Method for electrospray drying anaerobic bacteria and its composition

Abstract

The present disclosure relates to a method and system for electrospray drying anaerobic bacterial cells and / or Megasphaera elsdenii, its compositions and feed additives. The method, its compositions, and feed additives result in improved bacterial viability and yield after long-term storage. Such improvements are beneficial for a wide range of applications, including animal health and use in feed. The above method increases the stability and viability of anaerobic bacteria (e.g., M. elsdenii) and reduces or eliminates the disadvantageous features of conventional spray drying processes.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority based on U.S. Provisional Application No. 63 / 369,915, filed on July 29, 2022, which is hereby incorporated by reference in its entirety.

[0002] Background of the Disclosure Field of the Disclosure The present disclosure relates to methods and systems for electrospray drying anaerobic bacteria into dry powder by applying a small amount of heat.

Background Art

[0003] Background Megasphaera elsdenii (i.e., M. elsdenii) is an anaerobic, non - motile Gram - negative diplococcus that utilizes lactate as a preferred carbon source and can help prevent acidosis, a common digestive disorder that affects millions of beef and dairy cattle each year.

[0004] When beef and other ruminant animals consume large amounts of starchy diets (e.g., cereal grains) or simple sugars, opportunistic microorganisms in the stomach can rapidly ferment these compounds into lactic acid. Lactic acid is a strong organic acid that can cause lactic acidosis, which can disrupt normal digestive activity and cause extensive damage to the inner walls of the digestive tract of ruminants. Affected animals have sub - optimal capabilities. Furthermore, the most acute form of lactic acidosis can lead to irreversible damage to the animal's digestive and respiratory systems, as well as an increase in mortality.

[0005] M. elsdenii can help control lactic acidosis by converting lactic acid into volatile fatty acids (VFA), which are harmless organic compounds such as butyric acid, propionic acid, and acetic acid. However, the population of M. elsdenii in the digestive tract of ruminants is often at levels too low to prevent the risk of acidosis. Therefore, Lactipro® (a liquid culture of live cells derived from a strain of M. elsdenii) was developed to increase the rate of M. elsdenii colony formation in the digestive tract of ruminants. See, for example, U.S. Patent No. 7,550,139. However, there are practical limitations that restrict the use of products containing M. elsdenii, including the difficulty of maintaining M. elsdenii products under the anaerobic conditions required by the organism and the difficulty of transporting M. elsdenii products from the production facility to the end-use location within 14 days (beyond which the viability of M. elsdenii in the product significantly decreases). A product containing freeze-dried M. elsdenii (Lactipro NXT®) has a longer shelf life than Lactipro®, but there is a desire for further improved methods to scale up production and make it more cost-effective. See International Publication No. WO 2018 / 144653 A1, which is incorporated herein by reference in its entirety.

[0006] Methods such as spray drying typically use a heated drying fluid (e.g., heated air) to produce a dried powder, which can result in a reduction in the stability and / or viability of anaerobic bacteria (e.g., M. elsdenii). Accordingly, there is a need for new methods and apparatuses for performing a spray drying process that increases the stability and viability of anaerobic bacteria (e.g., M. elsdenii) and reduces or eliminates the disadvantageous features of conventional spray drying processes. There is also a need for stable powder formulations of other anaerobic bacteria including Bifidobacterium, e.g., B. breve, Lactobacillus, e.g., L. plantarum, Bifidobacterium, e.g., B. animalis subsp. lactis, Pediococcus, e.g., P. acidilactici, Lactobacillus, e.g., L. casei, Fibrobacter, e.g., F. succinogenes, and Butyrivibrio, e.g., B. fibrisolvens, as well as methods for producing the same that overcome existing limitations. There is also a need for stable powder formulations of other anaerobic bacteria including Bifidobacterium, e.g., B. breve, Lactobacillus, e.g., L. plantarum, Bifidobacterium, e.g., B. animalis subsp. lactis, Pediococcus, e.g., P. acidilactici, Lactobacillus, e.g., L. casei, Fibrobacter, e.g., F. succinogenes, and Butyrivibrio, e.g., B. fibrisolvens, Ruminococcus e.g., R. flavefaciens, as well as methods for producing the same that overcome existing limitations.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0008] Summary of the Disclosure In some aspects, a system for spray drying M. elsdenii cells into a dried powder, comprising a source of water, a carrier, and M. elsdenii cells; a tank comprising a stirring device, the tank being arranged to receive water, the carrier, and M. elsdenii cells from the source to form a slurry having a viscosity in the range of about 1 cP to about 500 cP; an electrode configured to apply an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; an atomizer; and a drying chamber having an inlet end, an outlet end, and an internal volume disposed between the inlet end and the outlet end, the internal volume being configured to hold the slurry and a drying fluid, is provided herein. In some aspects, the drying chamber is configured to dry the slurry. In some aspects, the atomizer is configured to (i) receive the slurry from the tank and (ii) discharge the slurry into the drying chamber to contact the drying fluid to form a dried powder comprising M. elsdenii cells. In some aspects, the drying fluid comprises nitrogen or argon at a temperature between about 50 °C and about 100 °C, and the entire system is under less than 2% oxygen.

[0009] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, the method comprising: preparing a culture comprising M. elsdenii cells, a growth medium, and an osmolyte protecting molecule; collecting the M. elsdenii cells; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between 50 °C and 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier. In some aspects, the dried powder contains less than 15% moisture content. In some aspects, the entire method is performed under less than 2% oxygen.

[0010] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, comprising the steps of preparing a culture comprising M. elsdenii cells, a growth medium, and an osmolyte protecting molecule, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; collecting the M. elsdenii cells; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier. In some aspects, the dried powder contains less than 15% moisture content. In some aspects, the entire method is performed under less than 2% oxygen.

[0011] In some aspects, the carrier has a final concentration of about 2 wt% to about 30 wt%.

[0012] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, the method comprising: preparing a culture comprising M. elsdenii cells and a growth medium; adding an osmolite protecting molecule; collecting the M. elsdenii cells; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of from about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of from about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier, wherein the dried powder has a moisture content of less than 15%, and the entire method is performed under less than 2% oxygen. The method is provided herein.

[0013] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, the method comprising: preparing a culture comprising M. elsdenii cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; adding an osmolyte protecting molecule; collecting the M. elsdenii cells; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of slurry droplets; introducing the spray of slurry droplets into a drying chamber; supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier, wherein the dried powder contains less than 10% moisture content and the entire method is performed under less than 2% oxygen. The method is provided herein.

[0014] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, comprising: preparing a culture comprising M. elsdenii cells and a growth medium; collecting the M. elsdenii cells; adding an osmolyte protecting molecule; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is carried out under less than 2% oxygen. The method is provided herein.

[0015] In one aspect, a method of electrospray drying M. elsdenii cells into a dried powder, the method comprising: preparing a culture comprising M. elsdenii cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; collecting the M. elsdenii cells; adding an osmolyte protective molecule; forming a slurry comprising water, a carrier, and the M. elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of slurry droplets; introducing the spray of slurry droplets into a drying chamber; and supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing M. elsdenii cells encapsulated within the carrier, wherein the dried powder contains less than 10% moisture content and the entire method is carried out under less than 2% oxygen. A method is provided herein.

[0016] In one aspect, a method of forming a dried powder comprising M. elsdenii cells encapsulated within a carrier, the method comprising: forming a slurry in a mixing tank comprising water, a carrier, and the M. elsdenii cells while stirring the mixing tank with a stirring device; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry in an atomizer and discharging the atomized slurry into a drying chamber to contact with a drying fluid to form a dried powder comprising M. elsdenii cells encapsulated within the carrier. A method is provided herein. In some aspects, the drying fluid comprises nitrogen or argon at a temperature between about 50°C and about 100°C. In some aspects, the dried powder contains less than 10% moisture content. In some aspects, the entire method is carried out under anaerobic conditions. In some aspects, the entire method is carried out under less than 2% oxygen.

[0017] In some embodiments, the drying fluid is selected from the group consisting of nitrogen and argon.

[0018] In some embodiments, M. elsdenii cells are concentrated about 20-fold to about 100-fold before collection.

[0019] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 0.1 kV to about 45 kV before supplying the drying fluid to the drying chamber.

[0020] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 10 kV to about 30 kV before supplying the drying fluid to the drying chamber.

[0021] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 11 kV to about 25 kV before supplying the drying fluid to the drying chamber.

[0022] In some embodiments, the drying fluid is at a temperature between about 60°C and about 90°C.

[0023] In some embodiments, the method further includes heating the drying fluid to between about 60°C and about 90°C by applying a voltage of about 10 kV to about 30 kV before supplying the drying fluid to the drying chamber.

[0024] In some embodiments, the voltage is applied continuously or in pulses.

[0025] In some embodiments, the carrier is selected from the group consisting of sugars, sugar alcohols, sugar derivatives, polysaccharides, encapsulating polymers, or nitrogen sources, and mixtures thereof. In some embodiments, the sugar is sucrose.

[0026] In some embodiments, the encapsulating polymer is alginate.

[0027] In some embodiments, the carrier is present in an amount of about 1% to about 40% (weight / volume).

[0028] In some embodiments, the dried powder contains about 1×10 3 ~about 1×10 13 CFU / gram of M. elsdenii cells.

[0029] In some embodiments, the dried powder contains about 1×10 3 CFU / gram of M. elsdenii cells.

[0030] In some embodiments, the volume of the culture is at least 2 to 50 liters. In some embodiments, the volume of the culture is at least 2 liters.

[0031] In some embodiments, the volume of the culture is at least 50 liters.

[0032] In some embodiments, there is provided a feed additive comprising electrospray-dried M. elsdenii cells produced by any of the methods described herein.

[0033] In some embodiments, the feed additive further comprises another microorganism.

[0034] In some embodiments, the feed additive is selected from the group consisting of powders, granules, microparticles, pellets, cakes, or combinations thereof.

[0035] In some embodiments, the feed additive is a probiotic.

[0036] In some embodiments, there is provided a composition comprising electrospray-dried M. elsdenii cells produced by any of the methods described herein. In some embodiments, the composition comprises any of the feed additives described herein.

[0037] In some embodiments, the composition is a capsule.

[0038] In some embodiments, a kit is provided herein that comprises electrospray dried M. elsdenii cells produced by any of the methods described herein, any of the feed additives described herein, or any of the compositions described herein.

[0039] In some embodiments, a method for treating or preventing a condition or disorder associated with lactic acid production in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of electrospray dried M. elsdenii cells produced by any of the methods described herein, any of the feed additives described herein, or any of the compositions described herein, is provided herein.

[0040] In some embodiments, the condition or disorder is acidosis.

[0041] In some embodiments, the condition or disorder is rumen acidosis. In some embodiments, the condition or disorder is hindgut acidosis.

[0042] In some embodiments, the condition or disorder is a respiratory disease.

[0043] In some embodiments, the condition or disorder is laminitis.

[0044] In some embodiments, the condition or disorder is an infectious disease.

[0045] In some embodiments, the infectious disease is caused by Salmonella or Campylobacter.

[0046] In some aspects, provided herein are methods for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by any of the methods described herein, any of the feed additives described herein, or any of the compositions described herein.

[0047] In some aspects, the opportunistic microorganism is pathogenic.

[0048] In some embodiments, the opportunistic microorganism is Salmonella or Campylobacter. In some embodiments, the opportunistic microorganism is Escherichia coli.

[0049] In some aspects, provided herein are methods for improving the bioavailability of plant-derived phosphorus in an animal's diet, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by any of the methods described herein, any of the feed additives described herein, or any of the compositions described herein.

[0050] In some aspects, provided herein are methods of improving growth performance in an animal, comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by any of the methods described herein, any of the feed additives described herein, or any of the compositions described herein. In some aspects, the improvement in growth performance in the animal is an improvement in feed intake, average daily gain, feed conversion ratio, meat gain, milk production in dairy animals, egg production in poultry, bone mineralization, or a combination thereof.

[0051] In some embodiments, the electrospray dried M. elsdenii cells, feed additive, or composition is administered before, simultaneously with, or after feeding the animal.

[0052] In some embodiments, the method further comprises mixing the electrospray dried M. elsdenii cells or feed additive with a liquid prior to administration.

[0053] In some embodiments, the liquid is administered orally or by spraying the liquid onto the animal.

[0054] In some embodiments, the method comprises a single administration of the electrospray dried M. elsdenii cells, feed additive, or composition.

[0055] In some embodiments, the method comprises daily administration of the electrospray dried M. elsdenii cells, feed additive, or composition.

[0056] In some embodiments, the method comprises administration of the electrospray dried M. elsdenii cells, feed additive, or composition more than once a day.

[0057] In some embodiments, the animal is a ruminant.

[0058] In some embodiments, the ruminant is selected from the group consisting of cattle, sheep, goats, deer, buffalo, and reindeer.

[0059] In some embodiments, the animal is a non-ruminant.

[0060] In some embodiments, the non-ruminant is selected from the group consisting of equine animals, poultry, and swine.

[0061] In some embodiments, the poultry is selected from the group consisting of chickens, ducks, geese, turkeys, pigeons, or doves.

[0062] In some embodiments, the poultry animal is selected from the group consisting of broilers, broiler breeders, and layers.

[0063] In some embodiments, the poultry animal is a chicken.

[0064] In some embodiments, the equine animal is a horse, pony, donkey, or mule.

[0065] In some embodiments, the carrier is selected from the group consisting of sugars, sugar alcohols, sugar derivatives, polysaccharides, encapsulating polymers, or nitrogen sources, and mixtures thereof. In some embodiments, the encapsulating polymer is alginate. In some embodiments, the sugar is sucrose.

[0066] In some embodiments, the dried powder contains about 1×10 3 ~ about 1×10 13 CFU / gram of M. elsdenii cells.

[0067] In some embodiments, the dried powder contains about 1×10 3 CFU / gram of M. elsdenii cells.

[0068] In some embodiments, provided herein is a dried powder comprising a plurality of dried particles formed by any of the methods described herein.

[0069] In some embodiments, provided herein are M. elsdenii cells encapsulated within a carrier and prepared by any of the methods described herein.

[0070] In one aspect, a system for spray drying anaerobic cells into a dried powder, comprising a source of water, a carrier, and anaerobic cells; a tank comprising a stirring device, the tank being arranged to receive water, a carrier, and anaerobic cells from the source to form a slurry having a viscosity in the range of about 1 cP to about 500 cP; an electrode configured to apply an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; an atomizer; a drying chamber having an inlet end, an outlet end, and an internal volume disposed between the inlet end and the outlet end, the internal volume being configured to hold the slurry and a drying fluid, is provided herein. In some aspects, the drying chamber is configured to dry the slurry. In some aspects, the atomizer is configured to (i) receive the slurry from the tank and (ii) discharge the slurry into the drying chamber to contact the drying fluid to form a dried powder containing anaerobic cells. In some aspects, the drying fluid comprises nitrogen or argon at a temperature between about 50°C and about 100°C. In some aspects, the entire system is under less than 2% oxygen.

[0071] In one aspect, a method of electrospray drying anaerobic cells into a dried powder, the method comprising: preparing a culture comprising anaerobic cells, a growth medium, and an osmolyte protecting molecule; collecting the anaerobic cells; forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing anaerobic cells encapsulated within the carrier. In some aspects, the dried powder contains less than 10% moisture content. In some aspects, the entire method is carried out under less than 2% oxygen.

[0072] In one aspect, a method of electrospray drying anaerobic cells into a dried powder, the method comprising: preparing a culture comprising anaerobic cells, a growth medium, and an osmolyte protecting molecule; collecting the anaerobic cells; forming a slurry comprising water, a carrier, and the anaerobic cells, the carrier having a final concentration of about 1 wt% to about 50 wt% and the slurry having a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing anaerobic cells encapsulated within the carrier. In some aspects, during the culturing, the pH, temperature, and / or weight osmolarity are changed for a predetermined time. In some aspects, the dried powder contains less than 15% moisture. In some aspects, the entire method is performed under anaerobic conditions. In some aspects, the entire method is performed under less than 2% oxygen.

[0073] In some aspects, the carrier has a final concentration of about 2 wt% to about 30 wt%.

[0074] In one aspect, a method of electrospray drying anaerobic cells into a dried powder, comprising the steps of preparing a culture comprising anaerobic cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; adding an osmolyte protective molecule; collecting the anaerobic cells; forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles encapsulating the anaerobic cells within the carrier, wherein the dried powder has a moisture content of less than 15% and the entire method is performed under less than 2% oxygen. The method is provided herein.

[0075] In one aspect, a method of electrospray drying anaerobic cells into a dried powder, comprising the steps of preparing a culture comprising anaerobic cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; adding an osmolyte protective molecule; collecting the anaerobic cells; forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; and supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles encapsulating the anaerobic cells within the carrier, wherein the dried powder has a moisture content of less than 10% and the entire method is performed under less than 2% oxygen. The method is provided herein.

[0076] In some embodiments, the osmolyte protecting molecule is added during cell growth, prior to collection, or after collection.

[0077] In some embodiments, a method of electrospray drying anaerobic cells into a dried powder, the method comprising: preparing a culture comprising anaerobic cells and a growth medium; collecting the anaerobic cells; adding an osmolyte protecting molecule; forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of from about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of from about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of slurry droplets; introducing the spray of slurry droplets into a drying chamber; supplying a drying fluid at a temperature between about 50 °C and about 100 °C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing anaerobic cells encapsulated within the carrier, wherein the dried powder has a moisture content of less than 15% and the entire method is performed under less than 2% oxygen, is provided herein.

[0078] In some embodiments, the osmolyte protecting molecule is added during cell growth, prior to collection, or after collection.

[0079] In one aspect, a method of electrospray drying anaerobic cells into a dried powder, the method comprising: preparing a culture comprising anaerobic cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; collecting the anaerobic cells; adding an osmolyte protective molecule; forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of slurry droplets; introducing the spray of slurry droplets into a drying chamber; and supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form a dried powder comprising a plurality of dried particles containing anaerobic cells encapsulated within the carrier, wherein the dried powder contains less than 10% moisture content and the entire method is performed under less than 2% oxygen. A method is provided herein.

[0080] In one aspect, the osmolyte protective molecule is added during cell growth, before collection, or after collection.

[0081] In one aspect, a method of forming a dried powder containing anaerobic cells encapsulated within a carrier, the method comprising: forming a slurry in a mixing tank containing water, a carrier, and anaerobic cells while stirring the mixing tank with a stirring device; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry in an atomizer and discharging the atomized slurry into a drying chamber to contact with a drying fluid to form a dried powder containing anaerobic cells encapsulated within the carrier. In one aspect, the drying fluid comprises nitrogen or argon at a temperature between about 50°C and about 100°C. In one aspect, the dried powder contains less than 15% moisture content. In one aspect, the entire method is performed under anaerobic conditions. In one aspect, the entire method is under less than 2% oxygen. A method is provided herein.

[0082] In some embodiments, the osmolyte-protecting molecule is added during cell growth, before harvesting, or after harvesting.

[0083] In some embodiments, the drying fluid is selected from the group consisting of nitrogen and argon.

[0084] In some embodiments, the anaerobic cells are concentrated about 25-fold to about 100-fold prior to harvesting.

[0085] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of between about 0.1 kV and about 45 kV prior to supplying the drying fluid to the drying chamber.

[0086] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of between about 10 kV and about 30 kV before supplying the drying fluid to the drying chamber.

[0087] In some embodiments, the method further includes heating the drying fluid to between about 50°C and about 100°C by applying a voltage of between about 11 kV and about 25 kV before supplying the drying fluid to the drying chamber.

[0088] In some embodiments, the drying fluid is at a temperature between about 60°C and about 90°C.

[0089] In some embodiments, the method further includes heating the drying fluid to between about 60°C and about 90°C by applying a voltage of between about 10 kV and about 30 kV before supplying the drying fluid to the drying chamber.

[0090] In some embodiments, the voltage is applied constant or pulsed.

[0091] In some aspects, the carrier is selected from the group consisting of sucrose, a sugar, a sugar alcohol, a sugar derivative, a polysaccharide, an encapsulating polymer, or a nitrogen source, and mixtures thereof.

[0092] In some embodiments, the encapsulated polymer is alginate.

[0093] In some embodiments, the dried powder contains anaerobic cells of about 1×10 3 ~ about 1×10 13 CFU / gram.

[0094] In some embodiments, the dried powder contains anaerobic cells of about 1×10 3 CFU / gram.

[0095] In some embodiments, provided herein is a dried powder comprising a plurality of dried particles formed by any of the methods described herein.

[0096] In some embodiments, provided herein are anaerobic cells encapsulated within a carrier, prepared by any of the methods described herein.

[0097] In some embodiments, provided herein is a method of improving growth ability in an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells produced by any of the methods described herein. In some embodiments, the improvement in growth ability in the animal is an improvement in feed intake, average daily gain, feed conversion ratio, meat gain, milk production in dairy animals, egg production in poultry, bone mineralization, or a combination thereof.

[0098] In some embodiments, provided herein is a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells produced by any of the methods described herein.

[0099] In some embodiments, provided herein are compositions comprising electrospray-dried anaerobic bacterial cells produced by any of the methods described herein.

[0100] In some embodiments, M. elsdenii cells are grown at about 30 °C to about 40 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C prior to collection.

[0101] In some embodiments, anaerobic cells are grown at about 30 °C to about 40 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C prior to collection.

[0102] In some embodiments, the slurry is processed at a rate of about 1 L / hour to about 10,000 L / hour. In some embodiments, the slurry is processed at a rate of about 3,000 L / hour. BRIEF DESCRIPTION OF THE DRAWINGS

[0103]

Figure 1

[0104]

Figure 2

[0105]

Figure 3

[0106]

Figure 4

[0107]

Figure 5

DETAILED DESCRIPTION OF THE INVENTION

[0108] DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure relates to a method for electrospray drying M. elsdenii into a dried powder by applying a small amount of heat. The present disclosure also relates to feed additives and compositions containing electrospray-dried M. elsdenii cells.

[0109] All publications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, the citation or identification of any reference in this application is not to be construed as an admission that such reference is available as prior art to the present invention. TERMS

[0110] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, singular terms shall include plurals and plural terms shall include singulars.

[0111] When section headings are used, the headings are not to be regarded as limiting.

[0112] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" includes a plurality of compounds, including mixtures thereof. The terms "a," "an," "the," "one or more," and "at least one" can be used interchangeably herein, for example.

[0113] As used herein, the term "about," when used to modify an amount associated with the present invention, refers to variability in numerical amounts that can occur, for example, through conventional testing and handling, inadvertent errors in such testing and handling, differences in the manufacture, source, or purity of the components utilized in the present invention, etc. The claims include equivalents of the recited amounts, whether or not modified by the term "about." In some embodiments, the term "about" means within 10% of the reported numerical value.

[0114] Throughout this application, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual numerical values within that range. For example, a range description such as 1 - 6 should be considered to have specifically disclosed sub-ranges such as 1 - 2, 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, 2 - 5, 2 - 6, 3 - 4, 3 - 5, 3 - 6, etc., as well as the individual numerical values within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0115] The terms "comprises", "comprising", "includes", "including", "having", and their conjugations are interchangeable and mean "including but not limited to". When an aspect is described herein using the word "comprising", it is understood that similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0116] The term "consisting of" means "including and limiting".

[0117] The term "consisting essentially of" means the specified materials of a composition or the specified steps of a method, as well as additional materials or steps that do not substantially affect the basic characteristics of the materials or method.

[0118] The term "and / or", as used herein, is to be construed as a specific disclosure that each of the two specified characteristics or components may or may not be accompanied by the other. Thus, the term "and / or", when used in a phrase such as "A and / or B" herein, is intended to include "A and B", "A or B", "A (alone)", as well as "B (alone)". Similarly, the term "and / or", when used in a phrase such as "A, B, and / or C", is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); as well as C (alone).

[0119] As used herein, the terms "culturing", "for culturing", and "cultivation" mean incubating cells under in vitro conditions that allow for cell growth or division or that allow for maintaining the 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).

[0120] As used herein, the term "probiotics" refers to one or more live microorganisms (bacteria and / or yeast) that, when administered in an appropriate amount, can provide a health benefit to an animal or subject, and may or may not include other components.

[0121] As used herein, the term "directly supplied microbial product" refers to a product that can be administered to an animal or subject as a feed mixture, bolus, and / or oral paste and that, when administered in an appropriate amount, can provide a health benefit to the animal or subject, and that includes one or more live microorganisms (bacteria and / or yeast), and may or may not include other components.

[0122] As used herein, the term "feed additive" refers to one or more components, products, or substances (e.g., cells) that are used alone or together in nutrients (e.g., to improve the quality of food (e.g., animal feed), to improve the ability and health of animals, and / or to enhance the digestibility of food or materials within food). A feed additive can be, for example, a probiotic.

[0123] As used herein, the terms "growth medium" and "culture medium" refer to solid (e.g., agar), semi-solid (e.g., agar), or liquid (e.g., broth) compositions that contain components that assist in the growth of cells.

[0124] As used herein, the terms "collection" and "collecting" refer to recovering cells from a culture, e.g., recovering cells from a culture into the growth medium, recovering cells by removing a volume of the growth medium from the cells (e.g., by concentrating the cells in a liquid culture or separating the cells from the growth medium), or stopping the culture of the cells. The term includes recovering or removing a volume of liquid containing cells from a liquid culture, including the volume in which the cells are concentrated.

[0125] As used herein, the term "isolated" does not necessarily reflect the degree to which the isolate is purified, but rather indicates isolation or separation from its natural form or natural environment. Isolates can include, but are not limited to, isolated microorganisms, isolated biomass, or isolated cultures.

[0126] As used herein, "excipient" refers to a component or mixture of components used to impart desired characteristics to a feed additive, food, composition, or pharmaceutical composition disclosed herein. Excipients of the present invention, when added to a pharmaceutical composition, can be described as "pharmaceutically acceptable" excipients, which means that the excipient is suitable for contact with the tissues of animals (i.e., humans and non-human animals) without undue toxicity, irritation, allergic response, or other problematic complications over the desired period of contact within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0127] As used herein, the term "yield" refers to the amount of viable or viable cells, including the amount per a specific volume (e.g., colony forming units per milliliter ("CFU / mL")) or the amount per a specific weight (e.g., CFU per gram ("CFU / g")).

[0128] As used herein, the term "viability" refers to a living organism (e.g., a living microbial cell). "Viability" refers to the ability to survive, particularly under certain specific conditions.

[0129] As used herein, "purify", "purified", and "purification" mean to make substantially pure or to be free of unwanted constituents, contaminating materials, mixtures, or impurities.

[0130] The term "animal" or "subject" refers to any organism belonging to the animal kingdom, including, without limitation, aquatic and terrestrial animals, such as, unless otherwise indicated: fish; commercial fish; ornamental fish; larval fish; bivalves; mollusks; crustaceans; shellfish; shrimp; larval shrimp; Artemia; rotifers; brine shrimp; filter feeders; amphibians; reptiles; mammals; non-human animals; livestock animals; animal husbandry animals; zoo animals; sports animals; breeding stock; race animals; show animals; native animals; rare or endangered animals; companion animals; pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, or horses; primates, such as monkeys (e.g., macaques, rhesus monkeys, African green monkeys, patas monkeys, cynomolgus monkeys, and long-tailed monkeys), anthropoid apes, orangutans, baboons, langurs, and chimpanzees; canids, such as dogs and wolves; felids, such as cats, lions, and tigers; equids, such as horses, ponies, donkeys, mules, and zebras; food animals, such as cows, buffalo, beef cattle, pigs, poultry, and sheep; ungulates, such as deer and giraffes; birds (i.e., avians); poultry, such as chickens, geese, ducks, quails, turkeys, pigeons, emus, ostriches, and any other birds used as food or animal husbandry animals, including broilers, broiler breeders, and layers; rodents, such as mice, rats, hamsters, and guinea pigs, etc. are included. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human subject. In some embodiments, the mammal excludes human subjects. Animal feeds include, but are not limited to, aquaculture feeds, livestock feeds including pet animal feeds, zoo animal feeds, working animal feeds, animal husbandry feeds, and combinations thereof. In some embodiments, the food includes animal feed and human food.

[0131] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate aspects, may also be provided in combination in a single aspect. Conversely, various features of the invention that are described in the context of a single aspect for brevity may be provided separately, or in any suitable sub-combination, or in any other suitable manner in the context of the invention's other described aspects. Certain features described in the context of various aspects are not considered essential features of those aspects unless the aspect would be inoperable without those elements.

[0132] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and claims. M. elsdenii

[0133] M. elsdenii cells derived from any strain or any combination of strains can be used in the present disclosure described herein.

[0134] M. elsdenii strains can be selected from a depository culture collection (e.g., American Type Culture Collection (“ATCC®”), National Collection of Industrial, Food and Marine Bacteria (“NCIMB”), National Collection of Type Cultures (“NCTC”), American Research Service (“ARC”) culture collection (i.e., “NRRL”), National Institute of Animal Health (NIAH) culture collection) or may be strains isolated from a natural source (e.g., from the digestive tract of ruminants).

[0135] Examples of M. elsdenii strains that can be selected from a culture collection include, but are not limited to, the strains listed by accession number in Table 1. Alternative notations for the accession numbers are also shown. Table 1. Examples of M. elsdenii strains and the source of each strain. [Table 1]

[0136] In some embodiments, the M. elsdenii cells are derived from strains having an accession 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, and combinations thereof, including any of the alternative notations in Table 1 or any commercial source of M. elsdenii.

[0137] In some embodiments, the M. elsdenii cells are derived from strains isolated from ruminants (e.g., cows). See, for example, U.S. Patent No. 7,550,139.

[0138] In some embodiments, the M. elsdenii cells are derived from strains isolated from non-ruminants (e.g., humans).

[0139] In some embodiments, the M. elsdenii cells are derived from strains selected for lactate utilization (e.g., strains that utilize lactate in the presence of sugars), resistance to ionophore antibiotics, relatively high growth rates, the ability to produce primarily acetate, the ability to grow at low pH values of less than 5.0 and about 4.5, production of volatile fatty acids (VFA), phytase activity, and combinations thereof. See, e.g., U.S. Patent No. 7,550,139.

[0140] In some embodiments, the strains selected for lactate utilization utilize lactate as the 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.

[0141] In some embodiments, the M. elsdenii cells are derived from strains that have a high growth rate compared to other strains. The growth rates of different strains can be determined, for example, by culturing the cells in a liquid medium and monitoring the increase in optical density over time.

[0142] In some embodiments, the M. elsdenii cells are derived from strains that have phytase activity.

[0143] In some embodiments, the M. elsdenii cells are derived from the M. elsdenii strain NCIMB 41125. This M. elsdenii strain has a high specific growth rate (0.94 generations / hour), can grow in a pH range of 4.5 - 6.5 or higher, not only uses D and L-lactate as its preferred substrates, but also has the ability to utilize glucose and other carbohydrates and is ionophore tolerant.

[0144] In some embodiments, the M. elsdenii cells are derived from M. elsdenii strain NCIMB 41787. In some embodiments, the M. elsdenii cells are derived from M. elsdenii strain NCIMB 41788.

[0145] In some embodiments, the M. elsdenii cells are derived from M. elsdenii strain ATCC® 25940.

[0146] In some embodiments, the M. elsdenii cells are derived from a strain selected from a stock culture collection or isolated from a natural source. Cells "derived from" a strain can be natural or artificial derivatives, such as partial isolates, mutants, variants, or recombinant strains.

[0147] In some embodiments, the M. elsdenii is lyophilized. See International Publication No. WO 2018 / 144653 A1, which is incorporated by reference in its entirety. Preparation of cultures containing anaerobic bacterial cells or M. elsdenii cells

[0148] Anaerobic bacteria, including M. elsdenii, should be cultured under anaerobic conditions for maximum yield and viability, hi some embodiments, anaerobic bacteria (e.g., M. elsdenii) should be cultured under less than 2% oxygen.

[0149] In some embodiments, the culture comprises M. elsdenii cells and a growth medium.

[0150] In some embodiments, the culture comprises one or more strains of M. elsdenii cells. In some embodiments, the culture comprises a single strain of M. elsdenii cells. In some embodiments, the culture consists of one or more strains of M. elsdenii cells (i.e., the cells in the culture consist of M. elsdenii cells, e.g., one or more strains of M. elsdenii cells). In some embodiments, the culture consists of a single strain of M. elsdenii cells.

[0151] 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, such as B. breve, Lactobacillus cells, such as L. plantarum, Bifidobacterium cells, such as B. animalis subsp. lactis, Pediococcus cells, such as P. acidilactici, Lactobacillus cells, such as L. casei, Fibrobacter, such as F. succinogenes, and Butyrivibrio, such as B. fibrisolvens, Ruminococcus, such as R. flavefaciens, and a growth medium.

[0152] Various fermentation parameters can be used to inoculate, grow, and collect anaerobic cells (e.g., M. elsdenii cells), including continuous fermentation (i.e., continuous culture) or batch fermentation (i.e., batch culture). See, for example, U.S. Patent No. 7,550,139.

[0153] The growth medium for anaerobic cells (e.g., M. elsdenii cells) can be solid, semi-solid, or liquid. The medium can contain nutrients that provide the essential elements and specific factors to enable growth. Various microbiological media and variants are well known in the art. The medium can be added to the culture at any time, e.g., at the start of the culture, during the culture, or intermittently / continuously.

[0154] Examples of growth media include: (1) a partially defined medium (this contains peptone, 3 g / L; yeast, 3 g / L; vitamin solution, 2 mL / L; mineral solution, 25 mL / L; indigo carmine (0.5%), 1 g / L; 12.5% L-cysteine, 2 g / L; 12.5% sodium sulfide, 2 g / L, and is supplemented with Na-lactate (partially defined lactate, SDL), glucose (partially defined glucose, SDG), or maltose (partially defined maltose, SDM)), (2) a modified reinforced Clostridium agar / broth medium (pre-reduced) (this contains peptone, 10 g / L; beef extract, 10 g / L; yeast extract, 3 g / L; dextrose 5 g / L; NaCl, 5 g / L; soluble starch, 1 g / L; L-cysteine HCl, 0.5 g / L; sodium acetate, 3 g / L; and resazurin (0.025%), 4 mL / L), (3) trypticase soy agar / broth containing defibrinated sheep blood, (4) a partially defined rumen fluid-free medium (this contains 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; thiamine 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, 0.07 mL / L, and monensin, 10 mg / L, add Na-lactate l and the mineral solution to the reservoir bottle and autoclave for 60 minutes; dissolve peptone in 300 mL of distilled H2O and autoclave separately; filter-sterilize the vitamin solution and the two reducing agents in advance; then autoclave and aerate the anaerobic gas into the reservoir bottle overnight; add the other components separately after cooling; and adjust the pH to the desired value with 5N HCl), and (5) Incubated rumen fluid lactate (「IRFL」) medium (this is 400 mL of incubated clarified rumen fluid derived from sheep fed alfalfa, 371 mL of distilled water, 2 g of peptone, 15 g of agar, 100 mL of 10% (weight / volume) sodium-D,L-lactate solution, 100 mL of 0.04% (weight / volume) bromocresol purple solution, and 25 mL of mineral solution containing 40 g / L of KH2PO4; 120 g / L of (NH4)2SO4; 8 g / L of MgSO4.7H2O, and 2.4 g / L of CaCl2.2H2O, where lactic acid (90% weight / volume) is used to adjust the pH to 5.5, then autoclave at 121 °C for 25 minutes, then aerate with an anaerobic gas mixture while cooling in a 50 °C water bath, and subsequently add 2 milliliters each of Na2S.9H2O (12.5% weight / volume) and cysteine.HCl.H2O (12.5% weight / volume)), but not limited to these.

[0155] In some embodiments, the culture comprises a growth medium comprising at least two carbon sources. In some embodiments, the at least two carbon sources are selected from the group consisting of casein, starch (e.g., gelatinized starch and / or soluble starch), lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sorbitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, lipids, oils, glycerol, sodium acetate, arabinose, soybean protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof.

[0156] In some embodiments, the at least two carbon sources consist of from about 1 to 99% of a first carbon source (e.g., any carbon source described herein) and from about 1 to 99% of a second carbon source (e.g., any carbon source described herein different from the first carbon source), and 100% of the at least two carbon sources consists of the first and second carbon sources. In some embodiments, the at least two carbon sources consist of from about 50 to 60% of a first carbon source and from about 40 to 50% of a second carbon source, from about 50 to 70% of a first carbon source and from about 30 to 50% of a second carbon source, from about 50 to 80% of a first carbon source and from about 20 to 50% of a second carbon source, or from about 50 to 90% of a first carbon source and from about 10 to 50% of a second carbon source. In other embodiments, the at least two carbon sources consist of from about 65 to 75% of a first carbon source and from about 25 to 35% of a second carbon source. In some embodiments, the first carbon source is lactate.

[0157] In some embodiments, the culture may further comprise an osmolyte protecting molecule. In some embodiments, the osmolyte protecting molecule is added before collecting the cells. In some embodiments, the osmolyte protecting molecule is added after collecting the cells. The osmolyte protecting molecule can protect anaerobic cells (e.g., M. elsdenii cells) from the stress caused by electrospray drying. Thereby, a long shelf life and high CFU / g after electrospray drying are obtained. Examples of classes of osmolyte protecting molecules include, but are not limited to, amino acids, sugars, polyols, methylamines, methylsulfonium compounds. In some embodiments, these classes are added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is methylamine. In some embodiments, methylamine is betaine. In some embodiments, the osmolyte protecting molecule is betaine. In some embodiments, betaine is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is urea. In some embodiments, urea is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is trimethylamine N-oxide (TMAO). In some embodiments, trimethylamine N-oxide (TMAO) is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is dimethylsulfoniopropionate. In some embodiments, dimethylsulfoniopropionate is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is sarcosine. In some embodiments, sarcosine is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is glycerophosphorylcholine. In some embodiments, glycerophosphorylcholine is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is myo-inositol. In some embodiments, myo-inositol is added to the culture between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is taurine.In some embodiments, taurine is added to the culture at between about 0.001 molar (M) and 0.5 M. In some embodiments, the osmolyte protecting molecule is glycine. In some embodiments, glycine is added to the culture at between about 0.001 molar (M) and 0.5 M.

[0158] In some embodiments, anaerobic cells (e.g., M. elsdenii cells) are grown at about 39°C to about 40°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0159] In some embodiments, the temperature of a culture containing anaerobic cells (e.g., M. elsdenii cells) may be increased for a predetermined time to induce the production of stress tolerance molecules (e.g., heat shock proteins and / or cold shock proteins) in the cells, and then the temperature may be returned to its original set point. This results in electrospray dried particles having a long shelf life and high CFU / g after electrospray drying. In some embodiments, the temperature is increased from 1 to 30 °C from the culture process set point. In some embodiments, the temperature is increased from 2 to 29 °C from the culture process set point. In some embodiments, the temperature is increased from 3 to 28 °C from the culture process set point. In some embodiments, the temperature is increased from 4 to 27 °C from the culture process set point. In some embodiments, the temperature is increased from 5 to 26 °C from the culture process set point. In some embodiments, the temperature is increased from 6 to 25 °C from the culture process set point. In some embodiments, the temperature is increased from 7 to 24 °C from the culture process set point. In some embodiments, the temperature is increased from 8 to 23 °C from the culture process set point. In some embodiments, the temperature is increased from 9 to 22 °C from the culture process set point. In some embodiments, the temperature is increased from 10 to 21 °C from the culture process set point. In some embodiments, the temperature is increased from 11 to 20 °C from the culture process set point. In some embodiments, the temperature is increased from 12 to 19 °C from the culture process set point. In some embodiments, the temperature is increased from 13 to 18 °C from the culture process set point. In some embodiments, the temperature is increased from 14 to 17 °C from the culture process set point. In some embodiments, the temperature is increased from 15 to 16 °C from the culture process set point. In some embodiments, the temperature is increased from 1 °C, 2 °C, 3 °C, 4 °C, or 5 °C, or 1 to 5 °C from the culture process set point.

[0160] In some embodiments, the period for increasing the temperature of the culture is from about 1 minute to 12 hours. In some embodiments, the period for increasing the temperature of the culture is from about 30 minutes to 11 hours. In some embodiments, the period for increasing the temperature of the culture is from about 1 to 10 hours. In some embodiments, the period for increasing the temperature of the culture is from about 2 to 9 hours. In some embodiments, the period for increasing the temperature of the culture is from about 3 to 8 hours. In some embodiments, the period for increasing the temperature of the culture is from about 4 to 7 hours. In some embodiments, the period for increasing the temperature of the culture is from about 5 to 6 hours.

[0161] In some embodiments, anaerobic cells (e.g., M. elsdenii cells) are cooled to about 18°C to about 25°C for storage.

[0162] In some embodiments, the pH of a culture containing anaerobic cells, e.g., M. elsdenii cells (e.g., during culture and / or at the time of collection) is between about 4.0 and about 8.0, between about 4.0 and about 7.5, between about 4.0 and about 7.0, between about 4.0 and about 6.5, between about 4.0 and about 6.0, between about 4.0 and about 5.5, between about 4.0 and about 5.0, between about 4.0 and about 4.5, between about 4.5 and about 8.0, between about 4.5 and about 7.5, between about 4.5 and about 7.0, between about 4.5 and about 6.5, between about 4.5 and about 6.0, between about 4.5 and about 5.5, between about 4.5 and about 5.0, between about 4.6 and about 6.9, between about 4.7 and about 6.8, between about 4.8 and about 6.7, between about 4.9 and about 6.6, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.0 and about 6.0, between about 5.0 and about 5.5, between about 5.1 and about 6.9, between about 5.2 and about 6.8, between about 5.3 and about 6.7, between about 5.4 and about 6.6, between about 5.5 and about 7.0, between about 5.5 and about 6.5, between about 5.1 and about 6.4, between about 5.2 and about 6.3, between about 5.3 and about 6.2, between about 5.4 and about 6.1, between about 5.5 and about 6.0, between about 5.0 and about 6.1, between about 5.0 and about 6.2, between about 5.0 and about 6.3, between about 5.0 and about 6.4, between about 5.1 and about 6.5, between about 5.2 and about 6.5, between about 5.3 and about 6.5, or between about 5.4 and about 6.5.

[0163] In some embodiments, the pH of a culture containing anaerobic cells (e.g., M. elsdenii cells) may be reduced for a predetermined time to induce the production of stress tolerance molecules (e.g., heat shock proteins and / or cold shock proteins) in the cells, and then the pH may be returned to its original set point. This results in electrospray-dried particles having a long shelf life and a high CFU / g after electrospray drying. In some embodiments, the pH is reduced from the culture process set point by 0.1 to 6.0 pH units. In some embodiments, the pH is reduced from the culture process set point by 0.5 to 5.5 pH units. In some embodiments, the pH is reduced from the culture process set point by 1.0 to 5.0 pH units. In some embodiments, the pH is reduced from the culture process set point by 1.5 to 4.5 pH units. In some embodiments, the pH is reduced from the culture process set point by 2.0 to 4.0 pH units.

[0164] In some embodiments, the pH of a culture containing anaerobic cells (e.g., M. elsdenii cells) may be increased for a predetermined time to induce the production of stress tolerance molecules (e.g., heat shock proteins and / or cold shock proteins) in the cells, and then the pH may be returned to its original set point. This results in electrospray-dried particles having a long shelf life and a high CFU / g after electrospray drying. In some embodiments, the pH is increased from the culture process set point by 0.1 to 6.0 pH units. In some embodiments, the pH is increased from the culture process set point by 0.5 to 5.5 pH units. In some embodiments, the pH is increased from the culture process set point by 1.0 to 5.0 pH units. In some embodiments, the pH is increased from the culture process set point by 1.5 to 4.5 pH units. In some embodiments, the pH is increased from the culture process set point by 2.0 to 4.0 pH units.

[0165] In some embodiments, the period for reducing or increasing the pH of the culture is from about 1 minute to 12 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from about 30 minutes to 11 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from about 1 to 10 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from about 2 to 9 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from 3 to 8 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from 4 to 7 hours. In some embodiments, the period for reducing or increasing the pH of the culture is from 5 to 6 hours.

[0166] In some embodiments, the weight osmolality of a culture containing anaerobic cells (e.g., M. elsdenii cells) may be increased for a predetermined time to induce the production of stress tolerance molecules (e.g., heat shock proteins and / or cold shock proteins) in the cells, and then the weight osmolality may be returned to its original set point. This results in electrospray dried particles having a long shelf life and high CFU / g after electrospray drying. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.2 M to 2 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.3 M to 1.9 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.4 M to 1.8 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.5 M to 1.7 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.6 M to 1.6 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.7 M to 1.5 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.8 M to 1.4 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 0.9 M to 1.3 M. In some embodiments, the weight osmolality of the culture can be increased by adding salt to the culture in the range of about 1.0 M to 1.2 M.

[0167] In some embodiments, the period for increasing the weight osmolarity of the culture is from about 1 minute to 12 hours. In some embodiments, the period for increasing the weight osmolarity of the culture is from about 30 minutes to 1 hour. In some embodiments, the period for increasing the weight osmolarity of the culture is from about 1 to 10 hours. In some embodiments, the period for increasing the weight osmolarity of the culture is from 2 to 9 hours. In some embodiments, the period for increasing the weight osmolarity of the culture is from 3 to 8 hours. In some embodiments, the period for increasing the weight osmolarity of the culture is from 4 to 7 hours. In some embodiments, the period for increasing the weight osmolarity of the culture is from 5 to 6 hours.

[0168] In some embodiments, the weight osmolarity of a culture containing anaerobic cells (e.g., M. elsdenii cells) may be decreased for a predetermined time to induce the production of stress tolerance molecules (e.g., heat shock proteins and / or cold shock proteins) in the cells and then the weight osmolarity may be returned to its original set point.

[0169] Fermenters of different sizes and designs that maintain anaerobic conditions can be used to culture anaerobic cells (e.g., M. elsdenii cells). In some embodiments, culturing the anaerobic cells needs to be performed under less than 2% oxygen. The fermenter may be capable of fermenting a culture volume sufficient for the commercial production of anaerobic cells (e.g., M. elsdenii cells), for example.

[0170] In some embodiments, the culture volume is about 2 liters, about 5 liters, about 10 liters, about 50 liters, about 100 liters, about 150 liters, about 200 liters, about 250 liters, about 300 liters, about 350 liters, about 400 liters, about 450 liters, about 500 liters, about 600 liters, about 800 liters, about 1,000 liters, about 1,200 liters, about 1,500 liters, about 1,800 liters, about 2,000 liters, about 2,200 liters, about 2,500 liters, about 2,750 liters, about 3,000 liters, about 4,000 liters, about 5,000 liters, about 6,000 liters, about 7,000 liters, about 8,000 liters, about 9,000 liters, about 10,000 liters, about 20,000 liters, about 50,000 liters, or about 75,000 liters.

[0171] In some embodiments, the culture volume is from about 2 liters to about 75,000 liters, from about 2 liters to about 70,000 liters, from about 2 liters to about 65,000 liters, from about 2 liters to about 60,000 liters, from about 2 liters to about 55,000 liters, from about 2 liters to about 50,000 liters, from about 2 liters to about 45,000 liters, from about 2 liters to about 40,000 liters, from about 2 liters to about 35,000 liters, from about 2 liters to about 30,000 liters, from about 2 liters to about 25,000 liters, from about 2 liters to about 20,000 liters, from about 2 liters to about 15,000 liters, from about 2 liters to about 10,000 liters, from about 2 liters to about 5,000 liters, from about 2 liters to about 2,500 liters, from about 2 liters to about 500 liters, from about 2 liters to about 250 liters, from about 2 liters to about 100 liters, from about 2 liters to about 50 liters, from about 2 liters to about 25 liters, or from about 2 liters to about 10 liters.

[0172] In some embodiments, the culture volume is from about 5 liters to about 75,000 liters, from about 5 liters to about 70,000 liters, from about 5 liters to about 65,000 liters, from about 5 liters to about 60,000 liters, from about 5 liters to about 55,000 liters, from about 5 liters to about 50,000 liters, from about 5 liters to about 45,000 liters, from about 5 liters to about 40,000 liters, from about 5 liters to about 35,000 liters, from about 5 liters to about 30,000 liters, from about 5 liters to about 25,000 liters, from about 5 liters to about 20,000 liters, from about 5 liters to about 15,000 liters, from about 5 liters to about 10,000 liters, from about 5 liters to about 5,000 liters, from about 5 liters to about 2,500 liters, from about 5 liters to about 500 liters, from about 5 liters to about 250 liters, from about 5 liters to about 100 liters, from about 5 liters to about 50 liters, from about 5 liters to about 25 liters, or from about 5 liters to about 10 liters.

[0173] In some embodiments, the culture volume is from about 10 liters to about 75,000 liters, from about 10 liters to about 70,000 liters, from about 10 liters to about 65,000 liters, from about 10 liters to about 60,000 liters, from about 10 liters to about 55,000 liters, from about 10 liters to about 50,000 liters, from about 10 liters to about 45,000 liters, from about 10 liters to about 40,000 liters, from about 10 liters to about 35,000 liters, from about 10 liters to about 30,000 liters, from about 10 liters to about 25,000 liters, from about 10 liters to about 20,000 liters, from about 10 liters to about 15,000 liters, from about 10 liters to about 10,000 liters, from about 10 liters to about 5,000 liters, from about 10 liters to about 2,500 liters, from about 10 liters to about 500 liters, from about 10 liters to about 250 liters, from about 10 liters to about 100 liters, from about 10 liters to about 50 liters, or from about 10 liters to about 25 liters.

[0174] In some embodiments, the culture volume is from about 250 liters to about 750 liters, from about 300 liters to about 800 liters, from about 350 liters to about 850 liters, from about 400 liters to about 900 liters, from about 450 liters to about 950 liters, from about 500 liters to about 1,000 liters, from about 750 liters to about 1,250 liters, from about 1,000 liters to about 2,000 liters, from about 2,000 liters to about 4,000 liters, from about 4,000 liters to about 8,000 liters, from about 5,000 liters to about 10,000 liters, from about 50 liters to about 75,000 liters, from about 50 liters to about 50,000 liters, from about 50 liters to about 25,000 liters, from about 50 liters to about 20,000 liters, from about 50 liters to about 15,000 liters, from about 50 liters to about 10,000 liters, from about 100 liters to about 10,000 liters, from about 100 liters to about 5,000 liters, from about 100 liters to about 4,000 liters, from about 100 liters to about 3,000 liters, from about 100 liters to about 2,900 liters, from about 100 liters to about 2,850 liters, from about 100 liters to about 2,800 liters, from about 100 liters to about 2,750 liters.

[0175] In some embodiments, the culture contains liquid and the method includes collecting anaerobic cells (e.g., M. elsdenii cells) by removing a percentage of the liquid. In some embodiments, the step of collecting the cells includes removing from about 5% to about 100%, from about 10% to about 100%, from about 15% to about 100%, from about 20% to about 100%, from about 25% to about 100%, from about 30% to about 100%, from about 35% to about 100%, from about 40% to about 100%, from about 45% to about 100%, from about 50% to about 100% of the liquid, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, or from about 95% to about 100% of the liquid. In some embodiments, the step of collecting the cells includes removing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the liquid. In some embodiments, the cells are concentrated about 1 - 100 fold. In some embodiments, the cells are concentrated about 5 - 100 fold. In some embodiments, the cells are concentrated about 10 - 100 fold. In some embodiments, the cells are concentrated about 15 - 100 fold. In some embodiments, the cells are concentrated about 20 - 100 fold. In some embodiments, the cells are concentrated about 25 - 100 fold. In some embodiments, the cells are concentrated about 30 - 100 fold. In some embodiments, the cells are concentrated about 35 - 100 fold. In some embodiments, the cells are concentrated about 40 - 100 fold.

[0176] In some embodiments, the culture contains a liquid, and the method includes the step of concentrating anaerobic cells (e.g., M. elsdenii cells) by removing a percentage of the liquid prior to collection. In some embodiments, concentrating the cells comprises removing 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% of the liquid, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the liquid. In some embodiments, concentrating the cells comprises removing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the liquid. In some embodiments, the cells are concentrated about 1 to 100-fold. In some embodiments, the cells are concentrated about 5 to 100-fold. In some embodiments, the cells are concentrated about 10 to 100-fold. In some embodiments, the cells are concentrated about 15 to 100-fold. In some embodiments, the cells are concentrated about 20 to 100-fold. In some embodiments, the cells are concentrated about 25 to 100-fold. In some embodiments, the cells are concentrated about 30 to 100-fold. In some embodiments, the cells are concentrated about 35 to 100-fold. In some embodiments, the cells are concentrated about 40 to 100-fold.

[0177] In some embodiments, the method includes collecting anaerobic cells (e.g., M. elsdenii cells) by concentrating the cells. In some embodiments, the step of collecting the cells includes concentrating the cells by at least one technique selected from the group consisting of centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. In some embodiments, the filtration includes clay filtration. In some embodiments, the filtration includes aggregation with clay. In some embodiments, the filtration includes aggregation using a combination of clay and chitosan. In some embodiments, the filtration includes tangential flow filtration, also known as crossflow filtration.

[0178] In some embodiments, the pH of the culture containing anaerobic cells (e.g., M. elsdenii cells) at the time of collection is between about 4.5 and about 7.0, between about 4.5 and about 6.5, between about 4.5 and about 6.0, between about 4.5 and about 5.5, between about 4.5 and about 5.0, between about 4.6 and about 6.9, between about 4.7 and about 6.8, between about 4.8 and about 6.7, between about 4.9 and about 6.6, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.0 and about 6.0, between about 5.0 and about 5.5, between about 5.1 and about 6.9, between about 5.2 and about 6.8, between about 5.3 and about 6.7, between about 5.4 and about 6.6, between about 5.5 and about 7.0, between about 5.5 and about 6.5, between about 5.1 and about 6.4, between about 5.2 and about 6.3, between about 5.3 and about 6.2, between about 5.4 and about 6.1, between about 5.5 and about 6.0, between about 5.0 and about 6.1, between about 5.0 and about 6.2, between about 5.0 and about 6.3, between about 5.0 and about 6.4, between about 5.1 and about 6.5, between about 5.2 and about 6.5, between about 5.3 and about 6.5, or between about 5.4 and about 6.5.

[0179] In some embodiments, the original pH of the culture containing anaerobic cells (e.g., M. elsdenii cells) at the time of collection is about 6.0. In some embodiments, the pH is increased to about 7.5 for about 5 minutes to about 60 minutes. In some embodiments, the pH is decreased to about 3.5 for about 5 minutes to about 60 minutes. In some embodiments, after about 5 minutes to 60 minutes, the pH is adjusted back to the original pH of about 6.0. In some embodiments, the adjustment to the original pH is performed using an osmoprotectant. In some embodiments, the osmoprotectant is selected from the group consisting of betaine, trehalose, proline, ectoine, or combinations thereof. In some embodiments, the osmoprotectant is a sugar, sugar alcohol, sugar derivative, polysaccharide, encapsulated polymer, or nitrogen source, and mixtures thereof. In some embodiments, the sugar is sucrose. In some embodiments, the adjustment to the original pH is performed without using an osmoprotectant.

[0180] In some embodiments, the pH is increased to about 7.5 for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the pH is increased to about 7.5 for about 5 minutes to about 60 minutes, about 10 minutes to about 60 minutes, about 15 minutes to about 60 minutes, about 20 minutes to about 60 minutes, about 25 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 35 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 25 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 5 minutes to about 10 minutes.

[0181] In some embodiments, the pH is decreased to about 3.5 over about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the pH is decreased to about 3.5 over about 5 minutes to about 60 minutes, about 10 minutes to about 60 minutes, about 15 minutes to about 60 minutes, about 20 minutes to about 60 minutes, about 25 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 35 minutes to about 60 minutes, about 45 minutes to about 60 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 25 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 5 minutes to about 10 minutes.

[0182] In some embodiments, the method comprises inoculating a growth medium in a fermenter with an inoculum comprising anaerobic cells (e.g., M. elsdenii cells) to prepare a culture, and incubating the culture at a temperature of about 39 °C until the pH of the culture reaches about 6.0. In some embodiments, the inoculum comprises anaerobic cells (e.g., M. elsdenii). In some embodiments, the method comprises inoculating a growth medium in a fermenter at a ratio of inoculum to medium of 1 / 50 to 1 / 4,000. In some embodiments, the ratio of inoculum to medium is 1 / 100. Electrospray drying of anaerobic bacterial cells and / or M. elsdenii cells

[0183] Spray drying is a method of producing a dry powder from a liquid or slurry by rapidly drying it with a hot gas (typically air or nitrogen).

[0184] The spray drying process starts with a liquid solvent, usually water, containing dissolved or suspended components such as an emulsion (e.g., an emulsion containing anaerobic cells and / or M. elsdenii). The suspension contains the material to be encapsulated (the load) and an amphiphilic carrier (typically, modified starch), which is homogenized as a suspension in the liquid solvent. The load is anaerobic cells and / or M. elsdenii, and the homogenized suspension is often referred to as a slurry.

[0185] The spray drying apparatus uses a type of atomizer, e.g., a spray nozzle, to disperse the slurry into a controlled spray with some relatively controlled droplet size. Depending on the process requirements, the droplet size can range from about 10 to 500 microns in diameter. The most common applications require droplet sizes in the range of 50 to 200 microns.

[0186] In conjunction with atomization, the slurry is fed into a drying chamber, usually a tower, where hot air is also introduced. The temperature of the air when it enters the drying chamber is typically within the range of 180 - 200 °C. The hot air supplies the energy for the evaporation of the volatile components of the liquid (water) from the droplets. As the water evaporates, the carrier forms a hardened shell around the load, producing a dried powder.

[0187] Anaerobic cells (e.g., M. elsdenii) are either emulsified in a carrier fluid system or dissolved therein to form a slurry. In some embodiments, the anaerobic cells are selected from the group consisting of Bifidobacterium cells, such as B. breve, Lactobacillus cells, such as L. plantarum, Bifidobacterium cells, such as B. animalis subsp. lactis, Pediococcus cells, such as P. acidilactici, Lactobacillus cells, such as L. casei, Fibrobacter, such as F. succinogenes, Ruminococcus, such as R. flavefaciens, and Butyrivibrio, such as B. fibrisolvens).

[0188] The slurry formed in the solution tank is delivered to the atomizer using a pump or other conveying means. The slurry enters the atomizer and exits the atomizer as a spray of droplets, and the droplets are introduced into the drying chamber. The feed of the drying fluid is heated by a process heater and supplied to the drying chamber by a blower. In some embodiments, the drying fluid is air, argon, or nitrogen. As the water evaporated from the droplets enters the heated air and the atomized droplets dry, solid particles are formed after exposure to the incoming heated air.

[0189] The dried powder exits the drying chamber together with the air containing water vapor and is conveyed to a cyclone separator, where the dried particles are removed from the circulating air stream by the separator and the particles are accumulated in a collection container. The air containing water vapor exits the collection container and enters a baghouse, where, after very fine particles are removed, the air containing water vapor is sent to a condenser by a blower. The condenser removes water vapor from the process air, and the recovered water may be reused or discarded.

[0190] One of the prominent attributes of conventional spray drying processes is the high temperature of the inlet gas (approximately 200 °C) exiting the heater and entering the drying chamber, as well as the temperature of the outlet gas exiting the drying chamber, which typically exceeds 100 °C. The droplets are injected into the high temperature environment within the chamber, but the droplets do not actually reach the inlet gas temperature. The droplets will, however, be heated to the point where a significant portion of the desired components of the droplets (i.e., portions of the load, anaerobic bacterial cells, and / or M. elsdenii) are undesirably modified. The undesired modification to the load (load loss) results in a reduction in the viability of the anaerobic cells and / or M. elsdenii. Thus, evaporation and thermal decomposition of the load degrade the performance characteristics of the final powder product, and thus result in a significant reduction in performance and a significant loss of revenue in commercial use.

[0191] The present disclosure provides an electrospray drying method using a low-temperature, e.g., "heat-free" spray drying process, which produces powder bacterial products (e.g., anaerobic cells and / or M. elsdenii) with excellent viability and stability. The spray drying method of the present disclosure does not utilize a heated gas to remove water from atomized fluid droplets, as in previously utilized spray drying operations. Instead, the spray drying method of the present disclosure uses unheated air, e.g., dehumidified air, to perform a high-throughput atomization process, and utilizes a unique drying device design and a slurry / emulsion with a high solid content (low water content) having a viscosity in the range of 1 to 2000 cP to produce dried powder at a low temperature, e.g., a temperature of about 50 to 100 °C. Instead, the spray drying method of the present disclosure uses unheated air, nitrogen, or argon, e.g., dehumidified air, nitrogen, or argon, to perform a high-throughput atomization process, and utilizes a unique drying device design and a slurry / emulsion with a high solid content (low water content) having a viscosity in the range of 1 to 2000 cP to produce dried powder at a low temperature, e.g., a temperature of about 50 to 100 °C. Additionally, an electrostatic charge is applied to the slurry or atomized droplets, thereby creating a force that repels adjacent droplets from each other due to their respective charges on the wet droplets.

[0192] In some aspects, the present invention is directed to a method of electrospray drying anaerobic cells and / or M. elsdenii cells.

[0193] In some aspects, the present invention is directed to electrospray dried anaerobic cells and / or M. elsdenii cells.

[0194] In some aspects, the present invention is directed to electrospray dried anaerobic cells and / or M. elsdenii cells produced by the methods or systems disclosed herein.

[0195] In some embodiments, the present disclosure provides a system for spray drying a liquid product into a dried powder using no heated air or using low-temperature heated air, according to one or more embodiments of the present invention. In some embodiments, the present disclosure provides a system for spray drying a liquid product into a dried powder using no heated air, nitrogen, or argon, or using low-temperature heated air, nitrogen, or argon, according to one or more embodiments of the present invention. This system includes some elements that are the same as or similar to those of a conventional spray drying system.

[0196] In some embodiments, this system includes a drying chamber (or equivalent conveyance mechanism) into which a slurry containing anaerobic cells and / or M. elsdenii is supplied using a pump. The slurry enters an atomizer and exits the atomizer as a spray of droplets, which are introduced into the drying chamber. A feed of unheated or low-temperature heated fluid (e.g., air, nitrogen, or argon, or other suitable gas) is supplied to the drying chamber by a blower. The supplied air, nitrogen, or argon can be subjected to dehumidification (through a dehumidification device) before being introduced into the drying chamber. The atomized droplets dry to form solid particles after exposure to the incoming air, nitrogen, or argon. Water evaporates from the droplets and enters the air, nitrogen, or argon in the drying chamber. The dried powder exits the drying chamber together with the air, nitrogen, or argon containing water vapor and is conveyed to a cyclone separator, where the dried particles are removed from the circulating air, nitrogen, or argon stream and the particles are accumulated in a collection vessel. The air, nitrogen, or argon containing water vapor exits the collection vessel and enters a baghouse, where, after very fine particles are removed, the air, nitrogen, or argon containing water vapor is sent to a condenser by a blower. The condenser removes water vapor from the process air, nitrogen, or argon, and the recovered water / gas may be reused or discarded.

[0197] Another reason that non-heated or low-heated air, nitrogen, or argon can be used in a spray drying system and process is that the slurry can be unconventional. In some embodiments, the slurry includes a liquid solvent, a carrier, and a live organism. In some embodiments, the liquid solvent is water, however, other suitable solvents may be utilized if required or desired. In some embodiments, the carrier is a carbohydrate. In some embodiments, the live organism can be an anaerobic bacterium and / or M. elsdenii.

[0198] The formation of the slurry is controlled such that adjustment of at least one variable, such as viscosity, the amount of liquid solvent (e.g., water), or other suitable metrics related to the water content of the slurry, is obtained. In some embodiments, the formation of the slurry can include controlling the viscosity of the slurry such that the viscosity in the atomization step is between about 1 and 2000 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 10 and 1950 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 50 and 1900 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 100 and 1800 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 150 and 1750 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 200 and 1700 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 250 and 1650 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 300 and 1600 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 350 and 1550 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 400 and 1500 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 450 and 1450 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 500 and 1400 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 550 and 1350 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 600 and 1300 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 650 and 1250 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 700 and 1200 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 750 and 1150 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 800 and 1100 centipoise (cP).In some embodiments, the viscosity in the atomization step is between about 850 and 1050 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 900 and 1000 centipoise (cP). In some embodiments, the viscosity in the atomization step is between about 925 and 975 centipoise (cP). In some embodiments, the viscosity in the atomization step is between 1 and 500 cP. In some embodiments, the viscosity in the atomization step is 5 to 50 centipoise (cP).

[0199] In some embodiments, forming the slurry may include controlling the ratio of water or liquid solvent in the slurry such that the ratio is at least one of between about 70 and 99 weight percent, between about 71 and 98 weight percent, between about 72 and 97 weight percent, between about 73 and 96 weight percent, between about 74 and 95 weight percent, between about 75 and 95 weight percent, between about 76 and 94 weight percent, between about 77 and 93 weight percent, between about 78 and 92 weight percent, between about 79 and 91 weight percent, between about 80 and 89 weight percent, between about 81 and 88 weight percent, between about 82 and 87 weight percent, between about 83 and 86 weight percent, and between about 84 and 85 weight percent during the atomization step.

[0200] Another reason non-heated air, nitrogen, or argon can be used in a spray drying system and process is because an electrostatic charging process is performed before, during, or after atomization. Specifically, an electrostatic charge is applied to the slurry or the atomized droplets. In this regard, the system and / or process includes a high voltage power supply (about 0.1 to 45 kV DC) connected to one or more electrodes. In some embodiments, the high voltage power supply is about 0.5 to 40 kV DC. In some embodiments, the high voltage power supply is about 1 to 35 kV DC. In some embodiments, the high voltage power supply is about 5 to 30 kV DC. In some embodiments, the high voltage power supply is about 10 to 25 kV DC. In some embodiments, the high voltage power supply is about 15 to 20 kV DC. In some embodiments, the high voltage power supply is about 15 kV DC.

[0201] In some embodiments, the system and / or process includes a programmable profile that uses a control module to provide a voltage gradient. In some embodiments, the voltage gradient starts at about 0.1 kV and increases to 45 kV. In some embodiments, the voltage gradient starts at about 0.5 kV and increases to 40 kV. In some embodiments, the voltage gradient starts at about 1 kV and increases to 35 kV. In some embodiments, the voltage gradient starts at about 5 kV and increases to 30 kV. In some embodiments, the voltage gradient starts at about 10 kV and increases to 25 kV. The use of the voltage gradient will, for example, distort the magnetic field and create a particle structure with high viability and / or stability.

[0202] In some embodiments, the system and / or process includes a programmable profile that uses a control module to provide an oscillating voltage. In some embodiments, the voltage will oscillate between 0.1 kV and 45 kV with a reference point of 25 kV. In some embodiments, the voltage will oscillate between 1 kV and 40 kV with a reference point of 25 kV. In some embodiments, the voltage will oscillate between 5 kV and 35 kV with a reference point of 25 kV. In some embodiments, the voltage will oscillate between 10 kV and 30 kV with a reference point of 25 kV. The use of the oscillating voltage will, for example, distort the magnetic field and create a particle structure with high viability and / or stability.

[0203] The polarity of the high voltage power supply can be configured as either positive or negative. The slurry (or atomized droplets) contacts the electrode and is charged there. In a preferred embodiment, the slurry is contacted with the electrode to obtain a charged slurry. Simultaneously or thereafter, the charged slurry is atomized to obtain a plurality of electrostatically charged wet particles (droplets).

[0204] The charges of each wet droplet create a force that tends to repel adjacent droplets from each other. In addition, the force on a given droplet opposes the surface tension of such a given droplet. When the charge on a given droplet exceeds a threshold level, the Rayleigh limit, the droplet becomes unstable and smaller satellite droplets are released from the given (parent) droplet. Since the surface charge density of the satellite droplets does not decrease even when evaporation occurs, one or more of the satellite droplets can also become unstable and generate further satellite droplets.

[0205] Electrostatically charged wet particles / droplets float in the drying chamber for a time sufficient for the aforementioned repulsive forces induced by the electrostatic charges of at least some of the wet particles / droplets to split at least some of such particles into wet sub-particles / droplets. The floating of the droplets is continued for a sufficient period of time without the presence of any heated drying fluid, and a sufficient amount of the liquid solvent is removed from most of the wet particles / droplets, leaving a plurality of dried particles (powder) each containing an active organism encapsulated in a carrier. Notably, by producing sub-particles / droplets from a given volume of atomized slurry (i.e., from a given droplet), a significant increase in the aggregated surface area of the sub-particles / droplets and, concomitantly, a reduction in the particle volume of each sub-particle / droplet after each fission event result in a faster drying of such volume.

[0206] The production of sub-particles / droplets can be referred to as Coulomb fission. The time scale of such Coulomb fission events is on the order of a few microseconds to a few milliseconds. By splitting from one given particle / droplet into approximately 10 sub-particles / droplets, the diameter of the given particle / droplet is reduced by approximately 30%. The amount of time (on the order of a few microseconds to a few milliseconds) required to achieve such a diameter reduction is an order of magnitude faster than diffusion evaporation in the presence of heated air, nitrogen, or argon, which occurs at a characteristic time t according to the following equation: t = do 2 / k, where do is the diameter of the particle and k is the evaporation-diffusion coefficient. For particles in the diameter range of 20 - 200 mm, the time to any significant diameter reduction by evaporation is on the order of seconds to a fraction of 10 seconds, which is much longer (by 1 - 2 orders of magnitude) than the diameter reduction by Coulomb fission.

[0207] Due to the relatively low water content in the slurry and the characteristics of the electrostatic charge on the droplets, either individually or in combination, significantly lower temperature conditions within the drying chamber are possible compared to heating air systems and processes. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 15 - 100 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 20 - 95 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 25 - 90 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 30 - 85 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 35 - 80 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 40 - 75 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 45 - 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 50 - 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is between 55 - 65 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is 62.5 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is 67.5 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is 80 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air) introduced into the drying chamber is 90 °C.

[0208] In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 15 and 100 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 20 and 95 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 25 and 90 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air or nitrogen or argon) introduced into the drying chamber is between 30 and 85 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 35 and 80 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 40 and 75 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 45 and 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 50 and 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is between 55 and 65 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is 62.5 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is 67.5 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is 70 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is 80 °C. In some embodiments, the temperature of the non-heated drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber is 90 °C.

[0209] In some embodiments, the drying fluid is a gas. In some embodiments, the drying fluid is argon. In some embodiments, the drying fluid is nitrogen. In some embodiments, the drying fluid is air.

[0210] Typically, the temperature of the inlet air, nitrogen, or argon results in the temperature of the outlet air from the drying chamber being lower than the temperature of the inlet air, nitrogen, or argon.

[0211] In some embodiments, the method further includes heating the air to between 50°C and 100°C by applying a voltage of 0.1 to 45 kV before supplying the air to the drying chamber. In some embodiments, the method further includes heating the air to between about 40°C and about 120°C by applying a voltage of 0.1 to 45 kV before supplying the air to the drying chamber. In some embodiments, the method further includes heating the air by applying a voltage before supplying the air to the drying chamber. In some embodiments, the air is heated to between about 40°C and about 120°C, between about 40°C and about 110°C, between about 40°C and about 100°C, between about 40°C and about 90°C, between about 40°C and about 80°C, between about 40°C and about 70°C, between about 40°C and about 60°C, between about 40°C and about 50°C, between about 50°C and about 120°C, between about 60°C and about 120°C, between about 70°C and about 120°C, between about 80°C and about 120°C, between about 90°C and about 120°C, between about 100°C and about 120°C, or between about 110°C and about 120°C. In some embodiments, the method further includes heating the air to between 50°C and 100°C by applying a voltage of 0.1 to 45 kV before supplying air, nitrogen, or argon to the drying chamber. In some embodiments, the method further includes heating air, nitrogen, or argon to between about 40°C and about 120°C by applying a voltage of 0.1 to 45 kV before supplying air, nitrogen, or argon to the drying chamber. In some embodiments, the method further includes heating air, nitrogen, or argon by applying a voltage before supplying air, nitrogen, or argon to the drying chamber. In some embodiments, air, nitrogen, or argon is heated to between about 40°C and about 120°C, between about 40°C and about 110°C, between about 40°C and about 100°C, between about 40°C and about 90°C, between about 40°C and about 80°C, between about 40°C and about 70°C, between about 40°C and about 60°C, between about 40°C and about 50°C, between about 50°C and about 120°C, between about 60°C and about 120°C, between about 60°C and about 90°C, between about 70°C and about 120°C, between about 80°C and about 120°C, between about 90°C and about 120°C, between about 100°C and about 120°C, or between about 110°C and about 120°C.In some embodiments, the voltage is about 0.1 kV, about 1 kV, about 5 kV, about 10 kV, about 15 kV, about 20 kV, about 25 kV, about 30 kV, about 35 kV, about 40 kV, or about 45 kV. In some embodiments, the voltage is between about 0.1 and about 45 kV. In some embodiments, the voltage is between about 0.1 and about 45 kV, about 0.1 and about 40 kV, about 0.1 and about 35 kV, about 0.1 and about 30 kV, about 0.1 and about 25 kV, about 0.1 and about 20 kV, about 0.1 and about 15 kV, about 0.1 and about 10 kV, about 0.1 and about 5 kV, about 0.1 and about 1 kV, about 1 and about 45 kV, about 5 and about 45 kV, about 10 and about 45 kV, about 15 and about 45 kV, about 20 and about 45 kV, about 25 and about 45 kV, about 30 and about 45 kV, about 35 and about 45 kV, or about 40 and about 45 kV.

[0212] In some embodiments, the method further includes heating the air to between 50°C and 100°C by applying a voltage of 15 kV before supplying the air to the drying chamber. In some embodiments, the method further includes heating the air, nitrogen, or argon to between 50°C and 100°C by applying a voltage of 15 kV before supplying the air, nitrogen, or argon to the drying chamber.

[0213] In some embodiments, the air is at a temperature between 70°C and 90°C. In some embodiments, the air, nitrogen, or argon is at a temperature between 70°C and 90°C.

[0214] In some embodiments, the method further includes heating the air to between 70°C and 90°C by applying a voltage of 15 kV before supplying the air to the drying chamber. In some embodiments, the method further includes heating the air, nitrogen, or argon to between 70°C and 90°C by applying a voltage of 15 kV before supplying the air, nitrogen, or argon to the drying chamber.

[0215] In some embodiments, the voltage is applied continuously or in pulses.

[0216] In some embodiments, it may be desirable to ensure that the drying fluid (e.g., air, nitrogen, or argon) introduced into the drying chamber has a relatively low water content. Thus, the system may include a process dehumidification device to remove some amount of water from the drying fluid prior to introduction into the drying chamber. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 1% and 20%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 2% and 19%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 3% and 18%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 4% and 17%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 5% and 16%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 6% and 15%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 7% and 14%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 8% and 13%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 9% and 12%. In some embodiments, after dehumidification, the unheated air, nitrogen, or argon introduced into the drying chamber can have a relative humidity between about 10% and 11%.

[0217] In some embodiments, the dew point temperature of the unheated air, nitrogen, or argon can be in the range of -20°C to 5°C, -15°C to 5°C, -12°C to 3°C, -12°C to 0°C, -12°C to -5°C, or any other suitable dew point temperature range suitable for spray drying operations.

[0218] The atomizer can be implemented using any of the known methods, apparatuses, and / or techniques. In some embodiments, the atomizer can be implemented using at least one of nozzle techniques, centrifugal techniques, pneumatic techniques, and ultrasonic techniques. For most atomization techniques, the slurry exits the atomization mechanism as thin liquid films or fragments of ligaments rather than as final droplets. The formation of droplets occurs due to the surface tension of the liquid immediately after the liquid exits the atomization mechanism. The size of the droplets obtained by a given type of atomization depends, for example, on the energy input that breaks up the slurry into fragments to increase the total effective surface area of the slurry.

[0219] In some embodiments, the average droplet size and distribution can be substantially constant with a given atomization technique and can be in the range of 1 to 500 microns. In some embodiments, the average droplet size is in the range of 10 to 450 microns. In some embodiments, the average droplet size is in the range of 50 to 400 microns. In some embodiments, the average droplet size is in the range of 100 to 350 microns. In some embodiments, the average droplet size is in the range of 150 to 300 microns. In some embodiments, the average droplet size is in the range of 200 to 250 microns.

[0220] In some embodiments, a single-nozzle (i.e., single-fluid) atomizer encapsulates cells by dispensing and mixing the cells, the carrier, and the encapsulation material. In some embodiments, a two-nozzle (i.e., dual-fluid) atomizer dispenses cells from one nozzle, dispenses the carrier and / or the encapsulation material from a second nozzle, and mixes the two fluids together to encapsulate the cells (by creating a coating of a core and an outer layer). In some embodiments, a single-nozzle (i.e., single-fluid) atomizer spray-dries the cells mixed with the carrier, and the encapsulation step is performed after spray-drying, or in a Wurster coating apparatus or other coating apparatus.

[0221] The electrostatic charging process and the resulting Coulomb fission process according to various aspects herein generally produce larger particles than conventional spray drying processes. However, these larger particles result from even larger parent particles that cannot be appropriately produced by conventional atomizers. The daughter particles produced according to the aspects herein are smaller, and the process tends to result in a bimodal size distribution for very viscous slurries.

[0222] Centrifugal (or rotary) atomization can be considered the most common form of atomization. Centrifugal atomization utilizes a rotating disk or wheel that breaks up a liquid slurry stream into droplets. In some aspects, a centrifugal atomization device can utilize a disk or wheel with a diameter of about 5 - 50 cm. In some aspects, a centrifugal atomization device can utilize a disk or wheel with a diameter of about 10 - 45 cm. In some aspects, a centrifugal atomization device can utilize a disk or wheel with a diameter of about 10 - 45 cm. In some aspects, a centrifugal atomization device can utilize a disk or wheel with a diameter of about 15 - 40 cm. In some aspects, a centrifugal atomization device can utilize a disk or wheel with a diameter of about 20 - 30 cm. In some aspects, the disk or wheel can rotate in the range of about 5,000 - 40,000 rpm. In some aspects, the disk or wheel can rotate in the range of about 10,000 - 35,000 rpm. In some aspects, the disk or wheel can rotate in the range of about 15,000 - 30,000 rpm. In some aspects, the disk or wheel can rotate in the range of about 20,000 - 30,000 rpm. The size of the droplets produced by a centrifugal atomization device is approximately inversely proportional to the peripheral velocity of the disk or wheel.

[0223] Nozzle atomization utilizes a pump that pressurizes the slurry and extrudes the slurry from the nozzle orifice to break up the liquid into fine droplets. In some embodiments, the orifice size is typically in the range of 0.5 to 30 mm. In some embodiments, the orifice size is typically in the range of 1 to 29 mm. In some embodiments, the orifice size is typically in the range of 5 to 25 mm. In some embodiments, the orifice size is typically in the range of 10 to 20 mm. In some embodiments, the orifice size is typically in the range of 13 to 17 mm. The size of the droplets depends on the size of the orifice and the pressure loss. The greater the pressure loss across the orifice, the smaller the droplets produced. Thus, for a given feed rate, smaller orifices and higher pump pressures may be utilized to reduce the particle / droplet size.

[0224] Two-fluid pneumatic atomization utilizes the interaction of a slurry with a fluid other than the slurry, typically compressed air, using a fluid nozzle for the compressed air and a fluid nozzle for the slurry. In some embodiments, the pressures of the air and the slurry can be in the range of about 200 - 350 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 210 - 340 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 220 - 330 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 230 - 320 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 240 - 310 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 250 - 300 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 260 - 290 kPa. In some embodiments, the pressures of the air and the slurry can be in the range of about 270 - 280 kPa. The particle size is controlled by varying the ratio of the compressed air flow to the slurry flow. In some embodiments, two-fluid pneumatic atomization utilizes the interaction of a slurry with a fluid other than the slurry, typically compressed air, nitrogen, or argon, using a fluid nozzle for the compressed air, nitrogen, or argon and a fluid nozzle for the slurry. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 200 - 350 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 210 - 340 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 220 - 330 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 230 - 320 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 240 - 310 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 250 - 300 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 260 - 290 kPa. In some embodiments, the pressures of the air, nitrogen, or argon and the slurry can be in the range of about 270 - 280 kPa.The particle size is controlled by varying the ratio of the compressed air, nitrogen, or argon stream to the slurry stream.

[0225] Ultrasonic atomization utilizes ultrasonic energy to vibrate the surface at ultrasonic frequencies. The slurry is brought into contact with the vibrating surface to generate particles / droplets.

[0226] In some embodiments, the present disclosure may use a centrifugal atomizer, a nozzle atomizer, a two-fluid atomizer, or an ultrasonic atomizer.

[0227] In some embodiments, the two-fluid atomizer comprises a body having a proximal end and a distal end. In some embodiments, the two-fluid atomizer has a channel extending through the body, typically including an inlet near the proximal end of the body and generally including an outlet near the distal end of the body. The channel is operative to convey the first of the two fluids, i.e., the slurry, from the inlet to the outlet.

[0228] In some embodiments, the two-fluid atomizer also includes at least one electrode operative to contact the slurry and apply an electrostatic charge thereto, such that the two-fluid atomizer is operative to produce a plurality of electrostatically charged wet particles / droplets. In some embodiments, the at least one electrode may be disposed within the body of the two-fluid atomizer such that the slurry contacts the electrode and becomes electrostatically charged while flowing from the inlet to the outlet of the channel. In some embodiments, the electrode may be disposed within the channel, preferably coaxially, such that most of the surface area of the electrode is available for contact with the slurry. In some embodiments, the electrode may be inserted into the channel using a threaded hole in the body and a threaded shaft of a complementary electrode, which engage to position the electrode within the channel. In some embodiments, a connection terminal may be electrically and mechanically coupled to the electrode to provide means for connecting to a high voltage power source and receiving a potential at the surface of the electrode.

[0229] In some embodiments, the two-fluid atomizer may include a nozzle in fluid communication with the outlet of the channel. In some embodiments, the outlet of the channel includes a tube sized and shaped to engage a complementary hole at the inlet end of the nozzle and be received therein. The engagement of the tube with the hole enables fluid communication of the slurry (charged with electrostatic charge) from the channel into an internal volume disposed directly within the nozzle. In some embodiments, a sealing ring may be utilized to ensure a tight fluid seal between the tube and the hole, even under fluid pressure. In some embodiments, the nozzle preferably includes a transition section (tapered surface) of decreasing diameter extending from the internal volume to the nozzle opening. In some embodiments, the nozzle opening preferably includes an internal hole sized to produce wet particles / droplets of a desired size and shape that are subject to the forces of surface tension, generally having a cylindrical shape.

[0230] In some embodiments, the two-fluid atomizer may further include a nozzle cap generally surrounding the nozzle and allowing the nozzle opening to extend through a hole at its distal end. In some embodiments, the nozzle cap includes an engagement feature at its proximal end that engages the distal end of the body. In some embodiments, the nozzle cap includes a threaded shank that fits into a complementary threaded hole in the body. In some embodiments, the sealing ring may be utilized to ensure a tight fluid seal between the inner surface of the nozzle cap and the outer surface of the nozzle, thereby forming an internal volume therebetween.

[0231] In some embodiments, the two-fluid atomizer includes another channel that extends through a body and typically includes an inlet near the proximal end of the body and an outlet near the distal end of the body. In some embodiments, the channel is operative to convey the second of the two fluids, i.e., unheated air, nitrogen, or argon, from the inlet to the outlet. In some embodiments, the outlet is in fluid communication with an internal volume (between the inner surface of the nozzle cap and the outer surface of the nozzle). Thus, the channel is operative to convey unheated air, nitrogen, or argon from the proximal end to the distal end of the two-fluid atomizer. In some embodiments, the flow of unheated air, nitrogen, or argon through the two-fluid atomizer can be about 5,100 m 3 / hour at an input pressure of about 130 psi. In some embodiments, the flow of unheated air, nitrogen, or argon through the two-fluid atomizer can be about 4,000 m 3 / hour at an input pressure of about 100 psi. In some embodiments, the flow of unheated air, nitrogen, or argon through the two-fluid atomizer can be about 4,500 m 3 / hour at an input pressure of about 115 psi. In some embodiments, the flow of unheated air, nitrogen, or argon through the two-fluid atomizer can be about 6,200 m 3 / hour at an input pressure of about 160 psi. In some embodiments, the flow of unheated air, nitrogen, or argon through the two-fluid atomizer can be about 5,500 m 3 / hour at an input pressure of about 145 psi.

[0232] In some embodiments, the nozzle includes a tapered surface on its outside, which is downstream of the outer surface and downstream of the internal volume. In some embodiments, the nozzle cap includes a complementary internal surface adjacent to the tapered surface. A number of grooves (recesses) are provided in the tapered surface and extend from the internal volume to the nozzle opening. When the complementary internal surface of the nozzle cap is adjacent to the tapered surface, the grooves provide fluid communication of non-heated air, nitrogen, or argon from the internal volume to the nozzle opening. The grooves terminate in an annular space between the peripheral end of the tapered surface and the outer surface of the nozzle opening, which is where the nozzle opening exits the nozzle. The annular space is in fluid communication with a hole, whereby a preferably sized hole (larger than the diameter of the nozzle opening) allows non-heated air, nitrogen, or argon to exit the nozzle and the nozzle cap under pressure. In some embodiments, the grooves terminate tangentially to the annular space, whereby they extend such that non-heated air, nitrogen, or argon causes a swirling fluid motion in the vicinity of the nozzle opening after exiting the space and the hole.

[0233] In some embodiments, the swirling fluid motion when non-heated air, nitrogen or argon exits the nozzle and the nozzle cap causes a swirling agitation to a plurality of wet particles / droplets when they exit the nozzle. This swirling agitation can suspend and agitate the wet particles / droplets to achieve the aforementioned breakup and evaporation. In some embodiments, the atomization approach described above allows for a relatively high slurry throughput of about 1 to 20 kg / h at an input pressure of about 20 to 100 psi. In some embodiments, the atomization approach described above allows for a relatively high slurry throughput of about 0.1 to 25 kg / h at an input pressure of about 15 to 125 psi.

[0234] In some embodiments, a drying chamber can be utilized in the system according to the present disclosure. In some embodiments, the drying chamber can include an inlet end, an outlet end, and an internal volume in which wet particles / droplets are dried. In some embodiments, the drying chamber is formed from a non-conductive material. In some embodiments, the material selection would be non-metallic in order to avoid conductive materials. However, in the prior art spray drying process, heating air (about 200 °C) is required, and as a result, a metal drying chamber (typically stainless steel) is required, otherwise the chamber will be distorted or broken.

[0235] In some embodiments, by using non-heated air, nitrogen, or argon, the temperature inside the drying chamber can be less than 50 °C, so that the drying chamber can be formed of a non-metallic material. In some embodiments, the drying chamber can be formed from materials such as polymer-based composite materials. Fiberglass composite material tanks wound with filaments (used for water storage, various foods, grain storage, brine, and a number of non-food-based applications) have excellent load-bearing characteristics and can be used to fabricate very large tanks. In some embodiments, a fiberglass composite material wound with filaments can be used to manufacture the drying chamber disclosed herein. Engineered plastics are typically low-cost in terms of base materials and manufacturing costs, for example, compared to similarly sized containers made from stainless steel. These materials also allow for great flexibility in the design of the drying chamber and enable the formation of complex shapes that are much more difficult and expensive to manufacture from stainless steel.

[0236] In some embodiments, the tank comprises an agitation device (e.g., a propeller or a stir bar).

[0237] In some embodiments, the tank is arranged to receive a liquid solvent (e.g., water), a carrier, and anaerobic cells and / or M. elsdenii cells.

[0238] By forming a drying chamber using a non-metallic non-conductive dielectric material (engineering plastic composite material), it becomes possible to use one or more electric fields inside the drying chamber itself to urge the particles / droplets along a desired trajectory and / or to urge such particles / droplets from the inlet end to the outlet end of the drying chamber. Different from a metallic conductive container, since all charges accumulate on the surface of the container, it is substantially impossible to generate an electric field inside.

[0239] In some embodiments, the drying chamber may include a first electrode located at or near its inlet end and a second electrode located at or near the outlet end of the drying chamber. By applying a power source between the first electrode and the second electrode, an electric field sufficient to urge the particles / droplets along a desired trajectory as they dry within the drying chamber is induced within the drying chamber. In some embodiments, the desired trajectory causes the lines of the electric field to generally extend parallel to the wall of the drying chamber, even when such wall tapers towards the outlet end. To achieve such a trajectory, the second electrode will need to be of a relatively smaller diameter compared to the first electrode. If the first and second electrodes are of generally the same diameter, the lines of the electric field will generally extend parallel to the wall of the drying chamber and then pass through the tapered wall at the outlet end. Other particle trajectories can be achieved based on the number, position, size, and shape of the electrodes. In some embodiments, the first and second electrodes may be disposed external to the drying chamber and still utilize the fact that it is formed from a non-conductive material to induce an electric field within the internal volume of the drying chamber.

[0240] In some embodiments, the morphology of the dried particles benefits from a non-thermal process in that the particles do not experience a rapid increase in temperature when entering the drying chamber and the particles do not exhibit cracks on the surface, volcanic structures on the surface, or hollow regions within the particles.

[0241] Generally, non-thermal processes exhibit high levels of preservation of starting viable organisms (e.g., anaerobic cells and / or M. elsdenii). In some embodiments, non-thermal processes resulted in more stable and viable anaerobic cells and / or M. elsdenii than thermal spray drying processes. In some embodiments, the occurrence of non-viable anaerobic cells and / or M. elsdenii was significantly reduced in non-thermal samples, resulting in a longer expected shelf life for powders containing anaerobic cells and / or M. elsdenii.

[0242] In some embodiments, the resulting dried powder comprises a plurality of dried particles, each of which individually encapsulates a final viable organism (e.g., anaerobic cells and / or M. elsdenii) within a carrier obtained by drying a slurry comprising an amount of starting viable organisms, a liquid solvent, and the carrier. In some embodiments, the starting viable organisms comprise one or more components, at least one of which is of one or more major molecular types from which at least one of bacteria, probiotics, etc. is obtained. In some embodiments, the bacteria are M. elsdenii as disclosed herein. In some embodiments, the bacteria are M. elsdenii NCIMB 41125.

[0243] In some embodiments, the carrier is a modified starch or a carbohydrate. In some embodiments, the carrier is selected from the group consisting of sucrose, maltodextrin, maltose, hydrophobic starch, sugars, sugar alcohols, sugar derivatives, or nitrogen sources (such as, but not limited to, ammonium salts and derivatives, yeast extract, skim milk, or corn steep liquor), and mixtures thereof. In some embodiments, the carrier is selected from the group consisting of sucrose, maltodextrin, maltose, polysaccharides, encapsulating polymers, hydrophobic starch, sugars, sugar alcohols, sugar derivatives, or nitrogen sources (such as, but not limited to, ammonium salts and derivatives, yeast extract, skim milk, or corn steep liquor), and mixtures thereof. In some embodiments, the encapsulating polymer is alginate. In some embodiments, the carrier has a concentration of 1 to 50 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 45 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 40 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 35 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 30 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 25 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 2 to 20 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 1 to 20 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 2 to 19 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 3 to 18 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 4 to 17 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 5 to 16 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 6 to 15 wt% of the total slurry and / or dried powder.In some embodiments, the carrier has a concentration of 7 - 14 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 8 - 13 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 9 - 12 wt% of the total slurry and / or dried powder. In some embodiments, the carrier has a concentration of 10 - 11 wt% of the total slurry and / or dried powder.

[0244] In some embodiments, the carrier has a concentration of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt% of the total slurry and / or dried powder.

[0245] In some embodiments, the moisture content is less than 1 wt%, less than about 2 wt%, less than about 3 wt%, less than about 4 wt%, less than about 5 wt%, less than about 6 wt%, less than about 7 wt%, less than about 8 wt%, less than about 9 wt%, less than about 10 wt%, less than about 11 wt%, less than about 12 wt%, less than about 13 wt%, less than about 14 wt%, less than about 15 wt%, less than about 20 wt%, less than about 25 wt%, less than about 30 wt%, less than about 35 wt%, less than about 40 wt%, less than about 45 wt%, or less than about 50 wt% of the dried powder. In some embodiments, the moisture content is about 1 - 30 wt% of the dried powder. In some embodiments, the moisture content is about 3 - 25 wt% of the dried powder. In some embodiments, the moisture content is about 5 - 20 wt% of the dried powder. In some embodiments, the moisture content is about 10 - 20 wt% of the dried powder.

[0246] In some embodiments, the final active organism comprises one or more of the components corresponding to those of the initial active organism that have been modified by drying the slurry. The weight percentage of at least one of one or more major molecular types in the final active organism is essentially the same (e.g., within about 5%, 4%, 3%, or 1% thereof) as the corresponding major molecular type in the initial active organism.

[0247] The use of high solid content emulsions (having a solid concentration of at least 40 wt%, preferably at least 50 wt% based on the total weight of the emulsion) in the low temperature, e.g., heat-free spray drying processes of the present disclosure provides several desirable attributes to the final powder produced by the process as another substantial economic advantage brought about by the low drying temperature and high solid content slurry / emulsion, including: (1) a high particle density such that the particles have a density greater than that of water (i.e., >1 g / cc) so that they readily sediment in an aqueous solution and rapidly dissolve or become suspended, (2) high resistance to oxidation obtained by the high solid content, (3) substantial energy efficiency due to the use of a high solid content slurry / emulsion associated with a low temperature as about half the amount of water evaporates compared to conventional spray drying processes, and (4) excellent retention of expensive active organisms such as M. elsdenii.

[0248] However, conventional high temperature drying processes cause a significant loss of viability of anaerobic cells and / or M. elsdenii, resulting in a powder with few viable anaerobic cells and / or M. elsdenii. Generally, powders obtained by conventional high temperature spray drying processes are, for example, of a small average diameter of about 60 - 100 micrometers, not sufficiently dense, and difficult to dissolve in an aqueous solution. In contrast, powders produced by the low temperature spray drying process of the present disclosure have a large average diameter of about 125 - 250 micrometers, are sufficiently dense, and readily form a solution.

[0249] Electrospray drying can be a continuous process. In some embodiments, the slurry is processed at a rate of about 0.1 L / hr to about 10,000 L / hr. In some aspects, the slurry is processed at a rate of between about 100 L / hour and about 900 L / hour. In some aspects, the slurry is processed at a rate of between about 200 L / hour and about 800 L / hour. In some aspects, the slurry is processed at a rate of between about 300 L / hour and about 700 L / hour. In some aspects, the slurry is processed at a rate of between about 400 L / hour and about 600 L / hour. In some aspects, the slurry is processed at a rate of between about 500 L / hour and about 600 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 0.25 L / hour to about 10,000 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 1 L / hour to about 10,000 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 10 L / hour to about 5,000 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 50 L / hour to about 1,000 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 100 L / hour to about 900 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 200 L / hour to about 800 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 300 L / hour to about 700 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 400 L / hour to about 600 L / hour. Electrospray drying may be a continuous process. In some embodiments, the slurry is processed at a rate of about 500 L / hour to about 600 L / hour.

[0250] Accordingly, spray-dried powders of the present disclosure can be produced that have at least one of the features that the final viable organism contains one or more of the components corresponding to those of the initial viable organism that are modified by the spray-drying process, and the spray-dried powder or fiber composition has (i) the weight percentage of at least one of one or more major molecular types (e.g., anaerobic cells and / or M. elsdenii) in the final organism is within about 15% of the weight percentage of the corresponding major molecular type in the initial viable organism, e.g., it can be within about 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the weight percentage of the corresponding major molecular type in the initial viable organism.

[0251] In some embodiments, about 1×10 3 ~1×10 13 CFU / g of electrospray-dried anaerobic cells and / or M. elsdenii cells are produced by the methods disclosed herein. In some embodiments, about 1×10 3 ~1×10 13 CFU / g of anaerobic cells and / or M. elsdenii cells are viable after electrospray drying.

[0252] In some embodiments, the amount of electrospray-dried anaerobic cells and / or M. elsdenii cells produced by the methods disclosed herein, and / or the amount of anaerobic cells and / or M. elsdenii cells that are viable after electrospray drying, is about 1×10 3 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 12 CFU / g, about 1×10 3 CFU / g to about 1×10 11 CFU / g, about 1×10 3 CFU / g to about 1×10 10 CFU / g, about 1×10 3 CFU / g to about 1×10 9 CFU / g, about 1×10 3 CFU / g to about 1×10 8 CFU / g, about 1×10 3 CFU / g to about 1×10 7 CFU / g, about 1×10 3 CFU / g to about 1×10 6 CFU / g, about 1×10 4 CFU / g to about 1×10 13 CFU / g, about 1×10 5 CFU / g to about 1×10 13 CFU / g, about 1×10 6 CFU / g to about 1×10 13 CFU / g, about 1×10 7 CFU / g to about 1×10 13 CFU / g, about 1×10 8 CFU / g to about 1×1013 CFU / g, about 1×10 9 CFU / g to about 1×10 13 CFU / g, about 1×10 10 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 5 CFU / g, about 1×10 4 CFU / g to about 1×10 6 CFU / g, about 1×10 5 CFU / g to about 1×10 7 CFU / g, about 1×10 6 CFU / g to about 1×10 8 CFU / g, about 1×10 7 CFU / g to about 1×10 9 CFU / g, about 1×10 8 CFU / g to about 1×10 10 CFU / g, about 1×10 9 CFU / g to about 1×10 11 CFU / g, or about 1×10 10 CFU / g to about 1×10 13 CFU / g.

[0253] In some embodiments, the electrospray dried anaerobic cells and / or M. elsdenii cells are viable at about -80°C, about -20°C, about 4°C, about 25°C, or combinations thereof for about 14 days to about 24 months. In some embodiments, the electrospray dried anaerobic cells and / or M. elsdenii cells are viable at about -80°C, about -20°C, about 4°C, about 25°C, or combinations thereof for at least 14 days, at least 1 month, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months.

[0254] In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about -20 °C, about 4 °C, about 30 °C, or combinations thereof for at least 75 days. In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about -20 °C for at least 6 months. In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about 37 °C for at least 28 days.

[0255] In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about -80 °C, about -20 °C, about 4 °C, about 25 °C, or combinations thereof for 14 days, 1 month, 6 months, 8 months, 10 months, 12 months, 15 months, 18 months, or 24 months.

[0256] In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about -20 °C, about 4 °C, about 30 °C, or combinations thereof for 75 days. In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about -20 °C for 6 months. In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are viable at about 37 °C for 28 days.

[0257] In some embodiments, about 1×10 3 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 12 CFU / g, about 1×10 3 CFU / mL to about 1×10 11 CFU / g, about 1×10 3 CFU / g to about 1×10 10 CFU / g, about 1×10 3 CFU / g to about 1×10 9 CFU / g, about 1×10 3 CFU / g to about 1×108 CFU / g, about 1×10 3 CFU / g to about 1×10 7 CFU / g, about 1×10 3 CFU / g to about 1×10 6 CFU / g, about 1×10 4 CFU / g to about 1×10 13 CFU / g, about 1×10 5 CFU / g to about 1×10 13 CFU / g, about 1×10 6 CFU / g to about 1×10 13 CFU / g, about 1×10 7 CFU / g to about 1×10 13 CFU / g, about 1×10 8 CFU / g to about 1×10 13 CFU / g, about 1×10 9 CFU / g to about 1×10 13 CFU / g, about 1×10 10 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 5 CFU / g, about 1×10 4 CFU / g to about 1×10 6 CFU / g, about 1×10 5 CFU / g to about 1×10 7 CFU / g, about 1×10 6 CFU / g to about 1×10 8 CFU / g, about 1×10 7 CFU / g to about 1×10 9 CFU / g, about 1×10 8 CFU / g to about 1×10 10 CFU / g, about 1×10 9 CFU / g to about 1×10 11 CFU / g, or about 1×10 10 CFU / g to about 1×10 13CFU / g of electrospray-dried anaerobic cells and / or M. elsdenii cells are viable after storage at temperatures of about -80°C, about -20°C, about 4°C, or combinations thereof for at least 14 days, at least 1 month, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months.

[0258] In some embodiments, about 1×10 3 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 12 CFU / g, about 1×10 3 CFU / g to about 1×10 11 CFU / g, about 1×10 3 CFU / g to about 1×10 10 CFU / g, about 1×10 3 CFU / g to about 1×10 9 CFU / g, about 1×10 3 CFU / g to about 1×10 8 CFU / g, about 1×10 3 CFU / g to about 1×10 7 CFU / g, about 1×10 3 CFU / g to about 1×10 6 CFU / g, about 1×10 4 CFU / g to about 1×10 13 CFU / g, about 1×10 5 CFU / g to about 1×10 13 CFU / g, about 1×10 6 CFU / g to about 1×10 13 CFU / g, about 1×10 7 CFU / g to about 1×10 13 CFU / g, about 1×10 8 CFU / g to about 1×10 13 CFU / g, about 1×10 9 CFU / g to about 1×10 13 CFU / g, about 1×10 10 CFU / g to about 1×10 13 CFU / g, about 1×10 3 CFU / g to about 1×10 5CFU / g, about 1×10 4 CFU / g to about 1×10 6 CFU / g, about 1×10 5 CFU / g to about 1×10 7 CFU / g, about 1×10 6 CFU / g to about 1×10 8 CFU / g, about 1×10 7 CFU / g to about 1×10 9 CFU / g, about 1×10 8 CFU / g to about 1×10 10 CFU / g, about 1×10 9 CFU / g to about 1×10 11 CFU / g, or about 1×10 10 CFU / g to about 1×10 13 CFU / g of electrospray-dried anaerobic cells and / or M. elsdenii cells are viable after storage at a temperature of about 25°C for at least 14 days, at least 1 month, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months. Feed additives, compositions, and kits

[0259] In some embodiments, the feed additive comprises electrospray-dried anaerobic cells and / or M. elsdenii cells disclosed herein. In some embodiments, the feed additive comprises lyophilized anaerobic cells and / or M. elsdenii cells produced by the methods disclosed herein.

[0260] In some embodiments, the feed additive is solid (i.e., "solid feed additive") or liquid (i.e., "liquid feed additive"). In some embodiments, the feed additive is semi-solid or gel (i.e., "semi-solid or gel feed additive"). The gel feed additive comprises an oxygen scavenger (e.g., ascorbic acid).

[0261] In some embodiments, the solid feed additive is a powder (e.g., a flowable powder), fine granules (i.e., pellets), particles (i.e., microparticles), pellets, cakes, water-soluble concentrates, pastes, boluses, tablets, dusts, these components, or combinations thereof.

[0262] In some embodiments, the liquid feed additive is a solution (e.g., an aqueous solution, an organic solution, or a water-soluble organic solution), a suspension, an emulsion, a drench, a spray, an injection, a beverage (e.g., a milk substitute), these components, or combinations thereof.

[0263] In some embodiments, the gel feed additive is an organogel. In some embodiments, the gel feed additive is an oral gel (i.e., a gel for oral administration).

[0264] In some embodiments, the feed additive is for use as a top dress (i.e., for adding to or mixing with the surface of food (e.g., animal feed)). In some embodiments, the feed additive is for administration as a liquid.

[0265] In some embodiments, the electrospray-dried anaerobic cells (e.g., Bifidobacterium cells, such as B. breve, Lactobacillus cells, such as L. plantarum, Bifidobacterium cells, such as B. animalis subsp. lactis, Pediococcus cells, such as P. acidilactici, Lactobacillus cells, such as L. casei, Fibrobacter, such as F. succinogenes, Ruminococcus, such as R. flavefaciens, and Butyrivibrio, such as B. fibrisolvens) and / or M. elsdenii cells disclosed herein can be used as a liquid feed additive by rehydrating, dissolving, solubilizing, and / or suspending the cells in a liquid.

[0266] In one aspect, the feed additive includes a feed additive carrier (i.e., one or more feed additive carriers).

[0267] Examples of suitable feed additive carriers include plant materials (i.e., whole plants or parts of plants (e.g., by way of example, seeds, stems, leaves, flowers, and / or roots), dried or processed plants or parts of plants), dried grains (e.g., distillers' dried grains), alfalfa, corn meal, citrus meal, fermentation residues, ground oyster shells, attapulgite clay, wheat short, molasses solution, corn cob meal, edible plant substances, toasted defatted soybean meal, soybean mill feed, antibiotic mycelis, vermiculite, soybean grits, whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice hulls, yeast cultures, dried starch, sodium aluminosilicate, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, essential oils, fatty acid monoglycerides and diglycerides, petroethyl fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc., as well as combinations thereof, but are not limited thereto.

[0268] In some embodiments, the feed additive comprises excipients (i.e., one or more excipients), including but not limited to microcrystalline cellulose; lactose; sodium citrate; calcium carbonate; dibasic calcium phosphate and glycine; disintegrants such as starch, sodium starch glycolate, croscarmellose sodium, and certain complex silicates; granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia; bulking agents such as maltodextrin; moisture scavengers such as silicon dioxide; oxygen scavengers such as ascorbic acid; and / or lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc.

[0269] In some embodiments, the feed additive is a granule comprising anaerobic cells and / or M. elsdenii cells and / or a core comprising the feed additive, and a coating on the core. In some embodiments, the coating is a hydrated barrier salt. The salt coating can provide improved heat tolerance, improved storage stability, and protection against other components within the granule that may otherwise have a deleterious effect (e.g., on stability) on the M. elsdenii cells and / or the feed additive.

[0270] In some embodiments, electrospray-dried anaerobic cells and / or M. elsdenii cells are mixed with a dry formulation of additives including, but not limited to, growth substrates, enzymes, sugars, carbohydrates, extracts, and growth-promoting micronutrients. Sugars can include, but are not limited to, lactose, maltose, dextrose, maltodextrin, sucrose, glucose, fructose, mannose, tagatose, sorbose, raffinose, amylose, starch, and galactose. The sugars can range from 50 to 95% either individually or in combination. Extracts can include, but are not limited to, yeast or dried yeast fermentation lysates in the range of 5 to 50%. In some embodiments, the extract includes a concentrated fermented corn extract component or corn steep liquor. Growth substrates can include, but are 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 can include, but are not limited to, mannitol, sorbitol, adonitol, and arabitol. The carbohydrates can range from 5 to 50% either individually or in combination. Micronutrients can 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 0.5 to 5.0%, manganese protein compound in the range of 0.25 to 1.00%, and manganese in the range of 0.25 to 1.00%.

[0271] In some embodiments, the anaerobic cell and / or M. elsdenii feed additive is prepared by mixing anaerobic cells and / or M. elsdenii cells, including a culture containing the cells and / or electrospray-dried cells (e.g., in a mixer), with any additional components of the feed additive, such as carriers and / or excipients. In some embodiments, the components are mixed to obtain a homogeneous mixture.

[0272] In some embodiments, the feed additive is a top-dress animal feed additive comprising the anaerobic cells and / or M. elsdenii cells (e.g., electrospray-dried cells) 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 hulls, yeast culture, dried starch, and sodium aluminosilicate, milk, water, and combinations thereof.

[0273] In some embodiments, the animal feed additive is a drench, spray, or supplement for milk substitutes comprising the anaerobic cells and / or M. elsdenii cells (e.g., electrospray-dried cells) disclosed herein and a water-soluble carrier. In some embodiments, the carrier is selected from the group consisting of whey, maltodextrin, sucrose, dextrose, dried starch, sodium aluminosilicate, milk, water, and combinations thereof.

[0274] In some embodiments, the present invention is directed to a food (e.g., animal feed) comprising anaerobic cells and / or M. elsdenii cells (e.g., electrospray-dried cells disclosed herein, e.g., electrospray-dried cells produced by the methods disclosed herein) and / or a feed additive disclosed herein. The food is any food for ingestion by an animal (i.e., non-human or human) and includes both solid and liquid compositions. Examples of foods include, but are not limited to, common foods; liquid products including water, milk, beverages, therapeutic beverages, and nutritional supplements; functional foods; supplements; nutraceuticals; formulations for infants (i.e., including non-human and human infants) including formulations for premature infants; foods for animals during pregnancy or lactation; foods for adult animals; and foods for the elderly. In some embodiments, the food comprises a liquid (e.g., a beverage, e.g., water, milk, or a milk substitute) comprising the feed additive.

[0275] In some embodiments, the present disclosure is directed to compositions comprising the anaerobic cells and / or M. elsdenii cells (e.g., electrospray dried cells) disclosed herein and / or feed additives. In some embodiments, the composition comprises electrospray dried anaerobic cells and / or M. elsdenii cells produced by the methods disclosed herein.

[0276] The compositions of the present disclosure may include one or more excipients. In some embodiments, the excipient can be, but is not limited to, an alkali agent, a stabilizer, an antioxidant, an adhesive, a separating agent, a coating agent, an external phase component, a controlled release component, a solvent, a surfactant, a water retention agent, a buffering agent, a filler, a softening agent, or a combination thereof. The excipients can include, but are not limited to, those listed in Remington: The Science and Practice of Pharmacy, 21 st ed. (2005), in addition to those considered herein. The inclusion of an excipient in a particular classification herein (e.g., “solvent”) is intended to be illustrative rather than limiting of the role of the excipient. A particular excipient can be included in multiple classifications.

[0277] In some embodiments, the composition is a pharmaceutical composition (e.g., for the treatment of non-human animals or humans). In some embodiments, the composition is a medical food (e.g., veterinary food). Medical foods include foods included in compositions consumed under the supervision of a physician (e.g., veterinarian) or administered externally, and foods intended for specific dietary management of conditions in which different nutritional requirements have been established by medical evaluation based on recognized scientific principles. In some embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients. In some embodiments, the term “pharmaceutically acceptable” means approved by a federal or state government regulatory authority or listed in the United States Pharmacopeia or other generally recognized international pharmacopeia for use in animals, more specifically in humans.

[0278] Regarding oral administration of the compositions, anaerobic cells and / or M. elsdenii cells (e.g., electrospray dried cells) or feed additives can be combined with excipients well known in the art. Such carriers can enable, for example, formulating the anaerobic cells and / or M. elsdenii cells or feed additives of the present invention into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject to be treated. In some embodiments, the composition is a tablet, pill, caplet, or capsule. Suitable excipients include, but are not limited to, sugars such as fillers, e.g., lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP), but are not limited thereto. If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or salts thereof such as alginic acid or sodium alginate, but are not limited thereto, may be added. Compositions that can be used orally include, but are not limited to, capsules made of gelatin, as well as soft-sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. In some embodiments, the dosage form is a plant-based dosage form in which the dosage form is not formed from an animal source and does not contain any components derived from an animal source. In some embodiments, the plant-based dosage form is a plant-based capsule.

[0279] In some embodiments, the present invention is directed to a kit or package comprising the anaerobic cells and / or M. elsdenii cells, feed additives, food, and / or compositions disclosed herein. The kit or package may include units (e.g., one or more units) of feed additives, food, compositions, or combinations thereof. In some embodiments, the kit includes freeze-dried cells produced by the methods disclosed herein, feed additives disclosed herein, or capsules disclosed herein. Method of administering electrospray-dried anaerobic bacterial cells and / or M. elsdenii to an animal

[0280] In some embodiments, the present invention is directed to a method of administering electrospray-dried M. elsdenii cells to an animal.

[0281] In some embodiments, the present invention is directed to a method of administering electrospray-dried anaerobic bacterial cells to an animal. In some embodiments, the present invention is directed to a method of administering electrospray-dried Bifidobacterium cells, such as B. breve, Lactobacillus cells, such as L. plantarum, Bifidobacterium cells, such as B. animalis subsp. lactis, Pediococcus cells, such as P. acidilactici, Lactobacillus cells, such as L. casei cells, Fibrobacter, such as F. succinogenes cells, Ruminococcus, such as R. flavefaciens, and Butyrivibrio, such as B. fibrisolvens cells to an animal.

[0282] In some embodiments, the method includes administering to the animal electrospray-dried M. elsdenii cells, feed additives, food, or compositions (e.g., capsules) described herein.

[0283] Administration may be by any suitable route, including, for example, oral (i.e., ingestible liquid or solid, oral drench, feed additive, food, composition, or capsule), spraying onto the body (i.e., spraying a mist), and / or injection.

[0284] In some embodiments, the method includes administering a solid, liquid, or gel comprising electrospray dried M. elsdenii or anaerobic cells.

[0285] In some embodiments, the method includes administering a solid feed additive comprising electrospray dried M. elsdenii or anaerobic cells. In some embodiments, the solid feed additive is a powder (e.g., a flowable powder), granule (i.e., pellet), particle (i.e., microparticle), pellet, cake, water-soluble concentrate, paste, bolus, tablet, dust, a combination of these components, or a combination thereof.

[0286] In some embodiments, the method includes administering a liquid feed additive comprising electrospray dried M. elsdenii or anaerobic cells. In some embodiments, the method includes administering electrospray dried M. elsdenii or anaerobic cells in a liquid. In some embodiments, the liquid is a solution (e.g., an aqueous solution, an organic solution, or a water-soluble organic solution), suspension, emulsion, drench, spray, injection, beverage (e.g., a milk substitute), a combination of these components, or a combination thereof. In some embodiments, the liquid is administered orally or by spraying the liquid onto the animal.

[0287] In some embodiments, the method includes combining a feed additive comprising electrospray dried M. elsdenii or anaerobic cells or cells with another animal feed additive to form an adjunct substance or premix for addition to animal feed. In some embodiments, the other feed additive comprises cells other than M. elsdenii.

[0288] In some embodiments, electrospray-dried M. elsdenii or anaerobic cells can be added to a feed additive as a liquid (e.g., in a broth or broth equivalent containing, for example, rehydrated electrospray-dried cells) or as a reconstituted cell paste. Dosage forms (e.g., a predetermined volume of drench or capsule) can also be formed, and if desired, electrospray-dried M. elsdenii or anaerobic cells can be added directly to animal feed, such as by sprinkling the liquid broth and / or electrospray-dried M. elsdenii or anaerobic cells onto the feed or mixing them into the feed.

[0289] In some embodiments, the method includes rehydrating a feed additive (e.g., powder, granule, microparticle, pellet, cake, electrospray-dried cells, or a combination thereof) to produce a liquid for administration.

[0290] In some embodiments, the method includes applying electrospray-dried M. elsdenii or anaerobic cells to animal feed through a delivery system that rehydrates the feed additive, including on a batch basis. For example, electrospray-dried powder is transferred from a polyvinyl hopper to a flushing system where the powder is diluted and then sprayed and mixed into the feed.

[0291] In some embodiments, the method includes applying electrospray-dried M. elsdenii or anaerobic cells to animal feed using a volumetric metering device having a storage bin. For example, electrospray-dried M. elsdenii or anaerobic cells (e.g., powder containing the cells) can be stored in the storage bin and discharged into a water bath or aqueous solution bath immediately before spraying onto the animal feed.

[0292] In one aspect, the present invention is a method for treating or preventing a condition or disorder associated with lactic acid production in the digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii disclosed herein, electrospray-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition (e.g., a capsule) disclosed herein.

[0293] In one aspect, the present invention is a method for treating or preventing a condition or disorder associated with lactic acid production in the digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition (e.g., a capsule) disclosed herein.

[0294] In some aspects, the condition or disorder is acidosis. In some aspects, the condition or disorder is rumen acidosis. In some aspects, the condition or disorder is a respiratory disease. In some aspects, the condition or disorder is laminitis. In some aspects, the condition or disorder is an infectious disease. In some aspects, the infectious disease is caused by Salmonella or Campylobacter. In some aspects, Salmonella is Salmonella enterica and / or Salmonella bongori. In some aspects, the Salmonella serotype is Salmonella Typhimurium and / or Enteritidis. In some aspects, Campylobacter is Campylobacter jejuni or Campylobacter coli. In some aspects, the condition or disorder is hindgut acidosis.

[0295] In some embodiments, the condition or disorder is a condition or disorder in which the levels of butyric acid, valeric acid, and / or propionic acid are increased.

[0296] In some embodiments, the present invention is directed to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells disclosed herein, electrospray-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein.

[0297] In some embodiments, the present invention is directed to a method for preventing or reducing the growth of opportunistic microorganisms in the gastrointestinal tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein.

[0298] In some embodiments, the opportunistic microorganism is pathogenic. In some embodiments, the opportunistic microorganism is Salmonella or Campylobacter. In some embodiments, Salmonella is Salmonella enterica and / or Salmonella bongori. In some embodiments, the Salmonella serotype is Salmonella Typhimurium and / or Enteritidis. In some embodiments, Campylobacter is Campylobacter jejuni or Campylobacter coli. In some embodiments, the opportunistic microorganism is Escherichia coli.

[0299] In some embodiments, the present invention is directed to a method for improving the bioavailability of plant-derived phosphorus in an animal's diet, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells disclosed herein, electrospray-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In some embodiments, the electrospray-dried M. elsdenii cells comprise phytase activity. In some embodiments, the method reduces environmental phosphorus waste generated by administration of the animal's diet in the absence of M. elsdenii cells.

[0300] In some embodiments, the present invention is directed to a method for improving the bioavailability of plant-derived phosphorus in an animal's diet, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In some embodiments, the electrospray-dried anaerobic cells comprise phytase activity. In some embodiments, the method reduces environmental phosphorus waste generated by administration of the animal's diet in the absence of anaerobic cells.

[0301] In some embodiments, the present invention is directed to a method for improving growth ability in an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells disclosed herein, electrospray-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein.

[0302] In one aspect, the present invention is directed to a method of improving growth ability in an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein.

[0303] In one aspect, the present invention is directed to a method of increasing the pH of the hindgut of an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein.

[0304] In one aspect, the improvement in growth ability in an animal is an improvement in feed intake, average daily gain, feed conversion ratio, meat gain, milk production in milk-producing animals, egg production in poultry, bone mineralization, or a combination thereof.

[0305] In one aspect, the present invention is directed to a method of acidifying the lower digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells disclosed herein, electrospray-dried M. elsdenii cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In one aspect, the lower digestive tract is the ceca of a poultry animal.

[0306] In one aspect, the present invention is directed to a method of acidifying the lower digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried anaerobic cells disclosed herein, electrospray-dried anaerobic cells produced by the methods disclosed herein, a feed additive disclosed herein, or a composition disclosed herein. In one aspect, the lower digestive tract is the ceca of a poultry animal.

[0307] In some embodiments, the electrospray-dried M. elsdenii or anaerobic cells disclosed herein, the electrospray-dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein are administered before, simultaneously with, or after feeding the animal.

[0308] In some embodiments, the method further comprises the step of mixing the electrospray-dried M. elsdenii or anaerobic cells disclosed herein, the electrospray-dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, or the solid feed additives disclosed herein with a liquid prior to administration.

[0309] In some embodiments, the liquid is administered orally (e.g., via an oral drench) or by spraying the liquid onto the animal (e.g., by spraying a mist).

[0310] In some embodiments, the method comprises a single administration of the electrospray-dried M. elsdenii or anaerobic cells disclosed herein, the electrospray-dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein.

[0311] In some embodiments, the method includes administering the electrospray dried M. elsdenii or anaerobic cells disclosed herein, the electrospray dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the administering includes sprinkling on an animal (e.g., a bird) the electrospray dried M. elsdenii or anaerobic cells disclosed herein, the electrospray dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the electrospray dried M. elsdenii or anaerobic cells disclosed herein, the electrospray dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein are ingested by the birds by preening.

[0312] In some embodiments, the method includes daily administering the electrospray dried M. elsdenii or anaerobic cells disclosed herein, the electrospray dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the administering is at least once a day, at least twice a day, at least three times a day, or more than three times a day. In some embodiments, the administering is performed freely (e.g., self-administration by drinking an available liquid containing or eating an available food containing the electrospray dried M. elsdenii or anaerobic cells, the electrospray dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additive, or the composition).

[0313] In some embodiments, the method comprises administering more than once a day the electrospray-dried M. elsdenii or anaerobic cells disclosed herein, the electrospray-dried M. elsdenii or anaerobic cells produced by the methods disclosed herein, the feed additives disclosed herein, or the compositions disclosed herein. In some embodiments, the administration is two, three, four, five, six, or more times a day. In some embodiments, the method comprises more than once a day administration followed by no administration for one or more days. In some embodiments, no administration for one or more days is no administration for one, two, three, four, five, or six days, one, two, three, or four weeks, one, two, three, four, five, or six months.

[0314] In some embodiments, the animal is a ruminant. In some embodiments, the ruminant can 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.

[0315] In some embodiments, the animal is a non-ruminant. In some embodiments, the non-ruminant can be, but is not limited to, equines, poultry, pigs, dogs, and cats. In some embodiments, the non-ruminant is selected from the group consisting of equines, poultry, and pigs.

[0316] In some embodiments, the animal is a zoo animal.

[0317] In some embodiments, the animal is a poultry animal. In some embodiments, the poultry animal is a bird (i.e., avian) used as a food animal, including but not limited to chickens, ducks, geese, turkeys, guinea fowl, pigeons, emus, or ostriches. In some embodiments, the poultry animal is selected from the group consisting of chickens, ducks, geese, turkeys, guinea fowl, or pigeons. In some embodiments, the poultry animal is selected from the group consisting of broilers, broiler breeders, and layers. In some embodiments, the poultry animal is a chicken.

[0318] In some embodiments, the animal is a member of the Equidae family. In some embodiments, the member of the Equidae family is a horse, pony, donkey, or mule.

Examples

[0319] Reference is now made to the following examples, which illustrate some embodiments of the invention in a non-limiting manner, together with the above description. (Example 1) Laboratory-scale production of electrospray-dried M. elsdenii

[0320] M. elsdenii NCIMB 41125 cultures were prepared in the laboratory using a laboratory-scale fermenter. Two 10-liter bioreactors of a partially defined medium consisting of two carbon sources were prepared to grow M. elsdenii NCIMB 41125.

[0321] The bioreactors were inoculated with an overnight culture of M. elsdenii NCIMB 41125 at a ratio of 1:100 and incubated at 39 °C for 15 hours under anaerobic conditions using a nitrogen blanket.

[0322] The resulting cultures were cooled to room temperature (approximately 25 °C), packaged in polyfoil bags with 900 mL aliquots (1x cell culture or fermentate), and stored at 4 °C until further processing. The stored cell cultures were used for Feedstock Numbers 1-6, 8, 10, and 11.

[0323] The cells prepared from the above-mentioned culture were placed in centrifuge bottles under anaerobic conditions (900 mL per bottle, a total of 11 bottles), and centrifuged at 5,200 rpm for 10 minutes at 20°C. After centrifugation, the supernatant was removed to obtain a 100-fold concentrate of the cells (9 mL per bottle, a total of 11 bottles). The concentrated cell pellet was resuspended in the supernatant to obtain a 10-fold cell concentration.

[0324] An additional culture bag (approximately 900 mL) was centrifuged under the same conditions. This concentrated cell pellet was resuspended in anaerobic reverse osmosis (RO) water to obtain a 1-fold cell concentration (9 mL of cell concentrate + 891 mL of RO water) for Feedstock No. 7.

[0325] The cell concentrates (10-fold) were combined, stirred well, and packaged in polyfoil bags (900 mL per bag) under sterile and anaerobic conditions. The cell concentrate (1-fold) was packaged in the same manner. Both bags were stored at 4°C until further processing for electrospray drying.

[0326] Prior to electrospray drying the cells to maximize cell viability, a stabilizer solution was added to the cell concentrate under sterile and anaerobic conditions. The stabilizer solution is a carrier solubilized in water, which also functions as a protectant for the cells, and this term is used interchangeably throughout this specification.

[0327] The carrier was left overnight in an anaerobic chamber to minimize the oxygen content and then packaged in a three-necked sterile anaerobic borosilicate glass bottle.

[0328] Sterile anaerobic reverse osmosis water ("anaerobic RO water") was prepared in a three-necked borosilicate glass bottle.

[0329] The final stabilizer solution was prepared by adding RO water to the carbohydrate powder in a borosilicate glass bottle under anaerobic conditions. Different stabilizer solutions and components are listed in Table 2. Table 2 - Components of the stabilizer solution

Table 2

[0330] To prepare different feed stocks (i.e., cell suspensions) for electrospray drying, the cell concentrate (1x or 10x) and the stabilizer solution were mixed at the ratios shown in Table 3. All additions were made under a nitrogen blanket to maintain an oxygen-free environment. Table 3 - Components of the feed stock for electrospray drying

Table 3

[0331] The concentration of M. elsdenii NCIMB 41125 cells was evaluated in liquid cultures (cell cultures, cell concentrates, cell suspensions / feed stocks) and in the dried samples obtained after electrospray drying.

[0332] Liquid samples were obtained at (1) 15 hours of incubation in a 10 L bioreactor (cell culture), (2) after collecting the cells using centrifugation (10x cell concentrate), and (3) after adding the stabilizer solution to the cell concentrate (final feed stock). All treatments were sampled in triplicate under sterile and anaerobic conditions, diluted in a sterile anaerobic diluent, and plated in duplicate on partially defined lactate agar (SDL agar). The agar plates were then incubated at 39 °C for 48 hours, after which the colony-forming units (CFU) were counted and the concentration per milliliter was calculated for each sample.

[0333] The dried sample obtained after electrospray drying was weighed, and 0.32 g of the dried sample was resuspended in 40 mL of sterile anaerobic diluent. The sample was rehydrated at room temperature for 2 hours, further diluted, and inoculated onto partially defined lactate agar. The plates were processed as described above to determine the final cell concentration in the dried sample.

[0334] The cell recovery rate after electrospray drying was calculated by dividing the total amount of M. elsdenii cells in the dried sample by the initial total amount of M. elsdenii cells in the sample before electrospray drying. Refer to the following equation.

Equation

[0335] The dry powder of M. elsdenii NCIMB 41125 was prepared using an electrostatic spray dryer. Drying was achieved with a heated nitrogen gas flow rate of 150 Nm 3 / hour and a normal inlet temperature in the range of 50 - 100 °C.

[0336] The feedstock (cell suspension) was maintained at 4 °C by delivering a low-temperature water stream to a jacketed beaker, where the feedstock was maintained and continuously stirred by an overhead stirrer. A nitrogen blanket was constantly flowed into the headspace of the container to maintain an oxygen-free environment.

[0337] The feedstock (cell suspension) was delivered through a peristaltic pump to a two-fluid nozzle at the top of the chamber of the electrostatic spray dryer, where the feedstock was atomized and charged. Atomization was achieved with a heated and pressurized nitrogen gas at 350 kPa and 35 °C.

[0338] Charging was delivered to the electrode in contact with the suspension in front of the nozzle through a voltage generator controlled by a pulse width modulation device. The atomized feedstock was simultaneously dried in the chamber, and the dried powder was collected at the end of the vertical chamber.

[0339] At the end of the electrospray drying process, the dried powder was recovered and stored in a metallized bag or moisture-protective container, with nitrogen flowing through it, and stored at 4 °C for long-term storage. The results of the process are shown in Table 4. Table 4 - Components of Electrospray Drying and Results after Electrospray Drying [Table 4]

[0340] Among the inlet temperatures tested for the electrospray drying process using the same carrier / stabilizer solution (1-fold sucrose), 70 °C resulted in the highest CFU recovery rate of 10.6%. Increases in inlet temperatures of 80 °C and 90 °C decreased the yield to 2.6% each. Additionally, electrospray drying at high temperatures (i.e., 80 °C and 90 °C) did not significantly reduce the moisture level in the resulting powder.

[0341] Among the carrier / stabilizer solutions tested (sucrose, maltodextrin + maltose, hydrophobic starch), sucrose resulted in the highest CFU, and 2-fold sucrose also further improved the CFU recovery rate % at 80 °C. Conducting electrospray drying at an inlet temperature of 70 °C using sucrose and hydrophobic starch reduced the CFU recovery rate %.

[0342] Cell concentration (1-fold or 10-fold) was not seen to affect the CFU recovery rate %. However, the 10-fold cell sample experienced a decrease in CFU during storage before electrospray drying, despite being 10-fold concentrated, resulting in a lower CFU concentration than predicted. (Example 2) Commercial-scale production of electrospray-dried M. elsdenii NCIMB 41125

[0343] Using a 10-liter bioreactor of a partially defined medium consisting of two carbon sources, an inoculum was prepared. The bioreactor was inoculated with an overnight culture of M. elsdenii NCIMB 41125 at a ratio of 1:100 and incubated at 39 °C for 14 hours. The resulting culture was cooled to room temperature and packaged into two 5L polyfoil bags.

[0344] Using one production tank, 2,100L of a partially defined medium consisting of two carbon sources was prepared for growing M. elsdenii NCIMB 41125 under sterile and anaerobic conditions. One of the 5L cultures of M. elsdenii NCIMB 41125 prepared above was inoculated into the production tank. After inoculation, the production tank was incubated at 39 °C for 14 hours and then cooled to room temperature. A 4L aliquot of the cell culture was obtained using sterile anaerobic techniques and the glucose level, OD, pH, contaminant levels (aerobic growth, yeast, and mold), M. elsdenii concentration, and specific gravity were evaluated.

[0345] The contents of the production tank (cell culture) were collected using tangential flow filtration (TFF). Briefly, the cell culture was circulated through the TFF system and the supernatant was discarded until only 21L of the retentate remained in the tank. At this point, the retentate was washed out and resuspended using 63L of RO water (prepared under anaerobic and sterile conditions) to a final retentate concentration of 25 times. The final retentate (84L) was packaged into 5L polyfoil bags under sterile and anaerobic conditions and stored at 4 °C until electrospray drying.

[0346] To prepare the stabilizer solution (carrier), sucrose powder was placed in an anaerobic chamber overnight to minimize the oxygen content and then packaged into a three-necked sterile and anaerobic borosilicate bottle. Sterile and anaerobic RO water was prepared in the three-necked borosilicate bottle. The stabilizer solution (sucrose solution) was prepared by adding RO water to the sucrose powder through the three necks under the anaerobic conditions shown in Table 5. Table 5 - Components of the stabilizer solution [Table 5]

[0347] By mixing the 25-fold cell concentrate (retentate) obtained after TFF and the stabilizer solution, different feed stocks (cell suspensions) for electrospray drying were prepared. All steps were carried out under a nitrogen blanket to maintain an oxygen-free environment at all times. The final sucrose concentration and cell concentration in the feed stock were the same for each sample (i.e., 62.5 mg / mL and approximately 22.5-fold, respectively, compared to the initial culture).

[0348] The concentration of M. elsdenii NCIMB 41125 cells was evaluated in liquid cultures (cell cultures, cell concentrates / retentates, cell suspensions / feed stocks), as well as in the dried samples obtained after electrospray drying.

[0349] Liquid samples were obtained (1) after 17 hours of incubation in the production tank (cell culture), (2) after collecting cells using a TFF system (25-fold cell concentrate / retentate), and (3) after adding the stabilizer solution (sucrose solution) to the cell concentrate (final feed stock, 22.5-fold). All treatments were sampled in triplicate under sterile and anaerobic conditions, diluted in a sterile anaerobic diluent, and seeded in duplicate on partially defined lactate agar (SDL agar). The agar plates were then incubated at 39 °C for 48 hours, after which the colony-forming units (CFUs) were counted and the concentration per milliliter was calculated for the samples.

[0350] The dried sample obtained after electrospray drying was weighed, and 0.3 g of the dried sample was resuspended in 60 mL of sterile anaerobic diluent or sterile anaerobic diluent supplemented with 10 g / L yeast and 5 g / L soybean peptone. The sample was rehydrated at room temperature for 2 hours and then further diluted in anaerobic diluent (with or without supplements) and seeded overnight on SDL agar or concentrated SDL agar supplemented with 1 mL of anaerobic diluent. The plates were processed as described above to determine the final cell concentration in the dried sample.

[0351] The cell recovery rate after electrospray drying was calculated by dividing the total amount of M. elsdenii in the dried sample by the initial total amount of M. elsdenii in the final feedstock sample (before electrospray drying).

Number

[0352] The dry powder of M. elsdenii NCIMB 41125 was prepared using an electrostatic spray dryer. Drying was achieved by heated nitrogen gas at a flow rate of 150 Nm 3 / h and a normal inlet temperature in the range of 50 - 100 °C.

[0353] The feedstock (cell suspension) was maintained at 4 °C by delivering cold water flow to a jacketed beaker, where the feedstock was maintained and continuously stirred by an overhead stirrer. A nitrogen blanket was constantly flowed into the headspace of the container to maintain an oxygen-free environment.

[0354] The feedstock (cell suspension) was delivered through a peristaltic pump to a two-fluid nozzle at the top of the chamber of the electrostatic spray dryer, where the feedstock was atomized and charged. Atomization was achieved by heated and pressurized nitrogen gas at 350 kPa and 35 °C.

[0355] Charging was delivered to the electrode in contact with the suspension in front of the nozzle through a voltage generator controlled by a pulse width modulation device. The atomized feedstock was simultaneously dried in the chamber, and the dried powder was collected at the end of the vertical chamber.

[0356] At the end of the electrospray drying process, the dried powder was collected, stored in a metal-treated bag or moisture-protected container, purged with nitrogen, and stored at 4 °C for long-term storage. The results of the process are shown in Table 6. Table 6 - Components of Electrospray Drying and Results after Electrospray Drying

Table 6

[0357] The results show that a CFU recovery rate % equivalent to Example 1 was obtained by electrospraying a 21.6-fold concentrated feedstock using a 1-fold sucrose solution at 70 °C. The results show that a more concentrated M. elsdenii feedstock can be electrospray dried without affecting the CFU recovery rate %. However, there was no improvement in the recovery rate by reducing the temperature to 65 °C and 62.5 °C, respectively. (Example 3) Conventional spray drying will result in a reduction in the recovery rate of viable M. elsdenii cells compared to electrospray drying.

[0358] M. elsdenii was cultured and processed for conventional spray drying as described in Example 2.

[0359] Using a conventional spray dryer, dried M. elsdenii powder was produced using sucrose, maltose, maltodextrin, sugar alcohol, or a starch mixture as a stabilizer under the following spray conditions (Table 7). Table 7 - Characteristics of the Electrospray Drying Process

Table 7

[0360] Conventional spray drying processes are expected to yield a sufficiently dried powder (water activity ≤ 0.3) at an inlet temperature higher than 120 °C while spray drying anaerobically with nitrogen. The CFU recovery rate % is expected to be less than 1%. If the same process is carried out using air as the drying gas, the CFU recovery rate % is expected to be less than 0.1%. In contrast, electrospray drying is expected to yield a 10 - 100 times higher CFU recovery rate %. (Example 4) Electrospray drying results in an increase in viable M. elsdenii and the production of dried powder compared to freeze drying.

[0361] M. elsdenii NCIMB 41125 cells were grown in a semi - defined medium consisting of two carbon sources in a 500 - liter tank under sterile and anaerobic conditions as described in Example 13 of International Publication No. WO 2018 / 144653 A1, which is incorporated herein by reference.

[0362] At the end of the cell growth phase, the cell culture was concentrated using a tangential flow filtration system (TFF) to remove 90% of the liquid (permeate) and recover a 10 - fold retentate.

[0363] The retentate was then mixed with 8% trehalose / 15% skim milk (T / SM) or 8% maltodextrin / 15% skim milk (M / SM). The retentate mixed with the appropriate cryoprotectant was sampled to determine the M. elsdenii concentration (i.e., viability count) before freeze - drying. The mixture was dispensed into vials (4 mL / vial), frozen at - 80 °C or in liquid nitrogen, and freeze - dried using a rapid cycle.

[0364] After the lyophilization process was completed, the viability of the bacteria was determined by resuspending the lyophilized product in an anaerobic chamber with an anaerobic diluent, rehydrating it at room temperature for 40 minutes, and then plating it on SDL20 agar.

[0365] Retention fluid samples were tested for cell viability during storage at 4 °C or 25 °C for 0, 2, 4, 8, 12, 16, 20, and 24 weeks under aerobic or anaerobic conditions using the spread plating technique. Briefly, stored products were sampled, serially diluted, and plated on SDL agar plates. The experiments were repeated on three different days, and all treatments were performed in triplicate.

[0366] The cell recovery rate was calculated by dividing the concentration of M. elsdenii cells recovered after lyophilization by the initial concentration of M. elsdenii cells measured in the corresponding retention fluid with or without cryoprotectant.

[0367] The results show that the recovery rate of M. elsdenii cells after lyophilization without cryoprotectant (control) was almost zero (Table 8). Samples flash-frozen in liquid nitrogen had higher recovery rates than their counterparts frozen at -80 °C, regardless of the use of cryoprotectant. The best recovery rates were observed for M / SM and T / SM frozen in liquid nitrogen, 5.82% and 3.96%, respectively. These recovery rates were still lower than those observed for electrospray drying performed at an inlet temperature of 70 °C in Examples 1 and 2. Table 8 - Recovery rates of M. elsdenii after lyophilization using various cryoprotectants and freezing temperatures

Table 8

[0368] In addition, electrospray drying enables the production of dried powders containing M. elsdenii cells that exceed lyophilization.

[0369] Assuming that the inlet feedstock stream has 10% solids, electrospray drying at a drying capacity / evaporation rate of 4 - 32 kg / hour can produce dried powder in the range of 16 - 384 kg (Figure 1). In contrast, freeze-drying equipment operates in a batch mode, and a freeze-dryer with a drying capacity / evaporation rate of 4 - 32 kg / hour produces 4 - 8 kg of dried powder in a 5-day week. This represents a 4 - 40-fold difference in dried powder productivity, indicating another advantage of using electrospray drying. (Example 5) Electrospray drying using a voltage gradient or oscillating voltage pattern will result in an increase in the recovery rate of viable M. elsdenii cells and an increase in shelf life.

[0370] Culture and process M. elsdenii cells for electrospray drying as described in Example 2, except that the voltage for charging the particles is set to (1) a gradually increasing voltage gradient from 1 kV (at the start of electrospray drying) to 15 kV (at the end of electrospray drying), or (2) an oscillating voltage of 1 - 15 kV within 0 minutes during electrospray drying. See Tables 9 and 10 respectively. The voltage gradient and / or oscillating voltage are predicted to increase the recovery rate of viable cells and the shelf life of the dried powder of M. elsdenii cells. Table 9 - Gradient Voltages for Electrospray Drying

Table 9

Table 10

[0371] M. elsdenii cells are cultured and processed for electrospray drying as described in Example 2, except that during the culturing process, at mid-logarithmic growth phase or later, the temperature is increased to 50 - 60 °C for several minutes to several hours and then cooled to ambient temperature. A temporary increase in temperature during culturing is expected to cause the production of stress proteins in the cells, thereby increasing the recovery rate of viable cells and the shelf life of the dried powder of M. elsdenii cells.

[0372] M. elsdenii cells are cultured and processed for electrospray drying as described in Example 2, except that during the culturing process, at mid-logarithmic growth phase or later, the pH is increased above 7.5 or decreased below 4 for several minutes to several hours and then returned to the original pH set point. A temporary increase or decrease in pH during culturing is expected to cause the production of stress proteins in the cells, thereby increasing the recovery rate of viable cells and the shelf life of the dried powder of M. elsdenii cells.

[0373] M. elsdenii cells are cultured and processed for electrospray drying as described in Example 2, except that during the culturing process, at mid-logarithmic growth phase or later, the weight osmolarity is increased by adding salts to induce osmotic stress for several minutes to several hours and then the additional salts are removed to return the weight osmolarity to the original set point. A temporary increase in weight osmolarity during culturing is expected to cause the production of stress proteins in the cells, thereby increasing the recovery rate of viable cells and the shelf life of the dried powder of M. elsdenii cells. (Example 7) Multiple M. elsdenii strains can be preserved by electrospray drying

[0374] The following experiments are conducted to determine the effects of various electrospray drying protocols on different M. elsdenii strains.

[0375] The following M. elsdenii strains were used in this experiment: (1) M. elsdenii NCIMB 41125, (2) M. elsdenii ATCC 25940, (3) M. elsdenii NCIMB 702261, (4) M. elsdenii NCIMB 702262, and (5) M. elsdenii NCIMB 702410.

[0376] The methods used in this experiment are the same as those described in Examples 1 and 2, except as shown below. It is predicted that similar results will be obtained for the amount of viable microorganisms recovered in the dried product after spray drying by electrospray drying of the M. elsdenii strains, compared to NCIMB 41125 (Examples 1 and 2). In addition, the electrospray drying process will have no effect on the viability of the microorganisms or on the growth ability of the microorganisms after drying.

[0377] Similar to M. elsdenii NCIMB 41125, among the inlet temperatures (i.e., 70 °C, 80 °C, and 90 °C) tested using the same carrier (sucrose), 70 °C is predicted to result in the highest cell recovery rate for all other M. elsdenii strains. At increased inlet temperatures of 80 °C and 90 °C, the cell yield decreases and it is not seen to significantly reduce the moisture level of the resulting powder. It is also predicted that reducing the temperature further to 65 °C and 62.5 °C will not improve the cell recovery rate.

[0378] Similar to M. elsdenii NCIMB 41125, among the carriers tested (i.e., sucrose, maltodextrin + maltose, and hydrophobic starch), sucrose is predicted to result in the highest cell recovery rate, and an increase in the sucrose concentration (50 - 150 g / L before electrospray drying) will also further improve the recovery percentage.

[0379] Similar to M. elsdenii NCIMB 41125, it is predicted that the feedstock cell concentration (1-fold or 10-fold) will not affect the cell recovery rate after electrospray drying. Similarly, it is predicted that feedstock cell concentrations of 22.5-fold and 1-fold will result in equivalent cell recovery rates that produce more concentrated products without affecting cell recovery efficiency. (Example 8) Anaerobic bacterial cells can be preserved by electrospray drying

[0380] The following examples are conducted to determine the effect of various electrospray drying protocols on different types of anaerobic bacteria.

[0381] Anaerobic bacteria can be classified into three categories: (1) obligate anaerobes, (2) aerotolerant anaerobes, and (3) facultative anaerobes. Obligate anaerobes are bacteria that do not survive at normal ambient oxygen concentrations. Some obligate anaerobes can survive under up to 8% oxygen, while others cannot survive unless the oxygen concentration is less than 0.5%. Aerotolerant anaerobes can survive in the presence of oxygen but do not utilize oxygen for growth. Facultative anaerobes can use oxygen for aerobic respiration but can also use anaerobic respiration when oxygen is absent.

[0382] Megasphaera, such as M. elsdenii, Fibrobacter, such as F. succinogenes, Butyrivibrio, such as B. fibrisolvens, and Bifidobacterium, such as B. breve are representative species of obligate anaerobes. Lactobacillus, such as L. plantarum and Bifidobacterium, such as B. animalis subsp. lactis are representative species of aerotolerant anaerobes. Pediococcus, such as P. acidilactici and Lactobacillus, such as L. casei are representative species of facultative anaerobes.

[0383] The method used in this experiment is the same as that described in Examples 1 and 2, except that media and growth conditions appropriate to allow optimal microbial cell growth are used. It is predicted that similar results to those obtained with M. elsdenii will be obtained with respect to the amount of viable cells recovered in the dried product after electrospray drying of anaerobic bacteria (i.e., Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, Ruminococcus flavefaciens, and Lactobacillus casei). In addition, the electrospray drying process will have no effect on the viability of the microorganisms nor on the growth ability of the microorganisms after drying.

[0384] Similar to all M. elsdenii strains, among the inlet temperatures tested (i.e., 70 °C, 80 °C, and 90 °C) using the same carrier (sucrose), 70 °C is predicted to result in the highest cell recovery rate for all other anaerobic bacteria. At increased inlet temperatures of 80 °C and 90 °C, the cell yield decreases and is not seen to significantly reduce the moisture level of the resulting powder. It is also predicted that decreasing the temperature further to 65 °C and 62.5 °C will not improve the cell recovery rate.

[0385] Similar to all M. elsdenii strains, among the carriers tested (i.e., sucrose, maltodextrin + maltose, and hydrophobic starch), sucrose is predicted to result in the highest cell recovery rate, and an increase in the concentration of sucrose will also further improve the recovery percentage.

[0386] Similar to all M. elsdenii strains, it is predicted that the feedstock cell concentration (1-fold or 10-fold) will not affect the cell recovery rate after electrospray drying. Similarly, it is predicted that feedstock cell concentrations of 22.5-fold and 1-fold will result in equivalent cell recovery rates that produce more concentrated products without affecting cell recovery efficiency. (Example 9) The voltage gradient during electrospray drying affects the M. elsdenii cell recovery rate

[0387] Applying voltage during spray drying is important for achieving effective spray drying at high temperatures in electrostatic spray drying. Conventionally, ESD is performed by maintaining the voltage at a constant level. In this example, ESD is performed by pulsating the voltage, demonstrating the feasibility of achieving effective drying and CFU recovery rate simultaneously.

[0388] Procedure: M. elsdenii NCIMB 41125 cells were grown in a 500-liter tank under sterile and anaerobic conditions in a partially defined medium consisting of two carbon sources as described in Example 13 of International Publication No. WO 2018 / 144653 A1.

[0389] At the end of the growth phase, the cell culture was concentrated using a tangential flow filtration system (TFF) to remove 99% of the liquid (permeate) and recover a 100-fold retentate. A stabilizer (sucrose) and a YEP solution (a sterile anaerobic diluent supplemented with 10 g / L of yeast and 5 g / L of soybean peptone) were added to the 100-fold retentate to achieve a 20-fold final cell concentration and the final additive concentrations listed in Table 11. Table 11 - Final Concentrations of Stabilizer Solutions

Table 11

[0390] The feedstock was electrospray dried in the ESD unit (PolarDry 001) in four runs using the following parameters. Table 12 - Characteristics of the electrospray drying process

Table 12

[0391] The electrospray dried powder was collected at the end of the run and the water activity, moisture content, viability (CFU / g), and intact cells / g were determined.

[0392] CFU analysis: The dried samples obtained after ESD were weighed and 0.3 g was resuspended in 60 mL of sterile anaerobic diluent or sterile anaerobic diluent supplemented with 10 g / L yeast and 5 g / L soybean peptone. The samples were left to rehydrate for 2 h at room temperature and then further diluted in anaerobic diluent (with or without supplement) and plated onto SDL agar or enriched SDL agar supplemented with 1 mL of anaerobic diluent overnight. The plates were processed as described above to determine the final cell concentration in the dried samples.

[0393] The cell recovery rate after ESD was calculated by dividing the total amount of M. elsdenii in the dried sample by the initial total amount of M. elsdenii in the final feedstock sample (before ESD).

Number

[0394] Analysis of intact cells: Intact cells (cells with an intact lipid membrane) were measured using a BactoBox® (SBT Instruments), a portable flow cytometer.

[0395] The rehydrated samples obtained from the CFU analysis were serially diluted (1:9 ratio) in PBS buffer to meet the target total particle / mL concentration of the Bactobox (total particles / mL from 10,000 to 5,000,000). The samples were then analyzed in the unit and the results were adjusted with respect to the dilution factor and sample weight.

[0396] The recovery rate of intact cells after ESD was calculated by dividing the amount of intact total M. elsdenii cells in the dry sample by the initial amount of intact total M. elsdenii cells in the final feedstock sample (before ESD).

Number

[0397] Table 13 provides the results obtained from the ESD protocol. Table 13 - Results from the ESD protocol

Table 13

[0398] In this example, we successfully demonstrated the effect of voltage pulsation on achieving ESD at low moisture content and water activity. Microbial powders with low moisture content generally correlate with long shelf lives because the dry material tends to be more stable. The recovery rate of intact cells is improved by voltage pulsation, while the CFU recovery rate is generally low. (Example 10) Electrospray drying using a complex nitrogen source as a stabilizer

[0399] In this example, we show the effect of utilizing rich complex nutrients as stabilizers to improve the CFU recovery rate and viability.

[0400] Procedure: M. elsdenii NCIMB 41125 cells were grown in a partially defined medium consisting of two carbon sources in a 500-liter tank at 39 °C under sterile and anaerobic conditions.

[0401] At the end of the growth phase, the cell culture was concentrated using a tangential flow filtration system (TFF) to remove 99% of the liquid (permeate) and recover a 100-fold retentate. Stabilizers and reverse osmosis (RO) water (with or without complex nitrogen source) were added to the 100-fold retentate to achieve a final cell concentration of 20-fold and the final additive concentrations listed in Table 14. Table 14 - Final Additive Concentrations in the Feedstock

Table 14

[0402] The feedstock was electrospray dried in an ESD unit (PolarDry 001) using the following parameters. Table 15 - ESD Parameters

Table 15

[0403] The electrospray dried powders were recovered at the end of each run, and the water activity, moisture content, viability (CFU / g), and intact cells / g were determined as described in Example 9.

[0404] Table 16 provides the results from the parameters measured from the ESD protocol. Table 16 - Results from the ESD Protocol

Table 16

[0405] These results demonstrate the feasibility and advantages of using a complex nitrogen source as a stabilizer during ESD to achieve high CFU and intact cell recovery rates. (Example 11) Accelerated and Short-Term Shelf Life Studies

[0406] This example shows the relative stability of dried M. elsdenii 41125 obtained from both the ESD and freeze-drying (FD) processes.

[0407] Procedure: The ESD powder obtained from Example 1 was packaged and stored in a metal-treated bag with nitrogen gas flowing through the headspace. The bag was heat-sealed and stored at -20 °C, 4 °C, or 37 °C. Concentrated cells (20-fold, from the same fermentation batch as in Example 1) solubilized in RO water with sucrose (50 g / L) used as a stabilizer were freeze-dried. The resulting freeze-dried powder was packaged in the same manner as the ESD powder and stored at 37 °C or 4 °C.

[0408] Cell viability was periodically analyzed according to the CFU analysis procedure described in Example 11 to measure the stability profiles at different temperatures. A storage temperature of 37 °C was used to mimic long-term storage effects and accelerate product degradation without inducing new changes (accelerated shelf life).

[0409] Results: Figure shows that both ESD and freeze-drying resulted in materials with equivalent stability profiles under accelerated high-temperature exposure. The freeze-dried powder had a higher initial CFU / g due to the lower filling amount of the solid excipient (125 g / L of sucrose vs. 50 g / L of sucrose). The decrease in viability of both the ESD and FD powders was at a similar rate. The stability is relatively comparable between the two drying techniques, but ESD offers a high throughput capacity due to its continuous production mode compared to the batch mode (freeze-drying equipment).

[0410] Figure 3 shows that both the ESD and FD powders showed an equivalent decrease in viability at 4 °C. Additionally, the ESD powder stored at -20 °C was not different from its counterpart stored at 4 °C. Storage of the ESD powder at 30 °C showed a decrease in viability, particularly after 30 days, compared to its counterparts stored at -20 °C or 4 °C. (Example 12) Extension of the Shelf Life of Freeze-Dried M. elsdenii Produced at Pilot Scale

[0411] In this example, the relative stability of freeze-dried M. elsdenii 41125 over a long storage period is shown.

[0412] Procedure: M. elsdenii NCIMB 41125 cells were grown in a partially defined medium consisting of two carbon sources under sterile and anaerobic conditions as described in Example 15 of International Publication No. WO 2018 / 144653 A1, freeze-dried, and this document is incorporated herein by reference in its entirety.

[0413] After freeze-drying was completed, the freeze-dried powder was recovered and mixed with maltodextrin as a bulking agent (0.16 g of freeze-dried powder + 2.34 g of maltodextrin). The resulting matrix was then filled into gelatin capsules and placed in a Mylar bag while maintaining anaerobic conditions.

[0414] Twenty-seven capsules were randomly selected from the beginning, middle, and end of the production run (81 capsules in total). The experiment was repeated 5 times for a total of 405 capsules (5 production batches). The M. elsdenii concentration in the capsules was determined by resuspending the freeze-dried product in an anaerobic chamber with 40 mL of anaerobic diluent, rehydrating at room temperature for 40 minutes, and then plating on partially defined lactate agar (i.e., viability counting). The M. elsdenii concentration was expressed as CFU / mL and log-transformed.

[0415] Metal-treated bags containing M. elsdenii capsules were stored at 4°C. Samples were obtained after storage for 0 months, 0.5 months, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months, processed in the same manner as described above (3 samples per treatment per time point, 405 samples in total), and the shelf life of M. elsdenii was determined.

[0416] Figure 4 shows that the M. elsdenii concentration was stable over time, with a logarithmic difference of 0.02 between the sample taken on day 0 and the sample taken after 24 months. (Example 13) Prolonging the Shelf Life of M. elsdenii Produced Using Different Inlet Temperatures during ESD

[0417] This example shows the relative stability of M. elsdenii NCIMB 41125 in ESD powder with respect to long-term storage, regardless of the inlet temperature used during processing.

[0418] Procedure: The M. elsdenii NCIMB 41125 feedstock (cell suspension) was prepared on a commercial scale by mixing the 25-fold cell concentrate (retentate) obtained after TFF, as highlighted in Example 2, with a 2-fold sucrose solution (stabilizer) to obtain a final sucrose concentration of 125 mg / mL and a cell concentration approximately 22.5-fold higher compared to the initial culture. The resulting cell suspension was then spray-dried using an electrostatic spray dryer as described in Example 2. Drying was achieved with heated nitrogen at a flow rate of 150 Nm 3 / h and an inlet temperature of 70 °C, 75 °C, or 80 °C.

[0419] Cell viability was analyzed periodically to measure the stability profiles of the ESD powders treated at different inlet temperatures. The procedure for CFU analysis was as described in Example 9, except that 0.3 g aliquots from each treatment were rehydrated with a sterile anaerobic diluent supplemented with 10 g / L yeast and 5 g / L soybean peptone in 30 or 40 mL instead of 60 mL. The final cell concentration was adjusted with respect to the amount of ESD powder and logarithmically transformed. ESD powder samples were tested at 0 months, 1 month, 2 months, 3 months, and 6 months. All samples were stored at -20 °C during the shelf life test.

[0420] Results: Figure 5 shows that all treatments resulted in stable products and accelerated shelf-life results were confirmed when stored at -20°C for up to 6 months, regardless of the inlet temperature used during ESD.

[0421] The concentration of M. elsdenii was stable over time, with log differences of 0.08, 0.27, and 0.45 between the sample collections on day zero and at 6 months for inlet temperatures of 80°C, 75°C, and 70°C, respectively. These results were not different from those observed for the FD M. elsdenii product in Example 3. (Example 14) ESD using encapsulated polymer

[0422] This example demonstrates the feasibility of performing spray drying and encapsulation of anaerobic bacteria in one step, while simultaneously achieving relatively high CFU and intact cell recovery rates.

[0423] Procedure: M. elsdenii NCIMB 41125 cells were grown in a semi-defined medium consisting of two carbon sources in a 500-liter tank at 39°C under sterile and anaerobic conditions.

[0424] At the end of the growth phase, the cell culture was concentrated using a tangential flow filtration system (TFF) to remove 99% of the liquid (permeate) and recover a 100-fold retentate. A stabilizer and an encapsulating polymer (alginate or gum arabic), as well as RO water, were added to the 100-fold retentate to achieve a 20-fold final cell concentration and the final additive concentrations listed in Table 17. Table 17 - Final additive concentrations in the feedstock

Table 17

[0425] The feedstock was electrospray dried in an ESD unit (PolarDry 001) using the parameters in Table 18. Table 18 - ESD Parameters [Table 18]

[0426] Electrospray-dried powder was recovered at the end of the run and the water activity, moisture content, viability (CFU / g), and intact cells / g were determined.

[0427] Cell viability was periodically analyzed according to the CFU analysis and intact cell analysis procedures described in Example 9 to measure the stability profiles at different temperatures.

[0428] Table 19 provides results from parameters measured from the ESD protocol. Table 19 - Results from the ESD Protocol [Table 19]

[0429] These results indicate the feasibility of encapsulating microorganisms within a soluble excipient with an encapsulating polymer in one step during ESD and maintaining high CFU and intact cell recovery rates. (Example 15) ESD Using a Mixture of Sugars and Sugar Alcohols

[0430] Sugar alcohols, such as sorbitol, xylitol, mannitol, are known as osmolyte protecting molecules. However, it is unclear whether it is effective as a protecting molecule for drying vegetative cells. This result indicates its feasibility.

[0431] Procedure: M. elsdenii NCIMB 41125 cells were grown in a 500-liter tank at 39 °C under sterile and anaerobic conditions in a partially defined medium consisting of two carbon sources.

[0432] At the end of the growth phase, the cell culture was concentrated using a tangential flow filtration system (TFF) to remove 99% of the liquid (permeate) and recover a 100-fold retentate. A stabilizer (sucrose + sorbitol) and a YEP solution (sterile anaerobic diluent supplemented with 10 g / L yeast and 5 g / L soy peptone) were added to the 100-fold retentate to achieve a 20-fold final cell concentration and the final additive concentrations listed in Table 20. Table 20 - Final Additive Concentrations in the Feedstock

Table 20

[0433] The feedstock was electrospray dried in an ESD unit (PolarDry 001) using the following parameters. Table 21 - ESD Parameters

Table 21

[0434] The electrospray dried powder was recovered at the end of the run and the water activity, moisture content, viability (CFU / g), and intact cells / g were determined. The CFU analysis and the analysis of intact cells were performed as described in Example 9.

[0435] Table 22 provides the results from the parameters measured from the ESD protocol. Table 22 - Results from the ESD Protocol

Table 22

[0436] In this example, it was successfully shown that this formulation can achieve a high CFU recovery rate and a recovery rate of intact cells. (Example 16) ESD of Gram-Positive Obligate Anaerobic Bacteria (Ruminococcus flavefaciens)

[0437] In this example, the applicability of the ESD process to another obligate anaerobe derived from different categories classified by its cell wall characteristics is shown. Ruminococcus flavefaciens is also an obligate anaerobe (similar to M. elsdenii), but it is gram-positive. The effectiveness of the spray drying process is closely related to its cell wall structure and integrity. Therefore, the demonstration of an effective ESD process in both gram-positive and gram-negative microorganisms is an important indicator of its scope of application.

[0438] Procedure: Ruminococcus flavefaciens ATCC 49949 cells were grown in a 1 L pyrex® bottle with a three-neck attachment under sterile and anaerobic conditions in a partially defined medium consisting of two carbon sources. The inoculated medium was incubated at 37 °C for 12 h in an anaerobic chamber.

[0439] At the end of the growth phase, a stabilizer (sucrose) was added to the fermentate to achieve the final additive concentrations listed in Table 23. Table 23 - Final additive concentrations in the feedstock

Table 23

[0440] The feedstock was electrospray dried in an ESD unit (PolarDry 001) using the following parameters. Table 24 - ESD parameters

Table 24

[0441] The electrospray-dried powder was recovered at the end of the run and the water activity, water content, viability (CFU / g), and intact cells / g were determined. CFU analysis and analysis of intact cells were performed according to the procedure described in Example 9.

[0442] Table 25 provides results from parameters measured from the ESD protocol. Table 25 - Results from the ESD protocol

Table

[0443] These results showed a substantially complete 100% CFU recovery rate and intact cell recovery rate by the ESD process for Ruminococcus flavefaciens ATCC 49949 cells. Ruminococcus flavefaciens showed a higher recovery rate compared to M. elsdenii NCIMB 41125. Based on the overall results obtained from both obligate anaerobes (M. elsdenii, a gram-negative microorganism; R. flavefaciens, a gram-positive microorganism), the described ESD process was shown to be effective in stabilizing and recovering viable anaerobic cells with different characteristics at high levels.

Claims

1. A system for spray drying Megasphaera elsdenii cells into a dried powder, the system comprising: a source of water, a carrier, and Megasphaera elsdenii cells; a tank comprising an agitation device, the tank being arranged to receive the water, the carrier, and the Megasphaera elsdenii cells from the source to form a slurry having a viscosity in the range of about 1 cP to about 500 cP; an electrode configured to apply an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; an atomizer; a drying chamber having an inlet end, an outlet end, and an internal volume disposed between the inlet end and the outlet end, the internal volume being configured to hold the slurry and a drying fluid; the drying chamber being configured to dry the slurry; the atomizer being configured to: (i) receive the slurry from the tank; (ii) discharge the slurry into the drying chamber to contact the drying fluid to form the dried powder comprising Megasphaera elsdenii cells; and the drying fluid comprising nitrogen or argon at a temperature between about 50°C and about 100°C, the system being under less than 2% oxygen.

2. A method for electrospray drying Megasphaera elsdenii cells into a dried powder, the method comprising: preparing a culture comprising Megasphaera elsdenii cells, a growth medium, and an osmolyte protecting molecule; collecting the Megasphaera elsdenii cells; forming a slurry comprising water, a carrier, and the Megasphaera elsdenii cells, the carrier having a final concentration of about 1 wt% to about 50 wt%, the slurry having a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber. ​ A step of supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder containing a plurality of dried particles containing the Megasphaera elsdenii cells encapsulated in the carrier, wherein the dried powder contains a water content of less than 15%, and the whole method is carried out under less than 2% oxygen, the method.

3. A method for electrospray drying Megasphaera elsdenii cells into a dried powder, comprising: A step of preparing a culture containing Megasphaera elsdenii cells, a growth medium, and an osmolyte protective molecule, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time, the step; A step of collecting the Megasphaera elsdenii cells; A step of forming a slurry containing water, a carrier, and the Megasphaera elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt%, and the slurry has a viscosity in the range of about 1 cP to about 500 cP, the step; A step of applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; A step of atomizing the slurry to generate a spray of droplets of the slurry; A step of introducing the spray of droplets of the slurry into a drying chamber; A step of supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder containing a plurality of dried particles containing the Megasphaera elsdenii cells encapsulated in the carrier, wherein the dried powder contains a water content of less than 15%, and the whole method is carried out under less than 2% oxygen, the method.

4. A method for electrospray drying Megasphaera elsdenii cells into a dried powder, comprising: A step of preparing a culture containing Megasphaera elsdenii cells and a growth medium; A step of adding an osmolyte protective molecule; A step of collecting the Megasphaera elsdenii cells; Forming a slurry comprising water, a carrier, and said Megasphaera elsdenii cells, wherein said carrier has a final concentration of from about 1 wt% to about 50 wt%, and said slurry has a viscosity in the range of from about 1 cP to about 500 cP; Applying an electrostatic charge to said slurry using a voltage gradient or an oscillating voltage; Atomizing said slurry to produce a spray of droplets of said slurry; Introducing said spray of droplets of said slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50 °C and about 100 °C to said drying chamber to dry said droplets and form said dried powder comprising a plurality of dried particles containing said Megasphaera elsdenii cells encapsulated within said carrier, wherein said dried powder contains a moisture content of less than 15%, and the overall method is carried out under less than 2% oxygen. Claim 5 A method for electrospray drying Megasphaera elsdenii cells into a dried powder, comprising: Preparing a culture comprising Megasphaera elsdenii cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; Adding an osmolyte protecting molecule; Collecting said Megasphaera elsdenii cells; Forming a slurry comprising water, a carrier, and said Megasphaera elsdenii cells, wherein said carrier has a final concentration of from about 1 wt% to about 50 wt%, and said slurry has a viscosity in the range of from about 1 cP to about 500 cP; Applying an electrostatic charge to said slurry using a voltage gradient or an oscillating voltage; Atomizing said slurry to produce a spray of droplets of said slurry; Introducing said spray of droplets of said slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50 °C and about 100 °C to said drying chamber to dry said droplets and form said dried powder comprising a plurality of dried particles containing said Megasphaera elsdenii cells encapsulated within said carrier, wherein said dried powder contains a moisture content of less than 15%, and the overall method is carried out under less than 2% oxygen. Claim 6 A method of electrospray drying Megasphaera elsdenii cells into a dried powder, comprising: preparing a culture comprising Megasphaera elsdenii cells and a growth medium; collecting the Megasphaera elsdenii cells; adding an osmolyte protecting molecule; forming a slurry comprising water, a carrier, and the Megasphaera elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets and form the dried powder comprising a plurality of dried particles containing the Megasphaera elsdenii cells encapsulated within the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is performed under less than 2% oxygen. **Claim 7** A method of electrospray drying Megasphaera elsdenii cells into a dried powder, comprising: preparing a culture comprising Megasphaera elsdenii cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; collecting the Megasphaera elsdenii cells; adding an osmolyte protecting molecule; forming a slurry comprising water, a carrier, and the Megasphaera elsdenii cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; atomizing the slurry to produce a spray of droplets of the slurry; introducing the spray of droplets of the slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder containing a plurality of dried particles containing the Megasphaera elsdenii cells encapsulated in the carrier, wherein the dried powder contains a water content of less than 10%, and the entire method is carried out under less than 2% oxygen. **Claim 8** A method for forming a dried powder containing Megasphaera elsdenii cells encapsulated in a carrier, the method comprising: In a mixing tank containing water, a carrier, and Megasphaera elsdenii cells, forming a slurry while stirring the mixing tank with a stirring device; Applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; Atomizing the slurry in an atomizer, discharging the atomized slurry into a drying chamber to contact with a drying fluid, thereby forming the dried powder containing the Megasphaera elsdenii cells encapsulated in the carrier, wherein the drying fluid contains nitrogen or argon at a temperature between about 50°C and about 100°C, the dried powder contains a water content of less than 15%, and the entire method is carried out under less than 2% oxygen. **Claim 9** The method according to any one of claims 2 to 7, wherein the Megasphaera elsdenii cells are concentrated about 20-fold to about 100-fold before collection. **Claim 10** The method according to any one of claims 2 to 8, further comprising heating the drying fluid to a temperature between about 50°C and about 100°C by applying a voltage between about 0.1 kV and about 45 kV before supplying the drying fluid to the drying chamber. **Claim 11** The method according to any one of claims 2 to 10, further comprising heating the drying fluid to a temperature between about 50°C and about 100°C by applying a voltage between about 10 kV and about 30 kV before supplying the drying fluid to the drying chamber. **Claim 12** The method according to any one of claims 2 to 8, wherein the drying fluid is at a temperature between about 60°C and about 90°C. **Claim 13** The method according to claim 12, further comprising heating the drying fluid to a temperature between about 60°C and about 90°C by applying a voltage between about 10 kV and about 30 kV before supplying the drying fluid to the drying chamber.

14. The method according to any one of claims 10, 11, and 13, wherein the voltage is applied in a constant or pulsed manner.

15. The method according to any one of claims 2 to 14, wherein the carrier is selected from the group consisting of sucrose, sugars, sugar alcohols, sugar derivatives, polysaccharides, encapsulating polymers, or nitrogen sources, and mixtures thereof.

16. The method according to claim 15, wherein the carrier is present in an amount of about 1% to about 40% (weight / volume).

17. The dried powder contains from about 1×10 ３ to about 1×10 １３ CFU / gram of Megasphaera elsdenii cells, the method according to any one of claims 2 to 16.

18. The dried powder contains about 1 × 10 ３ CFU / gram of Megasphaera elsdenii cells, the method according to claim 17.

19. The method according to any one of claims 1 to 18, wherein the volume of the culture is at least 50 liters.

20. A feed additive comprising electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19.

21. The feed additive according to claim 20, further comprising another microorganism.

22. The feed additive according to claim 20 or 21, selected from the group consisting of powders, granules, microparticles, pellets, cakes, or combinations thereof.

23. The feed additive according to any one of claims 20 to 22, which is a probiotic.

24. A composition comprising electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19 or the feed additive according to any one of claims 20 to 23.

25. The composition according to claim 24, which is a capsule.

26. A kit comprising electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19, the feed additive according to any one of claims 20 to 23, or the composition according to claim 24 or 25.

27. A method for treating or preventing a condition or disorder associated with lactic acid production in the digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19, the feed additive according to any one of claims 20 to 23, or the composition according to claim 24 or 25.

28. The method according to claim 27, wherein the condition or disorder is acidosis.

29. The method according to claim 27 or 28, wherein the condition or disorder is rumen acidosis.

30. The method according to claim 27, wherein the condition or disorder is a respiratory disease.

31. The method according to claim 27, wherein the condition or disorder is laminitis.

32. The method according to claim 27, wherein the condition or disorder is an infectious disease.

33. The method according to claim 32, wherein the infectious disease is caused by Salmonella or Campylobacter.

34. A method for preventing or reducing the growth of opportunistic microorganisms in the digestive tract of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19, a feed additive according to any one of claims 20 to 23, or a composition according to claim 24 or 25.

35. The method according to claim 34, wherein the opportunistic microorganism is pathogenic.

36. The method according to claim 34 or 35, wherein the opportunistic microorganism is Salmonella or Campylobacter.

37. A method for improving the bioavailability of plant-derived phosphorus in the diet of an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19, a feed additive according to any one of claims 20 to 23, or a composition according to claim 24 or 25.

38. A method for improving the growth ability in an animal, the method comprising administering to the animal an effective amount of electrospray-dried M. elsdenii cells produced by the method according to any one of claims 1 to 19, a feed additive according to any one of claims 20 to 23, or a composition according to claim 24 or 25, wherein the improved growth ability in the animal is an improvement in feed intake, average daily gain, feed requirement ratio, meat gain, milk production in dairy animals, egg production in poultry, bone mineralization, or a combination thereof.

39. The method according to any one of claims 27 to 38, wherein the electrospray-dried M. elsdenii cells, the feed additive, or the composition is administered before, simultaneously with, or after feeding the animal.

40. The method according to any one of claims 27 to 38, further comprising the step of mixing the electrospray-dried M. elsdenii cells or the feed additive with a liquid before administration.

41. The method according to claim 40, wherein the liquid is administered orally or by spraying the liquid onto the animal.

42. The method according to any one of claims 27 to 41, comprising a single administration of the electrospray-dried M. elsdenii cells, the feed additive, or the composition.

43. The method according to any one of claims 27 to 42, comprising daily administration of the electrospray-dried M. elsdenii cells, the feed additive, or the composition.

44. The method according to any one of claims 27 to 43, comprising administration of the electrospray-dried M. elsdenii cells, the feed additive, or the composition more than once a day.

45. The method according to any one of claims 27 to 44, wherein the animal is a ruminant.

46. The method according to claim 45, wherein the ruminant is selected from the group consisting of beef cattle, sheep, goats, deer, buffalo, and reindeer.

47. The method according to any one of claims 27 to 44, wherein the animal is a non-ruminant.

48. The method according to claim 47, wherein the non-ruminant is selected from the group consisting of equine animals, poultry animals, and pigs.

49. The method according to claim 48, wherein the poultry animal is selected from the group consisting of chickens, ducks, geese, turkeys, guinea fowls, or pigeons.

50. The method according to claim 48 or 49, wherein the poultry animal is selected from the group consisting of broilers, broiler breeders, and layers.

51. The method according to any one of claims 48 to 50, wherein the poultry animal is a chicken.

52. The method according to claim 48, wherein the equine animal is a horse, pony, donkey, or mule.

53. The system according to claim 1, wherein the carrier is selected from the group consisting of sucrose, sugars, sugar alcohols, polysaccharides, encapsulated polymers, sugar derivatives, or nitrogen sources, and mixtures thereof.

54. The dried powder contains from about 1×10 ３ to about 1×10 １３ CFU / gram of Megasphaera elsdenii cells, the system according to claim 1.

55. The dried powder contains about 1 x 10 ３ CFU / gram of Megasphaera elsdenii cells, the system according to claim 1.

56. A dried powder comprising a plurality of dried particles formed by the method according to any one of claims 2 to 19.

57. Megasphaera elsdenii cells encapsulated within a carrier, prepared by the method according to any one of claims 2 to 19.

58. A system for spray drying anaerobic cells into a dried powder, comprising a source of water, a carrier, and anaerobic cells, a tank equipped with a stirring device, arranged to receive the water, the carrier, and the anaerobic cells from the source to form a slurry having a viscosity in the range of about 1 cP to about 500 cP, an electrode configured to apply an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage, an atomizer, a drying chamber having an inlet end, an outlet end, and an internal volume disposed between the inlet end and the outlet end, the internal volume configured to hold the slurry and a drying fluid, the drying chamber being configured to dry the slurry, the atomizer being (i) configured to receive the slurry from the tank, (ii) configured to discharge the slurry into the drying chamber to contact the drying fluid to form the dried powder containing the anaerobic cells, wherein the drying fluid comprises nitrogen or argon at a temperature between about 50 °C and about 100 °C, and the entire system is under less than 2% oxygen.

59. A method for electrospray drying anaerobic cells into a dried powder, comprising preparing a culture comprising anaerobic cells, a growth medium, and an osmolyte protecting molecule, collecting the anaerobic cells, forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP, applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage, atomizing the slurry to produce a spray of droplets of the slurry, introducing the spray of droplets of the slurry into a drying chamber. Supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder comprising a plurality of dried particles containing the anaerobic cells encapsulated in the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is carried out under less than 2% oxygen. [

60. ] A method for electrospray drying anaerobic cells into a dried powder, comprising: Preparing a culture comprising anaerobic cells, a growth medium, and an osmolyte protecting molecule, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; Collecting the anaerobic cells; Forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; Applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; Atomizing the slurry to produce a spray of droplets of the slurry; Introducing the spray of droplets of the slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder comprising a plurality of dried particles containing the anaerobic cells encapsulated in the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is carried out under less than 2% oxygen. [

61. ] A method for electrospray drying anaerobic cells into a dried powder, comprising: Preparing a culture comprising anaerobic cells and a growth medium; Adding an osmolyte protecting molecule; Collecting the anaerobic cells; Forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt% and the slurry has a viscosity in the range of about 1 cP to about 500 cP; Applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; Atomizing the slurry to produce a spray of droplets of the slurry; Introducing the spray of droplets of the slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder comprising a plurality of dried particles containing the anaerobic cells encapsulated in the carrier, wherein the dried powder contains a water content of less than 15%, and the entire method is carried out under less than 2% oxygen. [

62. ] A method for electrospray drying anaerobic cells into a dried powder, comprising: Preparing a culture comprising anaerobic cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time; Adding an osmolyte protecting molecule; Collecting the anaerobic cells; Forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt%, and the slurry has a viscosity in the range of about 1 cP to about 500 cP; Applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; Atomizing the slurry to generate a spray of droplets of the slurry; Introducing the spray of droplets of the slurry into a drying chamber; Supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder comprising a plurality of dried particles containing the anaerobic cells encapsulated in the carrier, wherein the dried powder contains a water content of less than 15%, and the entire method is carried out under less than 2% oxygen. [

63. ] A method for electrospray drying anaerobic cells into a dried powder, comprising: Preparing a culture comprising anaerobic cells and a growth medium; Collecting the anaerobic cells; Adding an osmolyte protecting molecule; Forming a slurry comprising water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt%, and the slurry has a viscosity in the range of about 1 cP to about 500 cP; Applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage; Atomizing the slurry to generate a spray of droplets of the slurry; Introducing the spray of droplets of the slurry into a drying chamber; A step of supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder containing a plurality of dried particles including the anaerobic cells encapsulated in the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is carried out under less than 2% oxygen. [

64. ] A method for electrospray drying anaerobic cells into a dried powder, comprising: A step of preparing a culture containing anaerobic cells and a growth medium, wherein during the culture, the pH, temperature, and / or weight osmolarity are changed for a predetermined time. A step of collecting the anaerobic cells. A step of adding an osmolyte protecting molecule. A step of forming a slurry containing water, a carrier, and the anaerobic cells, wherein the carrier has a final concentration of about 1 wt% to about 50 wt%, and the slurry has a viscosity in the range of about 1 cP to about 500 cP. A step of applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage. A step of atomizing the slurry to generate a spray of droplets of the slurry. A step of introducing the spray of droplets of the slurry into a drying chamber. A step of supplying a drying fluid at a temperature between about 50°C and about 100°C to the drying chamber to dry the droplets, thereby forming the dried powder containing a plurality of dried particles including the anaerobic cells encapsulated in the carrier, wherein the dried powder contains less than 15% moisture content and the entire method is carried out under less than 2% oxygen. [

65. ] A method for forming a dried powder containing anaerobic cells encapsulated in a carrier, the method comprising: A step of forming a slurry in a mixing tank containing water, a carrier, and anaerobic cells, while stirring the mixing tank with a stirring device. A step of applying an electrostatic charge to the slurry using a voltage gradient or an oscillating voltage. A step of atomizing the slurry in an atomizer, discharging the atomized slurry into a drying chamber and contacting it with a drying fluid to form the dried powder containing the anaerobic cells encapsulated in the carrier, wherein the drying fluid contains nitrogen or argon at a temperature between about 50°C and about 100°C, the dried powder contains less than 15% moisture content, and the entire method is carried out under less than 2% oxygen.

66. The method according to any one of claims 59 to 64, wherein the anaerobic cells are concentrated about 20-fold to about 100-fold before collection.

67. The method according to any one of claims 59 to 66, further comprising heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 0.1 kV to about 45 kV before supplying the drying fluid to the drying chamber.

68. The method according to any one of claims 59 to 67, further comprising heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 10 kV to about 30 kV before supplying the drying fluid to the drying chamber.

69. The method according to any one of claims 59 to 65, wherein the drying fluid is at a temperature between about 60°C and about 90°C.

70. The method according to claim 69, further comprising heating the drying fluid to between about 60°C and about 90°C by applying a voltage of about 10 kV to about 30 kV before supplying the drying fluid to the drying chamber.

71. The method according to any one of claims 67, 68, and 70, wherein the voltage is applied continuously or in pulses.

72. The method according to any one of claims 59 to 71, wherein the carrier is selected from the group consisting of sucrose, sugars, sugar alcohols, polysaccharides, encapsulating polymers, sugar derivatives, or nitrogen sources, and mixtures thereof.

73. The dried powder contains anaerobic cells of about 1×10 ３ to about 1×10 １３ CFU / gram, and the method according to any one of claims 59 to 72.

74. The dried powder contains about 1 x 10 ３ CFU / gram of anaerobic cells, the method according to claim 73.

75. The system according to claim 58, wherein the carrier is selected from the group consisting of sucrose, sugars, sugar alcohols, sugar derivatives, polysaccharides, encapsulating polymers, or nitrogen sources, and mixtures thereof.

76. The dried powder contains anaerobic cells of about 1×10 ３ to about 1×10 １３ CFU / gram, and the system according to claim 58.

77. The dried powder contains anaerobic cells of about 1×10 ３ CFU / gram, and the system according to claim 58.

78. A dried powder comprising a plurality of dried particles formed by the method according to any one of claims 59 to 74.

79. Anaerobic cells encapsulated in a carrier, prepared by the method according to any one of claims 59 to 74.

80. A method for improving growth ability in an animal, comprising the step of administering to the animal an effective amount of electrospray-dried anaerobic cells produced by the method according to any one of claims 59 to 74, wherein the improved growth ability in the animal is an improvement in feed intake, average daily gain, feed conversion ratio, meat gain, milk production in dairy animals, egg production in poultry, bone mineralization, or a combination thereof.

81. A method for preventing or reducing the growth of opportunistic microorganisms in the digestive tract of an animal, comprising the step of administering to the animal an effective amount of electrospray-dried anaerobic cells produced by the method according to any one of claims 59 to 74.

82. A composition comprising electrospray-dried anaerobic bacterial cells produced by the method according to any one of claims 59 to 74.

83. The method according to any one of claims 2 to 7, wherein the Megasphaera elsdenii cells are grown at about 30 °C to about 40 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C before collection.

84. The method according to any one of claims 59 to 64, wherein the anaerobic cells are grown at about 30 °C to about 40 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C before collection.

85. The method according to any one of claims 2 to 7, wherein the carrier has a final concentration of about 2 wt% to about 30 wt%.

86. The method according to any one of claims 59 to 64, wherein the carrier has a final concentration of about 2 wt% to about 30 wt%.

87. The method according to any one of claims 1 to 7, 9 to 19, 59 to 64, 66 to 74, and 83 to 86, wherein the drying fluid is selected from the group consisting of nitrogen and argon.

88. The method according to any one of claims 2 to 7, further comprising the step of heating the drying fluid to between about 50 °C and about 100 °C by applying a voltage of about 11 kV to about 25 kV before supplying the drying fluid to the drying chamber.

89. The method according to any one of claims 59 to 64, further comprising heating the drying fluid to between about 50°C and about 100°C by applying a voltage of about 11 kV to about 25 kV before supplying the drying fluid to the drying chamber.

90. The method according to claim 15, wherein the encapsulated polymer is alginate.

91. The method according to claim 72 or 75, wherein the encapsulated polymer is alginate.

92. The method according to any one of claims 1 to 7, 9 to 19, 59 to 64, 66 to 74, and 83 to 86, wherein the slurry is processed at a rate of about 1 L / h to about 10,000 L / h.

Images