Encapsulated formulations, compositions, and uses for anaerobic bacteria

Abstract

The present disclosure relates to a composition comprising (a) (i) a Megasphaera elsdenii cell, an anaerobic bacterial cell, or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cell, the anaerobic bacterial cell, or the anaerobic bacterial cell in a trophic state is viable in the composition. The present disclosure also relates to feed additives, feeds, premixes, and kits comprising the composition described above.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority of U.S. provisional patent application No. 63 / 597,285 filed on November 8, 2023, the entirety of which is incorporated herein by reference.

[0003] Background of the disclosed content

[0004] Field of disclosure

[0005] The present disclosure relates to encapsulated formulations, compositions, and uses thereof for anaerobic bacteria. Background Technology

[0006] Megaspaera Elsdeni Megasphaera elsdenii )(i.e., M. elsdenyi) is an anaerobic, non-motile, and Gram-negative diplococcus that utilizes lactate as a desirable carbon source and can help prevent acidosis, a common digestive disorder affecting millions of beef and dairy cattle every year.

[0007] When cattle and other ruminants consume large amounts of starchy foods (e.g., grains) or monosaccharides, opportunistic microorganisms in the stomach can rapidly ferment these compounds into lactic acid. Lactic acid is a potent organic acid and can cause lactic acidosis, which disrupts normal digestive activity and can cause extensive damage to the lining of the digestive tract in ruminants. Affected animals exhibit suboptimal performance. Furthermore, the most acute forms of lactic acidosis can cause irreversible damage to the animal's digestive and respiratory systems, as well as lead to increased mortality.

[0008] M. elsdenyi can help control lactic acidosis by converting lactic acid into volatile fatty acids (VFAs, e.g., butyrate, propionate, and acetate) beneficial to animals. Volatile fatty acids are further metabolized by animals and other bacteria. However, the population of M. elsdenyi in the gastrointestinal tract (GIT) of ruminants is often too low to prevent the risk of acidosis. Therefore, to increase the colonization rate of M. elsdenyi in the gastrointestinal tract of ruminants, Lactipro, a liquid culture of viable cells derived from M. elsdenyi strains, is used. ® ...was developed. For example, refer to U.S. Patent No. 7,550,139. ​​However, there are substantial limitations restricting the use of products containing M. elsdeny, including the difficulty of maintaining M. elsdeny products under the anaerobic conditions required by the microorganism and the difficulty of transporting M. elsdeny products from the production facility to the final use site within 14 days, after which the viability of M. elsdeny in the product significantly decreases. Products containing freeze-dried M. elsdeny (Lactipro NXT ® and Lactipro FLX ® Although it has a longer shelf life than Lactipro®, there is a need for a more improved composition that is encapsulated and can be added to feed, as a feed additive, or alone. Refer to WO 2018 / 144653 A1, the entirety of which is incorporated herein by reference. These compositions will enable further use in the animal health market.

[0009] Cattle are at risk of developing acidosis if they fail to adapt properly to a high-starch diet or if feed intake is interrupted for any reason, such as weaning, transport, disease / injury, muddy barns, extreme heat or cold, storms, equipment failure, or treatment. The accumulation of lactic acid in the rumen can lead to acidosis, which has negative effects on the animals (i.e., reduced feed intake, decreased performance, and even death). Therefore, minimizing acidosis is important for producers, especially during the diet adaptation period when acidosis is most frequent. Traditionally, ruminants in feedlands are gradually adapted from a high-roughage diet to a high-concentration diet over a period of 3 to 4 weeks. The gradual increase in the high-concentration diet minimizes lactate accumulation in the rumen. M. elsdeni has the potential to alleviate acidosis by limiting lactic acid accumulation through the provision of active populations of M. elsdeni, thereby shortening the transition period for feedland cattle moving from a high-roughage diet to a high-concentration diet. M. elsdenii is a strictly anaerobic bacterium, and to date, the drench (LactiproNXT) that can be administered only during the limited period of handling or treating animals ® ) or bolus (LactiproFLX ® It is commercialized as ). This makes it unsuitable for daily or immediate administration required for situations where feed intake is interrupted, such as adverse weather conditions and / or equipment failure.

[0010] Similarly, M. elsdenyi has been considered as an option to reduce the incidence of metabolic diseases, such as subacute ruminal acidosis (SARA), during the transition from the dry to the lactation period in dairy cows. SARA can have severe effects on dairy cows, such as reduced milk production and laminitis, which is often associated with limping and can lead to early culling. Previous studies have demonstrated the importance of timely administration of M. elsdenyi. Using Drenchy or Bolus with limited administration periods prevents producers (e.g., dairy cows) from receiving the full benefits of M. elsdenyi.

[0011] Therefore, there is a need to develop a stable M. elsdenyi formulation that can withstand storage and feed mixing while still releasing bacteria in the bovine rumen. This is key for commercial feedlots and dairy cows, as it will enable daily or more targeted administration. In addition, Bifidobacterium ( Bifidobacterium ), for example, B. Brevet ( breve ), Lactobacillus( Lactobacillus ), for example, L. plantarum( plantarum ), Bifidobacterium ( Bifidobacterium ), for example, B. Animalis( animalis ) subspecies (subsp.) lactis( lactis ), Pediococcus( Pediococcus ), for example, P. acidilactis ( acidilactic acid ), Lactobacillus( Lactobacillus ), for example, L. Cajey( casei ), Fibrobacter( Fibrobacter ), for example, F. succinogenes( succinogenes ), Butyrivibrio( Butyrivibrio ), for example, B. fibrisolvense ( fibrisolvens ), and Luminococcus ( Ruminococcus ), for example, R. flavefaciens( flavefaciens ) , Blautia Blautia ) , For example, B. Obéum ( obeum) , Clostridium Clostridium ) , for example C. Butyricum butyricum ) , Akermansia Akkermansia ) , For example, A. mucinipilla ( muciniphila There is also a need for stable formulations of other anaerobic bacteria that can be released from the rumen, including ), as well as their compositions and uses. means of solving the problem

[0012] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells are viable in the composition.

[0013] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable after being added to a feed or feed additive.

[0014] In some embodiments, Megasphaera elsdenii cells in the composition can survive for up to 48 hours at 25 °C and pH 7.0 after being added to feed or feed additives.

[0015] In some embodiments, the present disclosure provides a composition comprising (a) (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0016] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a temperature of at least 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0017] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0018] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0019] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by a layer of one or more lipids and at least 10% of the Megasphaera elsdenii cells in the composition remain viable even after being processed through a micromachine system.

[0020] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by a layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable for up to 14 hours after being added to the microbin of a micromachine.

[0021] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by a layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable for up to 48 hours after being added to the microbin of a micromachine.

[0022] In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for about 14 to about 48 hours after being added to the microbins of the micromachine.

[0023] In some embodiments, the particle size of the Megasphaera elsdenii cells in the composition is less than about 1600 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells in the composition is less than about 400 μm.

[0024] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state is viable in the composition.

[0025] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after being added to a feed or feed additive.

[0026] In some embodiments, anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition can survive for up to 48 hours at 25 °C and pH 7.0 after being added to feed or feed additives.

[0027] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0028] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a temperature of 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0029] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0030] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0031] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable after being added to a micromachine.

[0032] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids, and in some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable for up to 14 hours after being added to the microbin of a micromachine.

[0033] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids, and in some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable for up to 48 hours after being added to the microbin of a micromachine.

[0034] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for about 14 hours to about 48 hours after being added to the microbin of the micromachine.

[0035] In some embodiments, the particle size of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition is less than about 1600 μm. In some embodiments, the particle size of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition is less than about 400 μm.

[0036] In some embodiments, at least 15%, 20%, 30%, 40%, 50%, 60%, about 70%, 80%, 90%, or 99% of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are viable in the composition.

[0037] In some embodiments, anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are dried.

[0038] In some embodiments, anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are dried by spray drying, electrospray drying, vacuum drying, jet drying, freeze drying, or a combination thereof.

[0039] In some embodiments, the composition comprises about 0.1% to about 15% (w / w) of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state.

[0040] In some embodiments, the composition is about 1 x 10 per gram. 4 to about 1 x 10 10 It includes anaerobic bacterial cells of CFU or anaerobic bacterial cells in a trophic state.

[0041] In some embodiments, the anaerobic bacterial cell or the anaerobic bacterial cell in a trophic state is selected from the group consisting of Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subs. lactis, Pediococcus acidilactis, Lactobacillus casei, Megasphaera elsdenii, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Luminococcus flavefaciens, Blautia obeum, Clostridium butyricum, Achermansia musiniphila, and combinations thereof.

[0042] In some embodiments, the composition is a granule, capsule, minicapsule, microcapsule, tablet, minitablet, or microtablet.

[0043] In some embodiments, the composition has a moisture content of about 5% (w / w) or less.

[0044] In some embodiments, one or more lipids of the core are selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, pewter, vegetable waxes, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic waxes, natural and synthetic resins and mixtures thereof.

[0045] In some embodiments, one or more lipids of the core are animal fats or oils and / or vegetable fats or oils.

[0046] In some embodiments, the vegetable fat or oil is selected from the group consisting of canola oil, cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil.

[0047] In some embodiments, the vegetable fat or oil is hydrogenated palm oil.

[0048] In some embodiments, the free fatty acid is myristic acid, lauric acid or stearic acid, or a combination thereof.

[0049] In some embodiments, one or more lipids of the core have a melting point of about 40 °C to about 85 °C.

[0050] In some embodiments, one or more lipids of the core have a melting point of about 55 °C to about 75 °C.

[0051] In some embodiments, one or more lipids coating the core are selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, pewter, vegetable waxes, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic waxes, natural and synthetic resins and mixtures thereof.

[0052] In some embodiments, one or more lipids coating the core are animal fats or oils and / or vegetable fats or oils.

[0053] In some embodiments, the vegetable fat or oil is selected from the group consisting of cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil.

[0054] In some embodiments, the vegetable fat or oil is hydrogenated palm oil or hydrogenated cottonseed oil.

[0055] In some embodiments, the free fatty acid is myristic acid, lauric acid, or stearic acid.

[0056] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C to about 80 °C.

[0057] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C to about 75 °C.

[0058] In some embodiments, at least one carrier comprises maltodextrin, sucrose, starch, cellulose, clay, biochar, lignin derivatives, sugar alcohols, or combinations thereof.

[0059] In some embodiments, the composition contains about 10% to about 99% (w / w) of total lipids.

[0060] In some embodiments, the composition contains about 70% to about 80% (w / w) of total lipids.

[0061] In some embodiments, the composition comprises at least one carrier of about 10% to about 30% (w / w).

[0062] In some embodiments, the composition comprises at least one carrier of about 15% to about 25% (w / w).

[0063] In some embodiments, the composition has a diameter of about 0.1 mm to about 3 mm.

[0064] In some embodiments, the composition has a diameter of about 0.2 mm to about 0.6 mm.

[0065] In some embodiments, the composition has a diameter of about 0.2 mm to about 0.4 mm.

[0066] In some embodiments, the core comprises about 1% to about 99% of the composition.

[0067] In some embodiments, the core comprises about 10% to about 90% of the composition.

[0068] In some embodiments, the core comprises about 25% to about 80% of the composition.

[0069] In some embodiments, one or more lipids coating the core comprise about 1% to about 99% of the composition.

[0070] In some embodiments, one or more lipids coating the core comprise about 5% to about 75% of the composition.

[0071] In some embodiments, the composition has a density of about 0.6 g / mL to about 1.2 g / mL.

[0072] In some embodiments, the composition has a porosity of about 10% to about 60%.

[0073] In some embodiments, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of Megasphaera elsdenii cells are viable in the composition.

[0074] In some embodiments, the Megasphaera elsdeni cell is dried.

[0075] In some embodiments, the Megaspaera elsdeni cells are dried by spray drying, electrospray drying, vacuum drying, jet drying, freeze drying, or a combination thereof.

[0076] In some embodiments, the composition comprises about 0.1% to about 15% (w / w) of Megasphaera elsdenii cells.

[0077] In some embodiments, the composition has a diameter of about 0.1 mm to about 1 mm.

[0078] In some embodiments, the composition is about 1 x 10 per gram. 4 to about 1 x 10 10 It includes CFU of Megasphaera elsdeni cells.

[0079] In some embodiments, anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are about 1 x 10 per gram of the composition. 4 to about 1 x 10 10 Includes CFU.

[0080] In some embodiments, the composition further comprises one or more of antibiotics, antimicrobial agents, anticoccidial agents, antiparasitic agents, sulfonamides, hormones, anti-bloat compounds, adrenergic receptor modulators, phages, prebiotics, probiotics, enzymes, essential oils, and / or carbohydrate immunostimulants.

[0081] In some embodiments, a feed additive composition comprising any of the compositions disclosed herein is provided herein.

[0082] In some embodiments, the feed additive composition is a powder, fine particles, pellets, cake, liquid, solid, suspension, emulsion, gel, or a combination thereof.

[0083] In some embodiments, a feed comprising any of the compositions disclosed herein or any of the feed additive compositions disclosed herein is provided herein.

[0084] In some embodiments, the feed further comprises animal protein, vegetable protein, corn, soybean meal, corn dried distillers grains with solubles (cDDGS), wheat, wheat protein, gluten, wheat byproduct, wheat bran, wheat dried distillers grains with solubles (wDDGS), corn byproduct including corn gluten meal, barley, oats, rye, triticale, whole soybeans, animal byproduct meal, alcohol-soluble protein, zein, corn zein, kafirin, rice, paddy rice, extruded paddy rice, oily seed protein, or a combination thereof.

[0085] In some embodiments, the animal protein or plant protein is selected from the group consisting of one or more of gliadin or an immunogenic fragment of gliadin, beta-casein, beta-lactoglobulin, glycinin, beta-conglycinin, cruciferin, nafin, hordain, keratin, feather meal or cauliflower meal, collagen, whey protein, fish protein, fish meal, meat protein, egg protein, soy protein and grain protein.

[0086] In some embodiments, the oily seed protein is selected from the group consisting of soybean seed protein, sunflower seed protein, rapeseed protein, canola seed protein, and combinations thereof.

[0087] In some embodiments, a premix comprising a) any of the compositions disclosed herein or any of the feed additive compositions disclosed herein and b) at least one mineral and / or at least one vitamin is provided herein.

[0088] In some embodiments, a kit is provided herein comprising a) i) any of the compositions provided herein, ii) any of the feed additive compositions provided herein, iii) any of the feeds provided herein and / or iv) any of the premixes provided herein and b) instructions for formulation and / or administration to a subject.

[0089] In some embodiments, a method for treating or preventing a pathological condition or disorder associated with lactic acid production in the gastrointestinal tract of a subject is provided herein, comprising the step of administering to a subject an effective amount of any of the feed additive compositions disclosed herein or any of the feeds disclosed herein.

[0090] In some forms, the pathological condition or disorder is acidosis.

[0091] In some forms, the condition or disorder is rumen acidosis.

[0092] In some forms, the pathological condition or disorder is a respiratory disease.

[0093] In some forms, the condition or disorder is laminitis.

[0094] In some embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal is provided, comprising the step of administering to an animal any of the feed additive compositions disclosed herein or any of the feeds disclosed herein an effective amount of any of the feeds disclosed herein.

[0095] In some forms, opportunistic microorganisms are pathogenic.

[0096] In some forms, the opportunistic microorganism is Salmonella, E. coli, or Campylobacter.

[0097] In some embodiments, a method for improving the growth performance of a subject is provided herein, comprising the step of administering to a subject an effective amount of any of the feed additive compositions disclosed herein or any of the feeds disclosed herein. In some embodiments, the improvement in the subject's performance includes one or more of the feed conversion ratio (FCR), body weight gain, feed efficiency, carcass quality, reduced mortality, reduced morbidity, feed intake, daily gain, carcass gain, bone mineralization, egg production, reduction in gastrointestinal microbial imbalance or dysfunction, milk composition (e.g., increased milk fat), and / or milk production, compared to the performance of a subject not administered the feed additive composition or feed.

[0098] In some embodiments, a method for increasing starch digestibility of a subject and lowering / or preventing a decrease in pH in the lower gastrointestinal tract is provided, comprising the step of adding an effective amount of any of the feed additive compositions disclosed herein to a feed to be administered to a subject, wherein the subject exhibits one or more of increased starch digestibility and / or lowered starch excretion in the feces compared to a subject not administered the feed additive composition.

[0099] In some embodiments, a method for increasing farm operational efficiency is provided, comprising the step of administering an effective amount of any of the feed additive compositions disclosed herein or any of the feeds disclosed herein to an animal on the farm. In some embodiments, the increased operational efficiency reduces labor costs, reduces forage transportation costs, and reduces the amount of forage added to the feed or feed additive.

[0100] In some modalities, the subject is a ruminant.

[0101] In some embodiments, ruminants are selected from a group consisting of cattle, goats, sheep, giraffes, deer, gazelles, buffalo, reindeer, and antelopes.

[0102] In some forms, the cow is a beef cow or a dairy cow.

[0103] In some modalities, the subject is a non-ruminant.

[0104] In some embodiments, non-ruminants are selected from a group consisting of horses, animals, poultry, and pigs.

[0105] In some embodiments, poultry is selected from a group consisting of chickens, geese, ducks, quails, turkeys, broilers, meat breeding hens, laying hens, or pigeons.

[0106] In some forms, poultry is chicken.

[0107] In some embodiments, the feed additive composition or feed is provided to the subject for daily or weekly administration. Brief explanation of the drawing

[0108] Fig. 1 This shows M. Elsdeny stability data for potato starch [PS], wheat starch [WS], and corn starch [CS] formulations stored in bulk at 4 °C or -20 °C. The WS formulation was not sampled after 4 months. Fig. 2 It shows M. elsdeny stability data (Log10 CFU / g of encapsulated M. elsdeny cells) during storage at 4 °C for 12 months. Fig. 3 After passing through a bowl or continuous flow micromachine system, or if not ('no micromachine'), and after 4 hours of aerobic exposure to a low pH and high moisture diet at room temperature M. Elsdeny Shows the recovery rate. Fig. 4 is using freeze-dried cultures, cores, 8(X1) formulations, 16(X1) formulations, and vegetable oil formulations in semi-limited lactate medium incubated at 39 °C for 18 hours. In vitro Shows the amount of gas produced (Ankom). Fig. 5 1.5 g aliquot (10) in Mylar pouch at -20 °C and 4 °C 6 Shows M. Elsdeny stability data for 8 encapsulated products (X1) stored in CFU). Dotted line = extrapolated data. Fig. 6 The encapsulated product is shown to have a recovery rate of M. elsdeny after mixing with a low pH / high water diet or a high pH / high water diet for up to 6 hours at 25 °C or 52 °C under aerobic conditions. Fig. 7 This shows the concentration of M. elsdenyi in crushed corn that has been sprayed with freeze-dried products and exposed to laboratory (indoor) or external solar atmospheric conditions. Interaction of exposure time * storage condition P = 0.0007, exposure time effect P < 0.0001, storage condition effect P < 0.0001. There is no significant difference between bars with common superscripts. Specific details for implementing the invention

[0109] The present disclosure relates to a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells are viable in the composition.

[0110] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable after being added to a feed or feed additive.

[0111] In some embodiments, the present disclosure provides a composition comprising (a) (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0112] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a temperature of at least 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0113] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0114] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0115] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state is viable in the composition.

[0116] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after being added to a feed or feed additive.

[0117] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0118] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a temperature of 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0119] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0120] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0121] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable after being added to a micromachine.

[0122] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids, and in some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 14 hours after being added to the microbin of a micromachine before being added to a feed truck.

[0123] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by one or more layers of lipids, and in some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 48 hours after being added to the microbin of a micromachine before being added to a feed truck.

[0124] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, just as each individual publication or patent application is specifically and individually stated to be incorporated by reference. Furthermore, any citation or identification of any reference in this application shall not be construed as an acknowledgment that such reference is available as prior art for the present invention.

[0125] definition

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of the people skilled in the art to which this invention pertains. In the event of a conflict, this application, including definitions, shall prevail. Unless otherwise required by the context, singular terms include plural forms and plural terms include singular forms.

[0127] If a section title is used, it must not be interpreted as restrictive.

[0128] As used in this application and the appended claims, the singular forms “one,” “one of,” and “he” include plural references unless the context clearly indicates otherwise. For example, the term “compound” or “at least one compound” may include plural compounds, including mixtures thereof. For example, the terms “one,” “one of,” “he,” “one or more,” and “at least one” may be used interchangeably herein.

[0129] As used herein, when used to modify quantities related to the present invention, the term “about” refers to a variation in quantity that may occur, for example, through routine testing and handling, through careless errors in such testing and handling, or through differences in the manufacture, source, or purity of the ingredients used in the present invention. Regardless of whether they are modified by the term “about,” the claims include equivalents of the cited quantities. In some embodiments, the term “about” means plus or minus 10% of the reported figure.

[0130] Throughout this application, various aspects of the invention may be presented in the form of a range. It should be understood that descriptions in the form of a range are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of the invention. Accordingly, descriptions of a range should be considered to include individual numerical values ​​within said ranges as well as all specifically disclosed possible sub-ranges. For example, a description of a range such as 1 to 6 should be considered to include individual numbers within said ranges, e.g., 1, 2, 3, 4, 5, and 6, as well as specifically disclosed sub-ranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, etc. This applies regardless of the width of the range.

[0131] The terms 'comprises', 'comprising', 'includes', 'including', 'having', and their variations are interchangeable and mean 'includes but is not limited thereto'. Wherever a mode is described in the language of 'comprising', it is understood that a similar mode described in the terms of 'constituting' and / or 'essentially constituting' is also provided.

[0132] The term 'constituting' means 'including and thereby limited'.

[0133] The term 'essentially constituting' means a designated step of a designated material or method of a composition and an additional material or step that does not have a significant effect on the basic properties of said material or method.

[0134] The term 'and / or' as used herein shall be construed as specifically disclosing each of two designated features or components together with or without the other feature or component. Accordingly, the term 'and / or' as used herein in phrases such as 'A and / or B' is intended to include 'A and B', 'A or B', 'A' (alone) and 'B' (alone). Likewise, the term 'and / or' as used in phrases such as 'A, B and / or C' is intended to encompass each of the following modes: A, B and C; A, B or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0135] As used herein, the terms 'culture,' 'culture for,' and 'culturing' mean incubating cells under in vitro conditions that allow for cell growth or division, or maintaining cells in a viable state. The term 'culture' may also be used herein to refer to cells incubated under in vitro conditions (e.g., cells incubated in a liquid growth medium).

[0136] As used herein, the term 'probiotic' refers to one or more viable microorganisms (bacteria and / or yeast) and may or may not include other ingredients that may provide health benefits to animals or subjects when administered in appropriate amounts.

[0137] As used herein, the term "direct-feeding microbial product" means a product containing one or more viable microorganisms (bacteria and / or yeast) and may or may not contain other ingredients that can be administered to an animal or subject as a feed mixture, bolus, and / or oral paste, and which can provide health benefits to an animal or subject when administered in appropriate amounts.

[0138] As used herein, the term "feed additive" refers to one or more ingredients, products, or substances (e.g., cells) used alone or together in the field of nutrition (e.g., to improve the quality of food (e.g., animal feed), to improve the performance and health of animals, and / or to improve the digestibility of food or substances within food). A feed additive may be, for example, a probiotic.

[0139] 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 containing components to support cell growth.

[0140] As used herein, the terms 'harvesting' and 'harvesting' refer to collecting cells from a culture, e.g., collecting cells in a growth medium from a culture, collecting cells by removing a certain amount of growth medium from the cells (e.g., by concentrating cells in a liquid culture or separating cells from the growth medium), or discontinuing the culture of the cells. The above terms include collecting or removing a certain amount of liquid containing cells from a liquid culture, which also includes a portion of the liquid in which the cells are concentrated.

[0141] As used herein, the term 'isolated' indicates isolation or separation from its original form or original environment, although it does not necessarily reflect the degree of purification of the isolate. The isolate may include, but is not limited to, isolated microorganisms, isolated biomass, or isolated cultures.

[0142] As used herein, the term “excipient” refers to a component or mixture of components used to impart desirable properties to a feed additive, food, composition, or pharmaceutical composition as disclosed herein. An excipient of the present invention may be described as a “pharmaceutically acceptable” excipient when added to a pharmaceutical composition, which means that the excipient is a compound, substance, composition, salt, and / or dosage form suitable for contact with tissues of animals (i.e., human and non-human animals) within the scope of sound medical judgment, without excessive toxicity, irritation, allergic reaction, or other problematic complications over a desired period of contact, in accordance with a reasonable benefit / risk ratio.

[0143] The term 'yield' as used herein refers to the amount of living or viable cells comprising a specific volume (e.g., colony-forming units per milliliter ('CFU / mL')) or a specific weight (e.g., CFU per gram ('CFU / g')).

[0144] As used herein, the term 'viable' refers to a living organism or organisms (e.g., a viable microbial cell or viable microbial cells). 'Viability' specifically refers to the ability to live under certain conditions.

[0145] As used herein, 'purify', 'purified', and 'purify' mean to make something substantially pure or clean from unwanted components, material contamination, mixtures, or imperfections.

[0146] As used herein, the terms ‘animal’ or ‘object’ refer to any organism belonging to the animal kingdom, and unless otherwise noted, aquatic and terrestrial animals, e.g., fish; commercial fish; ornamental fish; fry; bivalves; mollusks; crustaceans; shellfish; shrimp; shrimp larvae; Artemia; rotifers; brine shrimp; filter feeders; amphibians; reptiles; mammals; non-human animals; livestock; farm animals; zoo animals; sports animals; breeding livestock; racing animals; exhibition animals; hereditary animals; rare or endangered animals; companion animals; pets, e.g., dogs, cats, guinea pigs, rabbits, rats, mice, or horses; primates, e.g., monkeys (e.g., capuchin, rhesus, African green, patas, cynomolgus, and cercopithecus), apes, orangutans, baboons, gibbons, and chimpanzees; canids, e.g., dogs and wolves; Felidae animals, e.g., cats, lions, and tigers; equine animals, e.g., horses, ponies, donkeys, mules, and zebras; edible animals, e.g., cows, buffalo, cattle, pigs, poultry, and sheep; stray animals, e.g., deer and giraffes; birds (i.e., birds); poultry, e.g., chickens, geese, ducks, quails, turkeys, pigeons, emus, ostriches, and any other birds used as food or farm animals, including broilers, broiler breeders, and laying hens; rodents, e.g., mice, rats, hamsters, and guinea pigs; etc., but are not limited thereto. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal excludes a human subject. Animal feed includes, but is not limited to, aquaculture feed, livestock feed including pet food, zoo animal feed, working animal feed, livestock feed, and combinations thereof. In some embodiments, food includes animal feed and human food.

[0147] The term 'trophic state' used here refers to bacterial cells that actively metabolize nutrients and grow.

[0148] As used here, the term 'spore' refers to bacterial cells that do not grow or reproduce. This state helps bacteria survive in environments unfavorable for growth.

[0149] As used herein, the terms 'encapsulate', 'encapsulate', and 'encapsulated' refer to a trophozoite bacterial cell (e.g., Megasphaera elsdenii cell) forming at least one solid core surrounded by at least one continuous membrane or shell. In some embodiments, the core may be mixed with a carrier and coated with one or more lipids to form the core. In some embodiments, the core may be coated with at least one layer or one or more lipids.

[0150] For the sake of clarity, it is understood that specific features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of simplification, various features of the invention described in the context of a single embodiment may also be provided individually, in any suitable sub-combination, or as suitable in any other described embodiment of the invention. Specific features described in the context of various embodiments are not considered to be essential features of these embodiments, except where the embodiment would not operate without these elements.

[0151] Methods and materials similar or equivalent to those described herein may be used in the practice or instruction of the present invention, but suitable methods and materials are described below. Materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present invention will become apparent from the detailed description and claims.

[0152] Megaspaera Elsdeni

[0153] Cells of *Megasphaera elsdenii* (M. elsdenii) from any strain or any combination of strains may be used in the disclosures described herein.

[0154] M. elsdeny strains or strains may be selected from archived culture collections (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 collections (i.e., 'NRRL'), National Institute of Animal Health (NIAH) culture collections) or may be strains isolated from natural sources (e.g., from the gastrointestinal tract of ruminants).

[0155] Examples of M. elsdeni strains that can be selected from the culture collection are Table 1 Includes, but is not limited to, the strains listed by deposit number. Alternative names of deposit numbers also appear.

[0156] Table 1. Examples of M. elsdeni strains and sources for each strain.

[0157]

[0158] In some embodiments, M. elsdeny 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, CBS146325, CBS146326, CBS146327, CBS146328, CBS146329, CBS146330, including any of the alternative names in Table 1 or any commercially available source of M. elsdeny.

[0159] In some embodiments, M. elsdeny cells are derived from strains isolated from ruminants (e.g., cattle). See, for example, U.S. Patent No. 7,550,139.

[0160] In some embodiments, M. elsdeny cells are derived from strains isolated from non-ruminants (e.g., humans).

[0161] In some embodiments, M. elsdenyi cells are derived from strains selected for lactate utilization (e.g., strains utilizing lactate in the presence of sugars), resistance to ionophore antibiotics, relatively high growth rate, ability to produce mainly acetate, ability to proliferate at low pH values ​​of less than 5.0 and around 4.5, production of volatile fatty acids (VFAs), phytase activity, and combinations thereof. For example, see U.S. Patent No. 7,550,139.

[0162] In some embodiments, the strain selected for lactate utilization utilizes 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.

[0163] In some embodiments, M. elsdeny cells are derived from strains having a high growth rate compared to other strains. The growth rates of different strains can be determined, for example, by culturing cells in a liquid medium and monitoring the increase in optical density over time.

[0164] In some embodiments, M. elsdenyi cells originate from strains having phytase activity.

[0165] In some embodiments, M. elsdeni cells are derived from the Megaspaera elsdeni strain NCIMB 41125. This strain of Megaspaera elsdeni has a high specific growth rate (0.94 generations / hour), can grow in a pH range of 4.5 to 6.5 or higher, uses D- and L-lactate as its preferred substrates, but also has the ability to utilize glucose and other carbohydrates, and is resistant to ionophores.

[0166] In some embodiments, M. elsdeni cells are derived from Megaspaera elsdeni strain NCIMB 41787. In some embodiments, M. elsdeni cells are derived from Megaspaera elsdeni strain NCIMB 41788.

[0167] In some embodiments, M. elsdeni cells are derived from the Megaspaera elsdeni strain ATCC® 25940.

[0168] In some embodiments, M. elsdeny cells are selected from a collection of stored cultures or derived from strains isolated from natural sources. Cells 'derived' from the strain may be natural or artificial derivatives, such as, for example, sub-isolates, mutants, variants, or recombinant strains.

[0169] In some embodiments, M. Elsdeny is freeze-dried. See WO 2018 / 144653 A1, the entirety of which is incorporated herein by reference.

[0170] Preparation of a culture containing anaerobic bacterial cells or Megasphaera elsdenii cells

[0171] Anaerobic bacterial cells, including M. elsdeni, must be cultured under anaerobic conditions to obtain maximum yield and viability.

[0172] In some embodiments, the culture comprises M. elsdeni cells and a growth medium.

[0173] In some embodiments, the culture comprises one or more strains of M. elsdenyi cells. In some embodiments, the culture comprises a single strain of M. elsdenyi cells. In some embodiments, the culture consists of one or more strains of M. elsdenyi cells (i.e., the cells in the culture consist of M. elsdenyi cells, for example, one or more strains of M. elsdenyi cells). In some embodiments, the culture consists of a single strain of M. elsdenyi cells.

[0174] In some embodiments, the culture comprises one or more strains of anaerobic bacterial cells and a growth medium. In some embodiments, the culture comprises Bifidobacterium cells, e.g., B. breve; Lactobacillus cells, e.g., L. plantarum; Bifidobacterium cells, e.g., B. animalis subs. lactis; Pediococcus cells, e.g., P. acidilactis; Lactobacillus cells, e.g., L. casei; Fibrobacter, e.g., F. succinogenes; and Butyrivbrio, e.g., B. fibrisolvens; Luminococcus, e.g., Luminococcus flavefaciens; Blautia cells, e.g., B. obeum; Clostridium cells, e.g., C. butyricum; Achermansia cells, e.g., A. musiniphila; and a growth medium.

[0175] Various fermentation parameters may be used to inoculate, grow, and harvest anaerobic bacterial cells (e.g., M. elsdenyi cells), including continuous fermentation (i.e., continuous culture) or batch fermentation (i.e., batch culture). For example, refer to U.S. Patent No. 7,550,139.

[0176] Growth media for anaerobic bacterial cells (e.g., M. elsdenyi cells) can be solid, semi-solid, or liquid. The medium may contain nutrients that provide essential elements and specific factors enabling growth. Various microbial media and their modifications are well known in the field. The medium can be added to the culture at any time, including at the start of the culture, during the culture, or in an intermittent or continuous manner.

[0177] Examples of growth media include (1) a semi-limited medium containing 3 g / L peptone, 3 g / L yeast, 2 mL / L vitamin solution, 25 mL / L mineral solution, 1 g / L indigo carmine (0.5%), 2 g / L 12.5% ​​L-cysteine, and 2 g / L 12.5% ​​sodium sulfide, supplemented with Na-lactate (semi-limited lactate, SDL), glucose (semi-limited glucose, SDG) or maltose (semi-limited maltose, SDM), (2) 10 g / L peptone, 10 g / L beef extract, 3 g / L yeast extract, 5 g / L dextrose, 5 g / L NaCl, 1 g / L soluble starch, 0.5 g / L L-cysteine ​​HCl, 3 g / L sodium acetate, and 4 mL / L resazurin (0.025%) Modified fortified Clostridium agar / broth medium containing (pre-reduced), (3) trypticase soybean agar / broth medium with defibrinated sheep blood, (4) Na-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, thiaminium chloride 4 mg / l, nicotinamide 4 mg / l, Ca-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 / l), Na2S·9H2O 0.25 g / l, cysteine ​​0.25 g / l, antifoaming agent 0.A semi-limited rumen-free medium prepared by adding Na-lactate and mineral solution containing 0.7 ml / l and 10 mg / l monensin to a storage bottle and autoclaving for 60 minutes, dissolving peptone in 300 ml of distilled H2O and autoclaving separately, pre-filtering and sterilizing the vitamin solution and two reducing agents, injecting anaerobic gas into the storage bottle overnight after autoclaving, adding other components separately after cooling, and adjusting the pH to the desired value with 5N HCl, and (5) 400 ml of incubated purified rumen from an alfalfa-fed animal, 371 ml of distilled water, 2 g of peptone, 15 g of agar, 100 ml of 10% (w / v) sodium-D, L-lactate solution, 100 ml of 0.04% (w / v) bromocresol violet solution and 40 g / l It comprises, but is not limited to, an incubated rumen fluid lactate ('IRFL') medium containing 25 ml of a mineral solution containing KH2PO4; 120 g / l (NH4)2SO4; 8 g / l MgSO4·7H2O and 2.4 g / l CaCl2·2H2O, lactic acid (90% w / v) is used to adjust the pH to 5.5 before autoclaving at 121 °C for 25 minutes, then cooled in a 50 °C water bath while being gasmed with an anaerobic gas mixture, and then 2 ml each of Na2S·9H2O (12.5% ​​w / v) and cysteine·HCl·H2O (12.5% ​​w / v) are added.

[0178] In some embodiments, the culture comprises a growth medium comprising at least two carbon sources. In some embodiments, 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, fat, oil, glycerol, sodium acetate, arabinose, soy protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof.

[0179] In some embodiments, at least two carbon sources are composed of about 1 to 99% of a first carbon source (e.g., any carbon source described herein) and about 1 to 99% of a second carbon source (e.g., any carbon source described herein different from the first carbon source), and 100% of at least two carbon sources is composed of the first carbon source and the second carbon source. In some embodiments, at least two carbon sources are composed of about 50 to 60% of a first carbon source and about 40 to 50% of a second carbon source, about 50 to 70% of a first carbon source and about 30 to 50% of a second carbon source, about 50 to 80% of a first carbon source and about 20 to 50% of a second carbon source, or about 50 to 90% of a first carbon source and about 10 to 50% of a second carbon source. In some embodiments, at least two carbon sources consist of a first carbon source of about 65 to 75% and a second carbon source of about 25 to 35%. In some embodiments, the first carbon source is lactate.

[0180] In some embodiments, anaerobic bacterial cells (e.g., M. elsdenyi 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.

[0181] In some embodiments, anaerobic bacterial cells (e.g., M. elsdeny cells) grow at about 35 °C to about 40 °C.

[0182] To culture anaerobic bacterial cells (e.g., M. elsdeny cells), fermenters of different sizes and designs that maintain anaerobic conditions can be used. The fermenter can ferment a culture volume sufficient for the commercial production of, for example, anaerobic cells (e.g., M. elsdeny cells). In some embodiments, the culture volume is approximately 2 liters, approximately 10 liters, approximately 50 liters, approximately 100 liters, approximately 150 liters, approximately 200 liters, approximately 250 liters, approximately 300 liters, approximately 350 liters, approximately 400 liters, approximately 450 liters, approximately 500 liters, approximately 600 liters, approximately 800 liters, approximately 1,000 liters, approximately 1,200 liters, approximately 1,500 liters, approximately 1,800 liters, approximately 2,000 liters, approximately 2,200 liters, approximately 2,500 liters, approximately 2,750 liters, approximately 3,000 liters, approximately 4,000 liters, approximately 5,000 liters, approximately 6,000 liters, approximately 7,000 liters, It is 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 about 2 liters to about 75,000 liters, about 250 liters to about 750 liters, about 300 liters to about 800 liters, about 350 liters to about 850 liters, about 400 liters to about 900 liters, about 450 liters to about 950 liters, about 500 liters to about 1,000 liters, about 750 liters to about 1,250 liters, about 1,000 liters to about 2,000 liters, about 2,000 liters to about 4,000 liters, about 4,000 liters to about 8,000 liters, about 5,000 liters to about 10,000 liters, about 50 liters to about 75,000 liters, about 50 liters to about 50,000 liters, about 50 liters to about 25,000 liters, about 50 liters to about 20,000 liters, about 50 liters to about 15,000 liters, about 50 liters to about 10,000 liters, about 100 liters to about 10,000 liters, about 100 liters to about 5,000 liters, about 100 liters to about 4,000 liters, about 100 liters to about 3,000 liters, about 100 liters to about 2,900 liters, about 100 liters to about 2,850 liters, about 100 liters to about 2,800 liters, about 100 liters to about 2,750 liters, about 2 liters, about 10 liters, about 50 It is 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.

[0183] In some embodiments, the culture comprises a liquid, and the method includes the step of harvesting anaerobic bacterial cells (e.g., M. elsdenyi cells) by removing a certain proportion of the liquid. In some embodiments, the cell harvest is about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100% 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 It includes removing 95% to about 100% of the liquid. In some embodiments, cell harvesting includes removing at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, 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.

[0184] In some embodiments, the method comprises the step of harvesting anaerobic bacterial cells (e.g., M. elsdenyi cells) by concentrating the cells. In some embodiments, cell harvesting comprises 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, filtration comprises clay filtration. In some embodiments, filtration comprises tangential flow filtration, also known as cross-flow filtration.

[0185] In some embodiments, the pH of a culture containing anaerobic bacterial cells (e.g., M. elsdeny cells) 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.

[0186] In some embodiments, the method comprises the step of preparing a culture by inoculating an inoculum containing anaerobic bacterial cells (e.g., M. elsdeny cells) into the culture medium of a fermenter, 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 anaerobic bacterial cells (e.g., M. elsdeny cells) is a flask culture of anaerobic cells (e.g., M. elsdeny cells) or a portion thereof. In some embodiments, the method comprises the step of inoculating a growth medium in a fermenter at an inoculum-to-medium ratio of 1 / 50 to 1 / 4,000. In some embodiments, the inoculum-to-medium ratio is 1 / 100.

[0187] Encapsulated anaerobic bacteria and Megasphaera elsdenii cells

[0188] To help animals (e.g., cattle) avoid pathological conditions associated with changing their diet from a roughage-based diet to a high-starch diet, Megasphaera elsdenii cells can be administered to the animals. Since anaerobic bacterial cells and Megasphaera elsdenii cells cannot survive in the presence of oxygen, it is difficult to maintain viability until the cells can be administered. Accordingly, the present disclosure provides an encapsulated formulation that maintains the viability of cells for storage at various temperatures (e.g., about -20 °C, about 4 °C, about 25 °C, or about 52 °C), various pH levels (e.g., about 4.0, about 4.3, about 4.5, about 4.7, about 5, about 5.2, about 5.4, about 5.6, about 5.8, or about 6.0), and various moisture levels (e.g., about 5%, about 9%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%).

[0189] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells are viable in the composition.

[0190] In some embodiments, the composition disclosed herein is formulated to be delivered to and released from the rumen of a ruminant.

[0191] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable after being added to a feed or feed additive.

[0192] In some embodiments, Megasphaera elsdenii cells in the composition can survive for up to 48 hours at 25 °C and pH 7.0 after being added to feed or feed additives.

[0193] In some embodiments, the present disclosure provides a composition comprising (a) (i) a Megasphaera elsdenii cell, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0194] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a temperature of at least 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0195] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0196] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0197] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable after processing through a micromachine system.

[0198] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for up to 14 hours after being added to the microbin of a micromachine.

[0199] In some embodiments, the present disclosure provides a composition comprising (a) (i) Megasphaera elsdenii cells, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for up to 48 hours after being added to the microbin of a micromachine.

[0200] In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for about 14 to about 48 hours after being added to the microbins of the micromachine.

[0201] In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after being added to the microbin of the micromachine.

[0202] In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are viable for at least about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after being added to the microbin of the micromachine.

[0203] In some embodiments, at least 10% of the Megasphaera elsdenii cells in the composition are added to the microbin of a micromachine and then for about 1 hour to about 48 hours, about 2 hours to about 48 hours, about 4 hours to about 48 hours, about 6 hours to about 48 hours, about 8 hours to about 48 hours, about 10 hours to about 48 hours, about 12 hours to about 48 hours, about 14 hours to about 48 hours, about 16 hours to about 48 hours, about 18 hours to about 48 hours, about 20 hours to about 48 hours, about 22 hours to about 48 hours, about 24 hours to about 48 hours, about 26 hours to about 48 hours, about 28 hours to about 48 hours, about 30 hours to about 48 hours, about 32 hours to about 48 hours, about 34 hours to about 48 hours, about Survival is possible for 36 to about 48 hours, about 38 to about 48 hours, about 40 to about 48 hours, about 42 to about 48 hours, about 44 to about 48 hours, about 46 to about 48 hours, or about 48 hours.

[0204] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state is viable in the composition.

[0205] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after being added to a feed or feed additive.

[0206] In some embodiments, anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition can survive for up to 48 hours at 25 °C and pH 7.0 after being added to feed or feed additives.

[0207] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen.

[0208] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a temperature of 40 °C to 60 °C at pH 7.0 for 4 to 18 hours.

[0209] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a pH of 3 to 7 at a temperature of 25 °C for up to 48 hours.

[0210] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours.

[0211] In some embodiments, the present disclosure provides a composition comprising (a) (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by a layer of one or more lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after being added to a micromachine.

[0212] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier, (iii) a core comprising one or more lipids, and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 14 hours after being added to the microbin of a micromachine. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 14 hours after being added to the microbin of a micromachine before being added to a feed truck.

[0213] In some embodiments, the present disclosure provides a composition comprising (a) (i) anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids. In some embodiments, the core is coated by one or more layers of lipids. In some embodiments, the core is coated by a single layer of one or more lipids. In some embodiments, the core is coated by two or more layers of one or more lipids. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 48 hours after being added to the microbin of a micromachine. In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to 48 hours after being added to the microbin of a micromachine before being added to a feed truck.

[0214] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for about 14 to about 48 hours after being added to the microbin of the micromachine before being added to the feed truck.

[0215] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for up to about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after being added to the microbin of the micromachine.

[0216] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for at least about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after being added to the microbin of the micromachine.

[0217] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after being added to the microbin of the micromachine before being added to the feed truck.

[0218] In some embodiments, at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are added to the microbin of a micromachine before being added to a feed truck, and then for about 1 hour to about 48 hours, about 2 hours to about 48 hours, about 4 hours to about 48 hours, about 6 hours to about 48 hours, about 8 hours to about 48 hours, about 10 hours to about 48 hours, about 12 hours to about 48 hours, about 14 hours to about 48 hours, about 16 hours to about 48 hours, about 18 hours to about 48 hours, about 20 hours to about 48 hours, about 22 hours to about 48 hours, about 24 hours to about 48 hours, about 26 hours to about 48 hours, about 28 hours to about 48 hours, about 30 hours to about 48 hours, about 32 hours to about 48 hours, about Survival is possible for 34 to about 48 hours, about 36 to about 48 hours, about 38 to about 48 hours, about 40 to about 48 hours, about 42 to about 48 hours, about 44 to about 48 hours, about 46 to about 48 hours, or about 48 hours.

[0219] In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1600 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1500 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1400 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1300 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1200 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1100 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 1000 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 900 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 800 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 700 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 600 μm.In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 500 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 400 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 300 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 200 μm. In some embodiments, the particle size of the Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in the composition is less than about 100 μm.

[0220] In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1600 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1500 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1400 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1300 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1200 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1100 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 1000 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 900 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 800 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 700 μm.In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 600 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 500 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 400 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 300 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 200 μm. In some embodiments, the particle size of freeze-dried Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state in the composition is less than about 100 μm.

[0221] In some embodiments, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state are viable in the composition.

[0222] In some embodiments, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are viable in the composition. In some embodiments, about 15% to about 99%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 20%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or about 30% to about 60% of Megasphaera elsdeni cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state are viable in the composition.

[0223] In some embodiments, Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state are dried.

[0224] In some embodiments, Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state are dried by spray drying, electrospray drying, vacuum drying, jet drying, freeze drying, or a combination thereof.

[0225] In some embodiments, the composition comprises about 0.1% to about 15% (w / w) of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0226] In some embodiments, the composition is about 0.1% to about 15% (w / w), about 1% to about 15% (w / w), about 2% to about 15% (w / w), about 3% to about 15% (w / w), about 4% to about 15% (w / w), about 5% to about 15% (w / w), about 6% to about 15% (w / w), about 7% to about 15% (w / w), about 8% to about 15% (w / w), about 9% to about 15% (w / w), about 10% to about 15% (w / w), about 11% to about 15% (w / w), about 12% to about 15% (w / w), about 13% to about 15% (w / w), about 14% to about 15% (w / w), about 0.1% to It contains about 10% (w / w), about 1% to about 10% (w / w), about 5% to about 10% (w / w), about 0.1% to about 5% (w / w), about 1% to about 5% (w / w), or about 0.1% to about 1% (w / w) of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0227] In some embodiments, the composition comprises at least about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / w) of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0228] In some embodiments, the composition is about 1 x 10 per gram. 4 to about 1 x 10 10 It includes CFU of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0229] In some embodiments, the composition is about 1 x 10 per gram.4 to about 1 x 10 10 CFU, approximately 1 x 10⁶ per gram 5 to about 1 x 10 10 CFU, approximately 1 x 10⁶ per gram 6 to about 1 x 10 10 CFU, approximately 1 x 10⁶ per gram 7 to about 1 x 10 10 CFU, approximately 1 x 10⁶ per gram 8 to about 1 x 10 10 CFU, approximately 1 x 10⁶ per gram 9 to about 1 x 10 10 CFU, 1 x 10⁶ per g 4 to about 1 x 10 9 CFU, 1 x 10⁶ per g 4 to about 1 x 10 8 CFU, 1 x 10⁶ per g 4 to about 1 x 10 7 CFU, 1 x 10⁶ per g 4 to about 1 x 10 6 CFU, 1 x 10⁶ per g 4 to about 1 x 10 5 or 1 x 10 6 to about 1 x 10 8 It includes CFU of Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0230] In some embodiments, the composition is about 1 x 10 per gram. 4 CFU, approximately 1 x 10⁶ per gram 5 CFU, approximately 1 x 10⁶ per gram 6 CFU, approximately 1 x 10⁶ per gram 7 CFU, approximately 1 x 10⁶ per gram 8 CFU, approximately 1 x 10⁶ per gram 9 Approximately 1 x 10⁶ per CFU or gram 10 It includes CFU megagaspaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state.

[0231] In some embodiments, the anaerobic bacteria or anaerobic bacterial cells in a trophic state are selected from the group consisting of Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subs. lactis, Pediococcus acidilactis, Lactobacillus casei, Megasphaera elsdenii, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Luminococcus flavefaciens, Blautia obeum, Clostridium butyricum, Achermansia musiniphila, and combinations thereof.

[0232] In some embodiments, the composition is a granule, capsule, minicapsule, microcapsule, tablet, minitablet, or microtablet.

[0233] In some embodiments, the composition has a moisture content of about 5% (w / w) or less.

[0234] In some embodiments, the composition has a moisture content of about 5% (w / w), about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1%.

[0235] In some embodiments, one or more lipids of the core are selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, pewter, vegetable waxes, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic waxes, natural and synthetic resins and mixtures thereof.

[0236] In some embodiments, one or more lipids of the core are animal fats or oils and / or vegetable fats or oils.

[0237] In some embodiments, the vegetable fat or oil is selected from the group consisting of canola oil, cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil.

[0238] In some embodiments, the vegetable fat or oil is hydrogenated palm oil.

[0239] In some embodiments, the free fatty acid is myristic acid, lauric acid, or stearic acid or a combination thereof.

[0240] In some embodiments, Megasphaera elsdenii, anaerobic bacteria, or anaerobic bacterial cells in a trophic state may be encapsulated using one or more lipids disclosed herein by a spinning disc encapsulation method to form a core. Using the spinning disc encapsulation method, the spraying of droplets having a narrow size range can produce a core of cells coated with one or more lipids. Droplet formation depends on the fluid flow velocity ejected from the nozzle. Droplet formation may occur directly from the alignment of the edge of the spinning disc with the liquid flow, or indirectly through the dispersion of strands or sheets generated from the fluid flow over the spinning disc. In the spinning disc encapsulation process, many parameters must be considered, including liquid flow velocity, viscosity, and mass transfer phenomena from the microcapsules.

[0241] In some embodiments, a spinning disc device comprises a spinning disc / cup that rotates about its central axis to form a droplet and sprays the droplet radially outward, and a collection container(s) that surrounds the spinning cup and collects the core beads sprayed from the cup.

[0242] In some embodiments, on the day of treatment, maltodextrin (5 parts) is mixed with hydrogenated palm oil (HPO) (coating lipid; 11.2 parts) at 70 to 80 °C until a homogeneous mixture is obtained. Then, dried powder (1 part) (e.g., freeze-dried powder or electrospray-dried powder) (1 part) is added to the mixture (maltodextrin and HPO) at a temperature of 60 to 65 °C and flowed through a spinning disc / cup so that droplets are ejected radially outward. The droplets solidify immediately while cooling to below 55 °C to form a core (FD M. Elsdeny, maltodextrin and HPO), and the core beads are collected in a collection container.

[0243] In some embodiments, one or more lipids of the core have a melting point of about 40 °C to about 85 °C.

[0244] In some embodiments, one or more lipids of the core have a melting point of about 55 °C to about 75 °C.

[0245] In some embodiments, one or more lipids of the core are at about 40 °C to about 85 °C, about 45 °C to about 85 °C, about 50 °C to about 85 °C, about 55 °C to about 85 °C, about 60 °C to about 85 °C, about 65 °C to about 85 °C, about 70 °C to about 85 °C, about 75 °C to about 85 °C, about 80 °C to about 85 °C, about 55 °C to about 75 °C, about 60 °C to about 75 °C, about 65 °C to about 75 °C, about 70 °C to about 75 °C, about 40 °C to about 80 °C, about 40 °C to about 75 °C, about 40 °C to about 70 °C, about 40 °C to about 65 °C It has a melting point of °C, about 40 °C to about 60 °C, about 40 °C to about 55 °C, about 40 °C to about 50 °C, about 40 °C to about 45 °C, about 55 °C to about 70 °C, about 55 °C to about 65 °C, or about 55 °C to about 60 °C.

[0246] In some embodiments, one or more lipids of the core have a melting point of about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, or about 85 °C.

[0247] In some embodiments, one or more lipids coating the core are selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, pewter, vegetable waxes, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic waxes, natural and synthetic resins and mixtures thereof.

[0248] In some embodiments, one or more lipids coating the core are animal fats or oils and / or vegetable fats or oils.

[0249] In some embodiments, the vegetable fat or oil is selected from the group consisting of cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil.

[0250] In some embodiments, the vegetable fat or oil is hydrogenated palm oil.

[0251] In some embodiments, the free fatty acid is myristic acid, lauric acid, or stearic acid.

[0252] In some embodiments, the core of Megasphaera elsdenii, anaerobic bacteria, or anaerobic bacterial cells in a trophic state may be encapsulated in a Wurster fluid bed aggregation coater using one or more lipids disclosed herein. The Wurster technology is characterized by a spray nozzle located at the bottom of the fluid bed. Particles are moved along with a fluidizing airflow designed to induce upward circulation of the particles through the spray nozzle. The nozzle sprays a drop of the coating solution or suspension simultaneously with the particle flow, depositing the droplets onto the surface of the particles as they pass upward into the expansion chamber. This expansion chamber reduces the air velocity, causing the particles to recirculate back into the coating chamber. It also allows the particles to be temporarily separated from one another, thereby minimizing the possibility of particle aggregation and adhesion. As the particles move into and through the expansion chamber, the organic or aqueous coating solution evaporates, leaving non-volatile coating formulation components on the particle surface as part of the film coating being formed. Process parameters are designed for optimal solution evaporation and film coating characteristics. This batch process continues until each particle is uniformly coated to the desired coating percentage or film thickness. Additionally, the Wurster fluidized bed process can be used to apply hot-melt coatings such as lipids. The lipids are heated to a molten state and sprayed in the same manner as a solution suspension. Process parameters are adjusted to aggregate molten lipid droplets onto the surface of the circulating particles.

[0253] In some embodiments, the Wurster fluid bed aggregation coater has a special feature having a spray nozzle located at the bottom of a cylindrical Wurster tube within a chamber. Core particles (beads) are driven from the fluid bed into the cylindrical tube by an airflow difference and move in a circulating motion through the chamber, creating a core-shell structure by periodically crossing a spray zone where they collide with small droplets of the coating solution (hydrogenated cottonseed oil ['HCO']).

[0254] In some embodiments, the collected core beads are then spray-coated with cured cottonseed oil (HCO) in a Wurster fluid bed aggregation coater to obtain the final M. Elsdeny product. The core beads are driven from the fluid bed into a cylindrical Wurster tube by an air flow difference and move in a circulating motion through the chamber, periodically crossing a spray zone where they collide with small droplets of the coating solution (HCO) to create a core-shell structure (core 17.24 parts and coating 5.75 parts).

[0255] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C to about 80 °C.

[0256] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C to about 75 °C.

[0257] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C to about 80 °C, about 60 °C to about 80 °C, 65 °C to about 80 °C, 70 °C to about 80 °C, 75 °C to about 80 °C, 55 °C to about 75 °C, 55 °C to about 70 °C, 55 °C to about 65 °C, 55 °C to about 60 °C, about 55 °C to about 75 °C, about 60 °C to about 75 °C, about 65 °C to about 75 °C, about 70 °C to about 75 °C, about 55 °C to about 70 °C, about 55 °C to about 65 °C, or about 55 °C to about 60 °C.

[0258] In some embodiments, one or more lipids coating the core have a melting point of about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, or about 80 °C.

[0259] In some embodiments, at least one carrier comprises maltodextrin, sucrose, starch, cellulose, clay, biochar, lignin derivatives, sugar alcohols, or combinations thereof.

[0260] In some embodiments, the composition contains about 10% to about 99% (w / w) of total lipids.

[0261] In some embodiments, the composition comprises about 70% to about 80% (w / w) of total lipids. In some embodiments, the composition comprises about 60% to about 80% (w / w) of total lipids.

[0262] In some embodiments, the composition comprises a total of about 10% to about 99% (w / w) lipids, about 20% to about 99% (w / w) lipids, about 30% to about 99% (w / w) lipids, about 40% to about 99% (w / w) lipids, about 50% to about 99% (w / w) lipids, about 60% to about 99% (w / w) lipids, about 70% to about 99% (w / w) lipids, about 80% to about 99% (w / w) lipids, about 90% to about 99% (w / w) lipids, about 10% to about 90% (w / w) lipids, about 10% to about 80% (w / w) lipids, about 10% to about 70% (w / w) lipids, and about 10% to about 60 It includes % (w / w) lipids, about 10% to about 50% (w / w) lipids, about 10% to about 40% (w) lipids, about 10% to about 30% (w / w) lipids, about 10% to about 20% (w / w) lipids, about 40% to about 60% (w / w) lipids, about 30% to about 70% (w / w) lipids, or 20% to about 80% (w / w) lipids.

[0263] In some embodiments, the composition comprises a total of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% (w / w) of lipids. In some embodiments, the composition comprises a total of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% (w / w) of lipids.

[0264] In some embodiments, the composition comprises at least one carrier of about 10% to about 30% (w / w).

[0265] In some embodiments, the composition comprises at least one carrier of about 15% to about 25% (w / w).

[0266] In some embodiments, the composition comprises at least one carrier of about 10% to about 30% (w / w), about 15% to about 30% (w / w), about 20% to about 30% (w / w), about 25% to about 30% (w / w), about 10% to about 25% (w / w), about 10% to about 20% (w / w), about 10% to about 15% (w / w), about 15% to about 25% (w / w), about 20% to about 25% (w / w), or about 15% to about 20% (w / w).

[0267] In some embodiments, the composition has a diameter of about 0.1 mm to about 3 mm.

[0268] In some embodiments, the composition has a diameter of about 0.2 mm to about 0.6 mm.

[0269] In some embodiments, the composition has a diameter of about 0.2 mm to about 0.4 mm.

[0270] In some embodiments, the composition has a diameter size of about 0.1 mm to about 3 mm, about 0.2 mm to about 3 mm, about 0.5 mm to about 3 mm, about 1 mm to about 3 mm, about 1.5 mm to about 3 mm, about 2 mm to about 3 mm, about 2.5 mm to about 3 mm, about 0.1 mm to about 2.5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1.5 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.9 mm, about 0.1 mm to about 0.8 mm, about 0.1 mm to about 0.7 mm, about 0.1 mm to about 0.6 mm, about 0.1 mm to about 0.5 mm, about 0.1 mm to about 0.4 mm, about 0.1 mm to about 0.3 mm, about 0.1 mm to It is about 0.2 mm, about 0.2 mm to about 3 mm, about 0.2 mm to about 2.5 mm, about 0.2 mm to about 2 mm, about 0.2 mm to about 1.5 mm, about 0.2 mm to about 1 mm, about 0.2 mm to about 0.9 mm, about 0.2 mm to about 0.8 mm, about 0.2 mm to about 0.7 mm, about 0.2 mm to about 0.6 mm, about 0.2 mm to about 0.5 mm, about 0.2 mm to about 0.4 mm, or about 0.2 mm to about 0.3 mm.

[0271] In some aspects, the composition has a diameter size of about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, or about 3 mm.

[0272] In some embodiments, the core comprises about 1% to about 99% of the composition.

[0273] In some embodiments, the core comprises about 10% to about 90% of the composition.

[0274] In some embodiments, the core comprises about 25% to about 80% of the composition.

[0275] In some embodiments, the core comprises at least about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 25% to about 80%, about 25% to about 70%, about 25% to about 60%, about 25% to about 50%, about 25% to about It includes 40%, about 25% to about 30%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%. In some embodiments, the core comprises at least 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 25% to 80%, 25% to 70%, 25% to 60%, 25% to 50%, 25% to 40%, 25% to 30%, 30% to 80%, 40% to 80%, 50% to It includes 80%, 60% to 80%, or 70% to 80%.

[0276] In some embodiments, the core comprises at least about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the composition. In some embodiments, the core comprises at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the composition.

[0277] In some embodiments, one or more lipids coating the core comprise about 1% to about 99% of the composition.

[0278] In some embodiments, one or more lipids coating the core comprise about 5% to about 75% of the composition.

[0279] In some embodiments, one or more lipids coating the core are about 1% to about 99%, about 5% to about 99%, about 10% to about 99%, about 20% to about 99%, about 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 80% to about 99%, about 90% to about 99%, about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about It comprises 1% to about 5%, about 5% to about 75% of the composition, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, or about 5% to about 10%.

[0280] In some embodiments, one or more lipids coating the core comprise at least about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the composition. In some embodiments, one or more lipids coating the core comprise at least 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the composition.

[0281] In some embodiments, the composition has a density of about 0.6 g / mL to about 1.2 g / mL.

[0282] In some embodiments, the composition has a density of about 0.6 g / mL, about 0.7 g / mL, about 0.8 g / mL, about 0.9 g / mL, about 1.0 g / mL, about 1.1 g / mL, or about 1.2 g / mL.

[0283] In some embodiments, the composition has a porosity of about 10% to about 60%.

[0284] In some embodiments, the composition has a porosity of about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 30% to about 40%, or about 20% to about 50%.

[0285] In some embodiments, the composition has a porosity of at least about 10%, about 20%, about 30%, about 40%, about 50%, or about 60%. In some embodiments, the composition has a porosity of at least 10%, 20%, 30%, 40%, 50%, or 60%.

[0286] In some embodiments, the composition further comprises one or more of antibiotics, antimicrobial agents, anticoccidial agents, antiparasitic agents, sulfonamides, hormones, anti-bloat compounds, adrenergic receptor modulators, phages, prebiotics, probiotics, enzymes, essential oils, and / or carbohydrate immunostimulators.

[0287] Feed additives, feed, premixes, and kits

[0288] In some embodiments, a feed additive composition comprising any of the compositions disclosed herein is provided herein.

[0289] In some embodiments, the feed additive composition is a powder, fine particles, pellets, cake, liquid, solid, suspension, emulsion, gel, or a combination thereof.

[0290] In some embodiments, a feed comprising any of the compositions disclosed herein or any of the feed additive compositions disclosed herein is provided herein.

[0291] In some embodiments, the feed further comprises animal protein, vegetable protein, corn, soybean meal, corn distiller's dried grains (cDDGS), wheat, wheat protein, gluten, wheat byproduct, wheat bran, wheat distiller's dried grains (wDDGS), corn byproduct including corn gluten meal, barley, oats, rye, triticale, whole soybeans, animal byproduct meal, alcohol-soluble protein, zein, corn zein, caphyrin, rice, paddy rice, extruded paddy rice, oily seed protein, or a combination thereof.

[0292] In some embodiments, the animal protein or plant protein is selected from the group consisting of one or more of gliadin or an immunogenic fragment of gliadin, beta-casein, beta-lactoglobulin, glycinin, beta-conglycinin, cruciferin, nafin, hordain, keratin, feather meal or cauliflower meal, collagen, whey protein, fish protein, fish meal, meat protein, egg protein, soy protein and grain protein.

[0293] In some embodiments, the oily seed protein is selected from the group consisting of soybean seed protein, sunflower seed protein, rapeseed protein, canola seed protein, and combinations thereof.

[0294] In some embodiments, a premix comprising a) any of the compositions disclosed herein or any of the feed additive compositions disclosed herein and b) at least one mineral and / or at least one vitamin is provided herein.

[0295] In some embodiments, a kit is provided herein comprising a) i) any of the compositions provided herein, ii) any of the feed additive compositions provided herein, iii) any of the feeds provided herein and / or iv) any of the premixes provided herein and b) instructions for formulation and / or administration to a subject.

[0296] In some embodiments, the feed additive comprises encapsulated anaerobic cells and / or M. elsdenyi cells as disclosed herein. In some embodiments, the feed additive comprises encapsulated freeze-dried anaerobic cells and / or M. elsdenyi cells produced by the method disclosed herein.

[0297] In some embodiments, the feed additive is a solid (i.e., 'solid feed additive') or a liquid (i.e., 'liquid feed additive'). In some embodiments, the feed additive is a semi-solid or a gel (i.e. , It is a 'semi-solid or gel feed additive'. The gel feed additive may contain an oxygen scavenger (e.g., ascorbic acid). In some embodiments, the feed additive is sprayed onto the animal's hair (i.e. , 'Spray-type additive').

[0298] In some embodiments, the solid feed additive is a powder (e.g., fluid powder), granules (i.e., fine particles), particles (i.e., fine particles), pellets, cakes, water-soluble concentrates, pastes, boluses, tablets, dusts, components thereof, or combinations thereof.

[0299] In some embodiments, the liquid feed additive is a solution (e.g., aqueous, organic, or aqueous-organic solution), suspension, emulsion, drench, spray, injectable, beverage (e.g., milk substitute), a component thereof, or a combination thereof.

[0300] In some embodiments, the gel feed additive is an organic gel. In some embodiments, the gel feed additive is an oral gel (i.e., a gel for oral administration).

[0301] In some embodiments, the feed additive is intended for top application (i.e., to be added to the surface of food or mixed with food (e.g., animal feed). In some embodiments, the feed additive is intended for administration as a liquid.

[0302] In some embodiments, encapsulated anaerobic cells as disclosed herein (e.g., Bifidobacterium cells, e.g., B. breve; Lactobacillus cells, e.g., L. plantarum; Bifidobacterium cells, e.g., B. animalis subs. lactis; Pediococcus cells, e.g., P. acidilactis; Lactobacillus cells, e.g., L. casei; Fibrobacter, e.g., F. succinogenes; and Butyrivbrio, e.g., B. fibrisolvense; Luminococcus cells, e.g., R. flavefaciens; Blautia cells, e.g., B. obeum; Clostridium cells, e.g., C. butyricum; Achermansia cells, e.g., A. musiniphila and / or Megasphaera elsdenii cells) may be used as a liquid feed additive by rehydrating, dissolving, and / or suspending the cells in a liquid.

[0303] In some embodiments, the feed additive comprises a feed additive carrier (i.e., one or more feed additive carriers).

[0304] Examples of suitable feed additive carriers include plant material (i.e., whole plants or plant parts including dried or processed plants or plant parts (e.g., seeds, stems, leaves, flowers, and / or roots), dried grains (e.g., distiller's dried grains), alfalfa, cornmeal, citrus peel, fermentation residues, crushed oyster shells, atapulgus clay, horse meal, molasses, corn cob, edible plant material, roasted hulled soybean meal, soybean flour feed, antibiotic mycelium, vermiculite, soybean grit, whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice hull, yeast culture, dried starch, sodium silica aluminate, water, salt solution, alcohol, silicone, wax, petroleum jelly, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oil, fatty acid Monoglycerides and diglycerides, petroetral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof are included, but not limited to, combinations thereof.

[0305] In some embodiments, the feed additive comprises, but is not limited to, microcrystalline cellulose; lactose; sodium citrate; calcium carbonate; dibasic calcium phosphate and glycine; disintegrants such as starch, sodium starch glycolate, sodium croscarmellose and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; extenders such as maltodextrin; moisture removers such as silicon dioxide; oxygen removers such as ascorbic acid; and / or excipients such as magnesium stearate, stearic acid, glyceryl behenate and talc ( in other words It includes one or more excipients.

[0306] In some embodiments, the feed additive is a granule comprising a core containing anaerobic cells and / or M. elsdeny cells and / or the feed additive, and a coating on the core. In some embodiments, the coating is a hydrated barrier salt. The salt coating may provide improved heat resistance, improved storage stability, and protection against other components in the granule that would otherwise have an adverse effect (e.g., on stability) on the M. elsdeny cells and / or the feed additive.

[0307] In some embodiments, encapsulated anaerobic cells and / or M. elsdenyi cells are mixed with a dry formulation of an additive comprising, but not limited to, a growth substrate, enzymes, sugars, carbohydrates, extracts, and growth-promoting trace components. Sugars may include, but are not limited to, lactose, maltose, dextrose, maltodextrin, sucrose, glucose, fructose, mannose, tagatose, sorbose, raffinose, amylose, starch, and galactose. Sugars may be in the range of 50 to 95%, individually or in combination. The extract may include, but is not limited to, yeast or dried yeast fermentation solubles in the range of 5 to 50%. The growth substrate may include, but is not limited to, trypticase in the range of 5 to 25%, sodium lactate in the range of 5 to 30%, and Tween 80 in the range of 1 to 5%. Carbohydrates may include, but are not limited to, mannitol, sorbitol, adonitol, and arabitol. Carbohydrates may be in the range of 5 to 50%, either individually or in combination. Trace components may include, but are not limited to, calcium carbonate in the range of 0.5 to 5.0%, calcium chloride in the range of 0.5 to 5.0%, dipotassium phosphate in the range of 0.5 to 5.0%, calcium phosphate in the range of 0.25 to 1.00%, manganese protinate in the range of 0.25 to 1.00%, and manganese.

[0308] In some embodiments, the anaerobic cell and / or M. elsdeny feed additive is prepared by mixing (e.g., with a mixer) anaerobic cells and / or M. elsdeny cells, including a culture containing cells and / or encapsulated cells, with any additional component of the feed additive (e.g., a carrier and / or excipient). In some embodiments, the components are mixed to obtain a homogeneous mixture.

[0309] In some embodiments, the feed additive is an animal feed additive for top application comprising anaerobic cells and / or M. elsdeny cells (e.g., encapsulated cells) as disclosed herein and a carrier. In some embodiments, the carrier is selected from the group consisting of whey, maltodextrin, sucrose, dextrose, limestone (i.e., calcium carbonate), rice husk, yeast culture, dry starch, and sodium silica aluminate, milk, water, and combinations thereof.

[0310] In some embodiments, the animal feed additive is a drench, spray, or supplement of a milk substitute comprising anaerobic cells and / or M. elsdeny cells (e.g., encapsulated cells) as disclosed herein and a water-soluble carrier. In some embodiments, the carrier is selected from the group consisting of whey, maltodextrin, sucrose, dextrose, dry starch, sodium silica aluminate, milk, water, and combinations thereof.

[0311] In some embodiments, the present invention relates to a food (e.g., animal feed) comprising anaerobic cells and / or M. elsdeny cells (e.g., encapsulated cells as disclosed herein, e.g., encapsulated cells prepared by a method as disclosed herein) and / or a feed additive as disclosed herein. The food is any food intended for consumption by animals (i.e., non-human animals or humans) and comprises both solid and liquid compositions. The food includes, but is not limited to, general foods; liquid products including water, milk, beverages, therapeutic beverages and nutritional beverages; functional foods; supplements; health functional foods; infant formulas (i.e., including non-human and human infants) including formulas for premature infants; foods for pregnant or lactating animals; foods for adult animals; and foods for the elderly. In some embodiments, the food comprises a liquid (e.g., beverage, e.g., water, milk, or milk substitute) containing a feed additive.

[0312] In some embodiments, the present disclosure relates to a composition comprising anaerobic cells and / or M. elsdeny cells (e.g., encapsulated cells) and / or a feed additive as disclosed herein. In some embodiments, the composition comprises encapsulated anaerobic cells and / or M. elsdeny cells produced by the method disclosed herein.

[0313] The compositions of the present disclosure may include one or more excipients. In some embodiments, the excipients may be alkalis, stabilizers, antioxidants, adhesives, release agents, coating agents, external phase components, sustained-release components, solvents, surfactants, humectants, buffers, fillers, emollients, or combinations thereof, but are not limited thereto. In addition to those discussed herein, excipients may be, but are not limited thereto, described in the literature [ Remington: The Science and Practice of Pharmacy , 21 stIt may include excipients listed in [ed. (2005)]. Including excipients in a specific category of this invention (e.g., 'solvents') is intended to exemplify rather than limit the role of the excipients. Specific excipients may belong to multiple categories.

[0314] 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., a veterinary food). A medical food is in the form of a composition to be consumed or administered externally under the supervision of a physician (e.g., a veterinarian) and includes foods for the management of specific diets for pathological conditions in which unique nutritional requirements based on recognized scientific principles 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 federal or state regulatory agencies for use in animals, more specifically in humans, or listed in the United States Pharmacopoeia or other generally recognized international pharmacopoeias.

[0315] For oral administration of the composition, anaerobic cells and / or M. elsdeny cells (e.g., encapsulated cells) or feed additives may be combined with excipients well known in the art. Such carriers may enable, for example, the anaerobic cells and / or M. elsdeny cells or feed additives of the present invention to be formulated as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by a subject to be treated. In some embodiments, the composition is a tablet, pill, caplet, or capsule. Suitable excipients include fillers such as sugars comprising lactose, sucrose, mannitol, or sorbitol; It includes cellulose products such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrants such as, but not limited to, cross-linked polyvinylpyrrolidone, agar, or salts thereof such as alginic acid or sodium alginate may be added. Compositions suitable for oral use include capsules made of gelatin as well as soft sealing capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In some embodiments, the dosage form is a vegetarian dosage form, wherein the dosage form is not formed from animal origin and does not contain any components of animal origin. In some embodiments, the vegetarian dosage form is a vegetarian capsule.

[0316] In some embodiments, the present invention relates to a kit or package comprising anaerobic cells and / or M. elsdeny cells as disclosed herein, a feed additive, a food and / or a composition. The kit or package may comprise a unit of the feed additive, the food, or the composition, or a combination thereof (e.g., one or more units). In some embodiments, the kit comprises encapsulated freeze-dried cells produced by the method disclosed herein, a feed additive as disclosed herein, or a capsule as disclosed herein.

[0317] Method of administering encapsulated anaerobic bacterial cells and / or Megasphaera elsdenii to animals

[0318] In some embodiments, a method for treating or preventing a pathological condition or disorder associated with lactic acid production in the gastrointestinal tract of a subject is provided herein, comprising the step of administering to a subject an effective amount of any of a composition comprising encapsulated Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophozoic state as disclosed herein, a feed additive composition disclosed herein, or any of the feed disclosed herein.

[0319] In some forms, the pathological condition or disorder is acidosis.

[0320] In some forms, the condition or disorder is rumen acidosis.

[0321] In some forms, the pathological condition or disorder is a respiratory disease.

[0322] In some forms, the condition or disorder is laminitis.

[0323] In some embodiments, a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal is provided herein, comprising the step of administering to an animal any of the compositions disclosed herein comprising encapsulated Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state, any of the feed additive compositions disclosed herein, or any of the feeds disclosed herein.

[0324] In some forms, opportunistic microorganisms are pathogenic.

[0325] In some forms, the opportunistic microorganism is Salmonella, E. coli, or Campylobacter.

[0326] In some embodiments, a method for improving the growth performance of a subject is provided herein, comprising the step of administering to a subject an effective amount of any of the compositions disclosed herein comprising encapsulated Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state, the feed additive composition disclosed herein, or any of the feed disclosed herein. In some embodiments, the improvement in the subject's performance includes one or more of the following compared to the performance of a subject not administered the feed additive composition or feed: feed conversion ratio (FCR), body weight gain, feed efficiency, carcass quality, reduced mortality, reduced morbidity, feed intake, daily gain, carcass gain, bone mineralization, egg production, reduction in gastrointestinal microbial imbalance or dysfunction, milk composition (e.g., increased milk fat), and / or milk production.

[0327] In some embodiments, a method for increasing starch digestibility in a subject and lowering / lowering starch excretion in feces or preventing a decrease in pH in the lower gastrointestinal tract is provided, comprising the step of adding an effective amount of any of the compositions disclosed herein, including encapsulated Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state, to a feed for administration to a subject, wherein the subject exhibits one or more of increased starch digestibility and / or lowered starch excretion in feces compared to a subject not administered the composition, the feed additive composition, or the feed.

[0328] In some embodiments, a method for increasing farm operational efficiency is provided herein, comprising the step of administering to a farm animal an effective amount of any of the compositions disclosed herein, comprising encapsulated Megasphaera elsdenii cells, anaerobic bacterial cells, or anaerobic bacterial cells in a trophic state, any of the feed additive compositions disclosed herein, or any of the feeds disclosed herein. In some embodiments, increased operational efficiency reduces labor costs, reduces forage transport costs, and reduces the amount of forage added to the feed or feed additives.

[0329] In some modalities, the subject is a ruminant.

[0330] In some embodiments, ruminants are selected from a group consisting of cattle, goats, sheep, giraffes, deer, gazelles, buffalo, reindeer, and antelopes.

[0331] In some forms, the cow is a beef cow or a dairy cow.

[0332] In some modalities, the subject is a non-ruminant.

[0333] In some embodiments, non-ruminants are selected from a group consisting of horses, animals, poultry, and pigs.

[0334] In some embodiments, poultry is selected from a group consisting of chickens, geese, ducks, quails, turkeys, broilers, meat breeding hens, laying hens, or pigeons.

[0335] In some forms, poultry is chicken.

[0336] In some embodiments, the feed additive composition or feed is provided to the subject for daily or weekly administration.

[0337] In some embodiments, the method comprises the step of administering to an animal an encapsulated M. elsdenyi cell, a feed additive, a food, or a composition (e.g., a capsule) as described herein.

[0338] In some embodiments, the present invention relates to a method of administering encapsulated anaerobic bacterial cells to an animal. In some embodiments, the present invention relates to a method of administering to an animal encapsulated Bifidobacterium cells, e.g., B. breve; Lactobacillus cells, e.g., L. plantarum; Bifidobacterium cells, e.g., B. animalis subs. lactis; Pediococcus cells, e.g., P. acidilactis; Lactobacillus cells, e.g., L. casei cells; Fibrobacter, e.g., F. succinogenes cells; Butyrivibrio, e.g., B. fibrisolvense cells; Luminococcus cells, e.g., R. flavefaciens; Blautia cells, e.g., B. obeum; Clostridium cells, e.g., C. butyricum; Achermansia cells, e.g., A. musiniphila and / or Megasphaera cells, e.g., M. elsdeni.

[0339] Administration may be made by any compatible route, including, for example, oral method (i.e., ingestible liquid or solid, oral drench, feed additive, food, composition or capsule), body spray (i.e., mist spray) and / or injection.

[0340] In some embodiments, the method comprises the step of administering a solid, liquid, or gel containing encapsulated M. elsdeni cells or anaerobic bacterial cells.

[0341] In some embodiments, the method comprises the step of administering a solid feed additive comprising encapsulated M. elsdenyi cells or anaerobic bacterial cells. In some embodiments, the solid feed additive is a powder (e.g., fluid powder), granules (i.e., fine particles), particles (i.e., fine particles), pellets, cakes, water-soluble concentrates, pastes, boluses, tablets, dusts, components thereof, or combinations thereof.

[0342] In some embodiments, the method comprises the step of administering a liquid feed additive comprising encapsulated M. elsdenyi cells or anaerobic bacterial cells. In some embodiments, the method comprises the step of administering encapsulated M. elsdenyi cells or anaerobic bacterial cells as a liquid. In some embodiments, the liquid is a solution (e.g., aqueous, organic, or aqueous-organic solution), a suspension, an emulsion, a drench, a spray, an injectable, a beverage (e.g., a milk substitute), a component thereof, or a combination thereof. In some embodiments, the liquid is administered orally or by spraying the liquid onto an animal.

[0343] In some embodiments, the method comprises the step of combining a feed additive containing encapsulated M. elsdenyi cells or anaerobic bacterial cells or cells with another animal feed additive to form a supplement or premix for adding to animal feed. In some embodiments, the other feed additive comprises cells other than M. elsdenyi cells.

[0344] In some embodiments, encapsulated M. elsdeny cells or anaerobic bacterial cells may be added to a feed additive as a liquid (e.g., a broth or broth equivalent containing rehydrated electrospray-dried cells) or as a reconstituted cell paste. A dosage form (e.g., a drench or capsule of a predetermined volume) may also be formed, and if desired, the encapsulated M. elsdeny or anaerobic cells may be added directly to animal feed by spraying the liquid broth and / or the encapsulated M. elsdeny or anaerobic cells over the feed or mixing them into the feed.

[0345] In some embodiments, the method comprises the step of rehydrating a feed additive (e.g., powder, fine particles, pellets, cakes, electrospray-dried cells, or a combination thereof) to produce a liquid for administration.

[0346] In some embodiments, the method comprises the step of applying encapsulated M. elsdenyi cells or anaerobic bacterial cells to animal feed through a delivery system that rehydrates a feed additive, comprising a batch unit method. For example, electrospray-dried powder may be transferred from a polyvinyl hopper through an auger to a flushing system, and the flushing system dilutes the powder and sprays it onto the feed to be mixed.

[0347] In some embodiments, the method comprises the step of applying encapsulated M. elsdeni cells or anaerobic bacterial cells to animal feed using a volume measuring device having a storage container. For example, the encapsulated M. elsdeni cells or anaerobic bacterial cells (e.g., powder containing cells) may be stored in a storage container and discharged into water or an aqueous bath immediately before being sprayed onto animal feed.

[0348] In some embodiments, the present invention relates to a method for treating or preventing a pathological condition or disorder associated with lactic acid production in the gastrointestinal tract of an animal, comprising the step of administering to an animal an effective amount of encapsulated M. elsdenyi cells as disclosed herein, encapsulated M. elsdenyi cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition (e.g., capsule) as disclosed herein.

[0349] 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 respiratory disease. In some embodiments, the condition or disorder is laminitis. In some embodiments, the condition or disorder is infection. In some embodiments, the infection is caused by Salmonella or Campylobacter. In some embodiments, the Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella typhimurium and / or enteritidis. In some embodiments, the Campylobacter is Campylobacter jejuni or Campylobacter coli.

[0350] In some embodiments, the present invention relates to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to an animal an effective amount of encapsulated M. elsdenyi cells as disclosed herein, encapsulated M. elsdenyi cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein.

[0351] In some embodiments, the present invention relates to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, comprising the step of administering to an animal an effective amount of an encapsulated anaerobic bacterial cell as disclosed herein, an anaerobic cell produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein.

[0352] In some embodiments, the opportunistic microorganism is pathogenic. In some embodiments, the opportunistic microorganism is Salmonella or Campylobacter. In some embodiments, the Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella typhimurium and / or enteritidis. In some embodiments, the Campylobacter is Campylobacter jejuni or Campylobacter coli.

[0353] In some embodiments, the present invention relates to a method for improving the bioavailability of plant-derived phosphorus in an animal's diet, comprising the step of administering to an animal an effective amount of encapsulated M. elsdenyi cells as disclosed herein, encapsulated M. elsdenyi cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein. In some embodiments, the encapsulated M. elsdenyi cells possess phytase activity. In some embodiments, the method reduces environmental phosphorus waste resulting from the administration of an animal's diet without M. elsdenyi cells.

[0354] In some embodiments, the present invention relates to a method for improving the bioavailability of plant-derived phosphorus in an animal's diet, comprising the step of administering to an animal an effective amount of encapsulated anaerobic bacterial cells as disclosed herein, electrospray-dried anaerobic bacterial cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein. In some embodiments, the encapsulated anaerobic bacterial cells possess phytase activity. In some embodiments, the method reduces environmental phosphorus waste resulting from the administration of an animal's diet without anaerobic bacterial cells.

[0355] In some embodiments, the present invention relates to a method for improving the growth performance of an animal, comprising the step of administering to an animal an effective amount of encapsulated M. elsdenyi cells as disclosed herein, encapsulated M. elsdenyi cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein.

[0356] In some embodiments, the present invention relates to a method for improving the growth performance of an animal, comprising the step of administering to an animal an effective amount of an encapsulated anaerobic bacterial cell as disclosed herein, an encapsulated anaerobic bacterial cell produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein.

[0357] In some embodiments, the improvement in the growth performance of animals is an improvement in feed intake, daily weight gain, feed conversion ratio, carcass gain, milk composition in milk-producing animals (e.g., increase in milk fat), milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0358] In some embodiments, the present invention relates to a method for acidifying the lower gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of encapsulated M. elsdenyi cells as disclosed herein, encapsulated M. elsdenyi cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein. In some embodiments, the lower gastrointestinal tract is the cecum of a poultry animal.

[0359] In some embodiments, the present invention relates to a method for acidifying the lower gastrointestinal tract of an animal, comprising the step of administering to the animal an effective amount of encapsulated anaerobic bacterial cells as disclosed herein, encapsulated anaerobic bacterial cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein. In some embodiments, the lower gastrointestinal tract is the cecum of a poultry animal.

[0360] In some embodiments, encapsulated M. elsdeni cells or anaerobic bacterial cells as disclosed herein, encapsulated M. elsdeni cells or anaerobic bacterial cells produced by a method as disclosed herein, feed additives as disclosed herein, or compositions as disclosed herein are administered to animals before, at the same time as, or after feeding food.

[0361] In some embodiments, the method further comprises the step of mixing with a liquid before administration encapsulated M. elsdeni cells or anaerobic bacterial cells as disclosed herein, encapsulated M. elsdeni cells or anaerobic bacterial cells produced by the method as disclosed herein, or a solid feed additive as disclosed herein.

[0362] In some embodiments, the liquid is administered orally (e.g., oral drench) or by spraying the liquid onto the animal (e.g., mist spray).

[0363] In some embodiments, the method comprises a single dose of encapsulated M. elsdeni cells or anaerobic bacterial cells as disclosed herein, encapsulated M. elsdeni cells or anaerobic bacterial cells produced by the method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein.

[0364] In some embodiments, the present invention comprises daily administration of encapsulated M. elsdeny cells or anaerobic bacterial cells as disclosed herein, encapsulated M. elsdeny cells or anaerobic bacterial cells produced by a method as disclosed herein, a feed additive as disclosed herein, or a composition as disclosed herein. In some embodiments, administration is at least once daily, at least twice daily, at least three times daily, or more than three times daily. In some embodiments, administration is optional (e.g., self-administration by drinking an available liquid or consuming an available food comprising encapsulated M. elsdeny or anaerobic cells, encapsulated M. elsdeny or anaerobic cells produced by a method as disclosed herein, a feed additive, or a composition).

[0365] In some embodiments, the method comprises administering more than once a day of encapsulated M. elsdenyi cells or anaerobic bacterial cells as disclosed herein, encapsulated M. elsdenyi cells or anaerobic bacterial cells produced by the method as disclosed herein, feed additives as disclosed herein, or compositions as disclosed herein. In some embodiments, the administration is 2, 3, 4, 5, 6, or more times a day. In some embodiments, the method comprises a period of no administration of one day or more following the administration of more than once a day. In some embodiments, the period of no administration of one day or more is a period of no administration of 1 day, 2 days, 3 days, 4 days, 5 days 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.

[0366] In some embodiments, the animal is a ruminant. In some embodiments, the ruminant may be, but is not limited to, cattle, buffalo, sheep, goats, deer, reindeer, moose, giraffes, yaks, and elk. In some embodiments, the ruminant is selected from the group consisting of cattle, buffalo, sheep, goats, deer, and reindeer.

[0367] In some embodiments, the animal is a non-ruminant. In some embodiments, the non-ruminant may be, but is not limited to, horses, animals, poultry, pigs, dogs, humans, and cats. In some embodiments, the non-ruminant is selected from the group consisting of horses, animals, poultry, and pigs.

[0368] In some aspects, the animal is a zoo animal.

[0369] In some embodiments, the animal is a poultry animal. In some embodiments, the poultry animal is a bird (i.e., a bird) used as an animal for food, 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 broiler chickens, broiler breeder chickens, and laying hens. In some embodiments, the poultry animal is a chicken.

[0370] In some modalities, the animal is a horse and an animal. In some modalities, the horse and animal is a horse, a pony, a donkey, or a mule.

[0371] In some modalities, the animal is a pet. In some modalities, the pet is a dog, cat, guinea pig, rabbit, rat, mouse, or horse. In some modalities, the pet is a dog. In some modalities, the animal is a cat.

[0372] Examples

[0373] Now, together with the above description, refer to the following examples which illustrate some aspects of the present invention in a non-limiting manner.

[0374] Example 1: Formulation Evaluation

[0375] M. elsdenyi is a strictly anaerobic bacterium with high potential for use as a direct feed microorganism in the cattle industry. However, due to its anaerobic nature, M. elsdenyi has proven difficult to deliver reliably in feed in commercial feedlands / dairy facilities. Similar difficulties may arise with anaerobic bacteria that can be used in commercial feedlands / dairy facilities.

[0376] In the following examples, different types of encapsulation were tested on freeze-dried cultures of M. elsdenyi NCIMB 41125. All M. elsdenyi cultures were grown, cooled, concentrated, and freeze-dried (FD) according to the methods described in WO 2018 / 144653 A1 incorporated herein by reference. Test formulations were prepared using the resulting freeze-dried M. elsdenyi cultures and then evaluated as follows.

[0377] · Recovery rate after encapsulation - to determine commercial feasibility.

[0378] · Recovery rate after environmental exposure of micromachine empty - to determine the effect of environmental exposure before inclusion in feed via micromachine.

[0379] · Survival rate after passing through a micromachine – To determine the level of protection provided to M. Elsdeny after passing through a micromachine commonly used to distribute 'small' components into feed in commercial feedyards.

[0380] · Rumen release - To determine M. elsdeny release in the rumen

[0381] · Survival rate when mixed into feed – to determine the level of protection provided to M. elsdenie through mixing into feed before being ingested by animals.

[0382] · Stability Data (Shelf Life) - To determine the stability of M. elsdenyi in formulations stored for a long period at various temperatures.

[0383] Unless otherwise specified, the following test protocols were used for all formulations.

[0384] Recovery rate after encapsulation: Colony forming units (CFU) of M. elsdenyi were analyzed in the encapsulated product (formulation), and the recovery rate after encapsulation was determined by comparing this with the initial concentration of M. elsdenyi freeze-dried culture.

[0385] To briefly explain, the encapsulated material (formulation) was blended with a wettable powder under anaerobic conditions at room temperature. The samples were then plated in triple layers on semi-limited lactate (SDL) agar plates and incubated in an anaerobic chamber at 39 °C for 48 hours. Subsequently, CFU results were adjusted for the amount of freeze-dried culture in different formulations and expressed as CFU per gram of freeze-dried culture. The recovery rate after encapsulation was calculated by dividing the CFU / g after encapsulation by the CFU / g before encapsulation.

[0386] Recovery rate after micromachine empty environment exposure:Using a micromachine, low-concentration feed ingredients (i.e., less than 500 mg for direct-feeding microorganisms) were precisely added to feed batches / loads in commercial fattening facilities and dairy farms. Prior to addition to the micromachine, the trace ingredients were stored in individual bins connected to a scale and a computerized management system. The trace ingredients could remain in the micromachine bins for up to 14 hours before being mixed into the feed. To determine the loss occurring during exposure to the micromachine bin environment, the formulations were incubated at 25, 30, and 37 °C for up to 14 hours. This experiment was repeated three times.

[0387] A 20 g aliquot of the formulation was weighed in a weighing boat and placed in an incubator set to 25, 30, or 37 °C. After the appropriate incubation time, the weighing boat was retrieved and transferred to a humidity-controlled chamber to divide into additional 1.5 g aliquots. These aliquots were rehydrated with a rehydrating agent in an anaerobic chamber and blended. After blending, all samples were allowed to rehydrate for 2 hours before dilution and plating on semi-limited lactate agar. The CFU results were expressed as CFU per g of the formulation. The recovery rate after incubation in a micromachine bin was calculated by dividing the CFU / g after incubation by the CFU / g before incubation.

[0388] Survival rate after passing through micromachines: The weight of trace components was based on the product volume, the amount of feed / batch, and the number of animals fed per feed batch. The micromachine was calibrated to an estimate of an appropriate product volume with a tolerance. Once the computer pre-weighed the components, they were released from the bin and became a suspension in a water tank. The mixture was then flushed with water, which acted as a carrier to deliver the trace components to the final feed batch. The trace component / water mixture was applied to the feed in the feed truck using a spray bar and then thoroughly mixed into the feed.

[0389] Two methods of water administration were tested.

[0390] (1) Bowl System: Trace components were metered into a hopper, and the hopper discharged them into a slurry mixing bowl. An impeller was used to mix the slurry contents with water. To completely empty the tank, the mixture was flushed with water from the mixing tank. A pump transferred the component / water mixture to a spray bar.

[0391] (2) Continuous flow system: A trace amount of component is weighed and distributed into a continuous water flow that delivers the component / water mixture directly to the spray. This system has a higher component content and can be used for larger batch sizes.

[0392] Before starting the experiment, the system was flushed to prevent additional debris from being discharged into the sample. After flushing, a funnel with a mesh bag was placed inside the sprayer drain, and a collection bucket was placed underneath to collect the discharged sample and water.

[0393] A known amount of product (formulation) was slowly added to the micromachine, and water was passed through the system to flush until all the product was discharged. The operating time was recorded, and the system was continued to operate for an additional 15 to 30 seconds to clean any remaining product in the micromachine and hose.

[0394] The collected samples were transferred to an anaerobic chamber, rehydrated, and blended. After blending, all samples were allowed to rehydrate at room temperature for 2 hours before dilution and plating on SDL agar plates. The plates were incubated under anaerobic conditions at 39 °C for 48 hours.

[0395] Next, the CFU results were adjusted for the amount of product and expressed as CFU per gram of product (formulation). The recovery rate after micromachine was calculated by comparing it to the concentration (CFU / g) of the encapsulated product before passing through the micromachine.

[0396] In some embodiments, the generated samples were passed through a micromachine and mixed into feed to simulate feedfield application, in accordance with the described feed survival protocol. Subsequently, the CFU results were adjusted for the amount of product and expressed as CFU per gram of product (formulation). The recovery rate after incubation in feed (diet) was calculated by comparing it to the concentration of the encapsulated product before mixing into the feed.

[0397] Rumen release: The in vivo release of the formulation was determined using four fistula-forming steers. The animals were fed two different diets: a high roughage diet (80% roughage and 20% concentrate) and a high concentrate diet (20% roughage and 80% concentrate) (two steers per diet).

[0398] Empty Dacron bags were labeled, dried, and weighed before being filled with the encapsulated product (formulation). The bags were weighed again (initial weight) and heat-sealed. The Dacron bags were then placed in large nylon-mesh bags equipped with weights and incubated / suspended in the ventral rumen for a total of 24 hours (one bag per animal, two Dacron bags per treatment group per bag per animal). Two empty Dacron bags per castrated boar were also included as test samples.

[0399] Upon removal from castrated bulls, the nylon-mesh bags were rinsed and dried in an incubator set to 55 °C for 72 hours. After drying, the bags were weighed to determine the final weight. Subsequently, the rumen dry matter (DM) loss rate was calculated as follows.

[0400]

[0401] Lipid material present in the capsule must not decompose for 24 hours, and thus the initial weight was corrected for its non-lipid content. For the purposes of these examples, the amount of dry matter lost in the rumen was considered as the amount of dry matter released / available to the animal (rumen release amount).

[0402] Survival rate in feed: The following representative fattening farm diet was prepared and placed in individual containers (250 mL capacity) in 30-gram portions.

[0403] Table 2. Low pH and high moisture diet composition - 4.3 pH, 40% moisture.

[0404]

[0405] Table 3. High pH and high moisture diet composition - 5.6 pH and 43.6% moisture.

[0406]

[0407] Table 4. High pH and low moisture diet composition - 5.8 pH and 9% moisture.

[0408]

[0409] Samples of each formulation were weighed and placed on the diet within the containers. Each container was individually mixed by hand and placed on a bench at room temperature (25 °C) or incubated at 52 °C under aerobic conditions. Containers were sampled at designated times.

[0410] After 0, 2, and 4 hours, the designated containers were placed in an anaerobic chamber, their contents were rehydrated and blended, and allowed to rehydrate at room temperature for 2 hours. Subsequently, 1 mL samples were collected from each container, diluted, and plated onto SDL agar plates. The plates were incubated under anaerobic conditions at 39 °C for 48 hours.

[0411] Next, the CFU results were adjusted for the amount of freeze-dried powder in different formulations and expressed as CFU per gram of freeze-dried culture. The recovery rate after mixing (time 0) and after 2 or 4 hours of incubation in the diet (time 2 or 4) was calculated by comparing it to the concentration of the formulation (CFU / g) before mixing into the diet.

[0412] Stability / Storage Life: The stability of the formulation was tested in bulk by aliquoting samples into large resealable Mylar Foil bags (12" x 12"), or in individual bags by aliquoting samples into small Mylar Foil bags (single-use). Bags were appropriately labeled and stored at either -20 °C or 4 °C, and samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, and 12 months. Three bags per formulation, per temperature, and per time point.

[0413] On the day of sampling, samples (1.5 g, individual bag contents or aliquots from bulk bags) were weighed and transferred to an anaerobic chamber. The samples were then rehydrated, blended, and allowed to rehydrate for 10 minutes prior to plating on SDL agar. After 48 hours of incubation at 39 °C, the plates were counted, and the results were recorded as CFU per g of the formulation. Stability data were plotted against time.

[0414] Example 2: Effect of carrier on cell recovery rate after encapsulation and stability during storage at -20 °C or 4 °C.

[0415] Experiments were conducted to determine the effects of different carriers on the recovery rate and stability during storage of M. elsdeni cells after encapsulation.

[0416] A freeze-dried M. elsdeny culture obtained according to the method described in WO 2018 / 144653 A1 was mixed with one of three different types of starch from potato, wheat, or corn to form a matrix, and then encapsulated in a Wurster-type fluid bed coagulation coater with vegetable oil (a mixture of monoglycerides and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) at a 40 / 60 ratio (40% matrix and 60% coating) at 52 °C (Table 5).

[0417] Cell recovery rate (Table 5) Example 1Stability studies were performed on samples tested according to the protocols described in [document] and bulk-packaged at either -20 or 4 °C. The samples Example 1 Analysis was performed after storage for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, and 12 months according to the protocol described in (Fig. 1).

[0418] Table 5. Formulation composition and cell recovery rate after encapsulation when different types of starch are used as bulking agents.

[0419]

[0420] Formulations using corn and wheat starch as carriers had a lower cell recovery rate through the encapsulation process than formulations containing potato starch.

[0421] Storage temperature did not affect cell loss over 12 months. Most of the loss observed during the storage life occurred early in the storage period (within the first 15 days), regardless of the treatment group. Loss over the 12-month storage period was consistent in the range of 0.4 to 0.6 log CFU / g, regardless of the type of starch used as a carrier prior to encapsulation. Refer to Figure 1.

[0422] Example 19 As described, when performing two-step encapsulation, a similar or greater cell recovery rate through the encapsulation process is expected.

[0423] Likewise, the storage temperature is Example 19 Regardless of the type of starch used as a carrier prior to the two-step encapsulation as described, it is expected not to affect cell loss during the 12-month storage period.

[0424] Example 3: Vegetable oil encapsulation provides cell protection during exposure to low pH and high moisture feed.

[0425] To determine the level of protection provided to M. elsdenyi by vegetable oil encapsulation, encapsulated cells were mixed into a high-moisture, low-pH feeder diet at room temperature (25 °C) for up to 4 hours.

[0426] Freeze-dried M. elsdenyi cultures were obtained according to the method described in WO 2018 / 144653 A1. The resulting cultures were mixed with potato starch as a carrier to form a matrix, and then encapsulated in a Wurster-type fluidized bed coagulation coater with vegetable oil (a mixture of monoglycerides and diglycerides of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) in a 40 / 60 ratio (40% matrix and 60% vegetable oil) at 52 °C. The resulting formulation (vegetable oil (VO) formulation) was rapidly cooled to maximize cell recovery.

[0427] Example 1 Cell recovery rates after encapsulation (Table 6) were tested according to the protocol described. The freeze-dried culture, matrix, and vegetable oil (VO) formulations were mixed with a low pH and high moisture feeder diet (Table 2) at room temperature for 4 hours, and Example 1 and used in the survival protocol in the feed listed in Table 7.

[0428] Table 6. Sample composition

[0429]

[0430] Table 7. In-Feed Survival Rate Test - Contents Description and Rehydration Details.

[0431]

[0432] The recovery rate of M. elsdeny after mixing into the feed (T0) and after exposure to 25 °C for 4 hours (T4) was calculated by comparing the concentration of the formulation before mixing with the diet with the obtained concentration (Table 8).

[0433] Table 8. M. elsdeny recovery rate after mixing treatment groups with a low pH and high moisture diet for 0 or 4 hours at 25 °C under aerobic conditions.

[0434]

[0435] The addition of potato starch to FD M. elsdenii accelerated the death of M. elsdenii when mixed with a low pH and high moisture diet compared to FD M. elsdenii alone. Although the survival rate of M. elsdenii increased with vegetable oil encapsulation (VO formulation) compared to the freeze-dried culture and matrix treatment groups, the recovery rate after 4 hours of exposure to a low pH and high moisture diet remained low at approximately 20%. Refer to Table 8.

[0436] Example 4: Effects of fluidization time and temperature on M. elsdeny cell viability.

[0437] To determine the optimal temperature during the fluidization process, various temperatures and fluidization times are tested, and the recovery rates of M. Elsdeny cells at the end of the process are compared.

[0438] The fluidized bed device was preheated to meet the target process temperature. The process temperature was set a few degrees lower than the melting point of hydrogenated palm oil (HPO). Once the vessel reached the temperature, freeze-dried M. elsdenyi culture was added to the bottom of the vessel, and the filter pulse interval was set to 1 second before the process began. The air flow was set to 25 liters / min (LPM) and gradually increased to 160 LPM during fluidization. After performing fluidization for 30 or 60 minutes, the device was turned off, and the fluidized freeze-dried culture was collected in a sterile sample cup.

[0439] The generated product was aliquoted as a 0.16 g sample in a humidity-controlled chamber, transferred to an anaerobic chamber, rehydrated in 20 mL of anaerobic diluent, and plated on semi-limited lactate agar. After incubation at 39 °C for 48 hours, the recovery rate was calculated by comparing the concentrations of the freeze-dried culture before and after fluidization using CFU results (Table 9).

[0440] Table 9. M. elsdeny cell recovery rates after fluidization treatment for 30 or 60 minutes at 50 °C, 55 °C, 60 °C, and 65 °C.

[0441]

[0442] The cell recovery rate after fluidization (Table 9) was affected by the process duration and temperature. The highest recovery rate was obtained when fluidized at 50 °C for 30 minutes. M. elsdeny cells showed some degree of resilience despite the decrease in recovery rate, and 10 to 13% of the cells were able to survive the fluidization process at 65 °C for 30 to 60 minutes.

[0443] Example 5: Palm oil encapsulation alone did not provide sufficient moisture and heat protection to M. elsdenyi when mixed with low pH and high moisture feed.

[0444] Experiments were conducted to determine protection provided by spinning disc encapsulation alone, an external coating using a carnauba wax / cured cottonseed oil mixture sprayed in a Wurster fluid bed coagulation coater, or an additional coating layer of carnauba wax.

[0445] M. elsdeny cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. Subsequently, an aliquot (3.5 kg) of the resulting freeze-dried culture was encapsulated to form a core containing freeze-dried (FD) M. elsdeny and hydrogenated palm oil (HPO) using the spinning disc method. The cores were processed to produce small or large particles and then spray-coated with a mixture of carnauba wax (CW) and hydrogenated cottonseed oil (HCO) in a Wurster fluid bed coagulation coater. Additionally, a portion of the formulation was treated again with carnauba wax in a Wurster fluid bed coagulation coater to form a second outer coating. The final formulation compositions are listed in Table 10. The melting points of the oils and waxes are as follows.

[0446] · Hydrogenated palm oil = 58 °C to 62 °C

[0447] · Hardened cottonseed oil = 60 °C to 64 °C

[0448] · Carnauba wax = 82 °C to 86 °C

[0449] Next, the colony-forming units (CFU) of the encapsulated product were analyzed and compared with the initial freeze-dried powder. Example 1 As described in [document], the recovery rate after encapsulation was determined.

[0450] Table 10. Formulation composition and recovery rate after encapsulation.

[0451]

[0452] Formulations containing an additional coating around the core survived better during the encapsulation process than those prepared using the HPO and spinning disc methods alone. The particle size of the final product was found not to affect the cell recovery rate after encapsulation. The overall recovery rate after encapsulation Examples 2 and 3 It was lower compared to.

[0453] Next Example 1 To establish the survival rate in feed (Table 11) according to the procedure described, the formulation was tested in feed using a low pH and high moisture diet, which is a representative fattening farm diet prepared as shown in Table 2.

[0454] Table 11. M. Elsdeny recovery rate after mixing the encapsulated product with a low pH and high moisture diet at 25 °C or 52 °C for 0 or 4 hours under aerobic conditions.

[0455]

[0456] HPO (Core only, A When encapsulated with ), a low recovery rate was obtained after 4 hours of incubation on a low pH and high moisture diet at 25 or 52 °C (Table 11). This recovery rate was due to vegetable oil encapsulation (VO formulation, Example 3 It was similar to what was observed in ). CW / HCO( BWhen an external coating using ) was added, the recovery rate after 4 hours of exposure increased by 45% at 25 °C and by 25% at 52 °C. CW( E When another external coating using ) was added, the recovery rate was further improved at 25 °C, but a lower recovery rate was obtained at 52 °C. In the case where a second external coating was added to freeze-dried M. elsdenyi, which had a core formed by encapsulating it with hydrogenated palm oil (HPO) and then coated with a mixture of carnauba wax (CW) and hydrogenated cottonseed oil (HCO) (68%, B ) and when not added (83%, E The recovery rate observed in ) is the recovery rate previously observed when vegetable oil was used ( Example 3 It was higher than about 20%.

[0457] Example 6: A two-step encapsulation process is essential to provide sufficient protection to M. elsdenyi during exposure to low pH and high moisture feed.

[0458] Next, it was determined whether single-step encapsulation using only a spinning disc or a Wurster fluid bed coagulation coater could provide sufficient protection to M. elsdenii throughout encapsulation and mixing into feed.

[0459] M. elsdeni cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. An aliquot of the resulting freeze-dried culture was mixed with maltodextrin in a 1:5 ratio, and Example 5 It was encapsulated as described in [document]. The final formulation compositions are listed in Tables 12 and 13. The formulations were prepared to have a small final particle size (212 to 710 μm) to increase the surface area and maximize post-ingestion release (rumen release) in cattle. The colony-forming units (CFU) of the encapsulated product were analyzed and compared with the initial freeze-dried powder. Example 1 The recovery rate after encapsulation (Tables 12 & 13) was determined according to the protocol described in [Table].

[0460] Table 12. Formulation composition and recovery rate after encapsulation

[0461]

[0462] Example 5 As in, M. Elsdeny encapsulated using the HPO and spinning disk methods ( M. e. The recovery rate was approximately 16% (Table 12). The cell recovery rate was higher when the cells received only overcoating (a mixture of CW and HCO₃) without undergoing the spinning disc process. However, the cell recovery rate decreased as the amount of lipids in the overcoating increased, dropping from 65%, the highest lipid level tested, to 47%.

[0463] Table 13. Formulation composition and recovery rate after encapsulation.

[0464]

[0465] After spinning disc encapsulation using HPO, the addition of an external coating using Wurster did not significantly affect the cell recovery rate after encapsulation (Table 13), and all of them ranged from 19.2% to 22.9% compared to 16% for the formulation using only the spinning disc. The composition of the external coating, whether HCO alone or in combination with CW, did not significantly affect the cell recovery rate after encapsulation.

[0466] Next Example 1 To establish survival rates in feed according to the procedures described in [Table 2], formulations 5, 20, 8, 12, and 16 were tested in feed using a low pH and high moisture diet (Table 2).

[0467] Table 14. M. Elsdeny recovery rate after mixing the encapsulated product in a low pH and high moisture diet at 25 °C or 52 °C for 0 or 4 hours under aerobic conditions. *Carnauba wax (CW) and hydrogenated cottonseed oil (HCO) were mixed in the ratios indicated in parentheses. & Hydrogenated palm oil alone.

[0468]

[0469] Formulations prepared using only spinning discs or only Wursters did not provide sufficient protection to M. elsdenii during mixing in feed and resulted in a cell recovery rate of less than 10% after 4 hours of exposure regardless of the temperature or method used (Table 14).

[0470] Two-step encapsulation using a spinning disc and Wurster resulted in increased recovery rates at both 25 °C and 52 °C compared to either method used individually.

[0471] Example 7: The addition of a second overcoating after two-step encapsulation reduced the cell recovery rate after encapsulation.

[0472] It was determined whether adding an additional overcoating after two-step encapsulation provides additional protection for the recovery rate of M. elsdeny after encapsulation.

[0473] M. elsdeni cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. An aliquot of the resulting freeze-dried culture was mixed with maltodextrin in a 1:5 ratio, and Example 5 As described in [document], the lipid overcoating material was encapsulated with an additional coating to make up 33 or 50% of the total. The final formulation compositions are listed in Table 15. The colony-forming units (CFU) of the encapsulated product were analyzed and compared with the initial freeze-dried powder. Example 1 The recovery rate after encapsulation (Table 15) was determined according to the protocol described in [Table 15].

[0474] Table 15. Formulation composition and recovery rate after encapsulation.

[0475]

[0476] The recovery rate after encapsulation was low across all formulations. The addition of a second outer layer reduced cell viability during the process.

[0477] Next Example 1To establish survival rates in feed according to the procedures described in [Table 16], formulations 50 / 8B, 50 / 12B, and 50 / 16B were tested in feed using a low pH and high moisture diet (Table 2).

[0478] Table 16. M. Elsdeny recovery rate after mixing the encapsulated product with a low pH and high moisture diet at 25 or 52 °C under aerobic conditions for 0 or 4 hours.

[0479]

[0480] Formulation 50 / 8B had the highest cell recovery rate after 4 hours of incubation on a low pH and high moisture diet at either 25 °C or 52 °C. Increasing the amount of the second outer coating Example 6 Compared to formulations 8A, 12A, and 16A from, it increased cell viability when mixed into feed.

[0481] For formulations 33 / 8B, 33 / 12B and 33 / 16B Example 1 The storage life was further tested at 4 °C for up to 12 months according to the procedures described. The results were expressed as CFU per gram of the formulation. Refer to Figure 2. Regardless of the composition of the first or second coating, all formulations with a final 33% lipid coating exhibited similar performance when stored at 4 °C for up to 12 months.

[0482] Example 8: Optimal lipid content of coating in a two-step encapsulation formulation.

[0483] Next, the optimal level of lipid to be used in the external coating was determined to provide the highest level of protection within the feed while allowing the cells to be released in the rumen.

[0484] M. elsdeni cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. Aliquots of the resulting freeze-dried cultures were mixed with maltodextrin (as a carrier) in a 1:10 ratio or not mixed, as shown in Table 17 and Example 5As emphasized in [the text], it was encapsulated. The formulation was prepared to have a small particle size (213 to 704 μm) or a large particle size (704 to 1400 μm). The small particle size was assumed to increase the surface area and maximize release after ingestion (rumen release) in cattle.

[0485] Analyze the colony-forming units (CFU) of the encapsulated product and compare with the initial freeze-dried powder Example 1 The recovery rate after encapsulation (Table 17) was determined according to the protocol described in [Table 17].

[0486] Table 17. Formulation composition and cell recovery rate

[0487]

[0488] The presence of maltodextrin as a carrier within the core did not affect cell recovery rates after encapsulation (7A vs. 8A, 3A vs. 1A, 4A vs. 2A). Adding a coating, either alone or in combination with CW, resulted in a slight decrease in cell recovery rates compared to the corresponding core-only formulation. Cell recovery rates were lowest for formulations with larger particle sizes.

[0489] Next Example 1 The survival rate of the formulation in feed was tested for 4 hours at 25 °C and 52 °C using a low pH and high moisture diet representing a fattening farm diet (Table 2) according to the protocol described in [Table 18]. After 4 hours of incubation in the diet, the recovery rate was calculated by comparing it to the concentration of the encapsulated product before mixing it into the diet.

[0490] Table 18. After mixing the encapsulated product with a low pH and high moisture diet for 4 hours at 25 °C or 52 °C under aerobic conditions M. Elsdeny Recovery rate.

[0491]

[0492] All formulations showed a complete recovery rate after 4 hours of incubation at 25 °C in a low pH and high moisture diet, regardless of the treatment group (data not shown). The formulation containing 25% lipid in the outer coating had a slightly lower recovery rate compared to its counterpart containing 30%. The formulation with the smallest particle size showed a slight improvement in recovery rate after 4 hours of incubation at 52 °C in a low pH and high moisture diet.

[0493] Example 9: Optimal particle size of initial freeze-dried culture.

[0494] Next, an analysis was performed to determine whether standardizing the particle size of freeze-dried M. elsdeni cultures before two-step encapsulation resulted in higher cell recovery rates and protection.

[0495] M. elsdeni cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. The freeze-dried powder was sieved and divided into two groups, < 400 microns and < 1600 microns. The resulting freeze-dried powder was mixed with maltodextrin and Table 19 and Example 5 As emphasized in, it was encapsulated.

[0496] Analyze the colony-forming units (CFU) of the encapsulated product and compare with the initial freeze-dried powder Example 1 As described in [document], the recovery rate after encapsulation was determined.

[0497] Table 19. Formulation composition and cell recovery rate after encapsulation.

[0498]

[0499] The particle size of the freeze-dried M. elsdeny culture did not have a significant effect on the cell recovery rate after encapsulation. However, consistency in the particle size of the initial freeze-dried M. elsdeny culture is important for producing efficient encapsulation, as indicated by the increased cell recovery rate observed in this example compared to previous examples. Table 20 compares two-step formulations, all consisting of a 75% core made of maltodextrin and HPO and coated with 25% HCO, where the only difference is whether the initial freeze-dried culture was sieved (8(X1)A and 8(X2)) or not sieved (8A and 5A). The recovery rate in formulations with a homogeneous initial freeze-dried culture ranged from 37 to 46%, whereas the recovery rate in the same formulations without pre-screening of the freeze-dried culture was only 11.5 to 22%.

[0500] Table 20. Effect of freeze-dried culture pre-screening on final cell recovery rate after encapsulation.

[0501]

[0502] Example 1 The in-feed viability of Formulation 8 (X) was further tested at 25 °C and 52 °C for up to 14 hours according to the protocol described in [Table 21] and using a low pH and high-moisture diet (Table 2). After 4 hours of incubation in the diet, the recovery rate was calculated by comparing it to the concentration of the encapsulated product before mixing into the diet.

[0503] Table 21. After mixing the encapsulated product (8(X1)) into a low pH and high moisture diet under aerobic conditions at 25 °C or 52 °C for up to 14 hours, M. Elsdeny Recovery rate.

[0504]

[0505] After 4 hours of incubation on a low pH and high-moisture diet at 25 °C, over 92% of M. elsdenii were still alive, and up to 32% survived even after 14 hours, demonstrating the efficacy of encapsulation. At 52 °C, cell viability was lower, but the core alone ( Example 6 It was much better than ) or other previously tested encapsulations.

[0506] Example 10: Passing the formulation through a micromachine prior to exposure in feed had little effect on the recovery rate of M. elsdenyi.

[0507] Next, the effect of the passage of the micromachine system on the two-step formulation and the ability to protect cells when subsequently exposed to feed were determined.

[0508] Example 6 Formulations 8A and 16A from are processed individually using a micromachine (bowl system and continuous flow system), and then Example 1 It was mixed with a low pH and high moisture diet (Table 2) at room temperature for 4 hours according to the protocol described.

[0509] CFU results were adjusted for the amount of product and expressed as CFU per gram of product (formulation). Recovery after 4 hours of incubation in the diet was calculated by comparing the concentration of the encapsulated product before passing through the micromachine and before exposure in the feed. Results were compared to a formulation counterpart exposed only to the feed (not treated via the micromachine). Six samples per time point and per treatment group.

[0510] The recovery rate (Fig. 3) was not affected by the type of micromachine system (bowl vs. continuous) or the formulation used (when the outer coating contained carnauba wax (16A) or when it did not (8A)). Additionally, the recovery rate results for the micromachine system and the non-micromachine system were similar regardless of the formulation, which demonstrates that the passage through the micromachine system had little effect on the formulation's ability to protect M. elsdeny.

[0511] Example 11: External coating using HCO alone or CW following spinning disc HPO encapsulation enabled delayed but complete cell release after 7 to 12 hours in an in vitro fermentation model.

[0512] Next, we determined whether two-step encapsulation using a spinning disc and a Wurster system enables the complete release of bacteria (M. elsdenyi) in an in vitro fermentation model and whether the formulation yields the same level of gas production, which is an indicator of bacterial growth.

[0513] The ANKOM RF gas generation system provided an accurate method for monitoring and measuring in vitro gas generation. Freeze-dried M. elsdenyi culture, core, formulation 8(X1)( Example 9 ), Formulation 16(X1)( Example 10 ) and vegetable oil formulations( Example 3 Samples of ) were placed in Pyrex® bottles under sterile conditions. Then, 100 mL of semi-limited lactate medium was added to each bottle under sterile and anaerobic conditions. Subsequently, RF pressure sensor modules were fitted to the bottles, and the bottles were placed in a shaking incubator at 39 °C for 24 hours. All treatment groups were prepared in triplicate. The pressure in each bottle was measured and recorded at 5-minute intervals to generate a gas production curve in psi (Fig. 4). Sample weights were adjusted based on the initial sample concentration of M. elsdeny to aim for an equivalent amount of M. elsdeny in each bottle.

[0514] The formulation prepared by two-step encapsulation exhibited delayed release compared to the freeze-dried culture alone or the freeze-dried culture (core) encapsulated in a single layer of vegetable oil or hydrogenated palm oil (Fig. 4). The ability of cells to utilize lactate was unaffected, as indicated by similar gradients and total gas production across all encapsulated treatment groups. This experiment further demonstrated that the two-step formulation provides increased protection compared to the single-encapsulation formulation.

[0515] Example 12: Spinning disc HPO encapsulation followed by a hardened cottonseed oil-only external coating provides sufficient cell protection during exposure to high pH and low moisture feed.

[0516] Next, external coating with HCO was performed following spinning disc encapsulation to determine whether this process provides sufficient protection to M. elsdenyi when mixed with a low-moisture and high-pH diet and a high-moisture and low-pH diet for up to 4 hours.

[0517] Example 9 Except that Formulation 8(X1)A from was mixed with a finisher diet having low moisture and high pH (Table 4) and with a finisher diet having high moisture and low pH (Table 2), and the 15-gram portion of the diet was used instead of 30 grams Example 1 It was maintained at either 25 °C or 52 °C for 4 hours according to the protocol described. After 4 hours of incubation in the diet, the recovery rate (Table 22) was calculated by comparing it to the concentration of the encapsulated product before mixing it into the diet.

[0518] Table 22. M. Elsdeny recovery rate after mixing the encapsulated product (8(X1)) with a low-moisture and high-pH diet and a high-moisture and low-pH diet at 25 °C or 52 °C for 4 hours under aerobic conditions.

[0519]

[0520] All M. elsdenyi cells were recovered after exposure to a high-moisture and low-pH diet at room temperature (25 °C) for 4 hours (100%), and only 26% mortality was observed in the low-moisture and high-pH diet. In addition, the recovery rate after 4 hours in either diet at 52 °C was significantly higher compared to the previously tested formulation, which demonstrates that formulation 8(X1)A is superior to the previously tested formulation.

[0521] Example 13: Commercial-scale production of encapsulated M. elsdenyi using a two-step process with palm oil (HPO) and cottonseed oil (HCO).

[0522] We verified that two-step encapsulation using a hydrogenated palm oil core and a cottonseed oil coating is effective in protecting M. elsdenyi during storage, delivery, and exposure to a typical feedlot diet when produced on a commercial scale.

[0523] The final formulation was prepared by grinding freeze-dried M. elsdenyi cultures obtained according to the method described in WO 2018 / 144653 A1 to a small screen size (Sieve 024R01823) to obtain a uniform powder of less than 400 μm. The resulting powder was mixed with maltodextrin and encapsulated with hydrogenated palm oil (HPO) using a spinning disc to form a core. The core was then coated with hydrogenated cottonseed oil (HCO) in a Wurster fluid bed coagulation coater to obtain a final product with a particle size ranging from 212 to 710 μm. The composition of the final formulation is listed in Table 23. A total of three batches were manufactured on a commercial scale (25 kg). Example 1 According to the protocol described in [Table], the recovery rate after encapsulation (Table 23), storage life at -20 °C and 4 °C (Fig. 5), and survival rate in feed (Fig. 6) were tested.

[0524] Table 23. M. Elsdeny cell recovery rate after encapsulation process.

[0525]

[0526] The selected final formulation process yielded an average recovery rate of 55% for M. elsdeni cells after encapsulation, with a 9% variation across three batches.

[0527] The average loss of the three batches over the first month after storage at 4 °C or -20 °C was 0.2 and 0.1 log, respectively. Considering that most of the loss occurs within the first two months after storage, the data were extrapolated to the 12-month time point. Refer to Figure 5.

[0528] Next, the commercial batch was mixed with two high-concentration diets, namely low pH / high moisture (Table 2) and high pH / high moisture (Table 3). Example 1 As described in [Figure 6], survival rates in feed were tested at 25 °C and 52 °C for up to 6 hours.

[0529] The formulation provided protection against M. elsdenyi during feed mixing, exhibiting less than 25% mortality during the first 4 hours at 25 °C regardless of diet (Fig. 6). Additionally, M. elsdenyi showed a recovery rate of approximately 30% after 4 hours of exposure and was able to survive on the same diet when exposed to a higher temperature of 52 °C (Fig. 6). As expected, mortality of M. elsdenyi was greater after 6 hours of exposure, regardless of diet and temperature. Since the time between feed mixing and feeding to animals under typical conditions in commercial feedlands is less than 4 hours, this demonstrates that this formulation, manufactured with two-stage encapsulation using a hardened palm oil core and cottonseed oil coating, provides sufficient protection to M. elsdenyi to withstand feed mixing and feeding in commercial feedlands.

[0530] Example 1 The survival rate (Table 24) of commercial batches passing through a bowl micromachine system was further tested according to the protocol described in [Table 24].

[0531] Table 24. M. Elsdeny recovery rate after passing the formulation batch through a micromachine (bowl system).

[0532]

[0533] On average, 87% of the cells (M. elsdeny) survived after passing through the micromachine. Example 10 As in [example], the passage of the micromachine had little effect on the cell recovery rate.

[0534] also, Example 1 Formulation batches were tested by simulating storage in a micromachine bin at 25 °C, 30 °C, and 37 °C before inclusion in a diet according to the protocol described in (Table 25).

[0535] Table 25. Recovery rate after exposure to the empty environment of a micromachine at 25 °C, 30 °C, or 37 °C.

[0536]

[0537] Cell loss due to exposure to the empty environment of the micromachine was found to occur within the first 3 hours, with no significant difference regardless of temperature. An increase in temperature from 25 °C to 37 °C during environmental exposure was found to slightly decrease the cell recovery rate, with an additional 6% cell loss. However, minimal differences were observed when comparing incubation at 25 °C and 30 °C. These data demonstrate that the formulation can withstand storage in the bin for an extended period prior to delivery via the micromachine while still providing over 79% viable cells.

[0538] Example 14: Viability of freeze-dried M. elsdeni cultures mixed with sterile ground corn and exposed to environmental conditions (heat and humidity) for up to 4 hours.

[0539] To evaluate viable M. elsdeny potentially ingested by animals when freeze-dried M. elsdeny powder (unencapsulated) is mixed with ground corn and sprinkled on top of feed.

[0540] In the study presented in WO 2018 / 144653 A1, freeze-dried M. elsdenyi powder was sprinkled over sterile ground corn in an aluminum pan, and samples were collected after exposure to ambient air for 0, 1, 2, and 4 hours in the laboratory or under outdoor sunlight. This experiment was repeated four times in Wamego, Kansas, USA, during July and August 2016 (Fig. 7).

[0541] 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. elsdeny in freeze-dried products mixed with ground corn and exposed to air in the laboratory decreased numerically during 4 hours of exposure, but did not differ significantly from its initial concentration. The recovery rate after 4 hours was approximately 17.4%.

[0542] The concentration of M. elsdeny in samples exposed to outdoor conditions decreased much more rapidly than its counterparts stored indoors at room temperature, and was significantly different after 2 hours of exposure. After 4 hours, only 3.8% of the initial M. elsdeny cells were still viable on average. The M. elsdeny concentrations in these samples showed significant inter-experimental variability, as indicated by a large standard deviation. This large variability may be attributed to differences in outdoor conditions (heat and humidity), but it may also be due to the freeze-dried products used, which may have different degrees of resistance to heat and humidity.

[0543] Sterilized ground corn will have a much lower percentage of humidity than the low pH and high moisture diet or high pH and low moisture diet tested above and will be considered a less harsh condition; however, the encapsulated product showed recovery rates ranging from 75 to 78% at room temperature and 28 to 32% at 52 °C after 4 hours of exposure in either diet, significantly outperforming the results presented here. These results demonstrate the utility and efficiency of the formulation.

[0544] Example 15: Evaluation of Encapsulated M. elsdenyi NCIMB 41125 in an Accelerated Beef Cattle Step-Up Program and Acidosis Induction Model

[0545] The purpose of this study is LactiproNXT ® Drench M. Elsdeny 1x10 of NCIMB 41125 11 (1 CFU drench) or LactiproNXT ® The purpose was to evaluate the effects of daily feeding of drenched and encapsulated M. elsdenyi on dry matter intake (DMI), in vitro lactate utilization, rumen lactate, and volatile fatty acid (VFA) concentrations during an accelerated feed adaptation program, finishing period, and acidosis induction.

[0546] methodBritish crossbreed steers (n = 40, body weight (BW) 437 ± 98 kg) equipped with rumen cannulas were individually fed at the University of Nebraska. The treatment groups were composed as follows.

[0547] · Control group: Castrated bulls not fed M. elsdeni

[0548] · Mr. Drench: On the first day of the experiment, M. Elsdeny's 1x10 11 LactiproNXT CFU commercially available ® Castrated bulls that received Low Drenche and did not receive other M. Elsdeny

[0549] · Low-dose group: LactiproNXT on Day 1 of the experiment ® Receive a commercially available dose via drench and receive daily top-sprayed encapsulated M. elsdenyi, approximately 1 x 10 per person per day. 6 Castrated bulls to which CFU was provided to the rumen.

[0550] · Medium-dose group: LactiproNXT on Day 1 of the experiment ® Receive a commercially available dose via drench and receive daily top-sprayed encapsulated M. elsdenyi, approximately 1 x 10 per person per day. 7 Castrated bulls to which CFU was provided to the rumen.

[0551] · High-dose group: LactiproNXT on Day 1 of the experiment ® Receive a commercially available dose via drench and receive daily top-sprayed encapsulated M. elsdenyi, approximately 1 x 10 per person per day. 8 Castrated bulls to which CFU was provided to the rumen.

[0552] Control group cattle were acclimatized to late-finishing feed for 18 days, while the Drench group and low, medium, and high-dose groups were acclimatized for 9 days. Castrated cattle were fed once daily at 7:00 AM using a Calan gate system, and water was freely accessible. The experiment included five consecutive phases (Table 26): feed acclimatization period (days 1–19), fattening period (days 20–88), feed restriction (day 89, 24-hour complete feed restriction), induction period (day 90, feeding cattle 150% of the maximum fattening DMI), and recovery period (days 91–96).

[0553] Table 26. Diet and treatment group corresponding to the experimental period.

[0554]

[0555] Feed rejection samples were collected every 3 days during the feed adaptation period, every 7 days during the fattening period, and daily during the induction and recovery periods. Samples were collected at 6:00 AM and dried in a forced-air oven to determine dry matter intake (DMI) by adjusting for dry matter content (DM, Table 27).

[0556] Table 27. Diet composition from Stage 1 to fattening diet (% DM).

[0557]

[0558] The encapsulated product used in this experiment Example 3 It was prepared as described in [Table 28], and the amounts of freeze-dried M. elsdeni culture and carrier (potato starch) were varied to meet the daily target CFU per head (Table 28).

[0559] Table 28. Formulation composition.

[0560]

[0561] Rumen fluid samples were collected every 3 days during the feed acclimatization period, every 7 days during the fattening period, and daily at 1:00 PM during the induction and recovery period. During the induction and recovery period (Days 88, 90, 91, and 92), small amounts of collected rumen fluid tubes were stored at room temperature, and 0.1 mL of the fluid was inoculated into Hungate tubes containing semi-limited lactate medium to estimate lactate loss. A total of three tubes per animal were inoculated daily at 2:00 PM. The tubes were incubated in a 38 °C water bath for 0, 12, and 24 hours on Day 88, and for 0, 12, and 18 hours on Days 90 through 92, and then frozen for the analysis of lactate and VFA using gas chromatography.

[0562] Repeated measures were used within three stages: the feed adaptation period (days 1 to 19), the fattening period (days 20 to 88), and the recovery period (days 91 to 93). Data were used to test linear and quadratic effects of dose with the drench group as the intercept. Data were analyzed using covariate regression to test linear and quadratic effects of time and the time × treatment group interaction. The following contrasts were reported: control group versus Lactipro group (cattle fed M. elsdeny) and drench group versus daily feeding groups (low, medium, and high dose groups). Contrast coefficients for unequal intervals were obtained using Proc IML. Statistical significance was declared at P ≤ 0.10, and trends at P ≤ 0.15.

[0563] result : No differences in DMI were observed during the feed adaptation, fattening, or induction periods (Table 29). During the recovery period, DMI increased by 4.6% for the low, medium, and high dose groups compared to the drench group (P = 0.07), and DMI expressed as the percentage of intake before induction increased by an average of 17.5% for the low, medium, and high dose groups compared to the drench group (P = 0.05).

[0564] Table 29.Building intake of rumen cannula-equipped castrated bulls administered M. Elsdeny.

[0565]

[0566] In vitro lactate utilization on day 88 (before induction) was significantly increased in the daily administration group regardless of dose (low dose group: 98.3, medium dose group: 82.2, high dose group: 87.8 mmol / L) after 12 hours of incubation compared to the drench group (130.8 mmol / L) and the control treatment group (126.8 mmol / L) (P ≤ 0.05, Table 30).

[0567] Table 30. Lactate utilization rate before induction (Day 88).

[0568]

[0569] Treatment effects were observed at the 12-hour mark on days 90, 91, and 92 (Table 31), and castrated bulls in the low-dose (83.8 mmol / L), medium-dose (54.0 mmol / L), and high-dose (78.7 mmol / L) treatment groups showed higher lactate utilization rates than the control group (102.6 mmol / L) (P ≤ 0.05).

[0570] Table 31. Lactate utilization rate after induction (days 90, 91, and 92).

[0571]

[0572] During the feed adaptation period, cattle fed M. elsdeni had butyrate 3% higher (P < 0.01) and total VFA 4% lower (P = 0.06) compared to the control group (Table 32). During the fattening period, butyrate in cattle fed M. elsdeni tended to increase by 3% compared to the control group (P = 0.15). During the recovery period, cattle fed M. elsdeni daily had a 10% increase in total VFA concentration compared to steers in the Drench group (P = 0.08).

[0573] Table 32.Butyrate and total VFA concentrations in rumen cannula-fitted castrated steers administered M. elsdenyi during accelerated feed adaptation and acidosis induction.

[0574]

[0575] conclusion Regardless of the cell concentration used, accelerated feed adaptation (9 days compared to 18 days for the control group) was possible in both the Drenchy group and the M. elsdeny daily feeding group. However, the results showed that a single dose of LactiproNXT ® This suggests that drench does not persist in the rumen for up to 90 days; therefore, cattle experiencing acidosis in the later stages of fattening may benefit from receiving M. elsdeny daily as a topspray in addition to drench. Castrated steers fed M. elsdeny daily tended to have higher DMIs following the onset of acidosis. Additionally, they consumed more feed more quickly after a feed interruption, which was independent of the administered M. elsdeny concentration (low, medium, or high doses) and was likely due to higher lactate utilization. Daily feeding of M. elsdeny may be beneficial for commercial feedlands and dairy farmers during winter storms, revaccination dates, or any situations that alter or delay the cattle's feeding schedule.

[0576] The formulation used in this study is a single-layer encapsulation ( Example 3 It was manufactured using ), and was found to perform worse than two-step encapsulation using HPO and HCO in terms of recovery rate after encapsulation, survival rate in feed, and rumen release. Therefore, it can be expected that the two-step encapsulation formulation will yield similar or superior results when used in animal studies.

[0577] Example 16: Early drench administration of M. Elsdeny improved operational efficiency in commercial fattening facilities.

[0578] The main purpose of this embodiment is to shorten the feed adaptation period in commercial fattening facilities using LactiproNXT ®The goal was to quantify operational efficiency by using (M. Elsdeny).

[0579] Cattle farmers generally attempt to switch cattle to fattening compound feed as quickly as possible without inducing acidosis or bloat. The traditional feed adaptation period takes more than 21 days, which is intended to allow time for a normal population of lactic acid-utilizing bacteria to grow within the rumen and to process the lactic acid produced by lactic acid-producing bacteria during the starch breakdown process. During that feed adaptation period, the fattening facility (for the control group Example 15 (As exemplified in) by using a series of diets to gradually increase the level of starch and decrease the level of roughage, the number of mixtures (diets) to be manufactured, the number of deliveries to be carried out by feed trucks, and the amount of roughage to be ground and stored in the facility will be increased.

[0580] LactiproNXT ® Upon administration, it directly delivers an immediately viable population of M. elsdenyi to the rumen, thereby 'bypassing' the rumen adaptation period. LactiproNXT ® It functions as a management tool that enables feedlot managers to start feeding cattle fattening compound feed (low roughage, high starch) earlier, thereby improving operational efficiency at the facility. LactiproNXT ® A fattening facility manager who shortened the feed adaptation period using [method] reported the following while maintaining or exceeding animal performance: (1) a reduction in the volume of feed adaptation diet mixtures produced, (2) a reduction in movement within the facility to deliver the initial diet, (3) a reduction in the amount of roughage required, and (4) a reduction in the amount of hay to be ground.

[0581] Two studies on commercial fattening facilities (Table 33) were conducted using a similar protocol to evaluate the impact of the shortened feed adaptation period on operational efficiency. One independent company used LactiproNXT ®Changes at each facility were evaluated as a result of shortening the feed adaptation period by 10 days by adding [the product] as an initial drench (single application). Monitoring and evaluation were conducted on-site at each fattening facility for 3 to 4 days to assess all aspects of cattle feed administration, feed preparation, and delivery. The company's operations engineers gathered insights into their practices from fattening facility staff, collected baseline data, and developed a proprietary model to analyze the results. The presented case studies represent actual results and are based on conservative model estimates. Live and carcass performance was also measured as part of these studies but reported separately. Actual results may differ from the predicted model. This model continues to improve in terms of robustness, accuracy, and precision.

[0582] Table 33. Description of fattening facilities, assumptions, and operational efficiency for Case Studies 1 and 2.

[0583]

[0584] In conclusion, LactiproNXT ® Shortening the feed adaptation period using [method] can help fattening facilities significantly improve operational efficiency (Table 33). The fattening facilities analyzed in the case study used less hay, resulting in reduced maintenance and labor, reduced movement within the facility, reduced utility usage, and reduced feed time, thereby providing an opportunity to reallocate labor to other fattening facility tasks. The net savings were [values] for LactiproNXT ® Includes the annual cost of and the cost of the fattening diet to replace the initial diet. LactiproNXT ® The use of provided improved operational efficiency and net economic returns for fattening facilities.

[0585] Example 15As shown in [the text], daily administration of M. elsdenyi encapsulated in feed has been proven to provide additional benefits to animals, and additional operational savings are also expected because it can limit the number of feed changes during winter storms, revaccination days, or any situations that cause changes or delays in the cattle's feeding schedule.

[0586] Example 17: LactiproNXT ® M. Elsdeny's 1 x 10 in the on-arrival drench and castrated cattle feed upon arrival 6 or 1 x 10 7 An accelerated fattening diet program including daily administration of CFU is LactiproNXT ® Shows improvement in cattle performance compared to conventional fattening programs without.

[0587] Example 16 As mentioned, the need to grind multiple diets in forage acquisition, handling, feeding, and fattening facilities is a common challenge associated with traditional feed adaptation programs in commercial fattening facilities. In addition, Example 15 It demonstrates that cattle receiving M. elsdenyi daily as a top spray of feed had rumen fluids more efficient at metabolizing lactate in vitro, and that cattle receiving this bacterium daily were able to recover pre-induction intake more quickly compared to the control group and the Lactipro drench-alone treatment group. Therefore, accelerating the feed adaptation period with M. elsdenyi and including M. elsdenyi daily in the feed can improve not only operational efficiency but also animal performance.

[0588] The purpose of this study is to compare cattle that have undergone an existing feed program and LactiproNXT ® Upon arrival, 1 x 10 Drench and encapsulated M. Elsdeny per day 6 or 1 x 10 7 It is to compare the performance of cattle that have undergone a program including an accelerated fattening diet adaptation program involving the daily administration of CFUs via feed.

[0589] Approximately 1,600 to 2,000 castrated bulls aged 12 to 24 months and weighing 650 to 850 lb will be used in this experiment. The cattle will be housed in unfenced, in-house (dirt-floored) pens. The cattle will receive one of the following three treatments.

[0590] Control group - Traditional feed adaptation program without the use of M. elsdeny.

[0591] M. e . Low-dose group - M. Elsdeny (rehydrated Drench, M. Elsdeny NCIMB 41125 1 X 10 10 CFU, LactiproNXT ® ) will be administered during initial treatment, cattle will adapt to the feed within approximately 50% of the traditional feed adaptation protocol period, and encapsulated M. elsdenyi approximately 1 x 10 per head daily 6 It will be administered to deliver CFU.

[0592] M. e. High-dose group - M. Elsdeny (Rehydrated Drench, 1 x 10 of M. Elsdeny NCIMB 41125 10 CFU, LactiproNXT ® ) will be administered during initial treatment, cattle will adapt to the feed within approximately 50% of the traditional feed adaptation protocol period, and encapsulated M. elsdenyi approximately 1 x 10 per head daily 7 It will be administered to deliver CFU.

[0593] Encapsulated M. ElsdenyM. elsdenyi cultures were grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. Before forming a core containing encapsulated and freeze-dried (FD) M. elsdenyi, maltodextrin, and hydrogenated palm oil (HPO) using the spinning disc method, the freeze-dried powder was sieved to less than 400 micrometers and mixed with maltodextrin as shown in Table 34. The core was treated to produce small particles and then spray-coated with hydrogenated cottonseed oil (HCO) using a Wurster fluid bed coagulation coater. The final formulation composition is listed in Table 34. The final encapsulated cells were further mixed with a carrier (maltodextrin) to produce 1 x 10⁶ at a daily dose of 50 mg per head. 6 or 1x10 7 The target delivery amount of CFU was met.

[0594] Table 34. Formulation composition and cell recovery rate after encapsulation.

[0595]

[0596] The encapsulated product will be stored at -20 °C and dispensed daily using a continuous flow micromachine.

[0597] All diets will be formulated to meet or exceed the nutritional requirements of the animals. The dry matter (DM) composition and calculated and / or measured nutrient composition of the diet(s) will be reported. Dry matter and nutrient analysis (including starch content) of the compound feed will be performed. The weight and number of individuals of the feed delivered will be recorded daily in pen units during the study period. Any feed refused will be removed, weighed, and recorded. All cattle will have free access to water. Cattle will be fed through a four-stage diet adaptation as shown in Table 35.

[0598] Table 35. Feeding days and corresponding diets for each treatment group.

[0599]

[0600] The cattle will be divided into groups based on arrival date, source, and / or truckload until they are randomized. As soon as enough cattle are accommodated to fill one complete replicate (3 pens), the cattle will be randomly assigned to pens, with one pen allocated per treatment group. The process will be repeated 8 times to have 8 pens per treatment group. The experimental design will be a randomized complete block design. The block factor will be a replicate consisting of 3 pens, indicating that one pen is included in each treatment group per replicate (i.e., 8 blocks). The pens will serve as the experimental units, and the blocks will serve as either fixed or random effects, depending on the nature of the variable.

[0601] After randomization and before initial treatment, all pens within the replicate will be weighed using a platform scale, and this weight will serve as the initial study weight. Separate weights for each pen will also be collected between days 90 and 95 of feeding. After collecting these weights, feeding and water will be suspended for 4 hours before the cattle are returned to their original pens. This is intended to simulate revaccination or final treatment situations.

[0602] The test period will be approximately 180 days. On the 180th day of the test, all cattle pens will be weighed using our scales, and the value obtained by subtracting a 4% reduction factor from that weight will serve as the final weight for the test. Carcass data will be collected by a third-party data collection service.

[0603] Lactic acid utilization analysis - The lactic acid metabolic activity of rumen fluid will be measured in cattle 13 and 14 days after feeding. To briefly explain, the cattle are a 'control group' and ' MeAnimals will be randomly selected from the 'high-dose' treatment group pens (10 animals per treatment group, 1 to 2 animals per pen), transferred to the treatment facility, and rumen fluid samples will be collected via an esophageal tube using an electronic peristaltic pump. The samples will be filtered through four layers of gauze, transferred to pre-labeled 500 mL conical tubes, and tightly closed. Twelve 0.1 mL rumen fluid samples will be inoculated into twelve Hungate tubes containing semi-limited lactate medium. The tubes will be incubated in a water bath maintained at 40 °C. After 0, 3, 6, 9, 12, and 15 hours of incubation, two tubes will be removed to measure optical density, and the tubes will be placed in a -20 °C freezer. The frozen tubes will be sent to a third-party laboratory for evaluation of lactic acid concentration.

[0604] In addition to lactic acid utilization, the following response variables will be measured and analyzed: sorting (frequency and type), mortality rate, initial weight, final weight, daily gain, total gain, dry matter intake, feed efficiency, and carcass characteristics.

[0605] The difference between treatments is between the control treatment and Me low dose and Me A contrast phrase comparing the average of the high-dose group and Me low-dose group and Me It will be determined by using a comparative phrase that compares the high-dose group.

[0606] result Cattle treated with M. elsdeny will produce greater butyrate compared to the control group, regardless of the feeding period. They will also have a higher DMI after revaccination or final treatment compared to the control group. No differences in DMI will be observed during feed adaptation and the fattening period. In vitro lactate utilization will significantly increase in the M. elsdeny-treated group compared to the control group, regardless of dose.

[0607] conclusion Regardless of the concentration used, M. Elsdeny Accelerated feed adaptation is possible in both cases of daily feeding. Castrated steers fed M. elsdeni daily consumed more feed more quickly after a feed interruption, regardless of the administered M. elsdeni concentration (low or high dose), likely due to higher lactate utilization compared to the control group. Additionally, the low- and high-dose M. elsdeni treatment groups exhibited superior or equivalent cattle performance compared to the control group. Daily feeding of M. elsdeni is beneficial for commercial feeders and dairy farmers during winter storms, revaccination dates, or any situations that change or delay the cattle feeding schedule.

[0608] Example 18: Generation of encapsulated anaerobic cells by a two-step process using palm oil (HPO) and cottonseed oil (HCO).

[0609] Anaerobic bacteria can be divided into three categories: (1) obligate anaerobic bacteria, (2) acid-tolerant anaerobic bacteria, and (3) facultative anaerobic bacteria. Obligate anaerobic bacteria are bacteria that cannot survive at normal atmospheric oxygen concentrations. Some obligate anaerobic bacteria can survive at up to 8% oxygen, but others cannot survive unless the oxygen concentration is less than 0.5%. Acid-tolerant anaerobic bacteria can survive in the presence of oxygen, but do not use oxygen for growth. Facultative anaerobic bacteria can use oxygen for aerobic respiration, but may also use anaerobic respiration in the absence of oxygen.

[0610] Megasphaera, e.g., M. elsdenii; Fibrobacter, e.g., F. succinogenes; Butyrivibrio, e.g., B. fibrisolvens; Luminococcus, e.g., R. flavefaciens; and Bifidobacterium, e.g., B. breve are representative species of obligate anaerobic bacteria. Lactobacillus, e.g., L. plantarum; and Bifidobacterium, e.g., B. animalis subspecies lactis are representative species of acid-resistant anaerobic bacteria. Pediococcus cells, e.g., P. acidilactis; and Lactobacillus cells, e.g., L. casei are representative species of facultative anaerobic bacteria.

[0611] This example demonstrates the applicability of the formulation to other anaerobic bacteria listed above. It also demonstrates that two-step encapsulation using a hydrogenated palm oil core and a cottonseed oil coating is effective in protecting anaerobic bacteria during storage, delivery, and exposure to a typical feedlot diet.

[0612] A formulation was prepared by grinding a dried anaerobic bacterial culture to a small screen size (Sieve 024R01823) to obtain a uniform powder of less than 400 μm. The resulting powder was mixed with a carrier such as maltodextrin and encapsulated with hydrogenated palm oil (HPO) using a spinning disc to form a core. Subsequently, the core was coated with hydrogenated cottonseed oil (HCO) in a Wurster fluid bed coagulation coater to obtain a final product with a particle size in the range of 212 to 710 μm. The composition of the final formulation is listed in Table 36. Example 1 The formulation was tested for recovery rate after encapsulation (Table 36), shelf life, survival rate in feed, and rumen release according to the protocol described.

[0613] Table 36. Anaerobic cell recovery rate after encapsulation process.

[0614]

[0615] The final formulation yields an average recovery rate of at least 10% against anaerobic bacterial cells after encapsulation.

[0616] Package the formulation and store at -20 °C. Example 1 In order to evaluate the storage life according to the protocol described, sampling will be performed at a predetermined point in time.

[0617] Example 13Similar to M. elsdenyi, the average loss over the first month of storage at -20 °C will be less than 0.3 log. Considering that most loss occurs within the first few months of storage, the loss after 12 months of storage at -20 °C will be less than 0.5 log. Two-stage encapsulation will provide a stable shelf life regardless of the type of anaerobic bacteria tested.

[0618] Next, the formulation was mixed with two high-concentration diets, namely low pH / high moisture (Table 2) and high pH / high moisture (Table 3). Example 1 As described, survival rates in feed were tested at 25 °C and 52 °C for up to 6 hours.

[0619] The formulation will provide protection for anaerobic cells throughout feed mixing, exhibiting less than 25% mortality during the first 4 hours at 25 °C, regardless of the diet. Additionally, anaerobic cells will be able to survive within the same diet, showing a recovery rate of over 30% after 4 hours of exposure even when exposed to a higher temperature of 52 °C. Higher mortality of anaerobic cells is expected after 6 hours of exposure, regardless of diet and temperature. Under typical conditions in commercial feedyards, the time elapsed between feed mixing and distribution to animals is estimated to be less than 4 hours, demonstrating that the formulation provides sufficient protection for anaerobic cells to withstand feed mixing and distribution in commercial feedyards.

[0620] Next Example 1 The formulation will be tested for rumen release according to the protocol described to determine whether it is consistently released in the rumen after ingestion by anaerobic cells.

[0621] The measured rumen release for the formulation(s) will fall under the following commercial standards. Each is 50% released upon 24-hour field incubation in the rumen of castrated cattle fed high-concentration feed or high-rough feed.

[0622] The formulation will be further tested for survival rate upon passing through a micromachine and exposure to the environment inside the micromachine bin according to the protocol described in Example 1.

[0623] On average, at least 70% of the anaerobic cells will survive when passing through the micromachine, and at least 80% of the cells will be recovered when left in the bin for up to 14 hours at 25 °C, 30 °C, or 37 °C.

[0624] Example 19 - Generation of anaerobic encapsulated cells through a two-step process using freeze-dried or spray-dried cells.

[0625] This example describes M. elsdenii and other anaerobic bacteria produced by freeze-drying (PCT Publication No. WO 2018 / 144653 A1) or spray-drying (PCT Application No. PCT / US2023 / 028982). Example 18 Demonstrates the applicability and effectiveness of two-step encapsulation using a hardened palm oil core and cottonseed oil coating for (see reference).

[0626] Cultures of M. elsdenyi and other anaerobic bacteria were grown, cooled, and concentrated, then freeze-dried according to the method described in WO 2018 / 144653 A1 or spray-dried according to the method described in PCT application number PCT / US2023 / 028982. The resulting dried cultures were screen-grinded to obtain a homogeneous powder. The resulting powder was mixed with a carrier such as maltodextrin and encapsulated with hydrogenated palm oil (HPO) using a spinning disc to form a core. The core was then coated with hydrogenated cottonseed oil (HCO) to obtain a final product with a particle size ranging from 212 to 710 μm. The final formulation compositions are listed in Table 37. A formulation was prepared. Example 1 Recovery rate (Table 37), shelf life, survival rate in feed, and rumen release after encapsulation were tested according to the protocol described in [Table].

[0627] Table 37. Anaerobic cell recovery rate after encapsulation process.

[0628]

[0629] Regardless of the method used to produce the dried culture, the final formulation will yield an average recovery rate of at least 10% for all anaerobic bacterial cells tested after encapsulation.

[0630] Package the formulation and store at -20 °C. Example 1 In order to evaluate the storage life according to the protocol described, sampling will be performed at a predetermined point in time.

[0631] Example 13 As with M. Elsdeny, the average loss over the first month of storage at -20 °C will be less than 0.3 log. Considering that most of the loss occurs within the first few months after storage, the loss after 12 months of storage at -20 °C will be less than 0.5 log. The method used to produce the initial dried culture will not affect the shelf life regardless of temperature.

[0632] Next, the formulation was mixed with two high-concentration diets, namely low pH / high moisture (Table 2) and high pH / high moisture (Table 3). Example 1 As described, survival rates in feed will be tested at 25 °C and 52 °C for up to 6 hours.

[0633] The formulation will provide protection for anaerobic cells throughout feed mixing, exhibiting less than 25% mortality during the first 4 hours at 25 °C, regardless of the diet or the method used to dry the initial culture. Additionally, anaerobic cells will be able to survive within the same diet, showing a recovery rate of over 30% after 4 hours of exposure even when exposed to a higher temperature of 52 °C. As expected, anaerobic cell mortality will be higher after 6 hours of exposure, regardless of diet and temperature. Under typical conditions in commercial feedyards, the time elapsed between feed mixing and distribution to animals is estimated to be less than 4 hours, which demonstrates that the formulation provides sufficient protection for anaerobic cells to withstand feed mixing and distribution in commercial feedyards, regardless of the method used to dry the initial culture.

[0634] Next Example 1 The formulation will be tested for rumen release according to the protocol described to determine whether it is consistently released in the rumen after ingestion by anaerobic cells.

[0635] The measured rumen release for the formulation(s) will fall under the following commercial standards. Regardless of the method used to dry the initial culture, 50% is released upon 24-hour field incubation in the rumen of castrated steers fed either high-concentration feed or high-rough feed, respectively.

[0636] formulation Example 1 In accordance with the protocol described, further tests will be conducted on the survival rate upon passing through the micromachine and exposure to the environment inside the micromachine bin.

[0637] On average, regardless of the method used to dry the initial culture, at least 70% of the anaerobic cells will survive when passed through the micromachine, and at least 80% of the cells will be recovered when left in the bin for up to 14 hours at 25, 30, or 37 °C.

[0638] Example 20: The carrier used in the core had little effect on the efficiency of two-step encapsulation using palm oil (HPO) and cottonseed oil (HCO) in anaerobic cells.

[0639] This example will demonstrate that the use of maltodextrin, sucrose, or starch had little to no effect on the effectiveness of two-step encapsulation using a hydrogenated palm oil core and a cottonseed oil coating that protects anaerobic bacteria during storage, delivery, and exposure to a typical feedlot diet.

[0640] Cultures of M. elsdeni and other anaerobic bacteria according to the method described in WO 2018 / 144653 A1 ( Example 18 (Reference) was grown, cooled, concentrated, and freeze-dried. The resulting dried culture was screen-grinded to obtain a homogeneous powder. The resulting powder was mixed with a carrier such as maltodextrin, sucrose, or starch, and encapsulated with hydrogenated palm oil (HPO) using a spinning disc to form a core. The core was then coated with hydrogenated cottonseed oil (HCO) to obtain a final product with a particle size in the range of 212 to 710 μm. The final formulation composition is listed in Table 38. Example 1 The formulation was tested for recovery rate after encapsulation (Table 38), shelf life, survival rate in feed, and rumen release according to the protocol described.

[0641] Table 38. Anaerobic cell recovery rate after encapsulation process.

[0642]

[0643] All final formulations will yield an average recovery rate of at least 10% for anaerobic bacterial cells after encapsulation, regardless of the carrier used in the core.

[0644] Package the formulation and store at -20 °C. Example 1 In order to evaluate the storage life according to the protocol described, sampling will be performed at a predetermined point in time.

[0645] Example 13As with M. elsdenyi, the average loss of three batches over the first month of storage at -20 °C will be less than 0.3 log. Considering that most loss occurs within the first few months of storage, the loss after 12 months of storage at -20 °C will be less than 0.5 log. The carrier will not have a significant effect on the shelf life regardless of temperature or the bacteria used.

[0646] Next, the formulation was mixed with two high-concentration diets, namely low pH / high moisture (Table 2) and high pH / high moisture (Table 3). Example 1 As described, survival rates in feed were tested at 25 °C and 52 °C for up to 6 hours.

[0647] The formulation will provide protection for anaerobic cells throughout feed mixing, exhibiting less than 25% mortality during the first 4 hours at 25 °C, regardless of the diet. Additionally, anaerobic cells will be able to survive within the same diet, showing a recovery rate of over 30% after 4 hours of exposure even when exposed to a higher temperature of 52 °C. As expected, anaerobic cell mortality will be higher after 6 hours of exposure, regardless of diet and temperature. The time elapsed between feed mixing and distribution to animals under typical conditions in commercial feedyards is estimated to be less than 4 hours, which demonstrates that the formulation provides sufficient protection for anaerobic cells to withstand feed mixing and distribution in commercial feedyards, regardless of the carrier used within the core.

[0648] Next Example 1 The formulation will be tested for rumen release according to the protocol described to determine whether it is consistently released in the rumen after ingestion by anaerobic cells.

[0649] The measured rumen release for the formulation(s) will fall under the following commercial standards. Regardless of the carrier used in the core, 50% is released upon 24-hour field incubation in the rumen of castrated cattle fed high-concentration feed or high-rough feed, respectively.

[0650] formulation Example 1 In accordance with the protocol described, further tests will be conducted on the survival rate upon passing through the micromachine and exposure to the environment inside the micromachine bin.

[0651] On average, regardless of the carrier used in the core, at least 70% of the anaerobic cells will survive when passing through the micromachine, and at least 80% of the cells will be recovered when left in the bin for up to 14 hours at 25 °C, 30 °C, or 37 °C.

[0652] Example 21: All lipids tested to form a core provided sufficient protection to make the two-stage encapsulation of anaerobic cells commercially viable.

[0653] This example demonstrates that using palm oil, vegetable oil, stearic acid, or dritex to make the core did not affect the effectiveness of two-step encapsulation using a cottonseed oil coating that protects anaerobic bacteria during storage, delivery, and exposure to a standard feedlot diet.

[0654] M. elsdenii and other anaerobic bacterial cells according to the method described in WO 2018 / 144653 A1 ( Example 18 (Reference) was grown, cooled, concentrated, and freeze-dried. The resulting dried culture was screen-grinded to obtain a homogeneous powder. The resulting powder was mixed with a carrier such as maltodextrin and encapsulated with hydrogenated palm oil (HPO), vegetable oil, stearic acid, or dritex using a spinning disc to form a core. The core was then coated with hydrogenated cottonseed oil (HCO) to obtain a final product with a particle size in the range of 212 to 710 μm. The final formulation composition is listed in Table 39. Example 1The formulation was tested for recovery rate after encapsulation (Table 39), shelf life, survival rate in feed, and rumen release according to the protocol described.

[0655] Table 39. Anaerobic cell recovery rate after encapsulation process.

[0656]

[0657] All final formulations will yield an average recovery rate of at least 10% for anaerobic bacterial cells after encapsulation, regardless of the lipid source used to generate the core.

[0658] Package the formulation and store at -20 °C. Example 1 In order to evaluate the storage life according to the protocol described, sampling will be performed at a predetermined point in time.

[0659] Example 13 As with M. Elsdeny, the average loss of three batches over the first month of storage at 4 °C or -20 °C will be less than 0.3 log. Considering that most loss occurs within the first few months after storage, the loss after 12 months of storage at -20 °C will be less than 0.5 log. All lipid sources used to create the core will result in a formulation sufficiently stable for commercial products.

[0660] Next, the formulation was mixed with two high-concentration diets, namely low pH / high moisture (Table 2) and high pH / high moisture (Table 3). Example 1 As described, survival rates in feed were tested at 25 °C and 52 °C for up to 6 hours.

[0661] The formulation will provide protection for anaerobic cells throughout feed mixing, exhibiting less than 25% mortality during the first 4 hours at 25 °C, regardless of the diet. Additionally, anaerobic cells will be able to survive within the same diet, showing a recovery rate of over 30% after 4 hours of exposure even when exposed to a higher temperature of 52 °C. As expected, anaerobic cell mortality will be higher after 6 hours of exposure, regardless of diet and temperature. Under typical conditions in commercial feedyards, the time between feed mixing and distribution to animals is estimated to be less than 4 hours, demonstrating that the formulation provides sufficient protection for anaerobic cells to withstand feed mixing and distribution in commercial feedyards, regardless of the lipid source used to generate the core.

[0662] Next Example 1 The formulation will be tested for rumen release according to the protocol described to determine whether it is consistently released in the rumen after ingestion by anaerobic cells.

[0663] The measured rumen release for the formulation(s) will fall under the following commercial standards. Regardless of the lipid source used to create the core, 50% is released upon 24-hour field incubation in the rumen of castrated steers fed either high-concentration feed or high-rough feed, respectively.

[0664] formulation Example 1 In accordance with the protocol described, further tests will be conducted on the survival rate upon passing through the micromachine and exposure to the environment inside the micromachine bin.

[0665] On average, regardless of the lipid source used to generate the core, at least 70% of the anaerobic cells would survive when passing through the micromachine, and at least 80% of the cells were recovered when left in the bin for up to 14 hours at 25 °C, 30 °C, or 37 °C.

[0666] Example 22: Formulation Pore Size

[0667] Example 9 Samples of the core, formulation 8 (X1), and formulation 16 (X1) prepared as in [the example] were sent to a third-party laboratory for porosity analysis. The results are listed in Table 40.

[0668] Table 40. Porosity analysis

[0669]

[0670] The central pore size of the formulations consisting of two-stage encapsulation was 170.5 to 176 μm, which indicates that the tested formulations can release bacteria, namely M. elsdenyi cells, in the size range of 2.4 to 2.6 μm. In addition, these formulations had a porosity in the range of 35 to 40%, which further demonstrates that the formulations have the ability to release bacteria.

[0671] Finally, a small central pore diameter area in the range of 0.0053 to 0.0056 μm indicates the presence of numerous capillaries within the formulation, which means that the formulation is highly porous and interconnected.

[0672] In comparison, the core obtained by mixing freeze-dried M. elsdeni culture with maltodextrin and encapsulating it in hydrogenated palm oil (HPO) using the spinning disk method had a lower central pore diameter, but the porosity and central pore diameter area were equivalent.

[0673] Example 23: Alternative lipids for external coating

[0674] M. elsdenyi cultures will be grown, cooled, concentrated, and freeze-dried according to the method described in WO 2018 / 144653 A1. A fraction of the resulting freeze-dried culture will be mixed with maltodextrin in a 1:5 ratio and encapsulated as described in Example 5. The formulation will be produced to have a small final particle size (212 to 710 μm) to increase the surface area and maximize post-ingestion release (rumen release) in cattle. The colony-forming units (CFU) of the encapsulated product will be analyzed and compared with the initial freeze-dried powder to determine the post-encapsulation recovery rate according to the protocol described in Example 1.

[0675] We will analyze formulations using hydrogenated palm oil (58 °C to 62 °C, 27 stearin), beeswax (62 °C to 64 °C), hydrogenated soybean oil (66 °C to 71 °C, 17 stearin), paraffin (68.9 °C to 72.8 °C), stearic acid (69.3 °C) and candelilla (72.5 °C).

[0676] Example 5 As in, M. elsdeny cells will be encapsulated using the lipid and spinning disk method.

[0677] Example 1 In order to establish survival rates in feed according to the procedures described, the formulation will be tested in feed using a low pH and high moisture diet.

[0678] Example 24: Pets - Effects of daily supplementation of M. elsdenyi in pets (dogs)

[0679] Previous studies have demonstrated the clinical benefits of probiotic supplementation in companion animals, including the inhibition of pathogenic bacterial proliferation, the promotion of beneficial bacterial growth, the improvement of intestinal barrier integrity, and the modulation of immune function. Furthermore, some of these benefits are linked to positive behavioral changes by alleviating stress and anxiety.

[0680] The purpose of this experiment is to determine the effects of daily encapsulated M. elsdenyi supplementation on healthy animals and animals suffering from gastrointestinal diseases in companion animals.

[0681] Methods: At least 6 healthy dogs and 6 unhealthy dogs aged 2 to 9 years will be included in the study, with an equal ratio of males to females. The healthy control dogs will not have clinical signs of GI disease. The unhealthy dogs will have clinical signs of gastrointestinal disease (e.g., chronic enteropathy, exocrine pancreatic insufficiency, and / or diabetes mellitus) indicated by chronic diarrhea (stool score < 3) for at least 3 weeks.

[0682] All dogs will be housed in individual outdoor kennels with dirt floors that are cleaned twice a day. Exercise will be provided daily, but the dogs will be isolated by group (healthy group vs. unhealthy group).

[0683] All dogs will receive the same commercially available maintenance diet for 30 days during the adaptation period. For the next 30 days, dogs will receive either the control diet (maintenance diet without M. elsdeny) or the M. elsdeny diet (1 x 10 per dog per day). 5 Up to 1 x 10 9 The dogs will consume a maintenance diet supplemented with CFU of M. elsdeny. Subsequently, the dogs will be fed only the maintenance diet for 30 days (no M. elsdeny in either group, rest period) before being crossed over to the control group or the M. elsdeny diet for 30 days. The dogs will be fed using electronic feeders, and fresh food will be provided once a day in a calculated amount to maintain body weight. The study design is shown in Table 41.

[0684] Table 41. Research design.

[0685]

[0686] Blood, urine, and fecal samples will be collected at the end of the adaptation period (d30), as well as at approximately days 15 and 30 of each follow-up period (days 45, 60, 75, 90, 105, and 120). Metabolite levels and microbial composition will be evaluated at the end of each 30-day feeding period (days 30, 60, 90, and 120). Body weight measurements and behavioral assessments using validated questionnaires will be performed weekly.

[0687] Stool samples will be collected within 30 minutes of defecation and scored from 1 to 5, where a score of 1 indicates more than 75% liquid and 5 indicates more than 80% solid. The stool samples will then be homogenized in a mixer, aliquoted into vials, and frozen at -80 °C until further analysis.

[0688] Blood chemical components (e.g., glucose, ketones, hemoglobin A1, and lactate), cortisol, and inflammatory cytokines will be analyzed using enzymatic colorimetry. To identify metabolites and provide their relative quantification, overall plasma and fecal metabolite analysis will be performed using gas chromatography (for hydrophobic molecules) and liquid chromatography mass spectrometry (for hydrophilic molecules) platforms.

[0689] We will perform serum chemistry tests to measure triglyceride, creatinine, albumin, and cholesterol levels.

[0690] For all fecal samples, fecal pH analysis using a pH meter, short-chain fatty acid (SCFA) analysis using gas chromatography, and population analysis of Salmonella and Campylobacter species using selective culture methods will be performed.

[0691] We will analyze the fecal microbiome by extracting total DNA from thawed fecal samples and performing 16S rRNA gene amplification and sequencing of the V3-V4 region. The acquired raw sequences will be processed through a bioinformatics pipeline to remove sequences of insufficient quality and erroneous reads, as well as chimeric sequences, and the final sequences will be compared with available public databases. After the adaptation period, the microbial profiles of the healthy and unhealthy groups will be compared. The effect of M. elsdenyi supplementation on the abundance of unique taxonomic operational units will be evaluated by comparing the changes in their abundance after the treatment period.

[0692] Results: Unhealthy dogs will experience a significant improvement in stool quality after being fed M. elsdeni. This will be accompanied by changes in their fecal microbial population. The fecal microbiota of unhealthy dogs shifts to a healthy population profile after feeding food supplemented with M. elsdeni. This includes an increase in the abundance of bacteria belonging to Megasphaera, Blautia, Prevotella, and other beneficial bacteria, as well as a decrease in the abundance of gut bacteria and their members. In the absence of M. elsdeni supplementation, unhealthy dogs will have the highest fecal and blood lactate concentrations, suggesting that there is some accumulation of intestinal lactate in GI diseases. Lactate accumulation can occur when sugars are delivered to the colon due to carbohydrate dysdigestion, where the pH decreases due to the production of organic acids through fermentation. Consequently, acid-resistant Lactobacillus species grow preferentially while lactate-utilizing bacteria are inhibited, resulting in maintained lactate production but reduced lactate utilization. Unhealthy dogs supplemented with M. elsdeny daily will show lower fecal and blood lactate concentrations than when not receiving M. elsdeny.

[0693] SCFAs (e.g., butyrate, propionate, or acetate) will be higher in healthy animals than in unhealthy animals, regardless of the diet received. Additionally, M. elsdenyi supplementation will increase SCFA concentrations in animals compared to baseline levels in the absence of supplementation. SCFAs, including acetate, propionate, and butyrate, are well known to act as desirable fuel sources for colon cells and contribute to GIT health. M. elsdenyi supplementation will also result in changes in the ratio of SCFAs (butyrate, propionate, and acetate) that have inhibitory effects against pathogenic microorganisms (e.g., Salmonella enterica and E. coli). Furthermore, increases in butyrate and anti-inflammatory cytokines have been shown to have beneficial effects extending beyond the gastrointestinal tract, such as reducing anxiety and stress.

[0694] Microbiome analysis will show differences in bacterial populations between healthy and unhealthy groups. M. elsdeny supplementation will induce changes in bacterial populations, particularly in unhealthy dogs, resulting in a decrease in lactate-producing bacteria such as Bifidobacterium species, Enterococcus species, and Lactobacillus species.

[0695] Finally, the analysis of the validated questionnaire will show that unhealthy dogs reduced stress / anxiety after consuming food supplemented with M. elsdeny. Levels of stress hormones (i.e., cortisol) and other stress / anxiety-related metabolites (i.e., inflammatory markers) will be significantly lower after consuming food supplemented with M. elsdeny compared to the control group.

[0696] M. elsdeny supplementation will have a beneficial effect on animal health, and the effect will be much more significant in animals suffering from gastrointestinal diseases.

[0697] Example 25: Poultry - Evaluation of the application of encapsulated Megasphaera elsdeni in broiler chickens.

[0698] Objective: We will conduct a preliminary broiler performance study to evaluate the effect of daily administration of Megaspaera elsdeni NCIMB 41125 on the growth performance of broiler chicks.

[0699] Methods: 1-day-old broiler chicks (n=360) vaccinated with Coccivac-B spray vaccine on Day 0 will be randomly assigned to three different treatment groups: (1) a negative control group (nCON) not receiving Megaspaera, and (2) M. elsdenyi NCIMB 41125 (1 x 10 per chick) added to feed daily starting from Day 0. 3 Up to 1 x 10 6 M. Elsdeny group receiving CFU) and (3) positive control group (pCON) that does not receive M. Elsdeny NCIMB 41125 but receives early and growing feed treated with BMD (50 g / t) and late finishing feed treated with Starpak (20 g / t).

[0700] Each treatment group will consist of four cages, each containing 30 birds. The diets provided to the birds will be as follows: initial feed on days 0–18, growing feed on days 19–35, and finishing feed on days 36–39. Animal body weight, feed intake, and feed conversion ratio will be recorded over a 39-day trial period.

[0701] Results: Overall mortality rates will not differ between treatment groups. The feed conversion ratio for the M. elsdeni group will be significantly lower than that of the controls (nCON and pCON). Conversely, the body weight for the M. elsdeni group will increase compared to the two controls (nCON and pCON).

[0702] Example 26: Poultry - Effect of Megasphaera elsdeni on Salmonella and Campylobacter concentrations and prevalence in broiler chicks

[0703] Objective: This study will be conducted to evaluate the effect of daily administration of Megaspaera elsdenii NCIMB 41125 on the detection rate and concentration of Salmonella and Campylobacter in the cecum of broiler chicks.

[0704] Methods: 1-day-old broiler chicks (n=192) will be randomly assigned to two different treatment groups: 1) a control group not receiving M. elsdenyi NCIMB 41125 and 2) M. elsdenyi encapsulated in the daily diet (1 x 10 chicks per day). 3 Up to 1 x 10 6 Megaspaera Elsdeni NCIMB 41125 daily administration group receiving CFU.

[0705] Each of the two treatment groups will consist of 16 cages, each containing 6 birds. Animal body weight, feed intake, and feed conversion ratio will be recorded over a 15-day trial period. After the 15-day feeding period, two animals per cage will be randomly selected for slaughter, and their cecums will be collected to determine the detection rates of Salmonella and / or Campylobacter. Briefly, the cecums will be collected, placed in bags, and stored on ice. The cecums will then be washed with 70% ethanol and manually massaged to extract the contents. One milliliter of the collected contents will be serially diluted in phosphate buffer saline solution (PBS) and plated onto individual selective agar. The selective agar plates will be incubated. Probable Salmonella and Campylobacter colonies will be counted and confirmed using a rapid test kit. Additionally, one milliliter of the cecum contents sample will be added to 9 mL of individual selective enrichment medium. If no detectable growth is observed on the selective agar plate, the enrichment solution will be plated again on the selective agar plate for incubation and evaluated for the presence of Salmonella and Campylobacter. A random count of 9 (1 lower than the theoretical detection limit) will be assigned to samples that show no growth in the direct plating but positive growth in the enrichment method, and a count of 0 will be assigned to samples that show no growth in either the direct plating or the enrichment method.

[0706] Birds supplemented daily with encapsulated M. elsdeni will have lower cecal Salmonella and / or Campylobacter concentrations compared to the control group. The detection rate of Salmonella and / or Campylobacter in samples from the M. elsdeni group will also be reduced compared to the control group.

[0707] Example 27: Poultry - Effects of Megaspaera elsdeni on Growth Performance and Cecum Characteristics of Broilers

[0708] Experimental design and treatment groups

[0709] We will perform 24 replicates for two treatment groups using a randomized complete block design with 1-day-old Cobb 500 broiler chicks at the start of treatment. The treatment groups will be as follows: 1) a control group not receiving M. elsdenyi and 2) 1 x 10 per bird per day. 3 Up to 1 x 10 6 M. elsdeny group receiving daily supplementation of M. elsdeny strain NCIMB 41125 encapsulated in CFU in feed.

[0710] Birds will be housed in 48 pens, each containing 35 birds at the start of the experiment (a total of 1,680 birds). On the first day of the experiment, chicks will be assigned to groups of 35, and the weight of each group will be recorded. The bird groups will be processed by block, and the experimental treatment groups will be randomly assigned within each block.

[0711] To prevent cross-contamination with the treated birds, the control birds will be handled only by designated personnel who will not have contact with the treated birds, placed in designated transport containers, weighed, and transported to the pen.

[0712] The birds will have free access to fresh water. All diets will be supplied via gravity feeders suspended in the center of the cage. Feed will be added as needed to ensure free access throughout the study period. The birds will receive an initial diet (Days 1–16), a rearing diet (Days 17–30), and a late fattening diet (Day 31–end of study).

[0713] The total feed intake per pen for each stage (initial, growing, and late finishing) will be calculated as follows: Amount of feed administered - Amount of feed recovered.

[0714] The daily intake of the bird will be calculated as follows: Total feed consumption ÷ [Daily number of individuals in the pen x Total number of feeding days]

[0715] We will record our weight at the end of each feeding period (initial, growing, and late finishing).

[0716] Each week (days 7, 14, 21, 28, and 35), 1 to 3 birds will be randomly selected from each cage and euthanized by cervical dislocation. Cecum contents (0.5 g) will be collected to perform cecum pH, volatile fatty acid (VFA) analysis, and M. elsdeny quantitative analysis using quantitative real-time PCR.

[0717] To determine carcass measurements, birds will be slaughtered at 5 weeks of age. Feeding will be stopped approximately 4 hours prior to slaughter. Five average-sized birds will be selected from each pen, placed in capture boxes, and transported to the processing area. Immediately before slaughter by stunning, five birds per pen will be weighed to determine their live weight. The birds will be bled and their feathers plucked. The feet, heads, and shanks will be removed, and the internal organs will be extracted. Subsequently, the carcasses will be weighed per pen to determine the carcass yield.

[0718] Results: Broilers treated with M. elsdeni will exhibit similar or improved feed intake, feed efficiency, and average daily weight gain compared to the control group. Bird body weight and mortality rates in the M. elsdeni treatment group will be unaffected or improved.

[0719] The cecal pH of birds treated with M. elsdeni will show a significant difference compared to control birds.

[0720] Birds treated with M. elsdeny will have higher concentrations of acetate, butyrate, caproate, and total VFA in their cecal contents compared to control birds. The acetate:propionate (A:P) ratio will also be higher in the cecal contents of birds treated with M. elsdeny compared to the control group.

[0721] Example 28: Pigs - Effects of daily supplementation of M. elsdenyi on growth performance and fecal characteristics of sows and piglets.

[0722] Objective: This study was designed to determine the effects of supplementing M. elsdeni on growth performance and fecal characteristics in sows and piglets during the lactation and pre- and post-weaning periods.

[0723] Background: Weaning is a highly stressful event in which piglets undergo rapid changes in diet, environment, and social interactions. Consequently, weaning causes significant changes in the microbial population within the gastrointestinal tract and can lead to microbial imbalance.

[0724] Lactic acid-producing bacterial species (e.g., Lactobacillus, Bacillus, Enterococcus, or Pediococcus species) are commonly used probiotics in the swine industry. They have been shown to reduce pathogen occurrence and fecal excretion. One proposed mechanism of action is cross-feeding between lactic acid-producing bacteria and lactate-utilizing bacteria (e.g., M. elsdenii) in the colon, which results in the inhibition of Brachyspira hyodicenteria, a dysenteric pathogen; however, the exact mechanism by which this occurs has not yet been established. It can be hypothesized that the presence of lactate-utilizing bacteria in the colon may prevent the excessive growth of lactic acid-producing bacteria during stress periods and play a crucial role in preventing microbial imbalance by maintaining colonic pH and providing VFAs necessary for intestinal cell growth / proliferation and intestinal mucosal health. Consequently, piglets will experience improved pathogen resistance and nutrient absorption rates.

[0725] Sows often experience reduced feed intake during farrowing and mobilize stored body fat to meet lactation energy requirements, which can lead to excessive body weight loss and have detrimental effects on subsequent reproductive performance. Supplementing sows with *M. elsdenyi* can increase VFA production, thereby providing additional butyrate, which is known to be a desirable energy source for colon cells, prevent mucosal atrophy, stimulate mucin secretion, and help strengthen the intestinal barrier. Consequently, *M. elsdenyi* can improve feed intake by stimulating appetite through improved overall gut health and reduced gastrointestinal (GI) disorders. Intervention with *M. elsdenyi* can serve a dual purpose: increasing feed intake in sows to thus increase energy availability, and stabilizing the GI microbiome to prevent disease.

[0726] Method: A total of 28 sows will be blocked according to parity and assigned to one of four treatment groups consisting of a split plot design (Table 42), the overall plot will be whether sows are supplemented in Megaspaera elsdeni and the subplot will be whether piglets are supplemented in Megaspaera elsdeni.

[0727] Table 42. Split plot design.

[0728]

[0729] M. Elsdeni supplementation is 1 x 10 per sow 5 Up to 1 x 10 9 CFU, 1 x 10 per piglet 3 Up to 1 x 10 6 It will be provided daily in the form of M. elsdenyi encapsulated in CFU.

[0730] There will be 14 repetitions of the full plot, 14 repetitions of the subplot, and 7 repetitions of the interactive plot. Treatment will be administered daily to sows and / or piglets starting from the start of farrowing.

[0731] All sows will be fed the same common diet and managed according to standard procedures. On approximately the 21st day of nursing, piglets will be weaned and moved to rearing facilities. On the day of weaning, one piglet from the litter will be sacrificed to collect the entire cecal contents. Piglets will be housed in pens with 4 to 5 piglets per pen, and piglets from the same treatment group will be grouped into the same pen. The piglets will be fed the same common diet. Feed loss will be recorded, and daily feed intake will be calculated.

[0732] Sows will be weighed on days -3, -2, -1 before farrowing, and on day 21 (at weaning). Piglets will be weighed on days 2, 21 (at weaning), 28, 35, 42, and 49.

[0733] Fecal samples will be collected by rectal palpation on days 1, 7, 14, and 21 for sows, and on days 21, 28, 35, 42, and 49 for piglets.

[0734] Fecal samples will be analyzed for pH using a pH meter and for VFA concentration using a gas chromatograph. The collected whole cecal contents will be flash-frozen in liquid nitrogen and stored at -80 °C until PCR analysis.

[0735] We will perform quantitative real-time PCR analysis to quantify M. elsdenyi in cecum samples.

[0736] Results: Sows supplemented with M. elsdeny will exhibit less body weight loss and a higher average daily feed intake compared to their counterparts not supplemented with M. elsdeny. Additionally, sows supplemented with M. elsdeny will show higher cecal pH and increased VFA concentrations (e.g., increased propionate, butyrate, caproate, and total VFA) compared to control animals.

[0737] Piglets supplemented with M. elsdeny will have higher average daily weight gain, feed efficiency, and fecal pH compared to their unsupplemented counterparts, regardless of the sow's supplementation status. As with sows, M. elsdeny-treated piglets will show increased VFA concentrations, particularly butyrate, and lower mortality rates.

[0738] Overall, M. Elsdeny will improve the intestinal environment, including the composition of the final microbial fermentation product, intestinal pH, and intestinal microbial colonization.

[0739] Example 29: Long-term storage in a micromachine bin of M. Elsdeny encapsulated at a commercial scale in a two-step process using palm oil (HPO) and cottonseed oil (HCO).

[0740] This study demonstrated how effective two-step encapsulation using a hardened palm oil core and a cottonseed oil coating is in protecting M. elsdenyi during long-term storage in micromachine bins when produced on a commercial scale.

[0741] In this experiment, the final formulation composition was prepared as shown in Table 23 of Example 13. Three commercially produced batches (25 kg each) were tested by simulating long-term storage (up to 48 hours) in a micromachine bin at 25 °C, 30 °C, and 37 °C before inclusion in a diet according to the protocol described in Example 1 (Table 43).

[0742] Table 43. Recovery rate after long-term storage in a micromachine bin exposed to an environment of 25 °C, 30 °C, or 37 °C.

[0743]

[0744] Since there was no difference in cell loss due to exposure to the micromachine bin environment between batches, only the average is presented in Table 43.

[0745] Increasing the temperature from 25 °C to 37 °C during prolonged environmental exposure reduced the cell recovery rate, with an additional 25% cell loss at 48 hours. However, a minimal difference (4%) was observed when comparing 25 °C and 30 °C incubation at 48 hours. These data demonstrate that the formulation can withstand long-term incubation before delivery via micromachines while still providing over 56% viable cells.

[0746] Example 30 - Generation of anaerobic encapsulated cells through a two-step process using spray-dried cells.

[0747] This example is M. Elsdeny (produced by spray drying (WO 2024 / 026095 A1) Example 18 Using a hardened palm oil core and cottonseed oil coating for (see reference) Demonstrates the applicability and effectiveness of two-step encapsulation.

[0748] M. elsdeny was grown, cooled, concentrated, and spray-dried according to the method described in WO 2024 / 026095 A1. The resulting powder was encapsulated in hydrogenated palm oil (HPO) mixed with two amounts of carrier (maltodextrin 13.23% and 18.75%) using a spinning disc to form a core. The core was then coated with hydrogenated cottonseed oil (HCO) to obtain a final product with a particle size ranging from 212 to 710 μm. The composition of the final formulation is listed in Table 44. The formulation was prepared and Example 1 Recovery rate (Table 44), shelf life, survival rate in feed, and micromachine pass rate were tested after encapsulation according to the protocol described.

[0749] Rumen release will be tested using the Daisy rumen model. Briefly, rumen fluid collected from recently slaughtered castrated bulls fed a high-concentration diet will be sieved, and a 400 mL aliquot will be placed in a bottle containing 1600 mL of salivary buffer. A pre-weighed filter bag containing 0.5 g of formulation and a lead sinker (to sink the bag) will be added to the bottle. The contents of the bottle will be mixed and incubated at 39 °C under anaerobic conditions for 24 hours. After the incubation time is complete, the digestion bottle will be removed from the incubator, and the filter bag will be collected, dried, and weighed to determine the dry matter loss rate.

[0750] Table 44. M. Elsdeny cell recovery rate after formulation composition and encapsulation process.

[0751]

[0752] Regardless of the amount of carrier used, the final formulation yielded an average recovery rate of at least 30% for M. elsdenyi cells after encapsulation.

[0753] Package the formulation and store at 4 °C and -20 °C. Example 1 Samples were taken at predetermined points in time to evaluate the storage life according to the protocol described in [the document].

[0754] Example 13 As with M. Elsdeny, the average loss over the first month of storage at 4 °C or -20 °C was less than 0.3 log regardless of the amount of carrier (maltodextrin) used. Considering that most of the loss occurs within the first few months after storage, the loss after 12 months of storage at -20 °C will be less than 0.5 log.

[0755] Next, the formulation is mixed with a highly concentrated feed (Table 2) having a low pH and high moisture diet. Example 1 As described in [document], survival rates in feed at 25 °C and 52 °C for up to 4 hours were tested.

[0756] The formulation provided protection to M. elsdenyi cells throughout feed mixing, exhibiting a recovery rate of over 47% during the first 4 hours at 25 °C, regardless of the amount of maltodextrin used in the formulation. Furthermore, M. elsdenyi cells were able to survive within the same diet, exhibiting a recovery rate of over 18% after 4 hours of exposure even when exposed to a higher temperature of 52 °C. Since the time between feed mixing and distribution to animals under typical conditions in commercial feedlands is estimated to be less than 4 hours, this demonstrates that the formulation provides sufficient protection to M. elsdenyi cells to withstand feed mixing and distribution in commercial feedlands, regardless of the carrier used.

[0757] On average, at least 55% of M. elsdeny cells survived after passing through the micromachine.

[0758] The formulation will also be tested for ruminal release according to the Daisy rumen model protocol listed above to confirm whether it is consistently released from the rumen after ingestion by anaerobic cells.

[0759] Spray-dried M. elsdeny encapsulated via a two-step process exhibited similar results in cell viability, in-feed viability, and micromachine passage to those observed in freeze-dried M. elsdeny encapsulated using the same process; therefore, ruminal release is also expected to be similar. The ruminal release measured for the formulation(s) will fall within the following commercial standards. High-concentration diets regardless of the amount of maltodextrin used 50% is released from the rumen fluid of the fed castrated steer during 24-hour on-site incubation.

[0760] These data demonstrate that spray-dried M. elsdenyi cells, encapsulated via a two-step process, can generate viable cells capable of withstanding storage, feed mixing, and micromechanical passage, while still being released in the rumen. Since these results are not different from those observed when M. elsdenyi cells were freeze-dried and then encapsulated in the same manner, ( Example 13 The ), and two-step encapsulation process is successful in protecting M. elsdenyi cells regardless of the drying method used prior to encapsulation.

[0761] Example 31 - Effects of M. elsdeni oral drench on rumen pH dynamics in milking cows under subacute rumen acidosis induction

[0762] Subacute rumen acidosis (SARA) is a significant disorder in dairy cows that affects economic viability and animal welfare. A preventive strategy for SARA control is the oral drenching of probiotics such as *Megasphaera elsdenii*.

[0763] The purpose of this study was to evaluate the effects of M. elsdeni oral drench on rumen pH, milk production and composition, and feeding behavior in dairy cows under SARA induction.

[0764] This study consisted of two crossover trials with eight cattle each to determine the drenching efficacy of M. Elsdeny NCIMB 41125 viable cultures. The duration of each trial was eight days, with a four-week rest period between periods. Each animal was assigned to an individual feeder, which operated using each cattle's radio frequency identification (RFID) tag and recorded feed intake. Each animal ate from its assigned bin until the end of the trial period. The first three days of each period were considered the baseline days. On the fourth day of the period, the delivered feed was reduced by half based on the individual average dry matter intake (DMI) during the baseline days. On the fifth day of the period, all cattle received a provocation diet rich in highly fermentable carbohydrates, which was provided to the cattle for two hours to induce SARA. The induced mixture was composed of 2 kg of rolled barley, 2 kg of ground wheat, and 0.9 kg of molasses mixed with 4.3 kg of total mixed ration (TMR). The remaining feed was weighed and replaced with the standard diet provided freely. The last three days of each experimental period were considered recovery days.

[0765] The difference between each test is M. Elsdeny Drench (approximately 2×10 per mL). 8 LactiproNXT containing CFU ® The administration time was 100 mL; in Study 1, it was delivered 4 days prior to SARA induction (PRO-4), and in Study 2, it was delivered the day before SARA induction (PRO-1). Cattle were randomly assigned to the treatment group (PRO-4 or PRO-1) or control group (CON-4 or CON-1) drench, and during the subsequent period The assignment was made in reverse.

[0766] Milk production and composition, dry matter intake (DMI), feeding behavior (time spent feeding and number of feeder visits), and rumen pH were continuously recorded throughout the entire experimental period. Complete mixed feed samples were collected and analyzed during the reference days. Samples for nutrient and dry matter (DM) analysis were dried in a 55 °C oven for 48 hours. The dried samples were ground, passed through a 1 mm screen, and analyzed for acid detergent fiber (ADF) (AOAC International, 2000: method 973.18), neutral detergent fiber (NDF) with heat-resistant α-amylase and sodium sulfite (Van Soest et al., 1991), and crude protein (CP) (N x 6.25; AOAC International 2000: method 990.03; Leco FP-528 Nitrogen Analyzer, Leco, St. Joseph, MI).

[0767] During Experiment 1, PRO-4 cattle produced more milk (P < 0.01), had a higher protein percentage (P=0.03), and a lower fat-to-protein ratio (P=0.01) compared to control cattle. PRO-4 cattle also had a higher overall rumen pH, experienced acidosis for a shorter and less severe period (P < 0.05), and had a higher DMI (P=0.04) compared to control cattle. Feed Behavior was not affected by the treatment in Test 1 (P > 0.10).

[0768] In Experiment 2, only feeding time was affected by the treatment, and PRO-1 cattle spent more time per day feeding compared to control cattle (P < 0.01).

[0769] The results indicate the potential benefits of M. elsdenyi drenching on rumen pH dynamics, acidosis recovery, and potentially milk production and feed intake (Table 45). However, the timing of drenching administration appears to affect drenching efficacy. Stabilization of M. elsdenyi for daily administration in feed will facilitate adjustment of the treatment timing and ensure maximum product efficiency.

[0770] Table 45. Rumen pH kinetics, DMI, feeding behavior, milk production, and least squares mean (±SEM) of components in dairy cows (n=8) drenched with distilled water (control) or Megaspaera elsdeni 4 days prior to acidosis induction (Study 1) and 1 day prior to acidosis induction (Study 2).

[0771]

[0772] Example 32 - Effect of daily administration of encapsulated M. elsdenyi on rumen pH dynamics in milking cows under subacute rumen acidosis (SARA).

[0773] The purpose of this study is 1 x 10 per person per day 7 , 1 x 10 8 or 1 x 10 9 This study evaluates the effects of daily administration of encapsulated M. elsdenyi mixed into feed as CFU on rumen pH dynamics, milk production, milk composition, feed intake, feeding behavior, and rumen fluid composition in dairy cows under acidosis induction.

[0774] This experiment will use 32 Holstein multiparous cows that have entered their 90th day of milking (days in milk, DIM). The cows will be divided into four blocks and randomly assigned to one of four treatment groups (8 cows per treatment group).

[0775] - Control - M. Elsdeny was not administered.

[0776] - 10 7 (Low-dose group) - Encapsulated M. Elsdeny is about 2 x 10 per person daily 7 It will be administered daily to deliver CFU.

[0777] - 10 8 (Medium-dose group) - Encapsulated M. Elsdeny is about 2 x 10 per person daily 8 It will be administered daily to deliver CFU.

[0778] - 10 9 (High-dose group) - Encapsulated M. Elsdeny is about 2 x 10 per person daily 9 It will be administered daily to deliver CFU.

[0779] The experimental period will consist of a 10-day acclimatization period, a 1-day induction period, and a 10-day recovery period (a total of 22 days).

[0780] Encapsulated M. Elsdeny : The encapsulated product used in this experiment Example 13 It will be prepared as described in [the document], and Table 46 shows the final composition. Encapsulated M. elsdenyi will be further mixed with varying amounts of carrier (maltodextrin) to meet a daily CFU of 50 mg per head.

[0781] Table 46. Formulation composition

[0782]

[0783] The treatment agent will be administered to each feed trough before feeding by top-sprinkling a small amount of TMR. When the feed trough is emptied, the animals will receive the remaining TMR. Delivery of the treatment agent will begin on Day 0 of the adaptation period and continue until Day 22 of the recovery period.

[0784] On day 10, the cow will undergo a 24-hour feed restriction, receiving only 50% of the average intake from the previous 3 days. On day 11, the cow will be provided with 2 kg of rolled barley + 2 kg of ground wheat + 0.9 kg of molasses mixed with 4.3 kg of its standard TMR mixture for 2 hours. Afterward, the remaining feed will be weighed and then replaced with the remaining TMR mixture.

[0785] The cows will be housed in tie stall facilities consisting of tie stalls (1.2 x 2.4 m) lined with pine bedding. Each cow will be assigned one stall and will be tethered with a collar. During the experiment period, excluding the induction period, the cows will have access to feed through white feed troughs and free access to water through Ritchie watering troughs. The cows will be allowed to go outside three times a day to go to and from the milking parlor.

[0786] Dairy cow mixed feed will be fed as TMR once a day around 9 a.m. The total amount of feed delivered to each individual cow and the total amount rejected will be recorded daily to calculate the daily DMI.

[0787] On the day before the start of the experiment (Day 1), the cows will be equipped with an ear-mounted rumen sensor (Cow Manager) to measure activity and feeding behaviors, as well as a SmaXtec rumen pH bolus to measure rumen pH every 10 minutes.

[0788] Cows will be milked three times daily, and body weight, as well as milk production from each individual cow at each milking machine, will be recorded throughout the study period. Milk samples will be collected from individual cows during three consecutive milkings at the following intervals: Days 1-3, Days 7-9, Days 11-13, and Days 20-22. Samples from consecutive days will be mixed and preserved based on milk production and used for analysis of net protein, fat, lactose, solids-not-fat (SNF), milk urea nitrogen (MUN) concentrations, somatic cell counts (SCC), and milk fatty acid profiles.

[0789] Rumen fluid will be extracted via a Rumen-Mate pump 6 hours after feeding (or induction) on the day before the start of the experiment (-Day -1), Day 5, Day 9 (the day before feed restriction), Day 11 (after induction), Day 12, Day 13, Day 16, Day 23, and Day 30. The pH of the rumen fluid samples will be measured immediately after sampling using a portable pH meter (Ohaus ST20 pH Pen Meter). The rumen fluid samples will then be filtered through four layers of gauze. A 50 ml aliquot of the resulting fluid will be placed in pre-labeled Falcon tubes (two Falcon tubes per animal per sampling) and immediately frozen at -20 °C for subsequent VFA and lactate analysis. Another aliquot will be placed in SafeCollect TM Place in a collection tube (DNA / RNA Shield, Cat # R1211, Zymo Research, California), mix thoroughly, and freeze immediately at -20 °C for subsequent qPCR analysis.

[0790] On days 11, 12, and 13, using subsamples of collected rumen fluid Example 17 As previously stated, we will perform an analysis of the lactate utilization rate (loss rate).

[0791] Cattle receiving encapsulated M. elsdeny will exhibit a generally higher rumen pH and experience shorter and less intense attacks of acidosis. Additionally, the cattle will have a higher DMI and spend more time feeding compared to control cattle. In vitro measured lactate utilization (loss rate) will also be more efficient for cattle receiving encapsulated M. elsdeny.

[0792] Compared to Example 31, which followed a similar experimental design but tested a single dose of M. elsdenyi drench, this experiment will demonstrate the superiority of daily administration of encapsulated M. elsdenyi and its ability to further improve rumen pH dynamics, acidosis recovery, and potentially milk production and feed intake in dairy cows.

[0793] Example 33 - Effects of Daily Administration of Encapsulated M. elsdenyi on Early Lactation Performance in Dairy Cows

[0794] Similar to beef cattle, dairy cows transition from a low-energy diet during the dry period to a high-energy diet just before calving and during the early lactation period. Direct-feeding microorganisms such as M. elsdenyi have been regarded not only as an option to reduce the occurrence of metabolic disorders during this period, but also as a means to accelerate the transition, facilitate dairy management, and potentially reduce operating costs.

[0795] The purpose of this experiment is to determine the effects of daily administration of encapsulated M. elsdenyi in dairy cow feed on rumen pH, milk production, and overall health during the transition period (immediately before calving and early lactation).

[0796] A total of 80 cows in their second or later calving will be used from day 21 pre-calving to day 56 post-calving. Cows will be moved to BioControl pens in groups of 20 in four batches starting day 28 pre-calving, and a 7-day training period will be provided to allow them to adapt to individual feed stations. The cows will be used in a completely randomized block design and will be randomly assigned to one of the following four treatment groups.

[0797] - Control - M. Elsdeny was not administered.

[0798] - Low-dose group - Encapsulated M. elsdeny will be administered daily to deliver an estimated 0.5X of M. elsdeny per head daily.

[0799] - Medium-dose group - Encapsulated M. elsdeny will be administered daily to deliver an estimated 1X of M. elsdeny per head daily.

[0800] - High-dose group - Encapsulated M. elsdeny will be administered daily to deliver an estimated 2X of M. elsdeny per head daily.

[0801] The amount of M. Elsdeny in each group is Example 32It will be determined based on the results. Treatment will begin to be provided to each group 21 days before delivery and will continue for the first 21 days of breastfeeding.

[0802] The cattle will be housed in a large pen consisting of 24 sand litter free stalls (1.2 x 2.4 m) and will have access to feed through BioControl feeders and free access to water through Ritchie watering troughs.

[0803] All cattle will receive two basic compound feeds: a pre-calving diet formulated for the dry period and a lactation diet. Both compound feeds will be fed once daily as Total Mixed Ration (TMR) around 8:30 a.m. TMR (before treatment delivery) and individual components will be collected weekly throughout the study and stored at -20 °C until analysis.

[0804] Daily DMI and feeding behavior will be collected using BioControl feeders and analyzed individually before and after birth to determine whether the treatment affects feeding behaviors such as meal intervals, feeding and feeding speed and number of visits.

[0805] When the study begins, the cattle will be weighed and a SmaXtec rumen pH bolus will be used to record rumen pH every 10 minutes, and a CowManager® tag (Agis, Harmelen, Netherlands) will be fitted on the ear to monitor rumination and feeding behavior throughout the entire study period.

[0806] Rumen fluid will be extracted via a Rumen-Mate pump at the start of the experiment (Day -21) and, in relation to farrowing, on Days 3, 28, and 56, 6 hours after feeding. The pH of the rumen fluid samples will be measured immediately after sampling using a portable pH meter (Ohaus ST20 pH Pen Meter). The rumen fluid samples will then be filtered through four layers of gauze. A 50 ml aliquot of the resulting fluid will be placed in pre-labeled Falcon tubes (two Falcon tubes per animal per sampling) and immediately frozen at -20 °C for subsequent VFA and lactate analysis. Another aliquot will be placed in SafeCollect TM It will be placed in collection tubes (DNA / RNA Shield, Cat # R1211, Zymo Research, California). The tubes will be thoroughly mixed and immediately frozen at -20 °C for subsequent qPCR analysis of the M. elsdeni population.

[0807] After calving, cows will be milked three times daily, and milk production from each individual cow at each milking station will be recorded throughout the study period. Milk samples will be collected from individual cows weekly to analyze net protein, fat, lactose, SNF, MUN concentrations, and milk fatty acid profiles. For each cow, milk component production will be calculated by multiplying the component concentration on the test day by the milk production from the sampling during that period. The body weight of each individual cow will be recorded after each milking station.

[0808] Events in the cattle, including transition issues, health-related medical treatments, and non-medical observations, will be recorded. At the end of the study (Day 56), the cattle will be returned to a commercial herd. Lactation and service data will be monitored until the next lactation to determine the effects of daily administration of encapsulated M. elsdenyi on total milk production, culling rates, and reproduction.

[0809] Cows supplemented daily with encapsulated M. elsdeny immediately before calving and during the early lactation period will produce more milk, have a higher protein ratio, and a lower fat-to-protein ratio compared to control cows, regardless of the dose used (low, medium, or high). Animals administered M. elsdeny will reach the flow peak sooner and are likely to produce more milk at the flow peak. Cows supplemented daily with encapsulated M. elsdeny will also have a higher overall rumen pH compared to control animals and will experience acidosis attacks with rumen pH below 5.6 and 5.8 more briefly and less intensely. Rumen composition will differ, showing increased levels of propionate and butyrate in animals supplemented daily with encapsulated M. elsdeny. Additionally, the M. elsdeny group will exhibit higher DMI and increased feeding behavior compared to animals not receiving M. elsdeny. Finally, compared to the control group, the number of cattle culled in the M. Elsdeny group will decrease, gestation time will be shortened, and the pregnancy rate will increase.

[0810] These results support trends observed in previous dairy studies using a single dose of M. elsdenyi drench and will confirm that daily administration of encapsulated M. elsdenyi immediately before delivery and during early lactation can maintain benefits throughout the entire lactation period.

Claims

Claim 1 (a) (i) Megaspaera Elsdeni A composition comprising (ii) a cell, (iii) a core comprising at least one carrier and (iii) one or more lipids, and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells are viable in the composition. Claim 2 (a) (i) Megaspaera Elsdeni A composition comprising (ii) a cell, (iii) a core comprising at least one carrier and (iii) one or more lipids, and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable after being added to a feed or feed additive. Claim 3 A composition according to claim 2, wherein the Megasphaera elsdenii cells in the composition are viable for up to 48 hours at 25 °C and pH 7.0 after being added to the feed or feed additive. Claim 4 (a) (i) Megaspaera Elsdeni A composition comprising (ii) a cell, (iii) a core comprising at least one carrier and (iii) one or more lipids, and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and administration of the composition to a ruminant causes viable Megasphaera elsdenii cells to be released in the rumen. Claim 5 (a) (i) Megaspaera Elsdeni A composition comprising (ii) a cell, (iii) a core comprising at least one lipid, and (b) at least one layer of one lipid, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a temperature of at least 40 °C to 60 °C at pH 7.0 for 4 to 18 hours. Claim 6 (a) a composition comprising (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 at 25 °C for up to 48 hours. Claim 7 (a) a composition comprising (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours. Claim 8 (a) a composition comprising (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable after processing through a micromachine system. Claim 9 (a) a composition comprising (i) a Megasphaera elsdenii cell, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the Megasphaera elsdenii cells in the composition are viable for up to 48 hours after being added to the microbin of a micromachine. Claim 10 A composition according to claim 9, wherein at least 10% of the Megasphaera elsdenii cells in the composition are viable for about 14 hours to about 48 hours after being added to the microbin of a micromachine. Claim 11 A composition according to any one of claims 1 to 10, wherein the particle size of the Megasphaera elsdeni cells in the composition is less than about 1600 μm. Claim 12 A composition according to any one of claims 1 to 11, wherein the particle size of the Megasphaera elsdenii cells in the composition is less than about 400 μm. Claim 13 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state is viable in the composition. Claim 14 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after being added to a feed or feed additive. Claim 15 A composition according to claim 14, wherein the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state among the composition are capable of surviving for up to 48 hours at 25 °C and pH 7.0 after being added to the feed or feed additive. Claim 16 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and administration of the composition to a ruminant causes viable bacterial cells to be released from the rumen. Claim 17 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a temperature of 40 °C to 60 °C at pH 7.0 for 4 to 18 hours. Claim 18 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition are viable when the composition is exposed to a pH of 3 to 7 at 25 °C for up to 48 hours. Claim 19 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable when the composition is exposed to a pH of 3 to 7 and a temperature of 25 °C to 60 °C for up to 48 hours. Claim 20 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable after processing through a micromachine system. Claim 21 (a) a composition comprising (i) an anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state, (ii) at least one carrier and (iii) a core comprising one or more lipids and (b) at least one layer of one or more lipids, wherein the core is coated by the layer of one or more lipids, and at least 10% of the anaerobic bacterial cell or an anaerobic bacterial cell in a trophic state in the composition is viable for up to 48 hours after being added to the microbin of a micromachine. Claim 22 A composition according to claim 21, wherein at least 10% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state in the composition are viable for about 14 hours to about 48 hours after being added to the microbin of a micromachine. Claim 23 A composition according to any one of claims 13 to 22, wherein the particle size of the anaerobic bacterial cell or the anaerobic bacterial cell in a trophic state in the composition is less than about 1600 μm. Claim 24 A composition according to any one of claims 13 to 23, wherein the particle size of the anaerobic bacterial cell or the anaerobic bacterial cell in a trophic state in the composition is less than about 400 μm. Claim 25 A composition according to any one of claims 13 to 24, wherein at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are viable in the composition. Claim 26 A composition according to any one of claims 13 to 25, wherein the anaerobic bacterial cell or anaerobic bacterial cell in a trophic state is dried. Claim 27 A composition according to any one of claims 13 to 26, wherein the anaerobic bacterial cell or anaerobic bacterial cell in a trophic state is dried by spray drying, electrospray drying, vacuum drying, jet drying, freeze drying, or a combination thereof. Claim 28 A composition comprising about 0.1% to about 15% (w / w) of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state, in any one of claims 13 to 27. Claim 29 In any one of paragraphs 13 through 28, approximately 1 x 10 per g 4 to about 1 x 10 10 A composition comprising CFU of anaerobic bacterial cells or anaerobic bacterial cells in a trophic state. Claim 30 A composition according to any one of claims 13 to 29, wherein the anaerobic bacterial cell or anaerobic bacterial cell in a trophic state is selected from the group consisting of Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subs. lactis, Pediococcus acidilactis, Lactobacillus casei, Megasphaera elsdenii, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Luminococcus flavefaciens, Blautia oveum, Clostridium butyricum, Achermansia musiniphila, and combinations thereof. Claim 31 A composition according to any one of claims 1 to 30, wherein the composition is a granule, capsule, minicapsule, microcapsule, tablet, minitablet, or microtablet. Claim 32 A composition having a moisture content of about 5% (w / w) or less, in any one of claims 1 to 31. Claim 33 A composition according to any one of claims 1 to 32, wherein one or more lipids in the core are selected from the group consisting of animal oil or fat, vegetable oil or fat, triglycerides, free fatty acids, animal wax, beeswax, lanolin, shell wax, pewter, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic wax, natural and synthetic resins and mixtures thereof. Claim 34 A composition according to any one of claims 1 to 33, wherein one or more lipids in the core are animal fats or oils and / or vegetable fats or oils. Claim 35 A composition according to claim 34, wherein the vegetable fat or oil is selected from the group consisting of canola oil, cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil. Claim 36 In paragraph 34, the above vegetable fat or oil is hydrogenated palm oil, in the composition. Claim 37 A composition according to claim 33, wherein the free fatty acid is myristic acid, lauric acid, or stearic acid, or a combination thereof. Claim 38 A composition according to any one of claims 1 to 37, wherein one or more lipids in the core have a melting point of about 40 °C to about 85 °C. Claim 39 A composition according to any one of claims 1 to 38, wherein one or more lipids in the core have a melting point of about 55 °C to about 75 °C. Claim 40 A composition according to any one of claims 1 to 39, wherein one or more lipids in the core are selected from the group consisting of animal oil or fat, vegetable oil or fat, triglycerides, free fatty acids, animal wax, beeswax, lanolin, shell wax, pewter, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic wax, natural and synthetic resins and mixtures thereof. Claim 41 A composition according to any one of claims 1 to 40, wherein one or more lipids coating the core are animal fats or oils and / or vegetable fats or oils. Claim 42 A composition according to claim 41, wherein the vegetable fat or oil is selected from the group consisting of cottonseed oil, hydrogenated cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, hydrogenated soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, hydrogenated palm oil, linseed oil, tung oil, castor oil, and rapeseed oil. Claim 43 In paragraph 41, the vegetable fat or oil is hydrogenated palm oil or hydrogenated cottonseed oil, in the composition. Claim 44 A composition according to claim 40, wherein the free fatty acid is myristic acid, lauric acid, or stearic acid. Claim 45 A composition according to any one of claims 1 to 44, wherein the one or more lipids coating the core have a melting point of about 55 °C to about 80 °C. Claim 46 A composition according to any one of claims 1 to 45, wherein the one or more lipids coating the core have a melting point of about 55 °C to about 75 °C. Claim 47 A composition according to any one of claims 1 to 46, wherein at least one carrier comprises maltodextrin, sucrose, starch, cellulose, clay, biochar, lignin derivative, sugar alcohol, or a combination thereof. Claim 48 A composition comprising a total of about 10% to about 99% (w / w) of lipids in any one of claims 1 to 47. Claim 49 A composition comprising a total of about 70% to about 80% (w / w) of lipids in any one of claims 1 to 48. Claim 50 A composition comprising at least one carrier of about 10% to about 30% (w / w) in any one of claims 1 to 49. Claim 51 A composition comprising at least one carrier of about 15% to about 25% (w / w) in any one of claims 1 to 50. Claim 52 A composition according to any one of claims 1 to 51, wherein the diameter size is about 0.1 mm to about 3 mm. Claim 53 A composition according to any one of claims 1 to 52, wherein the diameter size is about 0.2 mm to about 0.6 mm. Claim 54 A composition according to any one of claims 1 to 53, wherein the diameter size is about 0.2 mm to about 0.4 mm. Claim 55 A composition according to any one of claims 1 to 54, wherein the core comprises about 1% to about 99% of the composition. Claim 56 A composition according to any one of claims 1 to 55, wherein the core comprises about 10% to about 90% of the composition. Claim 57 A composition according to any one of claims 1 to 56, wherein the core comprises about 25% to about 80% of the composition. Claim 58 A composition according to any one of claims 1 to 57, wherein the one or more lipids coating the core comprise about 1% to about 99% of the composition. Claim 59 A composition according to any one of claims 1 to 58, wherein the one or more lipids coating the core comprise about 5% to about 75% of the composition. Claim 60 A composition having a density of about 0.6 g / mL to about 1.2 g / mL in any one of claims 1 to 59. Claim 61 A composition having a porosity of about 10% to about 60% in any one of claims 1 to 60. Claim 62 In any one of paragraphs 1 through 12 and 31 through 61, the above Megaspaera Elsdeni A composition in which at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cells are viable in the composition. Claim 63 A composition according to any one of claims 1 to 12 and claims 31 to 62, wherein the Megasphaera elsdeni cells are dried. Claim 64 In any one of paragraphs 1 through 12 and 31 through 63, the above Megaspaera Elsdeni A composition in which cells are dried by spray drying, electrospray drying, vacuum drying, jet drying, freeze drying, or a combination thereof. Claim 65 A composition comprising about 0.1% to about 15% (w / w) of Megasphaera elsdenii cells in any one of claims 1 to 12 and claims 31 to 64. Claim 66 In any one of paragraphs 1 through 12 and 31 through 65, approximately 1 x 10 per g 4 to about 1 x 10 10 A composition comprising CFU of Megasphaera elsdenii cells. Claim 67 In claims 13 to 62, the anaerobic bacterial cells or anaerobic bacterial cells in a trophic state are about 1 x 10 per gram of the composition. 4 to about 1 x 10 10 A composition comprising CFU. Claim 68 A composition according to any one of claims 1 to 67, further comprising one or more of antibiotics, antimicrobial agents, anticoccidial agents, antiparasitic agents, sulfonamides, hormones, anti-bloat compounds, adrenergic receptor modulators, phages, prebiotics, probiotics, enzymes, essential oils, and / or carbohydrate immune stimulants. Claim 69 A feed additive composition comprising the above composition of any one of claims 1 to 68. Claim 70 In paragraph 69, a feed additive composition that is a powder, fine particles, pellets, cake, liquid, solid, suspension, emulsion, gel, or a combination thereof. Claim 71 Feed comprising the composition of any one of claims 1 to 67 or the feed additive composition of claim 69 or 70. Claim 72 In paragraph 71, feed further comprising animal protein, vegetable protein, corn, soybean meal, corn dried distillers grains with solubles (cDDGS), wheat, wheat protein, gluten, wheat byproduct, wheat bran, wheat dried distillers grains with solubles (wDDGS), corn byproduct including corn gluten meal, barley, oats, rye, triticale, whole soybeans, animal byproduct meal, alcohol-soluble protein, zein, corn zein, caphyrin, rice, paddy rice, extruded paddy rice, oilseed protein, or a combination thereof. Claim 73 In paragraph 72, the animal protein or plant protein is selected from the group consisting of one or more of gliadin or an immunogenic fragment of gliadin, beta-casein, beta-lactoglobulin, glycinin, beta-conglycinin, cruciferin, nafin, hordain, keratin, feather meal or cauliflower meal, collagen, whey protein, fish protein, fish meal, meat protein, egg protein, soy protein and grain protein. Claim 74 In paragraph 72, the above-mentioned oily seed protein is a feed selected from the group consisting of soybean seed protein, sunflower seed protein, rapeseed protein, canola seed protein, and combinations thereof. Claim 75 a) the composition of any one of claims 1 to 68 or the feed additive composition of any one of claim 69 or 70 and b) a premix comprising at least one mineral and / or at least one vitamin. Claim 76 A kit comprising a) i) the composition of any one of claims 1 to 68, ii) the feed additive composition of any one of claim 69 or 70, iii) the feed of any one of claims 71 to 74 and / or iv) the premix of claim 75, and b) instructions for formulation and / or administration to a subject. Claim 77 A method for treating or preventing a pathological condition or disorder associated with lactic acid production in the gastrointestinal tract of a subject, comprising the step of administering to the subject an effective amount of the composition of any one of claims 1 to 68, the feed additive composition of any one of claims 69 or 70, or the feed of any one of claims 71 to 74. Claim 78 In Paragraph 77, the above pathological condition or disorder is a living witness, method. Claim 79 In Paragraph 77, the above pathological condition or disorder is rumen acidosis, method. Claim 80 In paragraph 77, the above pathological condition or disorder is a respiratory disease. Claim 81 In paragraph 77, the above pathological condition or disorder is laminitis, method. Claim 82 A method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of a subject, comprising the step of administering to an animal an effective amount of the composition of any one of claims 1 to 68, the feed additive composition of any one of claims 69 or 70, or the feed of any one of claims 71 to 74. Claim 83 In paragraph 82, the above opportunistic microorganism is pathogenic, method. Claim 84 In paragraph 82, the method wherein the opportunistic microorganism is Salmonella, Escherichia coli, or Campylobacter. Claim 85 A method for improving the growth performance of a subject, comprising the step of administering to the subject an effective amount of the composition of any one of claims 1 to 68, the feed additive composition of any one of claims 69 or 70, or the feed of any one of claims 71 to 74, wherein the improvement in the performance of the subject comprises, when compared to the performance of a subject not administered the feed additive composition or feed, one or more of the following: feed conversion ratio (FCR), body weight gain, feed efficiency, carcass quality, reduction in mortality rate, reduction in morbidity rate, feed intake, daily weight gain, carcass gain, bone mineralization, egg production, reduction in digestive tract microbial imbalance or dysfunction, milk composition, and / or milk production. Claim 86 A method for increasing the starch digestibility of a subject and lowering / lowering the starch excretion in feces or preventing a decrease in pH in the lower gastrointestinal tract, comprising the step of adding the composition of any one of claims 1 to 68, the feed additive composition of any one of claim 69 or 70, to a feed for administration to a subject, wherein the subject exhibits one or more of increased starch digestibility and / or lowered starch excretion in feces compared to a subject not administered the feed additive composition. Claim 87 A method for increasing the operational efficiency of a farm, comprising the step of administering an effective amount of the composition of any one of claims 1 to 68, the feed additive composition of any one of claims 69 or 70, or the feed of any one of claims 71 to 74 to a subject of said farm, wherein the increased operational efficiency reduces labor costs, reduces forage transportation costs, and reduces the amount of forage added to the feed or feed additive. Claim 88 A method according to any one of paragraphs 77 to 87, wherein the subject is a ruminant. Claim 89 In paragraph 88, the method wherein the ruminant is selected from the group consisting of cattle, goats, sheep, giraffes, deer, gazelles, buffalo, reindeer, and antelopes. Claim 90 In paragraph 89, the method wherein the above-mentioned cow is a beef cow or a dairy cow. Claim 91 A method according to any one of claims 76 to 87, wherein the subject is a non-ruminant animal. Claim 92 In claim 91, the non-ruminant animal is selected from the group consisting of horses, animals, poultry, and pigs. Claim 93 In paragraph 92, the poultry is selected from the group consisting of chickens, geese, ducks, quails, turkeys, broilers, meat breeding hens, laying hens, or pigeons. Claim 94 In paragraph 92, the above poultry is a chicken, method. Claim 95 A method according to any one of claims 77 to 94, wherein the composition of any one of claims 1 to 66, the feed additive composition of claim 69 or 70, or the feed of any one of claims 71 to 74 is provided to the subject for daily or weekly administration.

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