阅读说明：本技术 使用埃氏巨型球菌生产青贮的植物材料的方法 (Method for producing ensiled plant material using giant coccus aegypti ) 是由 J·S·德鲁拉德 K·A·米勒 C·C·阿佩尔斯 T·M·霍恩 T·J·埃勒曼 G·R·赫 于 2018-10-19 设计创作，主要内容包括：本发明涉及使用厌氧细菌埃氏巨型球菌生产青贮的植物材料的方法及其青贮的植物材料。(The present invention relates to a method for producing ensiled plant material using the anaerobic bacterium megacoccus aegypti and ensiled plant material thereof.)

1. A method of producing ensiled plant material with improved aerobic stability, the method comprising:

(a) applying an effective amount of a cell of giant coccus aegypti (m.elsdeniii) to the plant material, and

(b) ensiling the plant material to produce an ensiled plant material,

wherein the ensiled plant material comprises improved aerobic stability compared to a control ensiled plant material produced in the absence of E.coli cells.

2. The method of claim 1, wherein the improved aerobic stability comprises a reduced pH.

3. A method of producing an increased amount of ensiled plant material, the method comprising:

(a) applying an effective amount of giant enterococcus ehmitis cells to a plant material, and

(b) ensiling the plant material to produce an ensiled plant material,

wherein the amount of ensiled plant material produced is increased compared to the amount of control ensiled plant material produced in the absence of E.coli cells.

4. A method of producing ensiled plant material, the method comprising:

(a) applying a giant coccus aegythi cell to a plant material, wherein the cell is selected from the group consisting of: ATCC @25940, ATCC @17752, ATCC @17753, NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof, and

(b) ensiling the plant material to produce an ensiled plant material.

5. The method of any one of claims 1 to 4, further comprising applying an additive to the plant material.

6. The method of any one of claims 1 to 5, wherein the applying is before harvesting the plant material, after harvesting the plant material, upon ensiling, or a combination thereof.

7. The method of claim 5 or 6, 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.

8. The method of any one of claims 1 to 3 or 5 to 7, wherein the E.maxima cells are selected from the group consisting of: ATCC @25940, ATCC @17752, ATCC @17753, NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof.

9. The method of any one of claims 1 to 8, wherein the giant coccus aegythii cell is a giant coccus aegythii NCIMB41125 cell.

10. The method of any one of claims 1 to 9, wherein the plant material is selected from the group consisting of: feed, crops, grasses, legumes, grains, fruits, vegetables, or combinations thereof.

11. The method of claim 10, wherein the plant material is corn, alfalfa, wheat, rye, barley, oats, triticale, millet, clover, sorghum, or combinations thereof.

12. The method of any one of claims 1 to 11, comprising applying the megacoccus aegypti cells in a liquid.

13. The method of any one of claims 1 to 12, wherein the method further comprises mixing the freeze-dried E.

14. The method of any one of claims 1 to 11, comprising applying the megacoccus aegythii cells as freeze-dried cells.

15. The method of claim 14, wherein the freeze-dried cells are encapsulated.

16. The method of claim 14, wherein a dried carrier comprises the freeze-dried cells.

17. The method of any one of claims 1 to 16, comprising applying at least about 10 per ton of plant material⁶To about 10¹⁴CFU of giant coccus aegypti cells.

18. Ensiled plant material produced by the method of any one of claims 1 to 17.

Technical Field

The present invention relates to a method for producing ensiled plant material using the anaerobic bacterium Megasphaera elsdenii. The invention also relates to ensiled plant material produced by the method.

Background

Ensiling is a fermentation process for storing plant material (e.g., feed and/or grain) consumed by animals, such as livestock. Stored plant material produced by the ensiling process is referred to as ensiled plant material. Examples of ensiled plant material include, but are not limited to, ensiled feed (also known as silage), ensiled grain (also known as fermented grain), and combinations of ensiled feed and grain.

Typically, the plant material is collected and sealed in a silo, which is any container that can maintain an anaerobic environment. Initially, the atmospheric oxygen entrained in the silo is reduced by the respiratory activity of the plant material and aerobic microorganisms. Fermentation begins when the conditions in the silo change to anaerobic conditions. This may be assisted by methods including compacting the plant material and preventing air from entering the silo.

By lactic acid fermentation in connection with lactic acid bacteria present on plant material, the growth of undesirable microorganisms such as clostridium, enterobacter and yeast during ensiling can be inhibited. Under favourable ensiling conditions, lactic acid bacteria will acidify the plant material and prevent competitive growth of undesirable microorganisms. However, if pH control is not successful during ensiling, undesirable microorganisms will proliferate and degrade amino acids, resulting in ensiled plant material of lower nutritional value. In addition, after opening the silo and exposing to air, further degradation of the ensilaged plant material may occur.

Thus, there is a need for improved methods of producing ensiled plant material.

Background

Summary of The Invention

The present disclosure relates to a method of producing ensiled plant material with improved aerobic stability comprising: (a) applying an effective amount of E.coli cells to a plant material, and (b) ensiling the plant material to produce an ensiled plant material, wherein the ensiled plant material comprises improved aerobic stability compared to a control ensiled plant material produced in the absence of E.coli cells.

In certain embodiments, the improved aerobic stability comprises a reduced pH.

The present disclosure relates to a method of producing increased amounts of ensiled plant material comprising: (a) applying an effective amount of E.coli cells to the plant material, and (b) ensiling the plant material to produce ensiled plant material, wherein the amount of ensiled plant material produced is increased compared to the amount of control ensiled plant material produced in the absence of E.coli cells.

The present disclosure relates to a method of producing ensiled plant material comprising: (a) applying a giant coccus aegythi cell to a plant material, wherein the cell is selected from the group consisting of: ATCC @25940, ATCC @17752, ATCC @17753, NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof, and (b) ensiling the plant material to produce ensiled plant material.

In certain embodiments, any of the methods further comprises applying an additive to the plant material.

In certain embodiments, the applying is before harvesting the plant material, after harvesting the plant material, at ensiling, or a combination thereof.

In certain embodiments, 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.

In certain embodiments, the megacoccus aegypti cell is selected from the group consisting of: ATCC @25940, ATCC @17752, ATCC @17753, NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof.

In certain embodiments, the megacoccus aegypti cell is a megacoccus aegypti NCIMB41125 cell.

In certain embodiments, the plant material is selected from the group consisting of: feed, crops, grasses, legumes, grains, fruits, vegetables, or combinations thereof.

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

In certain embodiments, any of the methods further comprises applying the megacoccus aegypti cells in a liquid.

In certain embodiments, any of the methods further comprises mixing the freeze-dried megacoccus aegythii cells with a liquid prior to applying the cells.

In certain embodiments, any of the methods further comprises applying the megacoccus aegythii cells as freeze-dried cells.

In certain embodiments, the dried carrier comprises freeze-dried cells.

In certain embodiments, any of the methods further comprises applying at least about 10⁶To about 10¹⁴CFU of megacoccus aegypti cells per ton of plant material.

The present disclosure relates to ensiled plant material produced by any of the methods.

Drawings

Figure 1 shows the temperature (° c) of plant material measured hourly over a 120 day period of ensiling. The "macrococcus" and "control" groups in FIGS. 1-14 are from a ratio of 50 milliliters per ton of plant material, respectively2x10 of⁸CFU/ml E.coli cells (E.coli strain NCIMB41125,wamego, Kansas) and plant material from untreated fresh corn ("control").

Figure 2 shows the cumulative weight loss (%) in the "supercooccus" and "control" groups at days 7, 14, 21, 28, 39, 90 and 120 of ensiling, as determined by weighing the bins containing plant material on the day and comparing with their respective starting weights at day 0 of ensiling. Effect of treatment, P < 0.01; effect of days, P > 0.10; treatment interacts with days, P > 0.10; standard error of mean 3.16.

FIG. 3 shows the average pH of samples taken from the "macrococcus" and "control" groups. Samples were taken from the silos on three open days (i.e., the days the silos were opened), including day 0 ("D0"), day 14 ("D14"), and day 120 ("D120") of ensiling. No effect of treatment, P > 0.6; standard error of mean 0.02.

FIG. 4 shows the average Volatile Fatty Acid (VFA) concentration (in millimoles) in samples taken from the "macrococcus" and "control" groups as described in FIG. 3. "SEM" is the standard error of the mean. "D" is open day effect, P < 0.05.

FIG. 5 shows the temperature (. degree.C.) measured for 14 days of exposure to ambient air in the "maxima" and "control" samples collected on day 14 of the open day as described in FIG. 3. At 72-110 hours, the temperature of "giant coccus" was higher than "control", P < 0.05. Treatment interacts with time, P < 0.01; effect of time, P < 0.01; effect of treatment, P < 0.01; standard error of the mean is 0.023.

Figure 6 shows the average pH of the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 14 of the open day as described in figure 3. Treatment interacts with days, P < 0.05; effect of time, P < 0.01; effect of treatment, P < 0.05; on day 14, treatments indicated by asterisks (—) differ from each other, P < 0.05; the standard error of the mean is 0.07.

Figure 7 shows the average millimolar ("mM") concentration of total volatile fatty acids in the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 14 of the open day as shown in figure 3. Figure 7 also shows the average concentration of volatile fatty acids in the pre-ensiled samples. Treatment interacts with the open day, P > 0.5; effect of inoculum, P > 0.1; effect on open days, P < 0.6; standard error of mean 6.65.

Figure 8 shows the average millimolar concentrations of volatile fatty acids ("VFA") in the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 14 of the open day as described in figure 3. "D" is open day effect, P < 0.05.

FIG. 9 shows the average weight in pounds ("lb") on days 0, 7 and 14 of exposure to ambient air for each "maxima" and "control" sample collected on day 14 of the open day as described in FIG. 3. Treatment interacts with days, P > 0.9; effect of days, P > 0.1; effect of treatment, P > 0.6; standard error of mean is 0.16.

FIG. 10 shows the temperature (. degree.C.) measured at 14 days of exposure to ambient air for the "maxima" and "control" samples collected on day 120 of the open day as described in FIG. 3. At 259-294 hours, the temperature of "macrococcus" was higher than that of "control", P < 0.10. Treatment-time interaction, P > 0.10; effect of time, P < 0.01; effect of treatment, P < 0.01; the standard error of the mean is 1.23.

Figure 11 shows the average pH of the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 120 of the open day as described in figure 3. Treatment interacts with days, P > 0.1; effect of time, P > 0.1; effect of treatment, P > 0.1; the standard error of the mean is 0.23.

Figure 12 shows the average millimolar ("mM") concentration of total volatile fatty acids in the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 120 on the open day as described in figure 3. Figure 12 also shows the average concentration of volatile fatty acids in the pre-ensiled samples. The treatment interacts with oxygen contact, P > 0.5; effect of inoculum, P > 0.15; effect on days of oxygen exposure, P < 0.01; standard error of mean 18.4.

Figure 13 shows the average millimolar concentrations of volatile fatty acids ("VFA") in the "macrococcus" and "control" samples measured at day 0 ("D0") and day 14 ("D14") of exposure to ambient air after collection on day 120 on the open day as described in figure 3. "D" is the effect on days of oxygen exposure; "I" is the interaction effect; "T" is the treatment effect; the letters indicate P < 0.05.

FIG. 14 shows the average weight in pounds ("lb") on days 0, 7 and 14 of exposure to ambient air for each "maxima" and "control" sample collected on day 120 of the open day as described in FIG. 3. Treatment interacts with days, P > 0.90; effect of days, P > 0.6; effect of treatment, P < 0.01; standard error of mean is 0.10.

FIG. 15 shows the effect of storage temperature on the viability of the E.coli NCIMB41125 liquid culture over a 28 day period, in terms of the yield of colony forming units per milliliter ("CFU/mL") of E.coli cells on a logarithmic ("Log 10") ratio.

FIG. 16 shows viability of liquid cultures of E.aegypti NCIMB41125 after 0, 7, 14, 21 and 28 days of storage at room temperature, as a function of yield of E.aegypti cells in terms of CFU/mL at Log10 ratio.

FIG. 17 shows a schematic of a tangential flow filtration ("TFF") system.

FIG. 18 shows the yield of E.coli cells in CFU/mL of retentate on the Y axis at the Log10 ratio after processing by the TFF system. The x-axis represents 70%, 80% or 90% volume reduction by the system. "target" refers to the theoretical recovery of giant enterococcus ehmitis after TFF processing. "conc." refers to the actual recovery of E.coli cells from the retentate, which is the volume of concentrated cells remaining after the indicated volume reduction.

FIG. 19 shows cell loss after freezing the cells at-80 ℃ or in liquid nitrogen (LiqN) and freeze-drying the cells using slow (38 hours at 135 mTorr) or fast (18.5 hours at 250 mTorr). Cells were from cultures with 8% maltodextrin/15% skim milk (M/SM) or 8% trehalose/15% skim (T/SM) as cryoprotectants using 70%, 80% or 90% volume reduced retentate of TFF.

FIG. 20 shows the yield of E.coli NCIMB41125 cells after initial rapid (liquid nitrogen) or slow (-20 ℃) freezing followed by lyophilization (in the presence of 15% maltodextrin and 0% or 7.5% trehalose).

FIG. 21 shows the cell loss observed in giant cells of E.aegypti NCIMB41125 frozen at-80 ℃ or in liquid nitrogen (LiqN) with or without 4% trehalose or 7.5% skim milk.

Figure 22 shows the cell loss observed on the resulting freeze-dried samples from 90% volume-reduced retentate mixed with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM), frozen at-80 ℃ or in liquid nitrogen (LiqN), and stored under anaerobic conditions for up to 24 weeks using rapid cycle freeze-drying and at room temperature or 4 ℃.

Figure 23 shows the cell loss observed on freeze-dried samples obtained from 90% volume-reduced retentate mixed with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM), frozen at-80 ℃ or in liquid nitrogen (LiqN), and stored under anaerobic conditions for 24 weeks at room temperature or 4 ℃ using rapid cycle freeze-drying. Bars without common superscript are statistically different, P < 0.02.

Figure 24 shows growth curves performed on non-freeze-dried or rehydrated freeze-dried products that were obtained after mixing of 90% volume-reduced retentate with 8% trehalose/15% skim milk (T/SM) or 8% maltodextrin/15% skim milk (M/SM), freezing in liquid nitrogen (LiqN), and storage under anaerobic conditions at 4 or 25 ℃ for 12, 16, 20, or 24 weeks using rapid cycle freeze-drying. The Optical Density (OD) at 600 nanometers (nm) measured from 0 to 20 hours is shown. Yellow-line not freeze-dried, red-line T/SM, blue-line M/SM, dotted-line stored at 4 ℃ and solid-line stored at 25 ℃.

Figure 25 shows the average millimolar concentrations of acetate in liquid extracts of untreated (control/untreated) and E.aegypti treated corn silage after 120 days of silage.

FIG. 26 shows the effect on pH levels of reconstituted high moisture maize with or without treatment with E.coli during ensiling.

FIG. 27 shows the effect of dry matter content in vitro cultures of mixed rumen microorganisms containing reconstituted high moisture maize with or without treatment with E.coli.

Figure 28 shows the effect of carcass value when feeding cattle with megacoccus aegypti treated silage compared to untreated silage during the background phase (before fattening). The value of each carcass is calculated using a standardized grid derived by averaging USDA weekly price data (premium and discount reported over a 10 year period from month 1 of 2008 to month 1 of 2018).

Detailed Description

The present invention relates to methods of producing ensiled plant material using megacoccus aegypti cells. The invention also relates to ensiled plant material produced by the method.

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

Term(s) for

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

To the extent that section headings are used, they are not to be construed as necessarily limiting.

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

As used herein, the term "about," when used to modify an amount associated with the present invention, refers to a change in value that can occur, for example, by conventional testing and handling; by inadvertent errors in such testing and handling; by differences in the manufacture, source or purity of the ingredients used in the present invention; and so on. The claims, whether or not modified by the term "about," include equivalents to the amounts recited. In some embodiments, the term "about" refers to plus or minus 10% of the reported numerical value.

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

The terms "comprising," including, "" containing, "" including, "" having, "and variations thereof are interchangeable and mean" including but not limited to. It should be understood that wherever the language "comprising" is used herein to describe aspects, similar aspects described as "consisting of and/or" consisting essentially of are also provided.

The term "consisting of means" including and limited to.

The term "consisting essentially of" refers to the specified materials or steps of a composition, as well as those other materials or steps that do not substantially affect the essential characteristics of the material or method.

The term "and/or" as used herein is to be understood as a specific disclosure of each of the two named features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone) and "B" (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "plant material" as used herein refers to any plant material that can be used as animal food and can be ensiled, including but not limited to feed, crops, grasses, legumes, grains, fruits, vegetables, or combinations thereof. Reference to "plant material" or a particular type of plant material (e.g., feed, crops, grasses, legumes, grains, fruits, vegetables, or combinations thereof) disclosed herein includes processed plant material (including but not limited to cut, chopped, or trimmed plant material, or combinations thereof), as well as a portion of plant material including but not limited to stems, stalks, leaves, skins, husks, cobs, ears, grains, crop residue, or combinations thereof.

As used herein, the terms "culturing," "for culturing," and "cultured" refer to incubating cells under in vitro conditions that allow the cells to grow or divide or maintain the cells in a viable state. The term "culture" may also be used herein to refer to cells incubated under in vitro conditions (e.g., cells incubated in a liquid growth medium). As used herein, the terms "growth medium" and "culture medium" refer to solid (e.g., agar), semi-solid (e.g., agar), or liquid (e.g., broth) compositions comprising components that support cell growth.

As used herein, the term "additive" 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, improve the performance and/or health of animals, and/or enhance the digestibility of ensiled plant material).

As used herein, the terms "harvest" and "harvested" with respect to cells refer to collecting cells from a culture, e.g., collecting cells in a growth medium from a culture, collecting cells by removing an amount of growth medium from a culture (e.g., by concentrating cells in a liquid culture or separating cells from a growth medium), or stopping a cell culture. The term includes collecting or removing a volume of liquid containing cells from a liquid culture, including a volume in which the cells have been concentrated. The terms "harvesting", "harvested" and "collecting" as used herein with respect to plant material refer to the collection of plant material by any manual or mechanical means.

As used herein, the term "isolated" does not necessarily reflect the degree to which an isolate has been purified, but rather denotes separation or isolation from the native form or native environment. An isolate may include, but is not limited to, an isolated microorganism, an isolated biomass, or an isolated culture.

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

As used herein, "excipient" refers to a component or mixture of components that is used to impart desired characteristics to the ensiled plant material disclosed herein. The excipients of the present invention may be described as "pharmaceutically acceptable" excipients, meaning that the excipients are compounds, materials, compositions, salts, and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of animals (i.e., human and non-human animals) without excessive toxicity, irritation, allergic response, or other problematic complications for the desired duration of contact, and at a reasonable benefit/risk ratio.

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

As used herein, the term "viable" refers to one or more living organisms (e.g., a living microbial cell or a plurality of living microbial cells). "viability" refers to the ability to survive, especially under certain conditions.

As used herein, "purified," "purified," and "purifying" refer to being substantially pure or clear apart from unwanted components, material contamination, blending, or imperfections.

The terms "invention" and "disclosure" may be used interchangeably when, for example, described or used in the phrases "invention" or "disclosure".

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiments are inoperable without those elements.

Although 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 not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Method for producing ensiled plant material

In one aspect, the present invention relates to a method of producing ensiled plant material with improved aerobic stability comprising: (a) applying an effective amount of E.coli cells to a plant material, and (b) ensiling the plant material to produce an ensiled plant material, wherein the ensiled plant material comprises improved aerobic stability compared to a control ensiled plant material produced in the absence of E.coli cells. In some embodiments, the improved aerobic stability comprises reduced pH, reduced weight loss, or both.

In another aspect, the invention relates to a method of producing increased amounts of ensiled plant material comprising: (a) applying an effective amount of E.coli cells to the plant material, and (b) ensiling the plant material to produce an ensiled plant material, wherein the amount of ensiled plant material is increased compared to the amount of control ensiled plant material produced in the absence of E.coli cells. The amount of ensiled plant material may be determined, for example, by weighing the ensiled plant material or weighing a container containing the ensiled plant material and subtracting the weight of the container.

In another aspect, the present invention relates to a method of producing ensiled plant material comprising: (a) applying a giant coccus aegythi cell to a plant material, wherein the cell is selected from the group consisting of: ATCC @25940,

NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof, and (b) ensiling the plant material to produce ensiled plant material.

Plant material in the process of the invention includes any plant material that can be ensiled for use in the production of ensiled plant material.

In some embodiments, the plant material in the methods of the invention is any plant material consumed by ruminants. In some embodiments, the ruminant may be, but is not limited to, a cow, a buffalo, a sheep, a goat, a deer, a reindeerMoose, giraffe, yak, and elk. In some embodiments, the ruminant is selected from the group consisting of: cattle, buffalo, sheep, goats, deer and reindeer. In some embodiments, the plant material in the process of the invention is any plant material consumed by a camelid. In some embodiments, the camelid may be, but is not limited to, alpacas (alabacas), llamas, guanacos (guanaco), llamasAnd camels.

In some embodiments, the plant material is selected from the group consisting of: feed, crops, grasses, legumes, grains, fruits, vegetables, or combinations thereof. In some embodiments, the plant material is corn (i.e., maize), alfalfa, wheat, rye, barley, oats, triticale, millet, clover, sorghum, or combinations thereof. In some embodiments, the plant material is crop residue, such as, but not limited to, sorghum, corn stover, or soybean stover. In some embodiments, the plant material is a weed. In some embodiments, the plant material is a mixture of grasses and legumes, including one or more grasses and one or more legumes.

Grass includes, but is not limited to: grass species (Agrostis spp.) -grass (bentgrass) (e.g., thin grass (Agrostis capitis) -common grass-clippings and stolon (Agrostis stolonifera) -stolon grass (creep bentgrass)); sandwort (Andropogon hallii) -saran stem; oat grass (arrhenthermum elatius) -pseudo oat grass; radicis bediensis (bostrichiochlo bladhii) -australian blue stem; glume grass (bothioochloa pertusa) -hurricane grass; brachypodium distachyon (Brachiaria decumbens) -surimi grass; brachiaria humilis (Brachiaria humicola) -Koroney grass; brome species (Bromus spp.) -brome; tribulus terrestris (Cenchrus ciliaris) -Bulbophyllum vulgare; saxifraga africana (Chloris gayana) -Roseby grass; bermuda grass (Cynodon dactylon) -bermudagrass; dactylis glomerata (Dactylis globorata) -orchard grass; pyramid-tare (Echinochloa pyradalis) -antelope grass; entolasia ibrica-bengomasa; festuca spp-Festuca species (Festuca spp.) -Festuca (e.g., Reed fescue (Festuca arundinacea) -Festuca arundinacea, Festuca pratensis-meadow Festuca, and Festuca rubra-Festuca fescue); pycnanthus extorum (Heteropogon continentalis) -Hemicentrotus nigra; lemongrass (Hymenachne amplexicaulis) -marshmallow occidentalis; erythrochloe (Hyporhenia rufa) -Erythrochloe (jaragua); leersia hexandra (Leeria hexandra) -south hay; lolium sp-Lolium perenne (e.g., Lolium multiflorum-italian Lolium perenne and Lolium perenne-perennial Lolium perenne); sorghum saccharatum maximus-Guinea grass; sugarcoated meadow grass (Melinis minutiflora) -sugarcoated meadow grass; paspalum villosum (Paspalum dialatum) -dallas grass (dallisgrass); phalaris arundinacea (phararis arundinacea) -canary reed canary Phalaris arundinacea (reed canarygrass); timothy grass (Phleum pratense) -Thymoxi; poa species (Poa spp.) such as Poa pratensis, Poa pratensis (e.g., Poa Texas Poa (Poa arachneifera) and Poa texatilis, Poa pratensis (Poa pratensis) and Poa cruzi, and Poa pratensis (Poa trivialis) and Poa rough Poa pratensis); setaria (Setaria sphacelata) -Setaria africana; yellow backthern arabic (Themeda triandra) -kangaroo grass; and thinopyrum intermedium (thinopyrum intermedium) -medium wheat grass.

Legumes include, but are not limited to, herbaceous legumes and tree legumes. Herbaceous legumes include, but are not limited to: perennial peanuts (Arachis pintoi) -pint peanuts; cassia tora (chamaecersita rotundifolia) -round leaf sensitive pea; butterflies (clinoria terrata) -butterfly peas; lotus corniculatus (Lotus corniculatus) -bird foot trefoil; all-grass of purple-flowered winged bean (macropurpureus) -purple-flowered cajan; macroptilium brachactatum-burgundy first bean; medicago species (Medicago spp.) -alfalfa (e.g., alfalfa (Medicago sativa) -alfalfa, alfalfa (lucerne) and tribulus alfalfa (Medicago truncatula) -barrel alfalfa); melilotus species (Melilotus pp.) -Melilotus (sweet clovers); neonotton bean (neontonia wightii) -perennial soybean; ormosia (Onobrychis vicifolia) -rubiaceae family; stigmaea species (Stylosanthes spp.) -Stylosanthes (stylo) (e.g. Stylosanthes humileis) -Stylosanthes (Townsville); stylosanthes guianensis (Stylosanthes scabra) -Stylosanthes guianensis; trifolium species (Trifolium spp.) -Trifolium (e.g., Trifolium hybridum-Asperger (alsike) Trifolium, Trifolium purpurea (Trifolium incarnatum) -Trifolium erythrosepala, Trifolium pratense-red, Trifolium pratense (Trifolium pratense) -Trifolium, Trifolium alba (Trifolium repens) -Trifolium alba); and Vicia spp-vetch (e.g. Vicia sativa-unifloral field pea, Vicia ervilia-bitter field pea, Vicia narbonensis-na vetch, Vicia sativa-common field pea, Vicia sativa, Rough field pea (Vicia sativa) -wild field pea); and cowpea parker (Vigna parkeri) -creeping cowpea). Tree leguminous plants include, but are not limited to: acacia (Acacia aneura) -Acacia; albizzia species (Albizia spp.) -silk trees; albizzia (Albizia candescens) -bellmont sieris (Belmont siris); albizzia lebbeck (Albzia lebbeck) -lebbeck (lebbeck); elephant's ear (enterobium cyclocarpum) -elephant's ear; and Leucaena leucocephala (Leucaenaleucocephala) -lead tree (LEAdtree).

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

The plant material may be harvested or collected at any suitable time, for example, when the moisture is at a suitable level. If the moisture level is too high, the plant material may be allowed to wither, for example, until the moisture reaches a suitable level.

In some embodiments, previously dehydrated plant material (e.g., dried grain) can be rehydrated for use in ensiling according to any method of the present invention.

Ensiling in any of the methods of the invention may be carried out according to any standard or known method for ensiling plant material. The plant material in the process of the invention is ensiled in a sealed container, also referred to herein as a "silo", to allow anaerobic fermentation of the plant material.

In some embodiments, the methods of the present invention further comprise applying an additive to the plant material.

In some embodiments, the applying (i.e., applying the E.coli cells, applying the additive, or both) in any of the methods of the invention is prior to harvesting the plant material, after harvesting the plant, at ensiling, or a combination thereof. When both the E.coli and the additive are applied, they may be applied together or separately at the same or different times.

The additive in the process of the invention may be any additive used during ensiling or applied to ensiled plant material.

In some embodiments, the additive in the method of the invention is selected from the group consisting of: microorganisms, enzymes, fermentable substrates, acids, preservatives, nutrients, and combinations thereof.

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

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

In some embodiments, the additive in the methods of the invention is a fermentation-stimulating microorganism, such as, but not limited to, lactic acid bacteria. In some embodiments, the microorganism is Lactobacillus (Lactobacillus), Pediococcus (Pediococcus), Enterococcus (Enterococcus), Propionibacterium (Propionibacterium), Lactococcus (Lactococcus), Streptococcus (Streptococcus), Leuconostoc (Leuconostoc), or Selenomonas (selenimonas). In some embodiments, the microorganism is Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus casei (Lactobacillus casei), Lactobacillus corynebacterium (Lactobacillus cornified), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus viridis (Lactobacillus virescens), Pediococcus acidilactici (Pediococcus acidilactici), Pediococcus pendula (Pediococcus pendula), pediococcus cerevisiae (Pediococcus cerevisiae), Enterococcus faecium (Enterococcus faecalis), Enterococcus faecalis (Enterococcus faecalis), Propionibacterium jensenii (Propionibacterium jensenii), Propionibacterium acidipropionici (Propionibacterium acidipropionici), Propionibacterium freudenreichii (Propionibacterium freudenreichii), Propionibacterium globosum (Propionibacterium globosum), Propionibacterium schermani (Propionibacterium shermanii), Lactococcus lactis (Lactococcus lactis), Streptococcus bovis (Streptococcus bovis), Leuconostoc mesenteroides (Leuconostoc mesenteroides) or Pseudomonas ruminata (Selenorum).

In some embodiments, the additive in the methods of the invention is an inhibitor of fermentation, 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, hexanoic acid, sorbic acid, benzoic acid, sulfuric acid, lactic acid, acrylic acid, calcium formate, propionic acid or propionate salts or other chemical inhibitors, such as but not limited to formaldehyde, paraformaldehyde, sodium nitrite, sodium metabisulfite sulfur dioxide, sodium hydroxide, sodium sulfate, sodium chloride, urea, or ammonia.

In some embodiments, the additive in the methods of the invention is an aerobic spoilage inhibitor, such as, but not limited to, propionic acid, propionate salt, acetic acid, hexanoic acid, or ammonia.

In some embodiments, the additive in the methods of the invention is a nutrient or nutrient source, such as, but not limited to, urea, ammonia, limestone (limestone), or other minerals.

In some embodiments, the additive in the process of the invention is an absorbent such as, but not limited to, grain, straw, bentonite, beet pulp, or polyacrylamide.

In some embodiments, an effective amount of S.aegypti in a method of the invention is at least about 10⁶At least about 10⁷At least about 10⁸At least about 10⁹At least about 10¹⁰About 10⁶To about 10¹⁴About 10⁶To about 10¹³About 10⁶To about 10¹²About 10⁶To about 10¹¹About 10⁷To about 10¹⁴About 10⁷To about 10¹³About 10⁷To about 10¹²About 10⁷To about 10¹¹About 10⁸To about 10¹¹About 10⁹To about 10¹¹About 10¹⁰To about 10¹¹About 10¹⁰To about 10¹²About 10¹⁰To about 10¹³Or about 10¹⁰To about 10¹⁴Colony Forming Units (CFU) per ton of plant material.

In some embodiments, the E.coli megacoccus cells of the methods of the invention are selected from the group consisting of:NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof.

In some embodiments, the megacoccus aegythii cell in the methods of the invention is a megacoccus aegythii NCIMB41125 cell.

In some embodiments, the methods of the invention comprise a megacoccus aegypti cell applied in a liquid.

In some embodiments, the methods of the invention comprise applying the E.coli cells as freeze-dried cells. In some embodiments, the dried carrier comprises freeze-dried cells. In some embodiments, the dry carrier includes, but is not limited to, calcium carbonate, milk powder, or sucrose.

In some embodiments, the methods of the invention further comprise mixing the freeze-dried E.coli cells with a liquid prior to applying the cells.

In some embodiments, the methods of the present invention comprise an additive applied in the liquid. When the E.coli cells and the additive are both applied in a liquid, they may be applied in separate liquids, may be applied in separate liquids at different times or simultaneously, or may be applied in a mixture in the same liquid.

In some embodiments, the methods of the present invention comprise applying the additive in a dry form, such as, but not limited to, a powder, a granule, or a freeze-dried form. When in dry form, the additive may be mixed with a dry carrier. When the E.coli cells and the additive are both applied in dry form, they may be applied in separate dry forms, may be applied in separate dry forms at different times or simultaneously, or may be applied as a mixture of dry forms, including a mixture of dry forms in a dry carrier.

In another aspect, the invention relates to ensiled plant material produced by any of the methods of the invention.

Giant coccus aegypti

Giant coccus aegypti cells from any strain or any combination of strains may be used in the invention described herein.

One or more strains of giant coccus aegypti may be selected from the stock culture collection (e.g., the American type culture collection)

The national center for Industrial, food and Marine bacteria ("NCIMB"), national center for type culture Collection ("NCTC"), the American center for research services ("ARC") culture Collection (i.e., "NRRL"), the National Institute of Animal Health (NIAH) culture Collection), or the strain may be isolated from a natural source (e.g., from the gastrointestinal tract of a ruminant).

Examples of E.coli strains that may be selected from the culture collection include, but are not limited to, the strains listed in Table 1 under the accession number. Alternative names for the deposit numbers are also indicated.

TABLE 1 examples of E.coli strains and the source of each strain.

In some embodiments, the megacoccus aegypti cell is from a strain with a deposit number selected from the group consisting of:NCIMB702261, NCIMB702262, NCIMB 702264, NCIMB702331, NCIMB702409, NCIMB702410, NCIMB41125, NCIMB 41787, NCIMB 41788, NRRL18624, NIAH1102, and combinations thereof, including any of the alternative designations in table 1.

In some embodiments, the megacoccus aegypti cell is from a strain isolated from a ruminant (e.g., a cow). See, for example, U.S. patent No. 7,550,139.

In some embodiments, the E.coli cells are from a strain isolated from a non-ruminant (e.g., human).

In some embodiments, the megacoccus aegypti cells are from a strain selected for lactate utilization (e.g., a strain that utilizes lactate in the presence of a sugar), resistance to ionophore antibiotics, relatively high growth rate, ability to produce primarily acetate, ability to proliferate at pH values below 5.0 and as low as 4.5, production of Volatile Fatty Acids (VFAs), phytase activity, and combinations thereof. See, for example, U.S. patent No. 7,550,139.

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

In some embodiments, the megacoccus aegypti cell is from a strain that has a high growth rate compared to other strains. The growth rate of different strains can be determined, for example, by culturing the cells in liquid medium and monitoring the increase in optical density over time.

In some embodiments, the megacoccus aegypti cells are from a strain capable of producing VFA, which can be determined, for example, by gas chromatography. In some embodiments, the VFA is a 6-carbon fatty acid.

In some embodiments, the megacoccus aegypti cell is from megacoccus aegypti strain NCIMB 41125. The E.coli strain has a high specific growth rate (0.94 generations/hr), is capable of growing at a pH range of 4.5 to 6.5 or higher, uses D-and L-lactate as its preferred substrate, but also has the ability to utilize glucose and other carbohydrates and to tolerate ionophores.

In some embodiments, the megacoccus aegypti cell is from the megacoccus aegypti strain NCIMB 41787. In some embodiments, the giant coccus aegypti is from the giant coccus aegypti strain NCIMB 41788.

In some embodiments, the E.coli cells are from a strain of E.coli

In some embodiments, the E.coli cells are derived from a strain selected from the group consisting of stock culture collections or isolated from natural sources. The cells "derived" from the strain may be natural or artificial derivatives, such as, for example, sub-isolates, mutants, variants or recombinant strains.

In some embodiments, the E.coli cells are commercial preparations, e.g.Products (e.g., Lactpro)

Wamego，Kansas)。

Preparation of a culture comprising E.coli cells

The E.coli cells used herein can be grown in liquid culture and used directly as liquid culture. Alternatively, cells may be isolated from liquid or solid cultures and resuspended in a suitable liquid or lyophilized prior to use.

Giant coccus aegypti is an anaerobic bacterium that must be cultured under strictly anaerobic conditions to obtain maximum yield and viability.

In some embodiments, the culture comprises a megacoccus aegypti cell and a growth medium.

In some embodiments, the culture comprises one or more strains of megacoccus aegypti cells. In some embodiments, the culture comprises a single strain of E.coli megacoccus cells. In some embodiments, the culture consists of one or more strains of megacoccus aegypti cells (i.e., the cells in the culture consist of megacoccus aegypti cells, e.g., one or more strains of megacoccus aegypti cells). In some embodiments, the culture consists of a single strain of E.coli megacoccus cells.

In some embodiments, the methods of the invention may further comprise growing, harvesting, and/or freeze-drying the megacoccus aegypti cells used in the methods.

A variety of fermentation parameters for inoculating, growing, and harvesting the E.coli cells may be used, including continuous fermentation (i.e., continuous culture) or batch fermentation (i.e., batch culture). See, for example, U.S. patent No. 7,550,139.

The growth medium for the E.coli cells may be solid, semi-solid, or liquid. The culture medium may contain nutrients that provide the necessary elements and specific factors to achieve growth. Various microbial culture media and variants are well known in the art. The medium may be added to the culture at any time, including at the beginning of the culture, during the culture, or intermittently/continuously.

Examples of growth media include, but are not limited to: (1) a semi-defined medium comprising peptone, 3 g/L; 3g/L of yeast; 2mL/L of vitamin solution; mineral solution, 25 mL/L; indigo carmine (0.5%), 1 g/L; 12.5% L-cysteine, 2 g/L; 12.5% of sodium sulfide, 2 g/L; and sodium lactate (semi-defined lactate, SDL), glucose (semi-defined glucose, SDG) or maltose (semi-defined maltose, SDM); (2) modified enhanced clostridial agar/broth medium (pre-reduced) containing peptone, 10 g/L; beef extract, 10 g/L; yeast extract, 3 g/L; dextrose 5 g/L; NaCl, 5 g/l; 1g/L of soluble starch; l-cysteine HCL, 0.5 g/L; 3g/L of sodium acetate; and resazurin (0.025%), 4 mL/L; (3) tryptose soy agar/broth with defibered sheep blood; (4) semi-defined rumen fluid medium containing sodium lactate (70%), 10 g/l; peptone, 2 g/L; KH (Perkin Elmer)₂PO₄1g/L；(NH₄)₂SO₄3g/l；MgSO₄7H₂O 0.2g/l；CaCl₂.2H₂O0.06 g/l; vitamins (pyridoxine 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, 0.2mg/l, biotin, 0.2mg/l, folic acid 0.1mg/l and cyanocobalamin, 0.02 mg/1); na (Na)₂S.9H₂O, 0.25 g/l; cysteine, 0.25 g/l; defoamer, 0.07ml/l and monensin, 10 mg/l; and it was prepared by: adding sodium lactate and mineral solution into a liquid storage bottle and autoclaving for 60 minutes; peptone was dissolved in 300ml of distilled H2O and autoclaved separately; filtering and sterilizing the vitamin solution and the two reducing agents in advance; after autoclaving, aerating the liquid storage bottle with anaerobic gas overnight; cooling and adding other components; and adjusting the pH to the desired value with 5N HCl; and (5) incubated rumen fluid lactate ("IRFL") medium containing 400ml of incubated clarified rumen fluid from an alfalfa-fed sheep, 371ml of distilled water, 2g of peptone, 15g of agar, 100ml of a 10% (w/v) solution of D, L-sodium lactate, 100ml of a 0.04% (w/v) solution of bromocresol purple and 25ml of mineral spiritsSubstance solution containing 40g/l KH₂PO₄；120g/l(NH₄)₂SO₄；8g/l MgSO₄.7H₂O and 2.4g/l CaCl₂.2H₂O, wherein the pH is adjusted to 5.5 using lactic acid (90% w/v), autoclaved at 121 ℃ for 25 minutes, then cooled in a water bath at 50 ℃ while aerating with an anaerobic gas mixture, and then two ml each of Na are added₂S.9H₂O (12.5% w/v) and cysteine HCl.H₂O(12.5％w/v)。

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

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 source described herein that is different from the first carbon source), wherein 100% of the at least two carbon sources consist of the first carbon source and the 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, about 50-70% of the first carbon source and about 30-50% of the second carbon source, about 50-80% of the second 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.

In some embodiments, the E.coli cells are cultured at about 39 ℃ to about 40 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, or about 40 ℃.

In some embodiments, the E.coli cells are cooled to about 18 ℃ to about 25 ℃ for storage.

In some embodiments, the pH of the culture comprising the escherichia coli cells (e.g., during culturing and/or at harvest) is about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, about 4.5 to about 5.0, about 4.6 to about 6.9, about 4.7 to about 6.8, about 4.8 to about 6.7, about 4.9 to about 6.6, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.0 to about 5.5, about 5.1 to about 6.9, about 5.2 to about 6.8, about 5.3 to about 6.7, about 5.4 to about 6.6, about 5.5 to about 7.0, about 5.5 to about 6.0, about 5.5 to about 6.5, about 5.5 to about 6.0, about 5.2 to about 5.0, about 5.5.5 to about 5.0, about 5.5 to about 6.0, about 5.5, about 5.5.0, about 5 to about 5.5, about 6.0, about 5.5.5.5 to about 6, about 6.0, about 5.5.5 to about 6, about 6.0, about 5.5.5.0, about 5 to about 6.5.0, about 6.5 to about 6.5.0, about 6.0, about 5.5.5..

To culture the E.coli cells, fermenters of different sizes and designs that maintain anaerobic conditions may be used. The fermentor can, for example, ferment a culture volume sufficient for commercial production of E.coli cells.

In some embodiments, the culture comprising the E.coli cells further comprises another microorganism (i.e., a microbial cell that is not an E.coli cell). In some embodiments, the culture comprises a megacoccus aegypti cell and another obligate anaerobic microorganism. In some embodiments, the culture comprises a megacoccus aegypti cell and another microorganism selected from the group consisting of: lactobacillus, Pediococcus, enterococcus, Propionibacterium, lactococcus, Streptococcus, Leuconostoc or Porphyromonas.

Freeze-dried megacoccus aegypti cells

In some embodiments, the megacoccus aegythii cells used in the methods and ensiled plant materials of the present invention are freeze-dried cells.

The freeze-dried E.coli megacoccus cells can be produced by a method comprising: preparing a culture comprising a megacoccus aegypti cell and a growth medium; harvesting the cells; freezing the cells; and freeze-drying the cells, wherein freeze-dried E.coli cells are produced. In some embodiments, the methods are performed in the order of preparing the culture, then harvesting the cells (i.e., harvesting the cultured cells), then freezing the cells (i.e., freezing the harvested cells), and then freeze-drying the cells (i.e., freeze-drying the frozen cells).

In some embodiments, the method is performed under anaerobic conditions. In some embodiments, the method comprises preparing the culture under anaerobic conditions, harvesting the cells, freezing the cells, freeze-drying the cells, or a combination thereof.

The methods may include any of the methods of preparing a culture described herein. The culture of the method may further comprise any property of the culture described herein.

In some embodiments, the cells in culture comprise E.coli cells. In some embodiments, the cells in culture consist of megacoccus aegypti cells.

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

In some embodiments, the method comprises harvesting the cells within 12 hours after the culture has ended its exponential growth phase. The culture may be cooled to room temperature to stop growth at the time of harvest.

In some embodiments, the culture comprises a liquid, and the method comprises harvesting the megacoccus aegythii cells (e.g., concentrated megacoccus aegythii cells) with a percentage of the liquid. In some embodiments, the method comprises harvesting cells having about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% liquid. In some embodiments, the method comprises harvesting cells having 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 1% liquid.

In some embodiments, the culture comprises a liquid and the method comprises harvesting the megacoccus aegythii cells (e.g., concentrated megacoccus aegythii cells) by removing a percentage of the liquid. In some embodiments, harvesting the cells comprises removing from about 50% to about 100% of the liquid, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, or from about 95% to about 100% of the liquid. In some embodiments, harvesting the cells comprises removing at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the liquid.

In some embodiments, the method comprises harvesting the megacoccus aegythii cells by concentrating the cells. In some embodiments, harvesting the cells comprises concentrating the cells by at least one technique selected from centrifugation, filtration, dialysis, reverse osmosis, and combinations thereof. In some embodiments, the filtering comprises clay filtering. In some embodiments, the filtration comprises tangential flow filtration, also known as cross-flow filtration.

In some embodiments, the pH of the culture comprising the escherichia coli cells at harvest is about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, about 4.5 to about 5.0, about 4.6 to about 6.9, about 4.7 to about 6.8, about 4.8 to about 6.7, about 4.9 to about 6.6, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.0 to about 6.0, about 5.0 to about 5.5, about 5.1 to about 6.9, about 5.2 to about 6.8, about 5.3 to about 6.7, about 5.4 to about 6.6, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.1 to about 6.5, about 5.5 to about 6.0, about 5.5 to about 6.5, about 5.1 to about 6.0, about 5.5.0, about 5.5 to about 6.0, about 5, about 5.0 to about 5.5, about 5.0, about 5 to about 6.0, about 5.0, about 5.5 to about 6.0, about 5, about 5.0 to about 6.0, about 5.0, about 6.0, about 5.0 to about 6.0, about 5.0, about 6.0, about 6.

In some embodiments, the method comprises inoculating an inoculum comprising megacoccus aegythii cells in a growth medium in a fermentor to prepare a culture, and incubating the culture at a temperature of about 39 ℃ until the pH of the culture is about 6.0. In some embodiments, the inoculum comprising the megacoccus aegypti cells is a shake flask culture of megacoccus aegypti cells or a portion thereof. In some embodiments, the method comprises inoculating the growth medium in the fermentor at an inoculum to medium ratio of 1/50 to 1/4,000. In some embodiments, the ratio of inoculum to culture medium is 1/100.

In some embodiments, the culture further comprises at least one cryoprotectant. In some embodiments, the 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, the at least one cryoprotectant is present in the range of about 1% to about 50% (w/v) of the culture, about 1% to about 40% (w/v) of the culture, about 1% to about 30% (w/v) of the culture, about 1% to about 20% (w/v) of the culture, about 1% to about 10% (w/v) of the culture, about 1% to about 5% (w/v) of the culture, about 10% to about 20% (w/v) of the culture, about 15% to about 25% (w/v) of the culture, about 20% to about 30% (w/v) of the culture, about 30% to about 40% (w/v) of the culture, about 40% to about 50% (w/v) of the culture, about 60% to about 70% (w/v) of the culture, 70% to 80% (w/v) of the culture. In some embodiments, the cryoprotectant is added by adding powdered cryoprotectant directly to the concentrated E.eggplantus cells. In some embodiments, the cryoprotectant is added by adding a solution of the cryoprotectant directly to the concentrated E.coli cells at a ratio of 1/1, at a ratio of 1/5, or at a ratio of 1/10.

In some embodiments, freezing the cells comprises placing the cells in a refrigerator or contacting the cells with dry ice, liquid nitrogen, or a combination thereof. Freezing the cells includes freezing the cells while the cells are in the container. Contacting the cells includes contacting a container containing the cells with a medium for freezing the cells. Media for freezing cells include, but are not limited to, a refrigerator, an acetone dry ice bath, liquid nitrogen, or combinations thereof.

In some embodiments, the method comprises freezing the cells at a temperature of about-20 ℃ to about-210 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-20 ℃ to about-80 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-80 ℃ to about-210 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-20 ℃ to about-196 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-80 ℃ to about-196 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-20 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-80 ℃. In some embodiments, the method comprises freezing the cells at a temperature of about-196 ℃. In some embodiments, the method comprises freezing the cells by contacting the cells with liquid nitrogen.

In some embodiments, the method comprises freezing the cells under anaerobic conditions.

In some embodiments, freezing produces frozen pellets comprising the cells. For example, freezing may be accomplished using a quick-freeze refrigerator.

In some embodiments, the frozen pellets have a diameter of about 0.001 to about 1.0 inch, about 0.01 to about 1.0 inch, about 0.1 to about 1.0 inch, about 0.2 to about 1.0 inch, about 0.3 to about 1.0 inch, about 0.4 to about 1.0 inch, about 0.5 to about 1.0 inch, about 0.6 to about 1.0 inch, about 0.7 to about 1.0 inch, about 0.8 to about 1.0 inch, about 0.9 to about 1.0 inch, about 0.001 to about 0.9 inch, about 0.01 to about 0.9 inch, about 0.1 to about 0.9 inch, about 0.2 to about 0.9 inch, about 0.3 to about 0.9 inch, about 0.4 to about 0.9 inch, about 0.5 to about 0.9 inch, about 0.6 to about 0.9 inch, about 0.0 to about 0.8 inch, about 0 to about 0.9 inch, about 0.0.3 to about 0.9 inch, about 0 to about 8 inch, about 0.9 inch, about 0 to about 0.9 inch, about 0.8 to about 0.9 inch, about 0 to about 0.9 inch, about 0.8 inch, about 0 to about 0.9 inch, about 0 to about 8, About 0.001 to about 0.7 inches, about 0.01 to about 0.7 inches, about 0.1 to about 0.7 inches, about 0.2 to about 0.7 inches, about 0.3 to about 0.7 inches, about 0.4 to about 0.7 inches, about 0.5 to about 0.7 inches, about 0.6 to about 0.7 inches, about 0.001 to about 0.6 inches, about 0.01 to about 0.6 inches, about 0.1 to about 0.6 inches, about 0.2 to about 0.6 inches, about 0.3 to about 0.6 inches, about 0.4 to about 0.6 inches, about 0.5 to about 0.6 inches, about 0.001 to about 0.5 inches, about 0.01 to about 0.5 inches, about 0.05 to about 0.5 inches, about 0.1 to about 0.5 inches, about 0.15 to about 0.5 inches, about 0.2 to about 0.5 inches, or about 0.5 inches.

The frozen E.coli cells may be stored frozen (e.g., below 0 ℃) prior to lyophilization or may be immediately lyophilized. In some embodiments, the frozen E.coli cells are stored at a temperature of less than about 0 ℃, less than about-10 ℃, less than about-20 ℃, less than about-50 ℃, less than about-80 ℃, or less than-196 ℃. In some embodiments, the frozen E.coli cells are stored at a temperature of about-20 ℃, about-30 ℃, about-40 ℃, about-50 ℃, about-60 ℃, about-70 ℃, about-80 ℃, about-90 ℃, about-100 ℃, about-150 ℃, about-196 ℃, or about-210 ℃.

In some embodiments, the frozen E.coli cells are lyophilized. In some embodiments, the frozen E.coli cells are freeze-dried. Lyophilization includes, for example, removing liquid from frozen cells.

In some embodiments, freeze-drying the E.coli cells comprises placing the frozen cells in a freeze-dryer. In some embodiments, freeze-drying comprises subjecting the frozen cells to reduced pressure and gradually heating the cells to room temperature.

In some embodiments, the method comprises freeze-drying the cells under anaerobic conditions.

In some embodiments, the lyophilized E.coli is produced on a commercial scale.

In some embodiments, lyophilization of the E.coli cells occurs at a pressure of about 50mTorr to about 2,000mTorr, about 100mTorr to about 1,950mTorr, about 150mTorr to about 1,900mTorr, about 200mTorr to about 1,850mTorr, about 250mTorr to about 1,800mTorr, about 300mTorr to about 1,750mTorr, about 350mTorr to about 1,700mTorr, about 400mTorr to about 1,650mTorr, about 450mTorr to about 1,600mTorr, about 500mTorr to about 1,550mTorr, about 550mTorr to about 1,500mTorr, about 600mTorr to about 1,500mTorr, about 650mTorr to about 1,450mTorr, about 700mTorr to about 1,400mTorr, about 750mTorr to about 1,350mTorr, about 1,500mTorr to about 1,900mTorr, about 1,450mTorr, about 700mTorr to about 1,400mTorr, about 1,900mTorr, about 1 mTorr to about 1,200mTorr, about 1,000mTorr, about 1,900mTorr, or about 1,900 mTorr. In some embodiments, the pressure during lyophilization is 135 mTorr. In some embodiments, the pressure during lyophilization is 250 mTorr.

In some embodiments, the time to complete the lyophilization process is about 5 hours to 15 days, about 6 hours to 15 days, about 7 hours to 15 days, about 8 hours to 15 days, about 9 hours to 15 days, about 10 hours to 15 days, about 11 hours to 15 days, about 12 hours to 15 days, about 18 hours to 15 days, about 24 hours to 15 days, about 36 hours to 14 days, about 48 hours to 13 days, about 3 days to 12 days, about 4 days to 11 days, about 5 days to 10 days, about 6 days to 9 days, about 7 days to 8 days. In some embodiments, the time to complete the lyophilization process is about 18.5 hours. In some embodiments, the time to complete the lyophilization process is about 38.5 hours.

In some embodiments, about 1 × 10 is produced by the methods disclosed herein³To 1 × 10¹²CFU/g of lyophilized cells of giant coccus aegypti in some embodiments, about 1 × 10 after lyophilization³To 1 × 10¹²CFU/g of giant cells of E.coli were viable.

Lyophilized teaA megacoccus megaterium cell can also be produced by a method comprising: (a) preparing a culture comprising E.coli cells and a growth medium comprising 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, fats, oils, glycerol, sodium acetate, arabinose, soy protein, soluble proteins, raffinose, and combinations thereof, (b) harvesting the cells under anaerobic conditions, (c) freezing the cells, and (d) freeze-drying the cells, wherein about 1x10 cells are produced³To about 1x10¹²CFU/g of lyophilized E.eggplantus cells.

Lyophilized E.coli megacoccus cells can also be produced by a method comprising: (a) preparing a culture comprising megacoccus aegypti cells and a growth medium, (b) harvesting the cells under anaerobic conditions within 12 hours after the end of the culture after exponential growth phase; (c) freezing the cells; (d) freeze-drying the cells; and optionally (e) encapsulating the freeze-dried cells, wherein freeze-dried E.coli cells or encapsulated freeze-dried E.coli cells are produced.

Lyophilized E.coli megacoccus cells can also be produced by a method comprising: (a) preparing a culture comprising a megacoccus aegythii cell and a growth medium, (b) harvesting the cell, (c) freezing the cell at a temperature of about-80 ℃ to about-210 ℃ within 5 hours after harvesting, (d) freeze-drying the cell, and optionally (e) encapsulating the freeze-dried cell, wherein a freeze-dried megacoccus aegythii cell is produced or an encapsulated freeze-dried megacoccus aegythii cell is produced.

In some embodiments, the present invention relates to an encapsulated freeze-dried composition comprising E.coli giant coccus cells, wherein the freeze-dried powder is encapsulated with: (a) oils including but not limited to vegetable oils, palm oil, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid (b) food grade polymers including but not limited to alginates, chitosans, carboxymethersCellulose, xanthan, starch, carrageenan, galatin and pectin (c) milk proteins including but not limited to casein and whey proteins, and (d) plant proteins from soybeans, beans (pulse) and cereals (cereal) including but not limited to zein. In some embodiments, the amount of freeze-dried megacoccus aegythii cells or encapsulated freeze-dried megacoccus aegythii cells and/or the amount of live megacoccus aegythii cells or encapsulated freeze-dried megacoccus aegythii cells after freeze-drying is about 1x10³CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10¹¹CFU/g, about 1x10³CFU/g to about 1x10¹⁰CFU/g, about 1x10³CFU/g to about 1x10⁹CFU/g, about 1x10³CFU/g to about 1x10⁸CFU/g, about 1x10³CFU/g to about 1x10⁷CFU/g, about 1x10³CFU/g to about 1x10⁶CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10¹²CFU/g, about 1x10⁵CFU/g to about 1x10¹²CFU/g, about 1x10⁶CFU/g to about 1x10¹²CFU/g, about 1x10⁷CFU/g to about 1x10¹²CFU/g, about 1x10⁸CFU/g to about 1x10¹²CFU/g, about 1x10⁹CFU/g to about 1x10¹²CFU/g, about 1x10¹⁰CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10⁶CFU/g, about 1x10⁵CFU/g to about 1x10⁷CFU/g, about 1x10⁶CFU/g to about 1x10⁸CFU/g, about 1x10⁷CFU/g to about 1x10⁹CFU/g, about 1x10⁸CFU/g to about 1x10¹⁰CFU/g, about 1x10⁹CFU/g to about 1x10¹¹CFU/g or about 1x10¹⁰CFU/g to about 1x10¹²CFU/g。

In some embodiments, the lyophilized E.coli cells or encapsulated lyophilized E.coli cells survive about 14 days to about 24 months at about-80 ℃, about-20 ℃, about 4 ℃, about 25 ℃, or a combination thereof. In some embodiments, the lyophilized E.coli cells or encapsulated lyophilized E.coli cells survive 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 ℃, about-20 ℃, about 4 ℃, about 25 ℃, or a combination thereof.

In some embodiments, about 1x10 after storage at a temperature of about-80 ℃, about-20 ℃, about 4 ℃, or a combination thereof for at least about 14 days, at least about 1 month, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 15 months, at least about 18 months, or at least about 24 months³CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10¹¹CFU/g, about 1x10³CFU/mL to about 1x10¹⁰CFU/g, about 1x10³CFU/g to about 1x10⁹CFU/g, about 1x10³CFU/g to about 1x10⁸CFU/g, about 1x10³CFU/g to about 1x10⁷CFU/g, about 1x10³CFU/g to about 1x10⁶CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10¹²CFU/g, about 1x10⁵CFU/g to about 1x10¹²CFU/g, about 1x10⁶CFU/g to about 1x10¹²CFU/g, about 1x10⁷CFU/g to about 1x10¹²CFU/g, about 1x10⁸CFU/g to about 1x10¹²CFU/g, about 1x10⁹CFU/g to about 1x10¹²CFU/g, about 1x10¹⁰CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10⁶CFU/g, about 1x10⁵CFU/g to about 1x10⁷CFU/g, about 1x10⁶CFU/g to about 1x10⁸CFU/g, about 1x10⁷CFU/g to about 1x10⁹CFU/g, about 1x10⁸CFU/g to about 1x10¹⁰CFU/g, about 1x10⁹CFU/g to about 1x10¹¹CFU/g or about 1x10¹⁰CFU/g to about 1x10¹²CFU/g lyophilized E.coli cells or encapsulated lyophilized E.coli cells survived.

In some embodiments, about 1x10 after storage at a temperature of about 25 ℃ 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 months³CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10¹¹CFU/g, about 1x10³CFU/g to about 1x10¹⁰CFU/g, about 1x10³CFU/g to about 1x10⁹CFU/g, about 1x10³CFU/g to about 1x10⁸CFU/g, about 1x10³CFU/g to about 1x10⁷CFU/g, about 1x10³CFU/g to about 1x10⁶CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10¹²CFU/g, about 1x10⁵CFU/g to about 1x10¹²CFU/g, about 1x10⁶CFU/g to about 1x10¹²CFU/g, about 1x10⁷CFU/g to about 1x10¹²CFU/g, about 1x10⁸CFU/g to about 1x10¹²CFU/g, about 1x10⁹CFU/g to about 1x10¹²CFU/g, about 1x10¹⁰CFU/g to about 1x10¹²CFU/g, about 1x10³CFU/g to about 1x10⁵CFU/g, about 1x10⁴CFU/g to about 1x10⁶CFU/g, about 1x10⁵CFU/g to about 1x10⁷CFU/g, about 1x10⁶CFU/g to about 1x10⁸CFU/g, about 1x10⁷CFU/g to about 1x10⁹CFU/g, about 1x10⁸CFU/g to about 1x10¹⁰CFU/g, about 1x10⁹CFU/g to about 1x10¹¹CFU/g or about 1x10¹⁰CFU/g to about 1x10¹²CFU/g lyophilized E.coli cells or encapsulated lyophilized E.coli cells survived.

Examples

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.