chickens were handled by designated personnel and placed on a designated platform for weighing, application, and transfer to the pens.

[0405] Eighteen pens (8 birds / pen; 144 birds total) were treated with a top dressing (a mixture of feed and freeze-dried Megasphaera elsdenii) containing 1.18×10 7 CFU / g of Megasphaera elsdenii strain NCIMB41125 at a rate of one quarter of a teaspoon per bird. Treatments were initiated on day 10 of the study and added daily at 1300 h directly to the trough feeders.

[0406] The remaining 18 pens (8 birds / pen; 144 birds total) served as controls and had no contact with the probiotic product. To minimize cross-contamination by the treated birds, control birds were handled by designated personnel and placed on a designated truck for weighing and transfer to the pens. B. Experimental Design and Treatments - Study 2

[0407] Day-old Cobb 500 broiler chicks (Cobb-Vantress, Siloam Springs, Arkansas) were used for 18 replications of two treatments blocked by battery and by stage. Treatments were control (no probiotic) or freeze-dried Megasphaera elsdenii strain NCIMB41125 (MS Biotec, Wamego, Kansas). Birds were housed in 108 pens, each pen containing 8 birds at the start of the experiment (1,152 birds total).

[0408] Birds were counted into eight groups and the weight of each group was recorded. Birds were processed by block and experimental treatments were randomly assigned to pens within each block. Eighteen pens (8 birds / pen; 144 birds total) were treated with a top dressing (a mixture of feed and freeze-dried Megasphaera elsdenii) containing 1.18×10 7 CFU / g of Megasphaera elsdenii strain NCIMB41125 at a rate of one quarter of a teaspoon per bird. Treatments were initiated on day 10 of the study and added daily at 1300 h directly to the trough feeders.

[0409] The remaining 18 pens (8 birds / pen; 144 birds total) served as controls and had no contact with the probiotic product. To minimize cross-contamination by the treated birds, the control birds were handled by designated personnel and placed on a designated platform for weighing and transfer to the pens. C. Feeding and Watering - Studies 1 and 2

[0410] Fresh water was available ad libitum. Before placing the birds in the pens, 9.5 kg of a common starter diet (Table 12) was placed in trough feeders along each pen. [Table 12]

[0411] Feed was replenished as needed to ensure free access throughout the study. At the end of the experiment (day 18), the unconsumed feed was removed from each feeder, weighed, and recorded. The total feed consumed per pen was calculated as the difference between the amount added to the feeder and the amount recovered from the feeder. The daily feed intake per bird was calculated as the total amount of feed consumed ÷ [number of birds per day in the pen × number of days fed]. D. Bird Weight - Studies 1 and 2

[0412] At the end of the study, all the birds in the pens were placed in buckets (50 cm length × 35 cm width × 40 cm depth) and weighed. The weight of the bucket was measured before placing the birds in it and subtracted from the total weight to determine the weight of the birds in the pen. The number of birds was also confirmed at this time. E. Statistical Analysis - Studies 1 and 2

[0413] Data were analyzed using the Mixed procedure of SAS® Software 9.4. The model included the fixed effect of treatment, the variable effect of block, and the pen as the experimental unit. Significance was indicated by P < 0.05. Differences between least squares means were determined using the PDiff option of SAS® Software. F. Results - Studies 1 and 2

[0414] Regarding Study 1, broilers across all treatment groups showed similar daily feed intakes, average daily weight gains, weight gain:feed ratio, and mortality rates.

[0415] However, as shown in Table 13, Study 2 indicated that both the average daily weight gain (P = 0.02) and weight gain:feed ratio (P = 0.04) were greater in birds receiving freeze - dried Megasphaera elsdenii compared to control birds. See Figure 21 showing the feed:weight gain ratio. Feed intakes and mortality rates did not differ among treatment groups.

Table 13

[0416] Eight quarter - horses, four mares and four geldings (average weight = 540 kg; SEM = 75 kg) with pre - installed cecal cannulas (Beard et al., JAS, Vol. 89(8):2425 - 2429 (2011)) were used in a 3×3 (treatment×horse) incomplete Latin square design repeated over three treatment periods. Each treatment period was separated by a 28 - day wash - out period. Treatments were: (1) negative control (without M. elsdenii; control), (2) 50 mL of fresh culture containing 1.97×10 9 CFU / mL of M. elsdenii strain NCIMB41125 (Lactipro Advance®, MS Biotec, Wamego, Kansas) administered by an oral drench, and (3) 7.02×10 8It was a 0.40 g freeze-dried culture containing M. elsdenii strain NCIMB41125 (MS Biotec, Wamego, Kansas) at CFU / mL. Horses were randomly assigned to treatment (Table 14).

Table 14

[0417] Horses were housed in individual stalls (3.05 × 3.66 m) within one shed and the shed was lined with pine shavings. To compensate for possible ventilation or temperature variations based on shed location, horses were randomly assigned to different stalls for each treatment period. Horses were walked daily for exercise during the treatment period.

[0418] For horses receiving the oral drencher, immediately prior to feeding on day 1 of each treatment period, a manual dosing device (60 mL variable automatic drench MKIII, NJ Phillips, NSW, Australia) was used to dose 50 mL of a fresh culture containing 1.97×10 9 CFU / mL of M. elsdenii strain NCIMB41125. Prior to administration of the probiotic culture, the 5 L bag of the fresh culture was vigorously shaken to homogenize the contents. A manual dosing device was attached to the bag using a Tygon tube and the reservoir was filled. Approximately 100 - 200 mL of the culture was discarded into a waste container to ensure there was no oxygen in both the tube and the device.

[0419] Horses in the freeze-dried probiotic treatment group were given two corns and a molasses-based treat containing the freeze-dried product every morning before feeding. Prior to the study, M. elsdenii strain NCIMB41125 was freeze-dried, with each having an average of 7.02×10 8Packaged in vacuum-sealed sachets containing approximately 0.40 g of freeze-dried bacteria along with M. elsdenii at CFU / mL. One sample was plated daily to ensure consistent bacterial viability throughout each treatment period. If the horse rejected the treatment, the freeze-dried product was administered manually as a bolus.

[0420] The remaining horses had no exposure to the probiotic during the period that served as the control. B. Feeding and Watering

[0421] During the treatment period, the horses were fed hay and concentrate twice a day, evenly divided between the two feedings. Each horse was given 1% of its body weight per day as feed in brome hay (Table 15).

Table 15

[0422] Each horse was increased from 0.2% of its body weight per day from day 1 to day 5 to 1% of its body weight in processed concentrate (analyzed in Table 15 above, composition in Table 16 below), and then maintained at 1% BW AF in grain from day 5 to day 7. All refusals were weighed and recorded. The stables were equipped with automatic waterers to provide fresh water freely. The waterers were cleaned and checked several times a day to ensure proper functioning.

Table 16

[0423] During each 7-day treatment period, cecal samples were collected every 4 hours using a cecal cannula. Horses were fed daily at 1000 hours and 2200 hours, and after collecting samples at 4, 8, and 12 hours after feeding, the next feeding was performed. On day 0 of each treatment period, samples were collected before dosing or feeding to establish baseline values for pH, VFA, and M. elsdenii population in the hindgut.

[0424] Samples were collected by removing the cannula stopper and capturing cecal contents as the sample flowed out of the cannula. Cecal fluid was filtered through four layers of cheesecloth and then placed in a 100 mL test cup. If sufficient sample was not collected by gravity flow, a hand-held pump was used to extract cecal contents. At 1000 hours on days 0, 1, 3, and 7, additional cecal samples were collected for PCR analysis. During the first treatment period, unfiltered samples were collected in 20 mL HDPE scintillation vials (Fischer Sci.; 03-337-23B). Since these unfiltered samples presented challenges in separating the samples for DNA extraction, for the remaining two treatment periods, filtered cecal fluid was collected in 50 mL Falcon conical centrifuge tubes (Corning Inc. 352070; Corning, NY) and immediately frozen at -80°C pending PCR analysis. Specialists changed gloves between each horse. D. Analysis of Cecal pH and Volatile Fatty Acids

[0425] The pH of the filtered cecal fluid was measured immediately after collection using a portable pH meter (Thermo Scientific Orion 3 Star Portable pH Meter, Waltham, MA; Accumet probe). After recording the pH, the sample was transferred in 1 mL aliquots to two microcentrifuge tubes and mixed with 0.25 mL of 25% metaphosphoric acid for protein removal. The sample was frozen at -18°C for at least 24 hours before VFA analysis.

[0426] The acidified and frozen cecal samples were thawed, homogenized using a vortex mixer, and centrifuged at 24 x g for 18 minutes. The aqueous supernatant was then transferred to a vial for gas chromatography. Volatile fatty acids were measured using an Agilent 7890 gas chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a DB-WAX capillary column (10 mm x 0.10 mm x 0.1 mm film thickness; Agilent and J&W columns, Santa Clara, CA) and a flame ionization detector. Hydrogen was used as the carrier gas at a flow rate of 46 cm / sec, with a 1 μL split injection and a split flow of 50:1. The initial oven temperature was 70°C, which was increased to 130°C at 15°C / min and then to 220°C at 60°C / min and held for 2 minutes. The inlet and detector temperatures were 260°C and 300°C, respectively. Volatile fatty acids were quantified by comparison with a known standard (Supelco Volatile Fatty Acid Standard Mix; Sigma-Aldrich, St. Louis, MO) containing acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, and heptanoate. E. Statistical analysis

[0427] Data were analyzed using the Glimmix procedure of SAS® software version 9.4. The model included the fixed effect of treatment and the random effects of horse, period, and the interaction of treatment by period. Horses served as the experimental units. The treatment by hour within day effect was not significant for any parameter and was therefore excluded from the model. Significance was indicated by P < 0.05, and trends were considered at 0.05 < P < 0.10. Differences between least squares means were determined using the PDiff option of SAS® software. F. Results

[0428] The pH of the cecum tended to be greater in horses treated with M. elsdenii as the amount of grain included in the diet increased compared to the control. See Figure 22. The pH of the cecum of horses administered M. elsdenii as an oral drencher increased above the control on day 5, i.e., the first day sufficient allotment of grain was given (7.00 and 7.19, respectively; P = 0.09). On day 7, horses receiving M. elsdenii as a freeze-dried treatment tended to have a greater cecal pH (7.19) than the control (6.99; P = 0.09).

[0429] Table 17 shows the VFA profiles of the treatment groups.

Table 17-1

Table 17-2

[0430] Supplementation with M. elsdenii had no effect on the concentration of acetate or propionate in the cecum (P > 0.10; Table **)。However, treatment - day interactions were detected in the acetate:propionate (A:P) ratio (P < 0.05). The cecal A:P ratio on day 7 was higher in horses receiving freeze - dried M. elsdenii (2.83) than in horses not receiving M. elsdenii (2.41) or horses receiving M. elsdenii as an oral drench (2.60; P < 0.05). Cecal valerate was higher in horses receiving freeze - dried M. elsdenii (0.15 mM) on day 7 than in control animals (0.04 mM) or horses receiving an oral drench of M. elsdenii (0.07 mM; P < 0.02). Cecal valerate was lower in drench - treated horses than in control animals (P < 0.01) on day 5 but was similar to that in horses treated with freeze - dried M. elsdenii (P > 0.10). Concentrations of heptanoate and isocaproate were negligible, and no treatment or interaction effects were detected. Thus, these VFAs were excluded from ** Table

[0431] Cecal pH and fermentation products were most affected by the supplementation of M. elsdenii from day 5 to day 7, on which days the largest amount of grain was consumed. (Example 11) Evaluation of the Administration of the Liquid Culture Megasphaera elsdenii NCIMB41125 (Lactipro®) in Broiler Chickens

[0432] At Virginia Diversified Research Corporation, a pilot broiler performance study was conducted to evaluate the effects of misted or force - fed Megasphaera elsdenii NCIMB41125 on the growth performance of broiler chicks.

[0433] On day 0, day-old broiler chicks (n = 720) inoculated with the spray vaccine Coccivac-B were randomly assigned to six different treatments: (1) a negative control group that did not receive Megasphaera (nCON), (2) a mist group that received M. elsdenii NCIMB41125 by mist (1 - 2 mL / bird) on day 0 if they were in their hatchery pen (d0 mist), (3) a day-7 gavaged group that received 2 mL of M. elsdenii NCIMB41125 by oral gavage after 2 h of feed withdrawal and 1 h of water withdrawal on day 7 (d7 GAV), (4) a day-14 gavaged group that received 5 mL of M. elsdenii NCIMB41125 by oral gavage after 2 h of feed withdrawal and 1 h of water withdrawal on day 14 (d14 GAV), (5) a day-21 gavaged group that received 10 mL of M. elsdenii NCIMB41125 by oral gavage after 2 h of feed withdrawal and 1 h of water withdrawal on day 21 (d21 GAV), and (6) a positive control group (pCON) that did not receive Megasphaera but received starter and grower feeds treated with BMD (50 g / t) and finisher feed treated with Stafac (20 g / t).

[0434] Each treatment was represented by four cages containing 30 birds each. The diet given to the birds was as follows: starter feed from 0 - 18 days, grower feed from 18 - 35 days, and finisher feed from 35 - 39 days. The weight of the animals, feed consumption, and feed conversion were recorded over the 39-day test period.

Table 18

[0435] The results are presented in Table 18. There was no difference in the overall mortality across all treatments (Table 18). Feed conversion in the d0 mist group was significantly lower than all other treatments on day 25, with a 5.3% improvement over pCON and a 6.5% improvement over nCON. After 39 days of feeding, feed conversion in the Megasphaera elsdenii treatment group was not significantly different from pCON, but numerically tended to be lower, except for d7 GAV. Feed conversion in the d21 GAV group was significantly lower than nCON, with a 7.7% improvement in feed conversion. (Example 12) Growth of various strains of M. elsdenii on a semi-defined growth medium supplemented with two carbon sources A. Experimental design

[0436] In this example, the following bacterial strains were used: (1) Megasphaera elsdenii NCIMB41125, (2) Megasphaera elsdenii ATCC25940, (3) Megasphaera elsdenii NCIMB702261, (4) Megasphaera elsdenii NCIMB702262, and (5) Megasphaera elsdenii NCIMB702410.

[0437] All strains were grown on semi-defined lactate growth medium in serum bottles. The resulting cultures were then used to inoculate 96-well plates containing a semi-defined growth medium supplemented with sodium lactate and glucose consisting of two carbon sources: 60% lactate and 40% glucose, 70% lactate and 30% glucose, or 40% lactate and 60% glucose.

[0438] The 96-well plates were then incubated at 39°C under anaerobic conditions, and the optical density (600 nm) was automatically recorded at 15-minute intervals. B. Analysis of the growth characteristics of M. elsdenii strains on various two-carbon media

[0439] To compare the growth characteristics, the growth curves obtained with various M. elsdenii strains in various semi-defined media were plotted with the incubation time on the x-axis and the optical density readings on the y-axis (Figs. 23 - 25).

[0440] All of the M. elsdenii strains tested in this experiment showed similar growth characteristics when grown on semi-defined media consisting of 60% lactate and 40% glucose, 70% lactate and 30% glucose, or 40% lactate and 60% glucose. (Example 13) Growth of various strains of M. elsdenii on semi-defined growth media supplemented with two carbon sources and subsequent freeze-drying of the cells A. Experimental design

[0441] In this example, the following bacterial strains were used: (1) Megasphaera elsdenii NCIMB41125, (2) Megasphaera elsdenii ATCC25940, (3) Megasphaera elsdenii NCIMB702261, (4) Megasphaera elsdenii NCIMB702262, and (5) Megasphaera elsdenii NCIMB702410.

[0442] All strains were grown on semi-defined lactate growth media in serum bottles. The resulting cultures were then used to inoculate a 5 L fermenter tank containing a semi-defined growth medium supplemented with sodium lactate and glucose consisting of two carbon sources: 60% lactate and 40% glucose, or 70% lactate and 30% glucose.

[0443] Samples of the culture were taken 8, 10, 12, 14, and 16 hours after inoculation, cooled to room temperature, and the cells were harvested aseptically and anaerobically by removing 99% of the liquid. Under aseptic and anaerobic conditions, the holding solution was mixed with a solution of cryoprotectant (sucrose) at a ratio of 1 / 5 to obtain a final sucrose concentration of 5% w / v. The mixture was sampled to determine the concentration of M. elsdenii (i.e., viable count) before freeze-drying.

[0444] An aliquot of the resulting mixture was transferred to 10 mL vials (4 mL / vial) and snap-frozen in liquid nitrogen. The vials were transferred to a freeze-dryer and freeze-dried according to a rapid cycle. When freeze-drying was complete, the freeze-dried product in the anaerobic chamber was resuspended in an anaerobic diluent, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar to determine bacterial viability (i.e., viable count).

[0445] Cell loss was calculated by subtracting the concentration of M. elsdenii recovered after freeze-drying from the initial (before freeze-drying) concentration of M. elsdenii. B. Comparison of post-freeze-drying cell loss of various M. elsdenii strains grown in various semi-defined media, harvested and freeze-dried at various times during growth

[0446] All strains of M. elsdenii tested in this experiment had viable cells after freeze-drying. The acceptable cell loss limit was set at 1.6 log CFU / mL. Regardless of strain or harvest time, the cell loss encountered during freeze-drying of cells grown on semi-defined medium containing 70% lactate and 30% glucose (Table 19) was all below the acceptable limit and in the range of 0.3 - 1.3 log.

Table 19

[0447] Cell losses encountered in the freeze-dry of cells grown on semi-defined medium containing 60% lactate and 40% glucose (Table 20) were more affected by the harvest time, but all strains still had cell losses below the acceptable limit for at least three of the harvest times. Optimization of the harvest time before freeze-drying can lead to further improvement in the recovery rate after freeze-drying.

Table 20

[0448] Overall, the methods described herein resulted in freeze-dried products containing viable cells, regardless of the Megasphaera elsdenii strain used. (Example 14) Encapsulation of freeze-dried M. elsdenii and determination of its stability

[0449] The M. elsdenii NCIMB41125 holding solution obtained through TFF was aseptically mixed with a cryoprotectant solution (sucrose) at a ratio of 1 / 5 to obtain a final concentration of 5% sucrose (w / v). The mixture was snap-frozen in liquid nitrogen and transferred to a freeze-dryer for freeze-drying using rapid cycles. When the freeze-drying was complete, the freeze-dried product in the anaerobic chamber was resuspended in an anaerobic diluent, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar to determine bacterial viability.

[0450] Subsequently, the obtained freeze-dried M. elsdenii powder was mixed with a carrier (bulking agent), and the powder was encapsulated by dispensing it into heated palm oil distillate or stearic acid. The mixture was then rapidly cooled, and the resulting product was sampled to determine the survival of the bacteria. The sample was resuspended in an anaerobic diluent in an anaerobic chamber, blended for 15 seconds, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar. This experiment was repeated three times (Methods 1, 2, and 3) using various heating temperatures and different types of oils. Method 1 used a heating temperature of 110°C and palm oil distillate as the encapsulating material. Method 2 used a heating temperature of 52°C and a mixture of mono- and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid as the encapsulating material. Method 3 used a heating temperature of 65°C and a mixture of mono- and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid as the encapsulating material.

[0451] The loss rate was calculated by subtracting the concentration of M. elsdenii recovered after encapsulation from the concentration recovered after freeze-drying and dividing this by the concentration recovered after freeze-drying.

[0452] Using Methods 2 and 3, additional samples were collected from the encapsulated product and stored at room temperature for 4 months under aerobic conditions to evaluate the stability of the product. The sample was resuspended in an anaerobic diluent in an anaerobic chamber, mixed for 15 seconds, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar. Figures 26 and 27 show that using Method 2 or 3, the stability of the encapsulated freeze-dried M. elsdenii was lost by only about 1 log CFU / g.

Table 21

[0453] Methods 2 and 3 used to encapsulate freeze-dried M. elsdenii resulted in a cell recovery rate higher than 67.9% (Table 21).

[0454] Furthermore, the freeze-dried samples encapsulated according to Method 2 or Method 3 had a 1-log decrease in cell viability during storage at room temperature under standard oxygen and humidity conditions for up to 4 months.

[0455] Encapsulation of freeze-dried M. elsdenii according to Methods 2 and 3 provided further protection of the bacteria from oxygen and moisture. This process enabled the storage of encapsulated freeze-dried M. elsdenii at room temperature without any specific packaging and allowed for the addition of the product to feed. (Example 15) Storage period of pilot-scale-produced freeze-dried M. elsdenii. A. Experimental design

[0456] A 600-liter batch of semi-defined medium consisting of 70% lactate and 30% glucose was used in this experiment to grow M. elsdenii NCIMB41125. After incubation at 39 °C for 14 hours, the resulting culture was cooled to room temperature, and the cells were harvested using the tangential flow filtration system described in Example 2. Ninety-nine percent of the liquid volume was removed from the culture, and the retentate was then resuspended in a sucrose cryoprotectant solution at a ratio of 1:5 to obtain a final sucrose concentration of 5% w / v.

[0457] The mixture was then frozen in liquid nitrogen, and the frozen pellet was transferred to a freeze-dryer and freeze-dried according to the rapid cycle. When the freeze-drying was complete, the freeze-dried powder was collected and packaged in Mylar pouches under anaerobic conditions, either alone (“M.e.”) or together with maltodextrin as a bulking agent (“M.e. + maltodextrin”) (Table 22).

Table 22

[0458] The concentration of M. elsdenii in the freeze-dried product (three samples per treatment) was determined by resuspending the freeze-dried product in an anaerobic diluent in an anaerobic chamber, rehydrating this at room temperature for 40 minutes, and then plating on semi-defined lactate agar (i.e., viable count). The M. elsdenii concentration was expressed as CFU / pouch and log-transformed. B. Study of Shelf Life

[0459] Mylar pouches containing various treatments were stored either at room temperature (75°F; 25°C) or at 40°F (4°C). At 0.5, 1, 2, 3, 4, and 6 months of storage, additional samples were obtained and processed as described previously (three samples per treatment per time point for 1 hour) to determine the shelf life of the product. The M. elsdenii concentration was expressed as CFU / pouch and log-transformed.

[0460] Shelf life data are presented in Figure 28. The M. elsdenii concentration over time was affected by the storage temperature. After 6 months of storage, samples stored at 40°F (4.4°C) were stable regardless of the presence or absence of maltodextrin, while samples stored at 75°F (23.9°C) lost approximately 1.6 log in the case of the "M.e." treatment and approximately 0.8 log in the case of the "M.e. + maltodextrin" treatment. (Example 16) Microbial Cell Growth, Medium, Temperature, and pH

[0461] Anaerobic bacteria can be divided into three categories: (1) obligate anaerobes; (2) aerotolerant anaerobes; and (3) facultative anaerobes. Obligate anaerobes are bacteria that cannot survive in oxygen at standard atmospheric concentration. Some obligate anaerobes can survive in up to 8% oxygen, while others require an oxygen concentration of less than 0.5% to survive. Aerotolerant anaerobes can survive in the presence of oxygen but do not utilize oxygen for growth. Facultative anaerobes can use oxygen for aerobic respiration but can also use anaerobic respiration when oxygen is absent.

[0462] Similarly, aerobic bacteria can be divided into two categories: (1) obligate aerobes; and (2) microaerophiles. Obligate aerobes require oxygen for the cells to respire and can survive in oxygen at standard atmospheric concentration. Microaerophiles require oxygen for cell growth but are damaged by oxygen at standard atmospheric concentration.

[0463] Yeasts are single-celled eukaryotic microorganisms classified as members of the fungal kingdom. Yeasts can be either obligate aerobes or facultative anaerobes.

[0464] Megasphaera such as M. elsdenii and Bifidobacterium such as B. breve are representative species of obligate anaerobes. Lactobacillus such as L. plantarum and Bifidobacterium such as B. animalis subsp. lactis are representative species of aerotolerant anaerobes. Pediococcus such as P. acidilactici and Lactobacillus such as L. casei are representative species of facultative anaerobes. Bacillus such as B. subtilis are representative species of obligate aerobes. Saccharomyces such as S. boulardii and S. cerevisiae are representative species of yeasts.

[0465] Aerobic bacteria, anaerobic bacteria, and yeast are grown on a medium containing at least one carbon source selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof. Also, aerobic bacteria, anaerobic bacteria, and yeast are grown on a medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof. Anaerobic bacteria are grown under anaerobic conditions or under the required oxygen conditions to promote anaerobic bacterial cell growth, and aerobic bacteria and yeast are grown at a temperature between 15°C and 45°C for L. plantarum and between 20°C and 45°C for B. breve, B. animalis subsp. lactis, P. acidilactici, L. casei, S. boulardii, and B. subtilis using a medium containing at least two carbon sources from the above under appropriate oxygen conditions. The optimal temperature for these strains is 37°C, except for S. cerevisiae, which prefers 30°C. The pH of the medium is between 4.0 and 9, more specifically between pH 4.0 - 4.5, 4.5 - 5.5, 5.5 - 6.5, 6.5 - 7.5, 7.5 - 8.5, or 8.5 - 9.0. The microorganisms are grown until the end of the logarithmic growth phase, i.e., for at least 1 hour - 6 hours, 6 hours - 12 hours, 12 hours - 24 hours, 24 hours - 36 hours, 36 hours - 48 hours, 48 hours - 72 hours, 72 hours - 96 hours, or 96 hours - 120 hours.

[0466] When the medium contains at least 1×10 3 CFU / g, the microorganisms are harvested under appropriate conditions and the microorganisms are freeze-dried and / or encapsulated for use in animal feed formulations. (Example 17) Use of tangential flow filtration for concentrating cultures of aerobic bacteria, anaerobic bacteria and yeast

[0467] The method presented in Example 2 is used in this example using the aerobic bacteria, anaerobic bacteria and yeast disclosed in Example 16 and an appropriate medium that allows optimal microbial cell growth. Similar to Megasphaera elsdenii, using tangential flow filtration on aerobic bacteria (Bacillus subtilis), anaerobic bacteria (Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici and Lactobacillus casei) and yeast (Saccharomyces boulardii and cerevisiae) results in similar results with respect to the amount of viable microorganisms recovered in the permeate and retentate over the concentration process. Furthermore, the filtration process has no effect on the viability of the microorganisms after filtration or on the ability of the microorganisms to grow. Thus, the microorganisms are prepared to be grown in liquid broth, filtered, frozen, freeze-dried and / or encapsulated. (Example 18) Freezing and freeze-drying parameters for aerobic bacteria, anaerobic bacteria and yeast

[0468] To determine the effect of various freezing and freeze-drying parameters on various types of aerobic bacteria, anaerobic bacteria and yeast, an example according to Example 17 is performed. Similar to Example 3, the acceptable cell loss threshold is set at 1.6 log CFU / mL.

[0469] The holding solutions of Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, Lactobacillus casei, Bacillus subtilis, Saccharomyces boulardii and Saccharomyces cerevisiae are resuspended in a cryoprotectant solution that does not contain a cryoprotectant, skim milk powder, trehalose, sucrose or combinations thereof before freeze-drying. Then, each mixture is transferred to a vial and slowly frozen at -80 °C or flash-frozen in liquid nitrogen, and then placed in a freeze dryer for freeze-drying using either a rapid cycle or a slow cycle.

[0470] All holding solutions that are not mixed with a cryoprotectant have higher cell loss and cell loss greater than the threshold compared to the holding solutions mixed with the cryoprotectant solution, regardless of the freeze-drying cycle or the freezing method.

[0471] Furthermore, all of the freeze-drying processes tested result in products that are able to maintain a sufficient survival rate to initiate the growth of the culture even after long-term storage at room temperature or at least 4 °C for 4 to 12 months after rehydration. (Example 19) Effect of storage conditions on the yield and stability of freeze-dried aerobic bacteria, anaerobic bacteria and yeasts

[0472] The test method for cell survival and microbial growth characteristics presented in Example 4 is used in this example using the aerobic bacteria, anaerobic bacteria and yeasts disclosed in Example 16 and using an appropriate medium that allows optimal microbial cell growth.

[0473] Next, to determine the effects of freeze-drying protocols and storage conditions on the growth characteristics and shelf life of freeze-dried cultures generated in the same manner as in Example 18 for Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, Lactobacillus casei, Bacillus subtilis, Saccharomyces boulardii, and Saccharomyces cerevisiae, growth curve analysis and spread plate culture techniques were used to test for microbial growth characteristics and cell survival during storage at 4°C or 25°C under aerobic or anaerobic conditions for 0, 2, 4, 8, 12, 16, 20, and 24 weeks.

[0474] Samples derived from freeze-dried anaerobic bacteria stored under aerobic conditions disintegrate more rapidly with additional cell loss compared to their anaerobically stored counterparts, regardless of treatment.

[0475] Samples stored at 25°C disintegrate faster than their counterparts stored at 4°C.

[0476] Samples frozen in liquid nitrogen and stored at 25°C after freeze-drying do not lose as many cells as their counterparts stored at 4°C over a 16-week storage period, but the differences between samples become significant between 25°C and 4°C storage after 20 and / or 24 weeks of storage.

[0477] On each sampling day, a growth curve experiment is conducted to compare the growth characteristics of freeze-dried products and non-freeze-dried products. The non-freeze-dried samples used for each growth curve are "new" (less than 2 days old). The freeze-dried products stored at 4°C have a shorter time lag than the freeze-dried products stored at 25°C. After 16 weeks of storage, all samples derived from anaerobic bacteria that were frozen in liquid nitrogen, freeze-dried, and stored under anaerobic conditions, containing cryoprotective substances, revived and were again viable. Similarly, all samples derived from aerobic bacteria and yeast that were frozen in liquid nitrogen, freeze-dried, and contained cryoprotective substances revived and were again viable. (Example 20) Effect of storage conditions on the yield and stability of encapsulated freeze-dried aerobic, anaerobic bacteria and yeast

[0478] The freezing and freeze-drying of the holding solutions of various aerobic bacteria, anaerobic bacteria, and yeasts are carried out as described in Examples 16-19. After freeze-drying, the anaerobic bacteria are mixed with a carrier (bulking agent), and the freeze-dried powder is dispensed into heated oil and encapsulated as described in Example 14. The mixture is then rapidly cooled, and the resulting product is sampled to determine the survival of the bacteria. The anaerobic microbial sample is resuspended in an anaerobic diluent in an anaerobic chamber, mixed for 15 seconds, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar. The aerobic microbial sample is resuspended in a diluent in oxygen at standard atmospheric concentration, mixed for 15 seconds, rehydrated at room temperature for 40 minutes, and then plated on semi-defined lactate agar. This experiment is repeated three times (Methods 1, 2, and 3) using various heating temperatures and various types of oil. Method 1 uses a heating temperature of 110°C and palm oil distillate as the encapsulating material. Method 2 uses a heating temperature of 52°C and a mixture of mono- and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid as the encapsulating material. Method 3 uses a heating temperature of 65°C and a mixture of mono- and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid as the encapsulating material.

[0479] The cell loss rate from encapsulation is less than 40% when Method 2 or 3 is used. After encapsulation of various aerobic bacteria, anaerobic bacteria, and yeasts, the encapsulated microorganisms stored at room temperature under standard atmospheric oxygen conditions have only a slight decrease in cell viability over storage (e.g., a decrease in cell viability of 0.5 - 2 log).

Claims

1. A method for generating freeze-dried Megasphaera elsdenii cells on a commercial scale, comprising: (a) preparing a liquid culture comprising M. elsdenii cells and a growth medium containing at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, and combinations thereof, under anaerobic conditions at a volume of about 2 liters to about 75,000 liters; (b) harvesting said cells under anaerobic conditions; (c) adding at least one cryoprotectant to the harvested cells; (d) freezing said cells; (e) freeze-drying said cells; comprising about 1×10 ７ to about 1×10 １２ CFU / g of freeze-dried M. elsdenii cells remain viable after freeze-drying, said method is carried out on a commercial scale, and said cryoprotectant is sucrose, trehalose, maltodextrin, or a combination thereof. Method.

2. The method according to claim 1, wherein said at least two carbon sources consist of about 50-90% of a first carbon source and about 10-50% of a second carbon source, said second carbon source being different from said first carbon source, and 100% of said at least two carbon sources consisting of said first carbon source and said second carbon source.

3. A method for generating freeze-dried Megasphaera elsdenii cells on a commercial scale, comprising: (a) preparing a liquid culture comprising M. elsdenii cells and a growth medium in a volume of from about 2 liters to about 75,000 liters; (b) harvesting the cells under anaerobic conditions within 12 hours after the culture has ended its logarithmic growth phase; (c) adding at least one cryoprotectant to the harvested cells; (d) freezing the cells; (e) freeze-drying the cells comprising from about 1×10 ７ to about 1×10 １２ CFU / g of freeze-dried M. elsdenii cells remain viable after freeze-drying, the method is carried out on a commercial scale, and the cryoprotectant is sucrose, trehalose, maltodextrin, or a combination thereof.

4. The harvesting step includes at least one technique selected from the group consisting of centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof, and optionally the filtration includes tangential flow filtration, and / or the culture contains a liquid, and the harvesting step includes removing from about 60% to about 100% of the liquid. The step of harvesting the cells under anaerobic conditions is carried out before the culture enters the stationary growth phase, and / or the freezing step is at a temperature of from about -80°C to about -210°C. The method according to any one of claims 1 to 3.

5. The freezing step includes contacting the container containing the M. elsdenii cells with liquid nitrogen, and / or the freezing step includes contacting the cells with liquid nitrogen, and / or The freezing step is at a temperature of about -196 °C, generating a frozen pellet containing the cells, and the diameter of the frozen pellet is about 0.001 to about 0.5 inches, and / or The pH of the M. elsdenii culture before harvesting is between about 4.5 and about 7.0, the method according to any one of claims 1 to 4.

6. About 1×10 ７ to about 1×10 １２ CFU / g of the freeze-dried M. elsdenii cells remain viable after storage at a temperature of about 25 °C for at least 2 weeks, or About 1×10 ７ to about 1×10 １２ CFU / g of the freeze-dried M. elsdenii cells remain viable after storage at about 4 °C for at least 1 month, The method according to any one of claims 1 to 5.

7. The at least one cryoprotective substance is sucrose and is present in an amount of about 1% to about 20% (w / v) of the culture, the method according to any one of claims 1 to 6.

8. The volume of the culture is about 50 liters to about 50,000 liters, the method according to any one of claims 1 to 7.

9. A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, improving the bioavailability of plant-derived phosphorus in the diet of an animal, or improving the growth performance in an animal, comprising administering to the animal an effective amount of freeze-dried M. elsdenii cells produced by the method according to any one of claims 1 to 8, wherein the improvement in the growth performance of the animal is an improvement in feed intake, average daily weight gain, feed conversion ratio, weight gain of carcass meat, milk production in milk-producing animals, egg production in poultry, bone mineralization or a combination thereof, and the animal is a non-human animal.

10. The method according to claim 9, wherein the animal is selected from the group consisting of cattle, sheep, goats, deer, buffalo, reindeer, chickens, geese, ducks, quails, pheasants, pigeons, broilers, broiler-breed chickens, egg-laying chickens, horses, ponies, donkeys and camels.

11. A method for generating freeze-dried encapsulated Megasphaera elsdenii cells on a commercial scale, comprising: (a) preparing a liquid culture comprising a growth medium containing Megasphaera elsdenii cells and at least two carbon sources selected from the group consisting of casein, lactate, dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract and combinations thereof, in a volume of from about 2 liters to about 75,000 liters under anaerobic conditions; (b) harvesting the cells under anaerobic conditions; (c) adding at least one cryoprotectant to the harvested cells; (d) freezing the cells; (e) freeze-drying the cells; (f) encapsulating the cells, and from about 1×10 ７ to about 1×10 １２ CFU / g of freeze-dried encapsulated Megasphaera elsdenii cells remain viable after freeze-drying, the method is carried out on a commercial scale, and the cryoprotectant is sucrose, trehalose, maltodextrin or a combination thereof. Method.

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