RU2799508C2 - Methods for obtaining silated plant materials using megasphaera elsdenii - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for obtaining ensiled plant materials using Megasphaera elsdenii anaerobic bacterium and to thus obtained ensiled plant materials, including introducing an effective amount of M. elsdenii NCIMB 41125 cells into the plant material and ensiling the plant material.

EFFECT: method allows to obtain plant material with improved aerobic stability.

14 cl, 28 dwg, 10 tbl

Description

fField of invention

[0001] The present invention relates to methods for producing ensiled plant materials using the anaerobic bacterium Megasphaera elsdenii. The present invention also relates to ensiled plant materials obtained by these methods.

State of the art

[0002] Ensiling is an enzymatic process used to preserve plant materials (eg, forage and/or grain) consumed by animals such as livestock. The preserved plant material obtained by the ensiling process is referred to as ensiled plant material. Examples of ensiled plant materials include, but are not limited to, ensiled forage, also known as silage, ensiled grain, also known as fermented grain, and combinations of ensiled forage and grain.

[0003] Typically, the plant material is collected and sealed in a silo, which can be any container capable of maintaining an anaerobic environment. First, the amount of atmospheric oxygen trapped in the silo is reduced by the respiration activity of plant material and aerobic microorganisms. Fermentation begins when conditions in the silo become anaerobic. This can be facilitated by methods including compacting the plant material and preventing air from entering the silo.

[0004] The growth of undesirable microorganisms during the ensiling process, such as clostridia, enterobacteria and yeast, can be inhibited by fermentation to form lactic acid associated with lactic acid bacteria present in plant materials. Under favorable ensiling conditions, lactic acid bacteria can oxidize plant materials and prevent competitive growth of unwanted microorganisms. However, if pH is not controlled successfully during the ensiling process, undesirable microorganisms can proliferate and degrade amino acids, resulting in ensiled plant material with lower nutritional value. In addition, further degradation of the ensiled plant material may occur after exposure to air when the silo is opened.

[0005] Thus, there is a need for improved methods for obtaining ensiled plant materials.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a method for producing ensiled plant material with improved aerobic stability, comprising: (a) introducing an effective amount of M. elsdenii cells into the plant material, and (b) ensiling the plant material to obtain ensiled plant material, where the ensiled plant material has improved aerobic stability compared to control ensiled plant material obtained in the absence of M. elsdenii cells.

[0007] In specific embodiments, the improved aerobic stability includes a reduced pH.

[0008] The present invention relates to a method for producing an increased amount of ensiled plant material, comprising: (a) introducing an effective amount of M. elsdenii cells into the plant material and (b) ensiling the plant material to obtain ensiled plant material, where the amount of ensiled plant material obtained is increased compared with the amount of control ensiled plant material obtained in the absence of M. elsdenii cells.

[0009] The present invention relates to a method for producing ensiled plant material, comprising: (a) introducing M. elsdenii cells into plant material, where the cells are selected from the group consisting of: ATCC® 25940, ATCC® 17752, ATCC® 17753, NCIMB 702261 and (b) ensiling plant material for obtaining ensiled plant material.

[0010] In specific embodiments, any of the methods further comprise adding an additive to the plant material.

[0011] In specific embodiments, the implementation is carried out before the collection of plant material, after the collection of plant material, during ensiling, or a combination of both.

[0012] In specific embodiments, the additive is selected from the group consisting of: another microorganism, an enzyme, a fermentable substrate, an acid, a preservative, a food additive, and combinations thereof.

[0013] In specific embodiments, the M. elsdenii cells are selected from the group consisting of: ATCC® 25940, ATCC® 17752, ATCC® 17753, NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331, NCIMB 702409, NCIMB 702 410 NCIMB 41125, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102, and combinations thereof.

[0014] In specific embodiments, the M. elsdenii cells are M. elsdenii NCIMB 41125 cells.

[0015] In specific embodiments, the plant material is selected from the group consisting of: forage, crop, grass, legume, grain, fruit, vegetable, or combinations thereof.

[0016] In specific embodiments, the plant material is corn, alfalfa, wheat, rye, barley, oats, triticale, millet, clover, sorghum, or combinations thereof.

[0017] In specific embodiments, any of the methods further comprises administering M. elsdenii cells in fluids.

[0018] In specific embodiments, any of the methods further comprises mixing the freeze-dried M. elsdenii cells with a liquid prior to introducing the cells.

[0019] In specific embodiments, any of the methods further comprises administering M. elsdenii cells in the form of freeze-dried cells.

[0020] In specific embodiments, the dry carrier contains freeze-dried cells.

[0021] In specific embodiments, any of the methods further comprises administering at least about 10 ⁶ to about 10 ¹⁴ CFU of M. elsdenii cells per ton of plant material.

[0022] The present invention relates to ensiled plant material obtained by any of the methods.

BRIEF DESCRIPTION OF THE DRAWINGS/ FIGURES

[0023] FIG. 1 shows the temperature (°C) of the plant material measured once per hour for 120 days of ensiling. Groups "Megasphaera" and "Control" in FIG. 1-14 were, respectively, plant material from fresh corn that was treated with 2×10 ⁸ cfu/ml of Megasphaera elsdenii cells (Megasphaera elsdenii strain NCIMB 41125, MSBiotec®, Wamego, Kansas) at a ratio of 50 milliliters per ton of plant material ("Megasphaera"), and plant material from fresh corn that was not treated ("Control").

[0024] FIG. 2 shows the total weight loss (%) in the "Megasphaera" and "Control" groups on days 7, 14, 21, 28, 39, 90 and 120 of ensiling, as determined by weighing the silos containing plant materials on that day and comparing with their respective initial weights on day 0 of ensiling. Treatment effect, P < 0.01; effect per day, P > 0.10; Dependence on the day of treatment, P > 0.10; Standard error of the mean=3.16.

[0025] FIG. 3 shows the average pH of samples taken from the "Megasphaera" and "Control" groups. Samples were taken from the silos on three opening days (i.e., days on which the silos were opened), including day 0 ("D0"), day 14 ("D14"), and day 120 ("D120") of ensiling. No treatment effect, P > 0.6; Standard error of the mean=0.02.

[0026] FIG. 4 shows the average concentrations of volatile fatty acids (VFA) (millimolar) in samples taken from the "Megasphaera" and "Control" groups, as described in FIG. 3. "SEM" is the standard error of the mean. "D" = effect of day of opening, with P < 0.05.

[0027] FIG. 5 shows the temperature (°C) measured during 14 days of exposure to ambient air on the "Megasphaera" and "Control" samples collected on opening day 14 as described in FIG. 3. The temperature for "Megasphaera" was higher than for "Control" after 72-110 hours, P < 0.05. Processing versus time, P < 0.01; Time effect, P < 0.01; Treatment effect, P < 0.01; Standard error of the mean=0.023.

[0028] FIG. 6 shows the mean pH of the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after harvest on opening day 14, as described in FIG. 3. Dependence on the day of treatment, P < 0.05; Time effect, P < 0.01; Treatment effect, P < 0.05; Treatments marked with an asterisk ("*") differ from each other on Day 14, P < 0.05; Standard error of the mean=0.07.

[0029] FIG. 7 shows the mean millimolar ("mM") total volatile fatty acid concentration in the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on opening day 14 as described in FIG. 3. In FIG. 7 also shows the average concentration of volatile fatty acids in the sample before ensiling. Dependence of processing on the day of opening, P > 0.5; Inoculant effect, P > 0.1; Effect of opening day, P < 0.6; Standard error of the mean=6.65.

[0030] FIG. 8 shows the average millimolar concentrations of volatile fatty acids ("VFA") in the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on opening day 14, as described in FIG. 3. "D" = Effect of Day of Opening, with P < 0.05.

[0031] FIG. 9 shows the average weight in pounds ("lb") on day 0, day 7, and day 14 of exposure to ambient air for each "Megasphaera" and "Control" sample collected on opening day 14, as described in FIG. 3. Dependence on the day of treatment, P > 0.9; Day effect, P > 0.1; Processing effect, P > 0.6; Standard error of the mean=0.16.

[0032] FIG. 10 shows the temperature (°C) measured after exposure to ambient air on day 14 in "Megasphaera" and "Control" samples collected on opening day 120 as described in FIG. 3. Temperature for "Megasphaera" was greater than for "Control" at 259-294 hours, P < 0.10. Processing versus time, P > 0.10; Time effect, P < 0.01; Treatment effect, P < 0.01; Standard error of the mean=1.23.

[0033] FIG. 11 shows the mean pH of the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after harvest on opening day 120 as described in FIG. 3. Dependence on the day of treatment, P > 0.1; Time effect, P > 0.1; Treatment effect, P > 0.1; Standard error of the mean=0.23.

[0034] FIG. 12 shows the mean millimolar ("mM") total volatile fatty acid concentration in the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after harvest on the day of opening 120 as described in FIG. 3. In FIG. 12 also shows the average concentration of volatile fatty acids in the sample before ensiling. Dependence of treatment on exposure to oxygen, P > 0.5; Inoculant effect, P > 0.15; Effect of a day of oxygen exposure, P < 0.01; Standard error of the mean=18.4.

[0035] FIG. 13 shows the average millimolar concentrations of volatile fatty acids ("VFA") in the "Megasphaera" and "Control" samples measured on day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on opening day 120, as described in FIG. 3. "D" = Effect of a day of oxygen exposure; "I" = Interaction effect; "T" = Processing effect; Letters indicate P < 0.05.

[0036] FIG. 14 shows the average weight in pounds ("lb") on day 0, day 7, and day 14 of exposure to ambient air for each "Megasphaera" and "Control" sample collected on opening day 120 as described in FIG. 3. Dependence on the day of treatment, P > 0.90; Day effect, P > 0.6; Treatment effect, P < 0.01; Standard error of the mean=0.10.

[0037] FIG. 15 shows the effect of storage temperature on the viability of liquid cultures of Megasphaera elsdenii NCIMB 41125 over a 28 day period, in terms of yield of M. elsdenii cells in colony forming units per milliliter (“CFU/mL”) on a logarithmic (“Log10”) scale.

[0038] FIG. 16 shows the viability of liquid cultures of M. elsdenii NCIMB 41125 after 0, 7, 14, 21 and 28 days of storage at room temperature, in terms of yield of M. elsdenii cells in cfu/ml on the Log10 scale.

[0039] FIG. 17 is a diagram of a continuous flow filtration ("TFF") system.

[0040] FIG. 18 shows the yield of M. elsdenii cells in cfu/ml of retentate on the y-axis in Log10 scale after passage through the TFF system. The x-axis indicates 70%, 80%, or 90% volume reduction by the system. "Target" refers to the theoretical isolation of M. elsdenii cells after processing by TFF. "Conc". refers to the actual isolation of M. elsdenii cells in the retentate, which is the volume of concentrated cells remaining after the marked reduction in volume.

[0041] FIG. 19 shows cell loss after freezing cells at -80°C or in liquid nitrogen (LiqN) and freeze-drying cells using slow (38 hours at 135 mTorr (18 Pa)) or fast (18.5 hours at 250 mTorr (33 Pa) )) cycle. Cells were derived from 70%, 80%, or 90% volume reduction retentates using post-TFF cultures containing either 8% maltodextrin/15% skim milk (M/SM) or 8% trehalose/15% skim milk (T/SM) as cryoprotectants.

[0042] FIG. 20 shows the yield of M. elsdenii NCIMB 41125 cells after initial fast (in liquid nitrogen) or slow (-20° C.) freezing by freeze drying (in the presence of 15% maltodextrin and 0% or 7.5% trehalose).

[0043] FIG. 21 shows the cell loss observed with M. elsdenii NCIMB 41125 cells frozen in the presence or absence of 4% trehalose or 7.5% skim milk, either at -80° C. or liquid nitrogen (LiqN).

[0044] FIG. 22 shows cell loss observed in freeze-dried samples prepared from a 90% volume reduction retentate blended with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM), frozen at -80°C or in liquid nitrogen (LiqN) and freeze-dried using a fast cycle, and stored at room temperature or 4°C under anaerobic conditions for up to 24 weeks.

[0045] FIG. 23 shows cell loss observed in freeze-dried samples prepared from a 90% volume reduction retentate blended with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM), frozen at -80°C or in liquid nitrogen (LiqN) and freeze-dried using a fast cycle, and stored at room temperature or 4°C under anaerobic conditions for 24 weeks. Bars without a common superscript are statistically distinct, P < 0.02.

[0046] FIG. 24 shows growth curves obtained for a non-freeze-dried or rehydrated freeze-dried product made from a 90% volume reduction retentate blended with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM ), frozen in liquid nitrogen (LiqN) and freeze-dried using after 12, 16, 20 or 24 weeks of storage under anaerobic conditions at 4 or 25°C. The optical density (OD) is shown at 600 nanometers (nm) as measured from 0 hours to 20 hours. Yellow lines=non-sublimated, Red lines=T/SM, Blue lines=M/SM, Dotted lines=preserved at 4°C, and solid lines=preserved at 25°C.

[0047] FIG. 25 shows the mean millimolar concentrations of acetate in a liquid extract of untreated (control/untreated) corn silage and Megasphaera elsdenii treated corn silage after 120 days of ensiling.

[0048] FIG. 26 shows the effect of treating reconstituted high moisture corn with or without Megasphaera elsdenii on pH levels during the ensiling process.

[0049] FIG. 27 shows the effect on dry matter content of an in vitro containing culture of mixed ruminants of high moisture reconstituted corn with or without treatment with Megasphaera elsdenii.

[0050] FIG. 28 shows the effect on carcass value when feeding livestock with silage treated with Megasphaera elsdenii compared to untreated silage during the pre-finishing phase (pre-finishing). The cost of each carcass was calculated using a standardized grid constructed by averaging weekly USDA price data for premiums and discounts published over a 10-year period between January 2008 and January 2018.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention relates to methods for using Megasphaera elsdenii cells to produce ensiled plant materials. The present invention also relates to ensiled plant materials obtained by these methods.

[0052] The entire contents of all publications, patents, and other references mentioned herein are incorporated by reference for all purposes, as if the contents of each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention.

Terminology

[0053] Unless otherwise defined, all technical and scientific terms, within the scope of the invention, have the same meaning as is generally accepted by a person skilled in the field to which this invention pertains. In case of conflict, the present application, including definitions, shall prevail. Unless the context otherwise requires, singular terms must include the plural, and plural terms must include the singular.

[0054] To the extent that section headings are used, they should not be construed as necessarily limiting.

[0055] For the purposes of the invention and in the appended claims, singular forms include plural reference objects unless the context clearly requires otherwise. For example, the term "compound" or "at least one compound" can include a variety of compounds, including mixtures thereof. The terms "one", "one or more" and "at least one", for example, can be used interchangeably in the present description.

[0056] In the context of the invention, the term "approximately", when used to modify an amount relating to the invention, refers to a variant numerical amount that may occur, for example, during conventional testing and manipulation; through inevitable error in such testing and manipulation; through differences in the manufacture, source, or purity of the ingredients used in the invention; and so on. Whether or not modified by the term "about", the claims include equivalents of the amounts recited. In some embodiments, the term "about" means plus or minus 10% of the specified numerical value.

[0057] Throughout this application, various embodiments of this invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered as specifically describing all possible subranges, as well as individual numerical values within that range. For example, a description of a range, such as 1 to 6, should be considered to include specifically described subranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, etc., as well as individual numerical values within this range, e.g. 1, 2, 3, 4, 5 and 6. This applies regardless of the bandwidth.

[0058] The terms "comprises", "comprising", "includes", "including", "having" and their conjugations are used interchangeably and mean "including, but not limited to". It will be appreciated that whenever aspects are described herein using the term "comprising", otherwise similar aspects described in terms of "consisting of" and/or "primarily consisting of" are also contemplated.

[0059] The term "consisting of" means "including and limited thereto".

[0060] The term "essentially consisting of" means the specified material of the composition or the specified steps of the method, and those additional materials or methods that do not significantly affect the essential characteristics of the material or method.

[0061] The term "and/or" within the scope of the invention should be taken as a specific description of each of the two specified features or components in the presence or absence of the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" in this specification is intended to include "A and B", "A or B", "A" (separately), and "B" (separately). Similarly, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to include each of the following: 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 (separately); B (separately); and C (separately).

[0062] The term "plant material", as used herein, refers to any plant material that can be used as animal feed and that can be ensiled, including, but not limited to, forage, crop, grass, legumes, grain, fruit, vegetable, or combinations thereof. Reference to "plant material" or a particular type of plant material described herein (e.g., forage, crop, grass, legumes, grain, cereal grain, fruit, vegetable, or combinations thereof) includes processed plant material (including but not limited to limit, cut, chopped, shredded, or beveled plant material, or combinations thereof), as well as parts of plant material, including, but not limited to, stems, petioles, leaves, skins, hulls, cobs, ears, grains, crop waste, or their combinations.

[0063] The terms "culture", "cultivate" and "cultivation", as used herein, refer to the incubation of cells under in vitro conditions to allow cell growth or division, or to keep the cells alive. The term "culture" may also, within the scope of the invention, refer to cells incubated under in vitro conditions (eg, cells incubated in a liquid growth medium). The terms "growth medium" and "culture medium", as used herein, refer to a solid (eg, agar), semi-solid (eg, agar), or liquid (eg, broth) composition that contains components to support cell growth.

[0064] The term “additive”, as used herein, refers to one or more ingredients, products, or substances (e.g., cells) used alone or together (e.g., to improve the quality of ensiled plant material, to improve the functioning and health of an animal, and/or to increase the digestibility of the ensiled plant material).

[0065] The terms "harvest" and "harvest" as used herein in relation to cells refer to the collection of cells from a culture, for example, the collection of cells in growth medium from a culture, the collection of cells by removing some of the growth medium from cells (eg, by concentrating the cells in liquid culture or separating the cells from the growth medium), or stopping the cell culture. The terms include the collection or removal of a certain volume of liquid containing cells from the liquid culture, including the volume in which the cells have been concentrated. The terms "harvest", "harvest" and "harvesting", in the context of the invention, in relation to plant material, refer to the collection of plant materials by manual or mechanical means.

[0066] The term "isolated", within the scope of the invention, does not necessarily reflect the degree to which the isolate is purified, but indicates isolation or separation from the natural form or natural environment. An isolate may include, but is not limited to, an isolated microorganism, an isolated biomass, or an isolated culture.

[0067] The term "effective amount", within the scope of the invention, refers to the amount that leads to the achievement of the desired result.

[0068] As used herein, "filler" refers to a component, or mixture of components, that is used to impart desirable characteristics to an ensiled plant material as described herein. An excipient of the present invention may be described as a "pharmaceutically acceptable" excipient, meaning that an excipient is a compound, material, composition, salt, and/or dosage form that, in the judgment of a physician, is suitable for contact with animal tissues (i.e., e. human and non-human animals) without undue toxicity, irritation, allergic response or other problematic complications for the desired duration of contact, in accordance with a reasonable benefit/risk ratio.

[0069] In the context of the invention, the term "yield" refers to the amount of live, or viable, cells, including the amount in a specific volume (for example, colony-forming units per milliliter ("CFU/ml")) or in a specific mass (for example, CFU per gram (“CFU/g”)).

[0070] As used herein, the term "viable" refers to a living organism or organisms (eg, a cell of a microorganism that is alive, or cells of a microorganism that are alive). "Vitality" refers to the ability to live, especially under certain conditions.

[0071] In the context of the invention, "purify", "purified" and "purification" means to make substantially clean or free from unwanted components, contamination, impurities or imperfections in the material.

[0072] The terms "invention" and "description" can be used interchangeably when describing or using, for example, in the phrases "the present invention" or "the present description".

[0073] It is understood that specific features of the invention, which, for clarity, are described in the context of separate embodiments, may also be presented in combination in one embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of one embodiment, may also be presented separately or in any suitable subcombination, or as appropriate in any other described embodiment of the invention. The specific features described in the context of the various embodiments should not be construed as necessary features of those embodiments unless the embodiment is inoperable without those elements.

[0074] While methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not limiting. Other features and advantages of the invention will become apparent from the detailed description and from the claims.

Methods for obtaining ensiled plant materials

[0075] In one aspect, the present invention relates to a method for producing ensiled plant material with improved aerobic stability, comprising: (a) introducing an effective amount of M. elsdenii cells into the plant material and (b) ensiling the plant material to obtain an ensiled plant material, where the ensiled the plant material has improved aerobic stability compared to the control ensiled plant material obtained in the absence of M. elsdenii cells. In some embodiments, improved aerobic stability includes reduced pH, reduced weight loss, or both.

[0076] In another aspect, the present invention relates to a method for producing an increased amount of ensiled plant material, comprising: (a) introducing an effective amount of M. elsdenii cells into the plant material, and (b) ensiling the plant material to obtain ensiled plant material, where the amount of obtained of ensiled plant material is increased compared to the amount of control ensiled plant material obtained in the absence of M. elsdenii cells. The amount of ensiled plant material can be determined, for example, by weighing the ensiled plant material or weighing the container containing the ensiled plant material and subtracting the weight of the container.

[0077] In another aspect, the present invention relates to a method for producing ensiled plant material, comprising: (a) introducing M. elsdenii cells into plant material, where the cells are selected from the group consisting of: ATCC® 25940, ATCC® 17752, ATCC® 17753 NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331, NCIMB 702409, NCIMB 702410, NCIMB 41125, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102 and (b) ensiling the plant material to obtain ensiled plant material.

[0078] Plant material in the methods of the present invention includes any plant material that can be ensiled to produce ensiled plant material.

[0079] In some embodiments, the plant material in the methods of the present invention is any plant material consumed by a ruminant. In some embodiments, the ruminant may be, but is not limited to, cattle, buffalo, sheep, goat, deer, reindeer, moose, giraffe, yak, and European elk. In some embodiments, the ruminant is selected from the group consisting of: cattle, buffalo, sheep, goat, deer, and reindeer. In some embodiments, the plant material in the methods of the present invention is any plant material consumed by camelids. In some embodiments, camelids may include, but are not limited to, alpacas, llamas, guanacos, vicunas, and camelids.

[0080] In some embodiments, the plant material is selected from the group consisting of: forage, crop, grass, legume, grain, fruit, vegetable, or combinations thereof. In some embodiments, the plant material is corn (ie, maize), alfalfa, wheat, rye, barley, oats, triticale, millet, clover, sorghum, or combinations thereof. In some embodiments, the plant material is crop waste, such as, but not limited to, sorghum, corn or soy straw. In some embodiments, the plant material is a weed. In some embodiments, the plant material is a mixture of grass and legumes, including one or more grasses and one or more legumes.

[0081] Herbs include, but are not limited to: Agrostis spp. - bent grass (eg, Agrostis capillaris - common bent grass and Agrostis stolonifera - marsh bent grass); Andropogon hallii - sandy bearded vulture; Arrhenatherum elatius - high ryegrass; Bothriochloa bladhii - Australian bearded vulture; Bothriochloa pertusa - hurricane grass; Brachiaria decumbens - Suriname grass; Brachiaria humidicola - coronivia; Bromus species are short-legged; Cenchrus ciliaris - buffalo grass; Chloris gayana - rhodes grass; Cynodon dactylon - Bermuda grass; Dactylis glomerata - crowded hedgehog; Echinochloa pyramidalis - antelope grass; Entolasia imbricata - bungoma herbs; Festuca species - fescue (for example, Festuca arundinacea - cane fescue, Festuca pratensis - meadow fescue, and Festuca rubra - red fescue); Heteropogon contortus - narrowed heteropogon; Hymenachne amplexicaulis - Westindian spartina; Hyparrhenia rufa - red hyparrenia; Leersia hexandra - six-stamen leersia; Lolium species - ryegrass (eg Lolium multiflorum - Italian ryegrass and Lolium perenne - pasture ryegrass); Megathyrsus maximus - guinea millet; Melinis minutiflora - treacle grass; Paspalum dilatatum - extended paspalum; Phalaris arundinacea - red canary grass; Phleum pratense - timothy; species of Poa - wheatgrass, meadow bluegrass (for example, Poa arachnifera - fusiform bluegrass, Poa pratensis - meadow bluegrass and Poa trivialis - common bluegrass); Setaria sphacelata - African foxtail; Themeda triandra - three-stamen temeda; and Thinopyrum intermedium - medium wheatgrass.

[0082] Legumes include, but are not limited to, herbaceous legumes and tree legumes. Herbaceous legumes include, but are not limited to: Arachis pintoi - pinto peanut; Chamaecrista rotundifolia - round-leaved bashful pea; Clitoria ternatea - butterfly pea; Lotus corniculatus - horned locust; Macroptilium atropurpureum - purple beans; Macroptilium bracteatum - Burgundy beans; Medicago species - alfalfa (for example, Medicago sativa - alfalfa, alfalfa and Medicago truncatula - tribulus alfalfa); Melilotus species - sweet clover; Neonotonia wightii - perennial soybean; Onobrychis viciifolia - sainfoin; species of Stylosanthes - stylosanthes (for example, Stylosanthes humilis - Townsville alfalfa); Stylosanthes scabra - shrub alfalfa; species of Trifolium - clover (for example, Trifolium hybridum - hybrid clover, Trifolium incarnatum - crimson clover, Trifolium pratense - red clover, Trifolium repens - white clover); and Vicia species - vetch (for example, Vicia articulata - single-flowered vetch, Vicia ervilia - clear vetch, Vicia narbonensis - Narbonne vetch, Vicia sativa - sowing vetch, intoxicating chaff, Vicia villosa - hairy vetch); and Vigna parkeri - creeping cowpea). Tree legumes include, but are not limited to: Acacia aneura - mulga; types of Albizia - silk acacia; Albizia canescens - grayish albizia; Albizia lebbeck - lebbeck; Enterolobium cyclocarpum - elephant's ear; and Leucaena leucocephala - leucena.

[0083] In some embodiments, the plant material in the methods of the present invention is plant material having a moisture content of from about 30% to about 90%, from about 35% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 45% to about 80%, from about 45% to about 75%, from about 45% to about 70%, from about 45% to about 65%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or about 50% to about 60%.

[0084] Plant material can be harvested or harvested at any suitable time, such as when the humidity is at a suitable level. If the moisture level is too high, the plant material may, for example, be allowed to wilt until the moisture reaches a suitable level.

[0085] In some embodiments, previously dehydrated plant material (eg, dried grain) can be rehydrated for use in ensiling, in accordance with any of the methods of the present invention.

[0086] Ensiling in any of the methods of the present invention can be carried out in accordance with any standard or known method for ensiling plant material. The plant material in the methods of the present invention is ensiled in a hermetically sealed container, also referred to herein as a "silo", to allow anaerobic fermentation of the plant material.

[0087] In some embodiments, the methods of the present invention further comprise adding an additive to the plant material.

[0088] In some embodiments, the use of any of the methods of the present invention (i.e., the use of Megasphaera elsdenii cells, the use of an additive, or both) occurs before the collection of plant material, after the collection of plant material, during ensiling, or in their combination. When both Megasphaera elsdenii cells and supplements are used, they can be used together or separately, at the same time or at different times.

[0089] The additive in the methods of the present invention can be any additive used in the ensiling process or added to the ensiled plant material.

[0090] In some embodiments, the additive in the methods of the present invention is selected from the group consisting of: a microorganism, an enzyme, a fermentable substrate, an acid, a preservative, a food additive, and combinations thereof.

[0091] In some embodiments, the additive in the methods of the present invention is a fermentable carbohydrate, including, but not limited to, a sugar source such as, but not limited to, molasses, sucrose, glucose, dextrose, whey, cereal grain, rice bran, wheat bran, citrus pulp, pineapple pulp or beet pulp.

[0092] In some embodiments, the additive in the methods of the present invention is an enzyme, such as, but not limited to, cellulase, hemicellulase, amylase, pectinase, protease, or xylanase.

[0093] In some embodiments, the additive in the methods of the present invention is a fermentation promoting microorganism, such as, but not limited to, lactic acid bacteria. In some embodiments, the microorganism is Lactobacillus, Pediococcus, Enterococcus, Propionibacterium, Lactococcus, Streptococcus, Leuconostoc, or Selenomonas. In some embodiments, the microorganism is Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cornyiformis, Lactobacillus curvatus, Lactobacillus salivarus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus viridescens, Pediococcus acidilactici, Pediococcus pentocace us, Pediococcus cerevisiae, Enterococcus faecium, Enterococcus faecalis, Propionibacterium jensenii, Propionibacterium acidipropionici, Propionibacterium freudenreichii, Propionibacterium globosum, Propionibacterium shermanii, Lactococcus lactis, Streptococcus bovis, Leuconostoc mesenteroides, or Selenomonas ruminantium.

[0094] In some embodiments, the additive in the methods of the present invention is a fermentation inhibitor, including, but not limited to, acids and organic salts such as, but not limited to, inorganic acids (e.g., hydrochloric acid), formic acid, acetic acid, caproic acid, sorbic acid, benzoic acid, sulfuric acid, lactic acid, acrylic acid, calcium formate, propionic acid or propionates, or other chemical inhibitors such as, but not limited to, formaldehyde, paraformaldehyde, sodium nitrite, sodium metabisulphite, sulfur dioxide, sodium hydroxide, sodium sulfate, sodium chloride, urea or ammonia.

[0095] In some embodiments, the additive in the methods of the present invention is an aerobic spoilage inhibitor, such as, but not limited to, propionic acid, propionates, acetic acid, caproic acid, or ammonia.

[0096] In some embodiments, the additive in the methods of the present invention is a nutritional supplement or nutrient source such as, but not limited to, urea, ammonia, limestone, or another mineral.

[0097] In some embodiments, the additive in the methods of the present invention is an absorbent such as, but not limited to, grain, straw, bentonite, sugar beet pulp, or polyacrylamide.

[0098] In some embodiments, the effective amount of Megasphaera elsdenii in the methods of the present invention is at least about 10 ⁶ , at least about 10 ⁷ , at least about 10 ⁸ , at least about 10 ⁹ , at least about 10 ¹⁰ , from about 10 ⁶ to about 10 ¹⁴ , from about 10 ⁶ to about 10 ¹³ , from about 10 ⁶ to about 10 ¹² , from about 10 ⁶ to about 10 ¹¹ , from about 10 ⁷ to about 10 ¹⁴ , from about 10 ⁷ to about 10 ¹³ , about 10 ⁷ to about 10 ¹² , about 10 ⁷ to about 10 ¹¹ , about 10 ⁸ to about 10 ¹¹ , about 10 ⁹ to about 10 ¹¹ , about 10 ¹⁰ to about 10 ¹¹ , from about 10 ¹⁰ to about 10 ¹² , from about 10 ¹⁰ to about 10 ¹³ , or about 10 ¹⁰ to about 10 ¹⁴ colony forming units (CFU) per ton of plant material.

[0099] In some embodiments, the M. elsdenii cells in the methods of the present invention are selected from the group consisting of: ATCC® 25940, ATCC® 17752, ATCC® 17753, NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331, NCIMB 702409 , NCIMB 702410, NCIMB 41125, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102, and combinations thereof.

[0100] In some embodiments, the M. elsdenii cells in the methods of the present invention are M. elsdenii NCIMB 41125 cells.

[0101] In some embodiments, the methods of the present invention include introducing M. elsdenii cells into a fluid.

[0102] In some embodiments, the methods of the present invention include administering M. elsdenii cells in the form of freeze-dried cells. In some embodiments, the dry carrier contains freeze-dried cells. In some embodiments, the dry carrier includes, but is not limited to, calcium carbonate, milk powder, or sucrose.

[0103] In some embodiments, the methods of the present invention further comprise mixing freeze-dried M. elsdenii cells with liquid prior to cell administration.

[0104] In some embodiments, the methods of the present invention include adding an additive to a liquid. When both the M. elsdenii cells and the supplement are added to a fluid, they may be administered in separate fluids, may be administered in separate fluids at separate times or at the same time, or may be administered as a mixture in the same liquids.

[0105] In some embodiments, the methods of the present invention include adding the additive in dry form, such as, but not limited to, powder, granules, or freeze-dried forms. When it is in dry form, the additive can be mixed with a dry carrier. When both the M. elsdenii cells and the supplement are administered in dry form, they may be in separate dry forms, they may be administered in separate dry forms at separate times or at the same time, or may be administered as a mixture of dry forms, including mixtures of dry forms in a dry carrier.

[0106] In another aspect, the present invention relates to ensiled plant material obtained by any of the methods of the present invention.

Megasphaera elsdenii

[0107] Cells of Megasphaera elsdenii from any strain or any combination of strains can be used according to the invention described in the present description.

[0108] The M. elsdenii strain or strains may be selected from a culture collection for storage (e.g., American Type Culture Collection (“ATCC®”), National Collection of Industrial, Food and Marine Bacteria (“NCIMB”), National Type Culture Collection ( "NCTC"), U.S. Research Service ("ARC") culture collections (i.e. "NRRL"), National Institute for Animal Health (NIAH) culture collections), or may be a strain isolated from a natural source ( e.g. from the gastrointestinal tract of a ruminant).

[0109] Examples of M. elsdenii strains that may be selected from the culture collection include, but are not limited to, the strains listed by deposit number in Table 1. Alternate deposit number designations are also indicated.

Table 1. Examples of M. elsdenii strains and the source of each strain.

Deposit number Alternative designations Strain Source ATCC® 25940 NCIMB 8927; BE2-2083 Sheep tripe ATCC® 17752 B159; NCIMB 702409; NCDO2409 N/A ATCC® 17753 T81; NCIMB 702410; NCDO2410 N/A NCIMB 702261 A17-2; A12-2; NCDO2261 Faeces of an adult NCIMB 702262 S17-3; NCDO2262 Feces of an immature pig NCIMB 702264 LC1 N/A NCIMB 702331 LC1; NCDO2263; NCDO2264; NCDO2331; N/A NCIMB 41125 Dairy cow tripe NCIMB 41787 Dairy cow tripe NCIMB 41788 Dairy cow tripe NRRL 18624 bull tripe NIAH 1102 N/A

[0110] In some embodiments, the M. elsdenii cells are derived from a strain having a deposit number selected from the group consisting of: ATCC® 25940, ATCC® 17752, ATCC® 17753, NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331 , NCIMB 702409, NCIMB 702410, NCIMB 41125, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102, and combinations thereof, including any of the alternative designations in Table 1.

[0111] In some embodiments, the M. elsdenii cells are derived from a strain isolated from a ruminant (eg, cow). See, for example, US Patent No. 7550139.

[0112] In some embodiments, the M. elsdenii cells are derived from a strain isolated from a non-ruminant animal (eg, human).

[0113] In some embodiments, the M. elsdenii cells are derived from a strain selected for lactate utilization (e.g., a strain that utilizes lactate in the presence of sugars), resistance to ionophore antibiotics, relatively high growth rate, ability to preferentially produce acetate, ability to proliferation at pH values below 5.0 and as low as 4.5, volatile fatty acid (VFA) production, phytase activity, and combinations thereof. See, for example, US Patent No. 7550139.

[0114] In some embodiments, the strain selected for lactate utilization utilizes lactate as the preferred carbon source in the presence of a soluble carbohydrate (eg, glucose and/or maltose). Utilization of lactate can be determined, for example, based on growth in a medium containing lactate and devoid of soluble carbohydrates compared to the same medium supplemented with soluble carbohydrates.

[0115] In some embodiments, the M. elsdenii cells originate from a strain with a high growth rate compared to other strains. Growth rates of various strains can be determined, for example, by culturing the cells in a liquid medium and monitoring the increase in optical density over time.

[0116] In some embodiments, the M. elsdenii cells are derived from a strain capable of producing VFAs, which can be determined, for example, by gas chromatography. In some embodiments, the VFA is a 6-carbon fatty acid.

[0117] In some embodiments, the M. elsdenii cells are derived from Megasphaera elsdenii strain NCIMB 41125. This strain of Megasphaera elsdenii has a high specific growth rate (0.94 generations/hour), is capable of growing in the pH range of 4.5 to 6.5 or more, uses D- and L-lactate as the preferred substrate, but also has the ability to utilize glucose and other carbohydrates, and is ionophore resistant.

[0118] In some embodiments, the M. elsdenii cells are derived from Megasphaera elsdenii strain NCIMB 41787. In some embodiments, the M. elsdenii cells are derived from Megasphaera elsdenii strain NCIMB 41788.

[0119] In some embodiments, the M. elsdenii cells are derived from Megasphaera elsdenii strain ATCC® 25940.

[0120] In some embodiments, the M. elsdenii cells are derived from a strain selected from a storage culture collection or isolated from a natural source. Cells "derived" from a strain may be a natural or artificial derivative, such as, for example, a subisolate, mutant, variant, or recombinant strain.

[0121] In some embodiments, the M. elsdenii cells is a commercial preparation, such as a Lactipro® product (eg, Lactipro advance®, MSBiotec®, Wamego, Kansas).

Obtaining a culture containing Megasphaera elsdenii cells

[0122] M. elsdenii cells for use in the present invention can be grown in liquid culture and used directly in liquid culture form. Alternatively, cells can be isolated from liquid or solid culture and either resuspended in a suitable liquid or freeze-dried prior to use.

[0123] M. elsdenii is an anaerobic bacterium that must be cultured under strict anaerobic conditions to maximize yield and viability.

[0124] In some embodiments, the culture contains M. elsdenii cells and a growth medium.

[0125] In some embodiments, the culture contains one or more M. elsdenii cell strains. In some embodiments, the culture contains a single M. elsdenii cell strain. In some embodiments, the culture consists of one or more M. elsdenii cell strains (ie, the cells in the culture consist of M. elsdenii cells, eg, one or more M. elsdenii cell strains). In some embodiments, the culture consists of a single strain of M. elsdenii cells.

[0126] In some embodiments, the methods of the present invention may also include growing, harvesting, and/or freeze-drying M. elsdenii cells for use in the methods.

[0127] Various fermentation parameters can be used to inoculate, grow, and harvest M. elsdenii cells, including continuous fermentation (ie, continuous culture) or batch fermentation (ie, batch culture). See, for example, US Patent No. 7550139.

[0128] The growth medium for M. elsdenii cells may be solid, semi-solid, or liquid. The medium may contain nutrients that provide the necessary elements, and specific factors that ensure growth. A variety of microbiological media and options are well known in the art. Media can be added to the culture at any time, including at the start of the culture, during the culture, or intermittently/continuously.

[0129] Examples of growth media include, but are not limited to: (1) partially defined media containing 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 supplemented with either Na-lactate (partially defined lactate, SDL), glucose (partially defined glucose, SDG) or maltose (partially defined maltose, SDM); (2) modified reinforced clostridial agar/broth medium (pre-reconstituted) containing peptone, 10 g/l; beef extract, 10 g/l; yeast extract, 3 g/l; dextrose 5 g/l; NaCl, 5 g/l; soluble starch, 1 g/l; L-cysteine HCl, 0.5 g/l; sodium acetate, 3 g/l; and resazurin (0.025%), 4 ml/l; (3) trypticase soy agar/broth with defibrinated sheep blood; (4) partially defined rumen juice-free medium containing Na-lactate (70%), 10 g/l; peptone, 2 g/l; KH ₂ PO ₄ 1 g/l; (NH ₄ ) ₂ SO ₄ 3 g/l; MgSO ₄ ⋅ 7H ₂ O 0.2 g/l; CaCl ₂ ⋅2H ₂ O 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); Na ₂ S⋅9H ₂ O, 0.25 g/l; cysteine, 0.25 g/l; antifoam, 0.07 ml/l and monensin, 10 mg/l; and which is obtained by adding Na-lactate and a mineral solution to a reservoir bottle and autoclaving for 60 minutes; dissolving peptone in 300 ml distilled H2O and autoclaving separately; sterilization by filtration of a solution of vitamins and two restorative agents in advance; after autoclaving, saturation of the reservoir bottle with anaerobic gas during the night; adding other components separately after cooling; and bringing the pH to the desired value using 5 N HCl; and (5) lactate-incubated rumen juice ("IRFL") medium containing 400 ml of incubated clarified rumen juice from fattened alfalfa sheep, 371 ml of distilled water, 2 g of peptone, 15 g of agar, 100 ml of 10% (wt./ vol.) a solution of D, L-sodium lactate, 100 ml of a 0.04% (wt./about.) solution of bromocresol purple and 25 ml of a mineral solution containing 40 g/l KH ₂ PO ₄ ; 120 g/l (NH ₄ ) ₂ SO ₄ ; 8 g/l MgSO ₄ ⋅7H ₂ O and 2.4 g/l CaCl ₂ ⋅2H ₂ O, where lactic acid (90% w/v) is used to bring the pH to 5.5 before autoclaving at 121° C. for 25 minutes, then cooled in a water bath at 50°C while saturated with anaerobic gas mixture, followed by the addition of each of Na ₂ S⋅9H ₂ O (12.5% w/v) and cysteine HCl⋅H ₂ O (12.5% w/v).

[0130] In some embodiments, the culture contains a growth medium containing at least two carbon sources. In some embodiments, at least two carbon sources are selected from the group consisting of: casein, starch (e.g., gelatinized starch and/or soluble starch), lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sucrose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soy protein, soluble protein, raffinose, and combinations thereof.

[0131] In some embodiments, the at least two carbon sources consist of about 1-99% of the first carbon source (e.g., any carbon source described herein) and about 1-99% of the second carbon source (e.g., any carbon described in the present description, other than the first carbon source), where 100% of at least two carbon sources consists of a first carbon source and a second carbon source. In some embodiments, the at least two carbon sources consist of about 50-60% of the first carbon source and about 40-50% of the second carbon source, of about 50-70% of the first carbon source and about 30-50% of the second carbon source, from about 50-80% of the first carbon source and about 20-50% of the second carbon source, or about 50-90% of the first carbon source and about 10-50% of the second carbon source. In other embodiments, the at least two carbon sources consist of about 65-75% of the first carbon source and about 25-35% of the second carbon source. In some embodiments, the first carbon source is lactate.

[0132] In some embodiments, the M. elsdenii cells are grown at about 39°C to about 40°C, at about 35°C, at about 36°C, at about 37°C, at about 38°C, at approximately 39°C or at approximately 40°C.

[0133] In some embodiments, the M. elsdenii cells are cooled to about 18°C to about 25°C for storage.

[0134] In some embodiments, the pH of the culture containing M. elsdenii cells (e.g., during culture and/or during harvest) 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.

[0135] For culturing M. elsdenii cells, fermenters of various sizes and designs that maintain anaerobic conditions can be used. The fermenter may be capable, for example, of fermenting culture volumes sufficient for commercial production of M. elsdenii cells.

[0136] In some embodiments, the culture containing M. elsdenii cells also contains another microorganism (ie, a microorganism cell that is not a M. elsdenii cell). In some embodiments, the culture contains M. elsdenii cells and another obligate anaerobic microorganism. In some embodiments, the culture contains M. elsdenii cells and another microorganism selected from the group consisting of: Lactobacillus, Pediococcus, Enterococcus, Propionibacterium, Lactococcus, Streptococcus, Leuconostoc, or Selenomonas.

Sublimated cells of Megasphaera elsdenii

[0137] In some embodiments, the Megasphaera elsdenii cells for use in the methods and ensiled plant materials of the present invention are freeze-dried cells.

[0138] Freeze-dried M. elsdenii cells can be obtained by a method including: obtaining a culture containing M. elsdenii cells and growth medium; collection of cells; cell freezing; and cell freeze drying, where freeze-dried M. elsdenii cells are obtained. In some embodiments, the method is carried out in the order of obtaining a culture, then harvesting the cells (i.e., harvesting the cultured cells), then freezing the cells (i.e., freezing the harvested cells), and then freeze-drying the cells (i.e., sublimation drying of frozen cells).

[0139] In some embodiments, the method is carried out under anaerobic conditions. In some embodiments, the method includes obtaining a culture, harvesting cells, freezing cells, freeze-drying cells, or combinations thereof under anaerobic conditions.

[0140] The method may include any of the methods for obtaining a culture, as described in the present description. The culture from the method may also have any of the culture properties described herein.

[0141] In some embodiments, the cells in culture contain M. elsdenii cells. In some embodiments, the cells in culture are composed of M. elsdenii cells.

[0142] In some embodiments, the growth medium contains at least two carbon sources selected from the group consisting of: casein, lactate (i.e., lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sucrose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soy protein, soluble protein, raffinose, and combinations thereof.

[0143] In some embodiments, the method includes harvesting the cells within 12 hours of the completion of the exponential growth phase of the culture. The culture can be cooled to room temperature to stop growth while harvesting.

[0144] In some embodiments, the culture contains liquid, and the method includes harvesting M. elsdenii cells (eg, concentrated M. elsdenii cells) with some percentage of liquid. In some embodiments, the method includes harvesting cells with about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, from about 1% to about 5%, about 10%, about 9%, about 8%, about 7%, about 6 %, about 5%, about 4%, about 3%, about 2%, or about 1% liquid. In some embodiments, the method includes harvesting cells with less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10 %, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% liquid.

[0145] In some embodiments, the culture contains liquid, and the method includes harvesting M. elsdenii cells (eg, concentrated M. elsdenii cells) by removing a percentage of the liquid. In some embodiments, harvesting cells includes removing about 50% to about 100% fluid, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, 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% liquid . In some embodiments, harvesting cells includes removing at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% liquid.

[0146] In some embodiments, the method includes harvesting M. elsdenii cells by cell concentration. In some embodiments, harvesting cells includes concentrating cells by at least one method selected from the group consisting of: centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. In some embodiments, the filtration includes filtration through clay. In some embodiments, the filtration includes in-line filtration along the flow, also known as cross-flow filtration.

[0147] In some embodiments, the pH of the culture containing M. elsdenii cells at the time of harvest 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 approximately 6.7, between approximately 4.9 and approximately 6.6, between approximately 5.0 and approximately 7.0, between approximately 5.0 and approximately 6.5, between approximately 5.0 and approximately 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 approximately 6.6, between approximately 5.5 and approximately 7.0, between approximately 5.5 and approximately 6.5, between approximately 5.1 and approximately 6.4, between approximately 5.2 and approximately 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.

[0148] In some embodiments, the method includes inoculating the growth medium in a fermenter using an inoculum containing M. elsdenii cells to obtain a culture, and incubating the culture at a temperature of about 39°C until the pH of the culture is about 6.0. In some embodiments, the inoculum containing M. elsdenii cells is a M. elsdenii cell culture flask or a portion thereof. In some embodiments, the method includes inoculating the growth medium in the fermenter at a ratio of inoculum to medium from 1/50 to 1/4000. In some embodiments, the ratio of inoculum to medium is 1/100.

[0149] In some embodiments, the culture further comprises at least one cryoprotectant. In some embodiments, at least one cryoprotectant is selected from the group consisting of: fructose, glucose, sucrose, milk powder, infant formula, skim milk, trehalose, maltodextrin, betaine, and combinations thereof. In some embodiments, at least one cryoprotectant is present in an amount of from about 1% to about 50% (w/v) of the culture, from about 1% to about 40% (w/v) of the culture, from about 1 % to about 30% (w/v) of the culture, from about 1% to about 20% (w/v) of the culture, from about 1% to about 10% (w/v) of the culture, from about 1% to about 5% (w/v) culture, about 10% to about 20% (w/v) culture, about 15% to about 25% (w/v) culture, from about 20% to about 30% (w/v) culture, about 30% to about 40% (w/v) culture, about 40% to about 50% (w/v) culture, from about 60% to about 70% (w/v) of the culture, from about 70% to about 80% (w/v) of the culture. In some embodiments, the cryoprotectant is added by adding cryoprotectant powder directly to the concentrated M. elsdenii cells. In some embodiments, the cryoprotectant is added by adding the cryoprotectant solution directly to the concentrated M. elsdenii cells at a ratio of 1/1, at a ratio of 1/5, or at a ratio of 1/10.

[0150] In some embodiments, freezing the cells includes placing the cells in a freezer or bringing the cells into contact with dry ice, liquid nitrogen, or a combination thereof. Cell freezing involves freezing cells while they are inside a container. Bringing the cells into contact includes bringing the container containing the cells into contact with the cell freezing medium. The cell freezing medium includes, but is not limited to, a freezer, an acetone-dry ice bath, liquid nitrogen, or a combination thereof.

[0151] In some embodiments, the method includes freezing the cells at a temperature of from about -20°C to about -210°C. In some embodiments, the method includes freezing the cells at a temperature of from about -20°C to about -80°C. In some embodiments, the method includes freezing the cells at a temperature of from about -80°C to about -210°C. In some embodiments, the method includes freezing the cells at a temperature of from about -20°C to about -196°C. In some embodiments, the method includes freezing the cells at a temperature of from about -80°C to about -196°C. In some embodiments, the method includes freezing the cells at a temperature of approximately -20°C. In some embodiments, the implementation, the method includes freezing the cells at a temperature of approximately -80°C. In some embodiments, the implementation, the method includes freezing the cells at a temperature of approximately -196°C. In some embodiments, the method includes freezing the cells by bringing the cells into contact with liquid nitrogen.

[0152] In some embodiments, the method includes freezing the cells under anaerobic conditions.

[0153] In some embodiments, freezing produces frozen pellets containing cells. For example, freezing can be performed using an ultra-fast freezer.

[0154] In some embodiments, the diameter of the frozen granules is from about 0.001 (0.0025 cm) to about 1.0 inches (2.54 cm), from about 0.01 (0.0254 cm) to about 1.0 in. (2.54 cm), approximately 0.1 (0.254 cm) to approximately 1.0 in. (2.54 cm), approximately 0.2 (0.508 cm) to approximately 1.0 in. (2.54 cm) ), from about 0.3 (0.762 cm) to about 1.0 inches (2.54 cm), from about 0.4 (1.016 cm) to about 1.0 inches (2.54 cm), from about 0, 5 (1.27 cm) to approximately 1.0 in (2.54 cm), approximately 0.6 (1.524 cm) to approximately 1.0 in (2.54 cm), approximately 0.7 (1.778 cm) ) to approximately 1.0 in. (2.54 cm), approximately 0.8 (2.032 cm) to approximately 1.0 in. (2.54 cm), approximately 0.9 (2.286 cm) to approximately 1.0 inch (2.54 cm), from about 0.001 (0.0025 cm) to about 0.9 inch (2.286 cm), from about 0.01 (0.0254 cm) to about 0.9 inch (2.286 cm), from about 0.1 (0.254 cm) to about 0.9 inches (2.286 cm), from about 0.2 (0.508 cm) to about 0.9 inches (2.286 cm), from about 0.3 (0.762 cm) to approximately 0.9 in. (2.286 cm), approximately 0.4 in. (1.016 cm) to approximately 0.9 in. (2.286 cm), approximately 0.5 in. (1.27 cm) to approximately 0.9 in. (2.286 cm) ), from about 0.6 (1.524 cm) to about 0.9 in (2.286 cm), from about 0.7 (1.778 cm) to about 0.9 in (2.286 cm), from about 0.8 (2.032 cm ) to approximately 0.9 in (2.286 cm), approximately 0.001 (0.0025 cm) to approximately 0.8 in (2.032 cm), approximately 0.01 (0.0254 cm) to approximately 0.8 in ( 2.032 cm), from about 0.1 (0.254 cm) to about 0.8 in (2.032 cm), from about 0.2 (0.508 cm) to about 0.8 in (2.032 cm), from about 0.3 ( 0.762 cm) to approximately 0.8 in (2.032 cm), approximately 0.4 (1.016 cm) to approximately 0.8 in (2.032 cm), approximately 0.5 (1.27 cm) to approximately 0.8 in. (2.032 cm), from about 0.6 (1.524 cm) to about 0.8 in. (2.032 cm), from about 0.7 (1.778 cm) to about 0.8 in. (2.032 cm), from about 0.001 ( 0.0025 cm) to approximately 0.7 in. (1.778 cm), approximately 0.01 (0.0254 cm) to approximately 0.7 in. (1.778 cm), approximately 0.1 (0.254 cm) to approximately 0 .7 in. (1.778 cm), approximately 0.2 (0.508 cm) to approximately 0.7 in. (1.778 cm), approximately 0.3 (0.762 cm) to approximately 0.7 in. 0.4 (1.016 cm) to approximately 0.7 in (1.778 cm), approximately 0.5 (1.27 cm) to approximately 0.7 in (1.778 cm), approximately 0.6 (1.524 cm) to 0.7" (1.778 cm), approximately 0.001" (0.0025 cm) to approximately 0.6" (1.524 cm), approximately 0.01" (0.0254 cm) to approximately 0.6" (1.524 cm) ), from about 0.1 (0.254 cm) to about 0.6 in (1.524 cm), from about 0.2 (0.508 cm) to about 0.6 in (1.524 cm), from about 0.3 (0.762 cm ) to approximately 0.6 in (1.524 cm), approximately 0.4 (1.016 cm) to approximately 0.6 in (1.524 cm), approximately 0.5 (1.27 cm) to approximately 0.6 in ( 1.524 cm), from about 0.001 (0.0025 cm) to about 0.5 inches (1.27 cm), from about 0.01 (0.0254 cm) to about 0.5 inches (1.27 cm), from about 0.05 (0.127 cm) to about 0.5 in (1.27 cm), from about 0.1 (0.254 cm) to about 0.5 in (1.27 cm), from about 0.15 ( 0.381 cm) to approximately 0.5 in (1.27 cm), approximately 0.2 (0.508 cm) to approximately 0.5 in (1.27 cm), approximately 0.3 (0.762 cm) to approximately 0 .5 inches (1.27 cm) or from about 0.4 (1.016 cm) to about 0.5 inches (1.27 cm).

[0155] Frozen M. elsdenii cells can be stored frozen (eg, below 0°C) prior to freeze-drying, or can be immediately freeze-dried. In some embodiments, the frozen M. elsdenii cells are stored at a temperature below about 0°C, below about -10°C, below about -20°C, below about -50°C, below about -80°C, or below about -196°C. In some embodiments, the frozen M. elsdenii cells are stored at about -20°C, about -30°C, about -40°C, about -50°C, about -60°C, about -70°C, about -80°C, approximately -90°C, approximately -100°C, approximately 150°C, approximately -196°C or approximately -210°C.

[0156] In some embodiments, the frozen M. elsdenii cells are lyophilized. In some embodiments, the frozen M. elsdenii cells are freeze-dried. Freeze-drying includes, for example, removing liquid from frozen cells.

[0157] In some embodiments, freeze-drying M. elsdenii cells includes placing the frozen cells in a freeze dryer. In some embodiments, freeze-drying involves subjecting the frozen cells to reduced pressure and gradually warming the cells to room temperature.

[0158] In some embodiments, the method includes freeze-drying the cells under anaerobic conditions.

[0159] In some embodiments, freeze-dried M. elsdenii is obtained on a commercial scale.

[0160] In some embodiments, the M. elsdenii cells are lyophilized at a pressure between about 50 mTorr (7 Pa) and about 2000 mTorr (267 Pa), between about 100 mTorr (13 Pa) and about 1950 mTorr (260 Pa), between approximately 150 mTorr (20 Pa) and approximately 1900 mTorr (253 Pa), between approximately 200 mTorr (27 Pa) and approximately 1850 mTorr (247 Pa), between approximately 250 mTorr (33 Pa) and approximately 1800 mTorr (240 Pa) , between approximately 300 mTorr (40 Pa) and approximately 1750 mTorr (233 Pa), between approximately 350 mTorr (47 Pa) and approximately 1700 mTorr (227 Pa), between approximately 400 mTorr (53 Pa) and approximately 1650 mTorr (220 Pa ), between approximately 450 mTorr (60 Pa) and approximately 1600 mTorr (213 Pa), between approximately 500 mTorr (67 Pa) and approximately 1550 mTorr (207 Pa), between approximately 550 mTorr (73 Pa) and approximately 1500 mTorr (200 Pa), between approximately 600 mTorr (80 Pa) and approximately 1500 mTorr (200 Pa), between approximately 650 mTorr (87 Pa) and approximately 1450 mTorr (193 Pa), between approximately 700 mTorr (93 Pa) and approximately 1400 mTorr ( 187 Pa), between approximately 750 mTorr (100 Pa) and approximately 1350 mTorr (180 Pa), between approximately 800 mTorr (107 Pa) and approximately 1300 mTorr (173 Pa), between approximately 850 mTorr (113 Pa) and approximately 1250 mTorr (167 Pa), between approximately 900 mTorr (120 Pa) and approximately 1200 mTorr (160 Pa), between approximately 950 mTorr (127 Pa) and approximately 1150 mTorr (153 Pa) or between approximately 1000 mTorr (133 Pa) and approximately 1100 mTorr (147 Pa). In some embodiments, the pressure during the lyophilization process is 135 mTorr (18 Pa). In some embodiments, the pressure during the lyophilization process is 250 mTorr (33 Pa).

[0161] In some embodiments, the time to completion of the lyophilization process is between about 5 hours and 15 days, between about 6 hours and 15 days, between about 7 hours and 15 days, between about 8 hours and 15 days, between about 9 hours and 15 days, between about 10 hours and 15 days, between about 11 hours and 15 days, between about 12 hours and 15 days, between about 18 hours and 15 days, between about 24 hours and 15 days, between about 36 hours and 14 between about 48 hours and 13 days, between about 3 days and 12 days, between about 4 days and 11 days, between about 5 days and 10 days, between about 6 days and 9 days, between about 7 days and 8 days. In some embodiments, the time to completion of the lyophilization process is approximately 18.5 hours. In some embodiments, the time to completion of the lyophilization process is approximately 38.5 hours.

[0162] In some embodiments, from about 1×10 ³ to 1×10 ¹² cfu/g of freeze-dried M. elsdenii cells is obtained by the method described in the present description. In some embodiments, from about 1×10 ³ to 1×10 ¹² CFU/g of M. elsdenii cells are viable after freeze-drying.

[0163] Freeze-dried Megasphaera elsdenii cells can also be obtained by a method comprising: (a) obtaining under anaerobic conditions a culture containing M. elsdenii cells and a growth medium containing at least two carbon sources selected from the group consisting of: casein, lactate (i.e. lactic acid), dextrose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, mannitol, sucrose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate , arabinose, soy protein, soluble protein, raffinose, and combinations thereof, (b) collecting cells under anaerobic conditions, (c) freezing cells, and (d) freeze-drying cells, where from about 1x10 ³ to about 1x10 ¹² CFU/g of sublimated M. elsdenii cells.

[0164] Freeze-dried Megasphaera elsdenii cells can also be obtained by a method including: (a) obtaining a culture containing M. elsdenii cells and growth medium, (b) collecting cells under anaerobic conditions within 12 hours after completion of the exponential growth phase of the culture, ( c) freezing the cells, (d) freeze-drying the cells, and optionally, (e) encapsulating the freeze-dried cells, where freeze-dried M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells are obtained.

[0165] Freeze-dried Megasphaera elsdenii cells can also be obtained by a method including: (a) obtaining a culture containing M. elsdenii cells and growth medium, (b) harvesting the cells, (c) freezing the cells at a temperature of from about -80°C to approximately -210° C. within 5 hours of collection, (d) cell freeze-drying, and optionally, (e) freeze-dried cell encapsulation, where freeze-dried M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells are obtained.

[0166] In some embodiments, the present invention relates to an encapsulated freeze-dried composition comprising M. elsdenii cells, wherein the freeze-dried powder is encapsulated using (a) oil, including, but not limited to, vegetable oil, palm oil, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid, (b) an edible polymer including, but not limited to, alginate, chitosan, carboxymethyl cellulose, xanthan gum, starch, carrageenan, gelatin, and pectin (c), milk proteins, including, but not limited to restrictions, casein and whey protein, and (d) vegetable protein from soy, legumes and cereals, including but not limited to zein. In some embodiments, the number of freeze-dried M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells obtained by the method described herein and/or the number of M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells that are viable after freeze-drying, is from about 1×10 ³ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ³ CFU/g to about 1×10 ¹¹ CFU/g, from about 1×10 ³ CFU/g to about 1×10 ¹⁰ CFU/g, from about 1×10 ³ CFU/g to about 1×10 ⁹ CFU/g, from about 1×10 ³ CFU/g to about 1×10 ⁸ CFU/g, from about 1× 10 ³ cfu/g to about 1x10 ⁷ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁶ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁵ cfu /g, from about 1x10 ⁴ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ⁵ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ⁶ cfu/g to about 1x10 ¹² CFU/g, from about 1x10 ⁷ CFU/g to about 1x10 ¹² CFU/g, from about 1x10 ⁸ CFU/g to about 1x10 ¹² CFU/g, from about 1×10 ⁹ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ¹⁰ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ³ CFU/g to about 1×10 ⁵ cfu/g, from about 1x10 ⁴ cfu/g to about 1x10 ⁶ cfu/g, from about 1x10 ⁵ cfu/g to about 1x10 ⁷ cfu/g, from about 1x10 ⁶ cfu /g to about 1x10 ⁸ cfu/g, from about 1x10 ⁷ cfu/g to about 1x10 ⁹ cfu/g, from about 1x10 ⁸ cfu/g to about 1x10 ¹⁰ cfu/g, from about 1x10 ⁹ CFU/g to about 1x10 ¹¹ CFU/g, or from about 1x10 ¹⁰ CFU/g to about 1x10 ¹² CFU/g.

[0167] In some embodiments, freeze-dried M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells are viable for about 14 days to about 24 months at about -80°C, about -20°C, about 4°C, approximately 25°C, or combinations thereof. In some embodiments, freeze-dried M. elsdenii cells or encapsulated freeze-dried M. elsdenii cells are viable for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months at about -80°C, about -20°C, about 4°C , approximately 25°C or combinations thereof.

[0168] In some embodiments, from about 1x10 ³ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ³ cfu/g to about 1x10 ¹¹ cfu/g, from about 1x 10 ³ cfu/ml to about 1x10 ¹⁰ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁹ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁸ cfu /g, from about 1×10 ³ CFU/g to about 1×10 ⁷ CFU/g, from about 1×10 ³ CFU/g to about 1×10 ⁶ CFU/g, from about 1×10 ³ CFU/g to about 1x10 ⁵ CFU/g, from about 1x10 ⁴ CFU/g to about 1x10 ¹² CFU/g, from about 1x10 ⁵ CFU/g to about 1x10 ¹² CFU/g, from about 1×10 ⁶ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ⁷ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ⁸ CFU/g to about 1×10 ¹² cfu/g, from about 1x10 ⁹ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ¹⁰ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ³ cfu /g to about 1x10 ⁵ cfu/g, from about 1x10 ⁴ cfu/g to about 1x10 ⁶ cfu/g, from about 1x10 ⁵ cfu/g to about 1x10 ⁷ cfu/g, from about 1x10 ⁶ CFU/g to about 1x10 ⁸ CFU/g, from about 1x10 ⁷ CFU/g to about 1x10 ⁹ CFU/g, from about 1x10 ⁸ CFU/g to about 1 x10 ¹⁰ cfu/g, from about 1 x 10 ⁹ cfu/g to about 1 x 10 ¹¹ cfu/g, or from about 1 x 10 ¹⁰ cfu/g to about 1 x 10 ¹² cfu/g of freeze-dried M. elsdenii cells, or encapsulated freeze-dried M. elsdenii cells are viable after storage at about -80°C, about -20°C, about 4°C, or combinations thereof for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months.

[0169] In some embodiments, from about 1x10 ³ CFU/g to about 1x10 ¹² CFU/g, from about 1x10 ³ CFU/g to about 1x10 ¹¹ CFU/g, from about 1x 10 ³ cfu/g to about 1x10 ¹⁰ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁹ cfu/g, from about 1x10 ³ cfu/g to about 1x10 ⁸ cfu /g, from about 1×10 ³ CFU/g to about 1×10 ⁷ CFU/g, from about 1×10 ³ CFU/g to about 1×10 ⁶ CFU/g, from about 1×10 ³ CFU/g to about 1x10 ⁵ CFU/g, from about 1x10 ⁴ CFU/g to about 1x10 ¹² CFU/g, from about 1x10 ⁵ CFU/g to about 1x10 ¹² CFU/g, from about 1×10 ⁶ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ⁷ CFU/g to about 1×10 ¹² CFU/g, from about 1×10 ⁸ CFU/g to about 1×10 ¹² cfu/g, from about 1x10 ⁹ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ¹⁰ cfu/g to about 1x10 ¹² cfu/g, from about 1x10 ³ cfu /g to about 1x10 ⁵ cfu/g, from about 1x10 ⁴ cfu/g to about 1x10 ⁶ cfu/g, from about 1x10 ⁵ cfu/g to about 1x10 ⁷ cfu/g, from about 1x10 ⁶ CFU/g to about 1x10 ⁸ CFU/g, from about 1x10 ⁷ CFU/g to about 1x10 ⁹ CFU/g, from about 1x10 ⁸ CFU/g to about 1 x10 ¹⁰ cfu/g, from about 1 x 10 ⁹ cfu/g to about 1 x 10 ¹¹ cfu/g, or from about 1 x 10 ¹⁰ cfu/g to about 1 x 10 ¹² cfu/g of freeze-dried M. elsdenii cells, or encapsulated, freeze-dried M. elsdenii cells are viable after storage at about 25° C. for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months.

EXAMPLES

[0170] Reference is now made to the following examples, which, together with the above descriptions, illustrate some embodiments of the invention in a non-limiting manner.

EXAMPLE 1

Evaluation of Megasphaera elsdenii NCIMB 41125 for ensiled plant material

A. Processing of plant material and ensiling

[0171] Fresh corn plant material was either treated with 2×10 ⁸ cfu/ml of Megasphaera elsdenii cells (i.e., Megasphaera elsdenii strain NCIMB 41125, MSBiotec®, Wamego, Kansas) injected at a rate of 50 ml per ton of plant material by means of a spray gun, or not treated (control).

[0172] Approximately 100 pounds (lb) (40 kilograms (kg)) of plant material treated with Megasphaera elsdenii cells or untreated plant material was packaged in individual 15-gallon (57-liter) drums (i.e., silos) and sealed closed. Prepared four repetitions for each treatment. Ensiling was carried out for 120 days under standard conditions.

[0173] Each silo contained a probe thermometer connected to a data logger. Temperature (°C) readings were collected once per hour for 120 days of ensiling, where the data is shown in FIG. 1 for treatment with Megasphaera elsdenii ("Megasphaera") or control ("Control") cells.

[0174] The silos were weighed on days 7, 14, 21, 28, 39, 90, and 120 to determine the percentage of total weight loss (e.g., respiration loss) during the ensiling period compared to the initial weight on day 0. See FIG. 2; Treatment effect, P < 0.01; Day effect, P > 0.10; Dependence on the day of treatment, P > 0.10; Standard error of the mean=3.16.

[0175] Additional tests were performed on three days of opening (ie, on the day on which the silos were opened), including day 0, day 14, and day 120 of ensiling.

B. pH and VFA tests

[0176] Samples were collected from the silo for each treatment on opening day 0, 14, and 120. The pH and millimolar concentrations of volatile fatty acids (VFA) in the samples were measured. Average values for pH are shown in FIG. 3; No treatment effect, P > 0.6; Standard error of the mean=0.02. Average values for VFA concentrations are shown in FIG. 4; "SEM" is the standard error of the mean. "D" = effect of day of opening, with P < 0.05.

C. Aerobic stability tests

[0177] For each opening day 14 and 120, approximately 10 lbs (4.5 kg) of ensiled plant material was transferred from the silo for each treatment to the respective 18 liter containers. The sample in each 18 L container was exposed to ambient air for 14 days.

[0178] FIG. 5 and FIG. 10 shows the mean temperature (°C) measured over 14 days of ambient air exposure for each treatment on opening day 14 (FIG. 5; Treatment versus time, P < 0.01; Effect of time, P < 0.01; Effect treatment, P < 0.01; standard error of mean=0.023) and day of opening 120 (FIG. 10; Treatment versus time, P > 0.10; Time effect, P < 0.01; Treatment effect, P < 0, 01; Standard error of the mean = 1.23).

[0179] FIG. 6 and FIG. 11 shows mean pH measurements on day 0 (“D0”) and day 14 (“D14”) of ambient air exposure for each treatment on opening day 14 (FIG. 6; Treatment day vs. treatment, P < 0.05; Effect of time, P < 0.01; Treatment effect, P < 0.05; Treatments marked with an asterisk (“*”) differ from each other by day 14 (P < 0.05; SEM=0.07), and day opening 120 (FIG. 11; Dependence on day of treatment, P > 0.1; Effect of time, P > 0.1; Effect of treatment, P > 0.1; Standard error of mean=0.23).

[0180] FIG. 7-8 and FIG. 12-13 shows the mean VFA concentrations (millimolar (mM)) measured on day 0 and day 14 of exposure to ambient air for each sample collected on opening day 14 (FIGS. 7-8) and opening day 120 (FIGS. 12-13 ). FIG. 7 and FIG. 12 shows total VFA concentrations including pre-ensiling sample. For FIG. 7, Dependence of treatment on the day of opening, P > 0.5; Inoculant effect, P > 0.1; Effect of opening day, P < 0.6; Standard error of the mean=6.65. For FIG. 8, "D" = Effect of Day of Opening, with P < 0.05. For FIG. 12, Dependence of treatment on exposure to oxygen, P > 0.5; Inoculant effect, P > 0.15; Effect of a day of oxygen exposure, P < 0.01; Standard error of the mean=18.4. For FIG. 13, "D" = Effect of a day of oxygen exposure; "I" = Interaction effect; "T" = Processing effect; Letters indicate P < 0.05.

[0181] FIG. 9 and FIG. 14 shows the mean weight (lb) on day 0, day 7, and day 14 of ambient air exposure for each treatment on opening day 14 (FIG. 9, Dependence on treatment day, P > 0.9; Effect of day, P > 0, 1; Treatment effect, P > 0.6; Standard error of mean=0.16) and day of opening 120 (FIG. 14, Dependence on treatment day, P > 0.90; Day effect, P > 0.6; Treatment effect , P < 0.01; Standard error of the mean = 0.10).

D. Generalization

[0182] Compared to controls, treatment of plant material with M. elsdenii cells prior to ensiling resulted in, for example, less weight loss during ensiling (i.e., during anaerobic fermentation), as well as reduced pH and less weight loss. after exposure to ambient air (i.e. increased aerobic stability). See, for example, FIG. 2, 6 and 9, respectively.

EXAMPLE 2

Effect of temperature on liquid culture yield of M. elsdenii

[0183] Storage temperatures of 4°C, 20°C, and 39°C were tested to evaluate cell viability in M. elsdenii NCIMB 41125 liquid cultures at 0, 7, 14, 21, and 28 days. The results indicated that product storage at 4°C significantly (P < 0.001) improved culture viability compared to storage at 20°C or 39°C, regardless of sampling day. After 28 days, the product stored at 4°C had 3.98×10 ⁶ colony forming units per milliliter (cfu/ml), compared to 1.26×10 ⁶ and 6.3×10 ⁵ cfu/ml for the product maintained at 20°C or 39°C, respectively (P <0.01; FIG. 15). Thus, the results show that reducing the storage temperature improves the viability of liquid cultures of M. elsdenii NCIMB 41125.

[0184] Additional data from a separate study show that the output of Megasphaera elsdenii NCIMB 41125 in liquid culture is reduced after 0, 7, 14, 21 and 28 days of storage at room temperature (FIG. 16).

EXAMPLE 3

Use of downstream filtration to concentrate cultures of M. elsdenii

[0185] Flow filtration along the flow (TFF) was investigated as a method of concentrating large volumes of culture with high productivity.

[0186] The pilot scale TFF system (see FIG. 17) was integrated into the M. elsdenii production line and tested on 500 liter (L) production runs to evaluate the pilot scale system at 70%, 80% and 90%. A TFF module was used with one 100 kDa RC membrane (channel height 0.875 mm). Three production cycles were performed for each reduction level, for a total of nine cycles. Samples were collected for each run and analyzed for pH, optical density (OD), presence or absence of aerobic contaminants, osmolarity, volatile fatty acid profile, M. elsdenii concentration and growth characteristics. Samples were collected from nine cultures of M. elsdenii ("Unsublimated") prior to filtration, from the permeate ("Permeate"), which is the volume removed by the system, and from the retentate ("Retentate"), which is the volume of the concentrated culture M .elsdenii containing cells remaining after volume reductions.

[0187] Permeate samples collected at the beginning, middle and end of the concentration process had consistent OD readings among volume reduction levels all below 0.045 (data not shown). The number of M. elsdenii isolated in the permeate increased during the concentration process (P=0.0006) but was still negligible (less than 3×10 ⁴ cfu/ml) compared to the number of cells collected in the retentate (< 0.002%). Volume reduction levels had no effect on the concentration of M. elsdenii isolated in the permeate (P >0.9; Table 2).

Table 2. Average recovery of M. elsdenii in samples collected during nine production runs conducted to evaluate the TFF system on a pilot scale at 70%, 80% and 90% volume reductions

CFU/ml, Log10 standard error Processing effect, P value Samples 70% 80% 90% permeate 1.19 1.07 1.12 0.40 0.958 Retentate 8.99 9.04 9.25 0.03 <0.0001

[0188] The yield of M. elsdenii in the retentate did not differ between the bags collected at the beginning, middle, or end of each pouching process (P=0.6088; data not shown). Retentate yield was affected by volume reduction levels (P < 0.0001; Table 2 and FIG. 18). The retentate after 90% volume reduction had a higher yield than the retentate after 70% and 80% volume reduction (P < 0.0001). However, the retentates for 70% and 80% did not differ from each other (P > 0.09).

[0189] The filtration process did not affect the ability of M. elsdenii cells to grow when inoculated back into SDL-20 media (Table 3). The slope angles in the exponential phase were not affected by the level of volume reduction (P > 0.3), but were affected by the sample type: "Retentate" versus "Unsublimated" (P < 0.001). Latency was influenced by both the level of volume reduction and sample type (P < 0.001). The latency period for the retentate decreased with increasing volume reduction, which was representative of the higher cell concentration.

Table 3. Comparison of slope and latency between stock M. elsdenii (before filtration=unsublimated) and retentate (after filtration) collected during different TFF cycles for volume reduction

sample type Effects, P-values Non-sublimated Retentate sample type Decrease volume Relationship 70% 80% 90% 70% 80% 90% Curve angle 0.39 0.36 0.37 0.44 0.42 0.43 < 0.001 0.329 0.979 Latent period, hour ^{1.42a 1.10b 1.07b} 0.32 ^s 0.29 ^s 0.24 ^s 0.001 <0.001 < 0.001

EXAMPLE 4

Freeze and freeze drying parameters for M. elsdenii

A. Freezing and freeze-drying of retentates

[0190] TFF retentates were used to test freezing methods, cryoprotectant incorporation, and various freeze-drying parameters.

[0191] The retentates were transferred to sterile whey bottles under degassing and mixed under aseptic conditions with various formulations of cryoprotectants (w/v): no cryoprotectant (Control), skimmed milk (SM), trehalose (T) and betaine. Samples were taken from the retentates mixed with the appropriate cryoprotectant to determine the concentration of M. elsdenii prior to freeze-drying (ie, the number of viable cells). The mixtures were transferred into 10 ml vials (4 ml/ampoules) and flash frozen in liquid nitrogen or slowly frozen at -80° C. overnight. The ampoules were transferred to a freeze dryer for lyophilization using either a slow or fast cycle. Upon completion of freeze-drying, bacterial survival was determined by resuspending the lyophilized product in an anaerobic chamber with an anaerobic diluent, allowing rehydration for 40 minutes at room temperature, and then plating onto SDL20 agar.

[0192] Cell loss was calculated by subtracting the concentration of M. elsdenii isolated after freeze-drying from the initial concentration of M. elsdenii measured in the appropriate retentate, mixed with cryoprotectants or not. SAS® software was used to calculate cell loss data by analyzing interactions between volume reduction levels (70%, 80% or 90%), freeze drying cycle (slow versus fast), freezing method (-80°C versus liquid nitrogen), cryoprotectants (no cryoprotectant, betaine, trehalose, skim milk, maltodextrin, trehalose/skimmed milk (T/SM) and maltodextrin/skimmed milk (M/SM)).

[0193] The cell loss observed in the control treatment (no cryoprotectants), regardless of other criteria, was 5 Log (cfu/ml) or higher. Similarly, the cell loss observed with betaine treatment, regardless of other criteria, was 3.96 Log CFU/mL or greater. The acceptable cell loss limit was set at 1.6 log cfu/ml. Retentates mixed with T/SM or M/SM were all below this threshold, regardless of the freeze drying cycle or freeze method used, with the exception of T/SM when frozen at -80°C and freeze dried using a slow or fast cycle (FIG. 19).

B. Effects of freeze drying conditions on storage

[0194] Megasphaera elsdenii NCIMB 41125 was concentrated 10x using a filtration device and freeze-drying assays were performed testing various characteristics: fast or slow initial freeze (liquid nitrogen vs. -20°C), presence or absence of trehalose (0%, 4 %, 7.5% and 10%) and mild or fast freeze drying cycles (38 hours at 2×10 ^-6 Torr (2.7 × 10 ^-4 Pa) compared to 16.5 hours at 135×10 ^-6 Torr (1.8×10 ^-2 Pa)). All freeze-drying methods tested produced products capable of maintaining sufficient viability to initiate culture growth after rehydration, even after prolonged storage of 4 to 12 months at room temperature. Differences in bacterial viability were nevertheless observed, depending on the freeze drying characteristics used (FIG. 20). Slow freezing, 7.5% trehalose incorporation, and mild freeze-drying steps maintaining a final water activity above 0.04 were associated with greater survival of Megasphaera elsdenii.

C. Effects of cryoprotectants on viability of freeze-dried M. elsdenii

[0195] Centrifuged concentrates of M. elsdenii NCIMB 41125 cells were resuspended in infant formula prior to lyophilization, resulting in only a 1-log reduction in cell viability when the cells were subsequently rehydrated. The addition of 4% trehalose and 7.5% skim milk to the M. elsdenii concentrate was tested before slow freezing at -80° C. or flash freezing in liquid nitrogen to determine the cell loss encountered during the initial freezing process. As shown in FIG. 21, flash freezing with 4% trehalose or 7.5% skim milk resulted in the greatest recovery of viable cells (0.79 log reduction in viable cells). The product without the addition of cryoprotectants, regardless of the freezing method used, lost between 2.34 and 1.95 log cfu/ml of Megasphaera elsdenii.

EXAMPLE 5

Effects of Storage Conditions on the Yield and Stability of Freeze-Dried M. elsdenii

[0196] To determine the effect of freeze drying methods and storage conditions on the growth characteristics and storage time of Megasphaera elsdenii NCIMB 41125, the retentate obtained after a 90% volume reduction was mixed with 8% trehalose/15% skimmed milk (T/SM) or 8 % maltodextrin/15% skim milk (M/SM), frozen at -80°C or in liquid nitrogen (LiqN) and freeze-dried using a fast cycle. The retentate samples were then tested for bacterial growth characteristics and cell survival during storage at 4°C or 25°C under aerobic or anaerobic conditions for 0, 2, 4, 8, 12, 16, 20 and 24 weeks using growth curve analysis. and method of distribution and sieving. Briefly, the products were sampled after storage, serially diluted and plated on an SDL agar plate. In addition, growth medium (SDL) was inoculated (1:100) with rehydrated freeze-dried products and optical density (OD, 600 nm) was recorded until the cultures reached stationary phase. The experiment was repeated on 3 different days, and all treatments were performed in triplicate. The rehydrated freeze-dried samples were diluted to obtain growth curves because without dilution, the optical density exceeded the limit due to the presence of skimmed milk. To facilitate comparison of growth curves, the same dilution was performed for non-sublimated samples used as controls.

[0197] RESULTS: Samples were freeze-dried and stored at room temperature or 4°C under aerobic or anaerobic conditions for 6 months. Samples stored under aerobic conditions, regardless of treatment, rapidly degraded with additional cell loss compared to their anaerobic equivalent, ranging from 0.4 to 3.2 Log, after only 2 weeks of storage. Based on these results, and to improve the clarity of the figure, only the cell loss observed in freeze-dried anaerobically stored products is shown in FIG. 22. During anaerobic storage, samples stored at room temperature degraded faster than their equivalents stored at 4°C, with the exception of T/SM samples frozen in liquid nitrogen. T/SM samples frozen in liquid nitrogen and stored at room temperature after freeze-drying did not statistically significantly lose more cells than their equivalent stored at 4°C over a 16-week storage period (P > 0.1). However, differences between samples became significant between storage at room temperature and 4°C after 20 weeks of storage (P=0.0002), with a difference of 0.84 log after 20 weeks and a difference of 0.89 log after 24 weeks of storage. All M/SM specimens degraded faster than their T/SM equivalent. After 24 weeks of storage (FIG. 23), cell loss in T/SM samples frozen in liquid nitrogen and stored at 4° C. under anaerobic conditions was significantly lower than with any of the other treatments (P < 0.02). T/SM samples frozen in liquid nitrogen and stored at 4°C under anaerobic conditions had a loss of 2.16 log compared to the concentration of M. elsdenii observed before freeze-drying, with a loss of 0.82 log after 24 weeks storage period. For each day of sampling, a growth curve experiment was performed to compare the growth characteristics of the sublimated products with the non-sublimated product. FIG. 24 shows growth curves obtained from samples frozen in liquid nitrogen, freeze-dried using a fast cycle (18.5 hours at 250 mTorr (33 Pa)), and stored anaerobically at 4° C. or room temperature. The non-sublimated samples used for each growth curve were "fresh" (not older than 2 days). The freeze-dried product stored at 4°C had a shorter latency period than the freeze-dried product stored at room temperature, consistent with the difference in M. elsdenii concentration observed in these samples (Table 4). After 16 weeks of storage, T/SM samples, regardless of storage temperature, had a shorter latency period than M/SM samples.

Table 4. Latent period observed for a non-freeze-dried or rehydrated freeze-dried sample prepared from a 90% volume reduction retentate blended with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/ SM) frozen in liquid nitrogen (LiqN) and freeze-dried using a fast cycle after 12, 16, 20 or 24 weeks of anaerobic storage at 4 or 25°C

Latent period (hour) after X weeks of storage 12 16 20 24 Average Art. off Lactipro 2.50 2.75 3.75 3.75 2.69 0.72 M/SM stored at 25°C 8.50 9.00 10.00 11.75 9.81 1.24 T/SM stored at 25°C 6.50 6.75 7.25 8.00 7.13 0.57 M/SM stored at 4°C 5.75 8.25 8.00 8.75 7.69 1.15 T/SM stored at 4°C 4.25 5.25 5.00 4.75 4.81 0.37

[0198] The slopes of the non-sublimated and MSM-treated samples kept at 25°C for 12 weeks and the MSM-treated samples kept at 20°C for 16 weeks were abnormally low (Table 5). After 20 and 24 weeks of storage, the slopes of the curves in the exponential phase for freeze-dried samples did not differ quantitatively from the non-sublimated control.

Table 5. Exponential phase slopes observed for non-freeze-dried or rehydrated freeze-dried sample prepared from 90% volume reduction retentate blended with 8% trehalose/15% skimmed milk (T/SM) or 8% maltodextrin/15% skimmed milk (M/SM) frozen in liquid nitrogen (LiqN) and freeze-dried using a fast cycle after 12, 16, 20 or 24 weeks of anaerobic storage at 4 or 25°C

Slopes of curves in the exponential phase after X weeks of storage 12 16 20 24 Average Art. off Lactipro 0.22 0.31 0.34 0.31 0.30 0.04 M/SM stored at 25°C 0.24 0.18 0.33 0.29 0.26 0.05 T/SM stored at 25°C 0.31 0.30 0.34 0.32 0.32 0.02 M/SM stored at 4°C 0.30 0.33 0.30 0.30 0.31 0.01 T/SM stored at 4°C 0.32 0.31 0.33 0.32 0.32 0.01

EXAMPLE 6

M. elsdenii fresh cut forage treatment changes organic acid profile in corn silage

A. Method for ensiling fresh cut forage and measuring dry matter, pH, VFA, lactic acid and acetic acid in samples

[0199] Whole grain corn (plant material) containing approximately 39% dry matter was reaped and treated with Megasphaera elsdenii or left untreated (control). The plant material was placed in four silage bags (100-125 tons/bag). Plant material was treated with either 2×10 ⁹ cfu/ml of Megasphaera elsdenii cells (i.e. Megasphaera elsdenii strain NCIMB 41125, MSBiotec®, Wamego, Kansas) injected at a rate of 50 ml per ton of plant material via the built-in forage header aerosol applicator, or left untreated (control). Treatment with M. elsdenii resulted in approximately 100 billion M. elsdenii colony forming units (CFU) per tonne of fresh plant material. The silage was then packed into silage bags.

[0200] After 120 days of ensiling, a forage probe (30” (76 cm) long) was used to extract 12 silage samples from each. Samples were taken from 12 points evenly distributed along the length of each bag, placed in foil sachets, which were sealed using vacuum and stored at -20°C in the freezer. Two sub-samples were taken from each foil sachet. The first was used to determine the dry matter content by weighing approximately 200 g into a foil cuvette and placing it in a forced ventilation oven at 105° C. for 24 hours. The remainder was pressed using a lever press to extract the liquid, and the pH of the liquid extract was measured. Four ml of the liquid extract was combined with 1 ml of a 25% (w/v) metaphosphoric acid solution in 10 ml conical tubes and frozen to facilitate deproteinization of the samples.

[0201] The extracts were then thawed, homogenized and centrifuged at 14,000 x g for 15 minutes. The clear supernatant was transferred to chromatographic vials and volatile fatty acids (VFA) concentrations were determined using an Agilent 7890 gas chromatograph equipped with a flame ionization detector and a capillary column (Nukol, 15 m long × 0.5 mm diameter × 0.5 µm film thickness ) using hydrogen as the carrier gas.

[0202] The colorimetric method was used to analyze the concentrations of lactic acid in liquid extracts. Briefly, lactic acid standards were prepared at concentrations of 0, 2.5, 5, 10, and 15 mg/dL. Deionized water was combined with the acidified liquid extract to obtain a 40:1 dilution. The resulting solutions (0.5 ml) were combined with 0.5 ml of 20% CuSO ₄ ·5H ₂ O, 4 ml of deionized water and 0.5 g of Ca(OH) _2. The solutions were vigorously shaken and left to stand for approximately 30 min, then centrifuged at 1000 xg for 10 min, after which 0.5 ml of the supernatant was transferred into a glass tube containing 0.25 μl of 4% CuSO ₄ ·5H ₂ O. Three ml of concentrated sulfuric acid was added to the solutions, and the solutions were homogenized using shaking mixer, heated in a boiling water bath for 5 minutes and then cooled to below 20°C by immersion in an ice bath. After cooling the solution, 50 μl of p-hydroxydiphenol was added, the tubes were shaken, heated in a water bath at 30°C for 30 min, then returned to the boiling water bath for 1.5 minutes to burn off excess alkali. The tubes were cooled in an ice bath and 30 μl of the solutions were pipetted into one of the wells in a 96-well plate. The absorbance was determined at a wavelength of 560 nm using a plate reader. Values were evaluated using computer software based on a regression curve derived from 5 standards.

[0203] All data was analyzed using the MIXED procedure from SAS (version 9.2). Treatment was used as a fixed effect and repetition was used as a random effect. The least squares means were compared using the pdiff function in SAS.

B. Results for dry matter content, pH, VFA content, lactic acid and acetic acid in samples

[0204] Corn silage made using Megasphaera elsdenii contained slightly less dry matter than control silage (38.8% vs 40.3%; P<0.02) (Table 6). Additional fermentation characteristics are shown in Table 6. Silage pH was slightly lower for Megasphaera elsdenii treated silage compared to control (untreated) silage (3.77 vs 3.81; P=0.01). Compared to control silage, product treated with Megasphaera elsdenii contained higher concentrations of propionate (6.1 mM vs 10.0 mM; P=0.04), isobutyrate (3.6 mM vs 5.1 mM; P <0.01) and total volatile fatty acids (297.6 mM vs 359.1 mM; P<0.001), but contained less lactic acid (4.36 M vs 4.75 M; P=0.01). Most of the volatile fatty acids present in both silages consisted of acetic acid, which was higher in corn silage treated with Megasphaera elsdenii (342.6 mM vs 286.6 mM; P<0.001; figure 25).

Table 6. Fermentation characteristics of liquid extracts from silage treated with Megasphaera elsdenii. Organic acid concentrations and pH values reflect the characteristics of liquid extracts after 120 days of ensiling.

Control Megasphaera SEM P value Volatile fatty acid, mM Total 297.6 359.1 17.95 <0.001 Propionate 6.13 10.00 1.815 0.04 Isobutyrate 3.55 5.08 0.898 <0.01 Butyrate 0.76 0.93 0.281 0.54 lactate; M 4.75 4.36 0.14 0.01 pH 3.81 3.77 0.016 0.01 Dry matter, % 40.3 38.8 0.66 0.02

[0205] Corn silage treated with Megasphaera elsdenii had a lower pH and lower lactic acid concentration, but contained higher concentrations of volatile fatty acids, compared to the untreated control group. Most notably, acetic acid concentrations were greater for silages treated with Megasphaera elsdenii, which could improve the aerobic stability of corn silage when the silage is exposed to ambient air and aerobic organism activity increases, potentially spoiling the silage (finishing phase). It is believed that silos with increased concentrations of acetic acids tend to improve the aerobic stability of the silage. Treatment of plant material with Megasphaera elsdenii increased acetic acid concentrations by approximately 20% compared to the untreated control (FIG. 25), indicating that this treatment increased the aerobic stability of the treated silage.

EXAMPLE 7

Treatment of reconstituted high moisture corn with M. elsdenii alters fermentation characteristics compared to untreated control

A. Method for Ensiling Reconstituted High Moisture Corn and Measuring Dry Matter Content, pH, VFA Content, Lactic Acid, Acetic Acid, and Digestibility in Samples

[0206] The corn kernel was dry-packed to a geometric mean particle size of approximately 3000 microns (3 mm) and then combined with water to achieve a final moisture content of 32%. Experimental treatments consisted of untreated control (Control) and corn treated with Megasphaera elsdenii strain NCIMB 41125 (MS Biotec, Wamego, KS). Megasphaera elsdenii was pre-bred in an anaerobic diluent and added to the grain as an aerosol, yielding approximately 45 million CFU per pound of grain. The mixture was then homogenized by grinding for 3 minutes using a planetary mixer (Univex M20, Salem, NH). The ground mixtures were then divided into 2 lb (0.9 kg) aliquots, which were then placed in a foil sachet and sealed using vacuum. This process was repeated four times for each treatment. Bags within each treatment and repeat were distributed for the duration of the fermentation of 0, 7, 14, 21 or 28 days. Day 0 bags were immediately placed in the freezer for storage. The rest of the bags were placed in an isothermal chest and stored for the respective ensiling periods of 7, 14, 21 or 28 days. After ensiling, the bags were removed from the chest and placed in the freezer.

[0207] After fermentation, the pH of the resulting products was determined by mixing 25 grams of sample (based on dry matter) with 70 ml of distilled water. The mixtures were placed in an orbital shaker at 100 rpm for 30 minutes and then filtered through fibrous paper. The permeate was collected and the pH was determined using a calibrated pH meter. 4 ml of the permeate was then combined with 1 ml of a 25% metaphosphoric acid solution, homogenized using a shaking mixer and frozen. The samples were then thawed, homogenized using a shaking mixer, and 2 ml fractions of liquid were transferred to centrifuge tubes and centrifuged at 14,000×g for 15 minutes. The supernatant was removed and placed in chromatographic vials. The concentrations of volatile fatty acids, including acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate and heptanoate, were determined by gas chromatography using an Agilent 7890 gas chromatograph equipped with a flame ionization detector and a Nukol capillary column (length 15 m × diameter 0.53 mm × film thickness 0.50 µm). Samples were measured in duplicate.

[0208] Colorimetric laboratory assays were used to determine the concentration of lactic acid in corn extracts. Lactic acid standards were prepared at concentrations of 0, 2.5, 5, 10, and 15 mg/dl. The filtered, acidified and centrifuged permeate was diluted by combining 20 parts of distilled water with 1 part of the permeate, and homogenizing using a shaking mixer. The diluted sample (0.5 ml) was mixed with 4 ml of deionized water, 0.5 g of calcium hydroxide and 0.5 ml of a 20% copper sulfate solution. The mixtures were centrifuged for 10 minutes at 1000×g. The resulting supernatant (0.5 ml) was mixed with 25 μl of a 4% copper sulfate solution and 3 ml of sulfuric acid, stirred, and immediately placed in a boiling water bath for 5 minutes. The samples were then placed in an ice bath and cooled to room temperature. Fifty (50) μl of p-hydroxybiphenyl was added to the solution, the mixtures were homogenized using a shaking mixer and then placed in a bath at 86°F (30°C) for 30 minutes. The samples were then placed in a boiling water bath and removed from the bath after 90 seconds and allowed to cool to room temperature. Two hundred (200) μl of the solution was placed in a well of a 96-well plate and the absorbance was read at 560 nm using a plate reader. Samples were analyzed in quadruplicate.

[0209] The digestibility of corn treated with Megasphaera elsdenii was evaluated using an in vitro culture system. Rumen juice was collected from cannulated Jersey mixed breed bulls kept at the Kansas State University Beef Cattle Research Center. The rumen juice was filtered through cheesecloth, poured into a separating funnel, sparged with nitrogen gas, and placed in an incubator at 102°F (39°C) for approximately 45 minutes to ensure separation into layers. The bottom layer was discarded and the middle layer was collected and used as an inoculum of microorganisms for in vitro assays. Ten (10) ml of filtered rumen juice was added to culture bottles containing 140 ml of McDougall's buffer and 3 grams of grain (based on dry matter). The contents were sparged with nitrogen, the bottles were sealed using ANKOM pressure sensitive modules (ANKOM RF Gas Production System, Macedon, NY), placed in an orbital incubator at 102°F (39°C) and shaken continuously for 16 hours. At the end of the incubation cycle, the bottles were removed from the shaker and the pH was measured using a calibrated pH meter. Four (4) ml of the liquid layer was transferred into vials, mixed with 1 ml of a 25% metaphosphoric acid solution, homogenized using a shaking mixer, and then placed in a freezer. The remaining contents of each bottle were emptied into a flat aluminum cuvette and dried at 221°F (105°C) for 48 hours. The weight of the dried mass was recorded, and the disappearance of dry matter was determined for each culture. Organic acid profiles were determined for each crop by gas chromatography using the methods described previously for grain extracts.

B. Results for dry matter content, pH, VFA content, lactic acid and acetic acid in samples

[0210] Changes in grain pH during the 28-day ensiling period are shown in FIG. 26. Treatment of grain with Megasphaera elsdenii resulted in a faster decrease in pH from day 0 to 7 compared to control (untreated) grain (P < 0.01), and pH differences persisted on days 14, 21 and 28 of ensiling (Effect processing, effect of time, and relationship between processing and days of ensiling, P < 0.01). Organic acid profiles for grain are summarized in Table 7. Compared to control grain, total volatile fatty acid concentrations were higher for reconstituted grain treated with Megasphaera elsdenii on days 7 and 14 of ensiling (P < 0.05), but did not differ after that (P > 0.50). Acetic acid and propionic acid concentrations were similar for grain ensiled with and without treatment with Megasphaera elsdenii (P > 0.10). The concentrations of isovaleric, valeric, isocaproic, caproic, and heptanoic acids were generally higher in grains treated with Megasphaera elsdenii compared to controls (P < 0.05), but lactic acid concentrations were similar in grains with or without no treatment with Megasphaera elsdenii (P > 0.10).

Table 7 Organic acid concentrations in liquid extracts from reconstituted high moisture corn treated with Megasphaera elsdenii or untreated controls. ‡ = Standard error of the mean. § = probability of treatment effect (P < 0.05). M=Processing effect. D=Ensiling day effect. I=Relationship between processing and ensiling days.

Ensiling days Organic acid, mM 0 7 14 21 28 S.E.M‡ P § value Total concentrations of volatile fatty acids Control 14.2 34.3 40.9 55.3 60.0 10.2 M, D Megasphaera 18.2 59.0 59.4 59.0 56.7 Acetic acid Control 8.3 28.1 34.6 43.5 48.6 7.5 D Megasphaera 11.9 42.1 41.9 42.3 43.7 propionic acid Control 0.6 0.6 0.0 2.6 2.1 1.0 - Megasphaera 0.5 0.0 1.3 0.0 1.6 Butyric acid Control 0.0 0.0 0.0 0.4 1.4 0.3 D Megasphaera 0.0 0.0 0.0 0.0 0.4 0.4 isobutyric acid Control nd nd nd nd nd - - Megasphaera nd nd nd nd nd - - Valeric acid Control 0.0 2.5 2.9 3.6 3.3 1.2 M, D Megasphaera 0.0 8.0 7.5 8.6 5.2 Isovaleric acid Control 0.0 0.9 0.8 1.9 1.5 0.9 M, D Megasphaera 0 3.1 2.9 2.0 1.6 Caproic acid Control 0.0 0.9 0.8 1.0 1.0 0.6 M, D Megasphaera 0.0 2.4 2.3 1.6 1.4 Isocaproic acid Control 0.0 0.0 0.0 0.3 0.0 0.4 M Megasphaera 0.0 0.5 0.5 1.3 0.5 Heptanoic acid Control 5.3 1.4 1.8 2.0 2.1 0.5 M, D Megasphaera 5.9 2.9 2.9 3.1 2.4 Lactic acid Control 1.1 39.8 50.9 52.4 57.5 5.4 D Megasphaera 1.1 42.2 49.7 48.4 51.7

[0211] In vitro dry matter changes are summarized for Megasphaera elsdenii treated corn and untreated corn in FIG. 27. Treatment of corn with Megasphaera elsdenii did not change the susceptibility of the corn grain to microbial degradation compared to untreated control grain. The decrease in dry matter content in crops tended to increase with day of ensiling, but there was no apparent relationship between days of ensiling and maize treatment with Megasphaera elsdenii.

[0212] This example shows that high moisture grain treatment with Megasphaera elsdenii alters the fermentation characteristics of ensiled grain compared to control (untreated) grain. This method constitutes a way to accelerate fermentation activity in high moisture reconstituted grains.

EXAMPLE 8

M. elsdenii treatment of fattened corn silage improves carcass value compared to untreated control

A. Livestock fattening method, feed composition and carcass collection

[0213] Mixed breed bulls (336 head; 633 ± 14.1 lb (287.1 ± 6.4 kg)) were used in a fattening study to evaluate the effect of Megasphaera elsdenii as a silage treatment on growth efficiency. Approximately 24 hours after arrival at the Beef Cattle Research Center, bulls were vaccinated with Bovishield Gold 5 and Ultrabac 7/Somnubac (Zoetis Animal Health, Florham Park, NJ), treated with liquid topical ectoparasites (StandGuard, Elanco Animal Health, Greenfield, IN) were identified using numbered ear tags, implanted with Thylane Component TE-200 cartridges (Elanco Animal Health) and weighed.

[0214] The bulls were stratified by initial weight and distributed, within the stratum, to 28 fattening pens with 14 heads per pen and 14 pens for processing. Treatments consisted of feeder rations (Table 8) derived from untreated corn silage (Control) or corn silage treated with 1 billion colony forming units of Megasphaera elsdenii (NCIMB strain 41125; MS Biotec, Wamego, KS) per ton of fresh forage material. The bulls were given unlimited access to food once a day for 112 days.

Table 8. Composition of diets for preparation for fattening and final fattening. ¹ Moist Corn Gluten Food, Cargill Starches & Sweeteners North America; Minneapolis, MN. ² Megasphaera elsdenii strain NCIMB 41125 was applied to forage in the header using an inline aerosol applicator at a rate of 50 ml of fresh culture per ton of freshly cut forage material. The inoculant provided 100 billion colony forming units per ton of feed. The microbial inoculant was not used to produce control corn silage. ³ Contained soybean meal, limestone, salt, trace elements, vitamins, and nutritional premixes, and provided (based on total dietary DM) 0.25% salt, 0.15 ppm. cobalt, 10 ppm copper, 0.50 ppm iodine, 20 ppm manganese, 0.10 ppm selenium, 30 ppm zinc, 1000 IU/lb vitamin A, 10 IU/lb vitamin E, and 30 grams/tonne rumenzine (Elanco Animal Health). ⁴ Contained limestone, salt, urea, trace elements, vitamins, and nutritional supplement premixes and provided (based on total dietary DM) 0.25% salt, 0.15 ppm. cobalt, 10 ppm copper, 0.50 ppm iodine, 20 ppm manganese, 0.10 ppm selenium, 30 ppm zinc, 1000 IU/lb vitamin A, 10 IU/lb vitamin E, and 30 grams/tonne rumenzine (Elanco Animal Health). Tylosin phosphate (Thylan, Elanco Animal Health) was introduced into the feed at a ratio of 0 or 8 grams/ton during the finishing phase (evenly distributed among treatments in the pre-finishing phase). Ractopamine hydrochloride (Optaflexx, Elanco Animal Health) was added to feed at a rate of 24 grams/tonne during the last 34 days prior to harvest.

Finishing phase Finishing phase Control Megasphaera Ingredient, % dry matter Steam-rolled corn grain - - 57.68 Sweet bran ¹ - - 30.00 Control corn silage (no inoculant) 57.21 - - Corn silage inoculated with Megasphaera elsdenii - 57.21 - alfalfa hay 38.36 38.36 10.00 Additive ³ 4.43 4.43 - Additive ⁴ - - 2.32 Nutrient composition (based on DM) Total protein, % 12.50 12.50 13.30 Useful energy to maintain mass, Mcal/cwt 66 66 0.97 Useful energy for weight gain, Mcal/cwt 40 40 0.66 Neutral detergent fiber, % 39.1 39.1 19.06 Calcium, % 0.65 0.65 0.66 Phosphorus, % 0.27 0.27 0.49

[0215] At the end of the fattening phase, cattle were weighed, re-implanted with Thylane Component TE-200 cartridges, and switched to a general finisher ration (Table 8). Cattle were given unlimited access to feed once a day for a total of 115 days during the finishing phase. At the end of the finishing phase, the pens were weighed and the bulls were transported to a commercial slaughterhouse, where data on fresh carcass weight and incidence of liver abscess were obtained on the day of collection.

[0216] Dorsal area, back fat thickness at ¹² rib, marbling score, and USDA quality and yield scores were collected after 36 hours of refrigeration. Final live weight was calculated as fresh carcass weight divided by typical carcass yield after cutting of 63%, and average daily gain and feed efficiency were calculated using live weight corrected for slaughter weight. Carcass value was determined using a standardized grid constructed using 10-year averages of base price and carcass premiums and discounts as published by the USDA (January 2008 - January 2018).

[0217] Data was analyzed in SAS version 9.2 (SAS Inst. Inc., Cary, NC) using the MIXED procedure for continuous data and the GLIMMIX procedure for categorical data. The finisher treatment was a fixed effect, the block was a random effect, and the pen was the experimental unit.

B. Results for Livestock Growth and Feed Consumption, and Carcass Characteristics

[0218] Efficiency during the feeder and finisher phases of livestock are summarized in Table 9. Average daily gain, dry matter intake, and feed:gain ratio during rearing and finisher phases were similar for cattle fed with training diets. for fattening containing corn silages in the presence and absence of the inoculant (P > 0.10).

Table 9. Pre-finishing and finishing performance of corn silage-fed bulls with or without Megasphaera elsdenii as a silage treatment during the pre-finishing phase (pre-finishing pen). Megasphaera elsdenii strain NCIMB 41125 was applied to the header using an inline aerosol applicator at a rate of 50 ml of fresh culture per tonne of fresh forage material. The inoculant provided 100 billion colony forming units per ton of feed. During the control treatment of silage, no inoculant was introduced. ^¥ Standard error of the mean. ^‡ Final weight calculated as fresh carcass weight divided by standardized meat carcass yield after cut of 63%.

Control Megasphaera ^S.E.M. P value Efficiency of preparation for fattening Initial weight, f 635 632 1.4 0.10 Final mass, f 1010 1005 17.7 0.83 Average daily gain, f 3.39 3.40 0.028 0.71 Dry matter intake, f/day 19.14 19.51 0.411 0.28 Feed: Growth 5.65 5.73 0.092 0.36 Finisher Efficiency Final weight corrected for slaughter weight, f‡ 1361 1377 16.9 0.35 Average daily gain adjusted for slaughter weight, f‡ 3.05 3.24 0.132 0.16 Dry matter intake, f/day 23.72 23.99 0.370 0.48 Feed: Growth Corrected for Carcass Weight‡ 8.03 7.50 0.359 0.16

[0219] Carcass characteristics are shown in Table 10. Individual carcass performance scores were similar for the two treatments (P > 0.10); however, the cumulative impact of non-significant changes in carcass characteristics tended (P < 0.07) to increase total carcass cost for cattle fed silage inoculated with Megasphaera elsdenii compared to cattle fed untreated silage ($1628.68 vs $1597.98) ( FIG. 28). This difference was largely due to non-significant but significant increases in carcass weight for the Megasphaera elsdenii silage group, as well as smaller differences in carcass quality and yield. The inventors of the present invention believe that these differences actually appeared during the fattening phase, but were probably masked by differences in the filling of the gastrointestinal tract. Thus, this experiment shows that feeding livestock with silage treated with Megasphaera elsdenii increases the overall cost of the carcass.

Table 10 Carcass characteristics of bulls fed corn silage with or without Megasphaera elsdenii as silage inoculant during the pre-finishing phase (before the finisher pen). ^† Megasphaera elsdenii strain NCIMB 41125 was applied to the header using the inline aerosol applicator at a rate of 50 ml of fresh culture per tonne of fresh forage material. The inoculant provided 100 billion colony forming units per ton of feed. During the control treatment of silage, no inoculant was introduced. ^¥ Standard error of the mean. ^‡ Marbling index determined by computer visualization system (VBG 2000, E+V Technology GmbH & Co. KG, Oranienburg, Germany). Small (400-499). ^§ Consists of USDA or commercial graded carcasses.

Control Megasphaera ^S.E.M. P value Weight of steam carcass, f 857 868 9.1 0.23 The thickness of the spinal fat at the ^12th rib, inch 0.53 0.53 0.019 0.94 Dorsal pulp area, inch ² 14.28 14.36 0.200 0.69 Marbling Index ^‡ 407 409 7 0.78 USDA Quality Index, % Gradation "Choice" 50.6 57.3 5.43 0.21 Gradation "Select" 48.2 42.1 5.42 0.26 Gradation below "Select" ^§ 1.2 0.6 0.85 0.15 Total USDA exit rate, % 2.42 2.44 0.090 0.86 Yield 1, % 10.4 12.4 6.71 0.77 Yield 2, % 46.3 38.7 8.60 0.38 Yield 3, % 34.7 42.2 7.96 0.34 Yield 4, % 8.0 6.0 2.91 0.50 Yield 5, % 0.6 0.7 0.85 0.99 Liver abscess, % 12.7 16.7 5.20 0.44

Claims (18)

1. A method for obtaining ensiled plant material with improved aerobic stability, including: (a) introducing an effective amount of M. elsdenii NCIMB 41125 cells into the plant material, and (b) ensiling the plant material to obtain ensiled plant material. 2. The method of claim 1 wherein the improved aerobic stability includes a reduced pH. 3. A method for obtaining an increased amount of ensiled plant material, including: (a) introducing an effective amount of M. elsdenii NCIMB 41125 cells into the plant material, and (b) ensiling the plant material to obtain ensiled plant material. 4. The method according to any one of claims 1 to 3, further comprising adding the additive to the plant material. 5. The method according to claim 4, where the introduction is carried out before the collection of plant material, after the collection of plant material, during ensiling, or a combination of both. 6. The method of claim 4 or 5, wherein the additive is selected from the group consisting of: another microorganism, an enzyme, a fermentable substrate, an acid, a preservative, a nutrient, and combinations thereof. 7. The method according to any one of claims 1-6, wherein the plant material is selected from the group consisting of: forage, crop, grass, legumes, grains, fruits, vegetables, or combinations thereof. 8. The method of claim 7 wherein the plant material is corn, alfalfa, wheat, rye, barley, oats, triticale, millet, clover, sorghum, or combinations thereof. 9. A method according to any one of claims 1 to 8, comprising administering M. elsdenii NCIMB 41125 cells in liquid. 10. The method according to any one of claims 1 to 9, wherein the method further comprises mixing freeze-dried M. elsdenii NCIMB 41125 cells with liquid prior to cell administration. 11. A method according to any one of claims 1 to 8, comprising administering M. elsdenii NCIMB 41125 cells in the form of freeze-dried cells. 12. The method of claim 11 wherein the freeze-dried cells are encapsulated. 13. The method according to claim 11, where the dry carrier contains freeze-dried cells. 14. The method of any one of claims 1-13, comprising administering at least about 10 ⁶ to about 10 ¹⁴ CFU of M. elsdenii NCIMB 41125 cells per ton of plant material.

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