USE OF ISOLATED ENZYME-PRODUCING STRAPS OF BACILLUS AND AN ANIMAL FEEDING

专利摘要:
enzyme-producing strains of bacillus, their use, composition and feeding an animal. The present invention relates to enzyme-producing strains of bacillus that provide benefits to animals and to methods of using these strains. in one embodiment, the present invention relates to compositions comprising the enzyme-producing strains of bacillus. in yet another embodiment, the present invention relates to an animal feed comprising enzyme-producing strains of bacillus.
公开号:BR112014003950B1
申请号:R112014003950-0
申请日:2012-08-24
公开日:2022-01-11
发明作者:Mari Ellen Davis；Justin Sawall；Anthony Neumann；Greg Siragusa；Luis Romero
申请人:Dupont Nutrition Biosciences Aps.；
IPC主号:

专利说明:

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[001] This patent application claims priority to 35 USC § 119(e) to US Interim Patent Application No. 61/526, 881 filed Aug. 24, 2011 and US Interim Patent Application No. 61/ 527, 371 filed August 25, 2011, the entirety of both patent applications is incorporated by reference. BIBLIOGRAPHY
[002] Full bibliographic citations of references referred to in the present invention by the first author's surname in parentheses can be found in the Bibliography section immediately before the claims. FIELD
[003] The description refers to the strains of Bacillus that produce the enzymes that provide benefits to animals and the methods of using these strains. In one embodiment, the present invention relates to methods for improving the growth performance of an animal. In another embodiment, the present invention relates to a direct microbial feed, and to the feeding of an animal supplemented with a direct microbial feed. In another embodiment, the present invention relates to methods for improving manure storage units. In yet another embodiment, the present invention relates to methods of alleviating an inflammatory response. BACKGROUND
[004] The global swine industry has seen an increase in feed by-products (Dry Distillers with Soluble Grains (DDGS), Wheat Bran, etc.), initially from 0 to 10% to current extremes of 30 to 60% . These diet cost savings have been a huge opportunity for the industry with regards to a savings in feed input costs, but it comes with a set of challenges as well. The fermentation process to extract ethanol from corn removes almost all of the starch, leaving the resulting DDGS feed by-product containing approximately 40% fiber. This higher fiber content compared to corn results in reduced dry matter digestibility and about 10 percent less digestibility of most amino acids compared to corn DDGS (Stein and Shurson, 2009).
[005] Therefore, the inclusion of DDGS in livestock diets can have negative impacts on animal performance and carcass traits. In addition to the negative effects on animal growth and carcass quality, changes in nutrient digestibility as a result of adding DDGS with a high fiber content have implications for the handling, storage and decomposition of swine manure. The commercial swine industry has indicated that manure holding capacity is lower in anaerobic deep pit swine manure storage units, and that manure from swine fed high level of DDGS has more solids accumulation as well as ammonia, methane and hydrogen sulfide gas emissions.
[006] In view of the foregoing, it would be desirable to provide the Bacillus strains that produce the enzymes that provide benefits to animals and the methods of using these strains. SUMMARY
[007] The description refers to the production of the Bacillus enzyme. In one embodiment, the strains are Bacillus subtilis. In another embodiment, the strains are Bacillus pumilus.
[008] In at least some embodiments, the B. subtilis strain(s) is (are) from Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, and Bacillus subtilis AGTP BS 1069, and Bacillus subtilis AGTP 944, and strains having all of the same characteristics, any derivative or variant thereof, and mixtures thereof. In some embodiments, the B. pumilus strain(s) is/are Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX1 1-1, and those strains having all of the same characteristics, any derivative or variant thereof, and their mixtures.
[009] In one embodiment, the present invention relates to methods comprising administering an effective amount of enzyme-producing strain(s), one or more combination(s) of the strain(s), one or more plus supernatant(s) from a culture of the strain(s), feed including one or more strain(s) or mixtures thereof to an animal, wherein administration improves at least one of the following body weight options, average daily gain, average daily feed intake, feed conversion, carcass traits, nutrient digestibility and manure residue problems.
[0010] In another embodiment, the enzyme that produces the strains can be administered to an animal to improve at least one of the components of the feed breakdown complex, manure residue problems, production efficiency, carcass characteristics, and performance while feeding high levels of DDGS.
[0011] In one embodiment, one or more enzyme producing strain(s) is (are) administered as a direct microbial feed (DFM). Direct microbial feed includes one or more Bacillus strain(s). The strain(s) that produces the enzyme is (are) effective at breaking down indigestible foods in another way, such as DDGS. This allows for an increase in nutrient availability, resulting in a better growth response of the animals. In addition, the production of enzyme (s) strain (s) of slaughterhouse manure (s) associated with odors, thus improving the quality of operational ambient air. In at least some embodiments, odor reduction is through the reduction of volatile fatty acids, ammonia, and/or methane and the production of hydrogen sulfide gas.
[0012] In other embodiments, the present invention relates to a method comprising administering an effective amount of the enzyme-producing strain(s), combination of one or more of the strain(s), a or more supernatant(s) from a culture of the strain(s), the feed including one or more strain(s) or mixtures thereof to a swine in an amount effective to improve the storage unit of manure. In certain embodiments, the swine manure storage unit is a manure pit. In at least some modalities, administration improves at least one of the following characteristics: lower incidence of foaming, less solids accumulation, and less nitrogen, sulfur, phosphorus, fiber-bound nitrogen, total protein, fat, fiber content and when compared to a control manure pit.
[0013] In certain embodiments, the enzyme producing strain(s) is (are) directly applied to a manure storage unit, such as a manure pit. The improvements resulting from contacting the (S) enzyme-producing (S) strain directly to a manure storage unit include at least a lower incidence of foaming, less solids build-up, and less nitrogen, sulfur, phosphorus, fiber-bound nitrogen, total protein, fat, and fiber content from control manure wells.
[0014] In another embodiment, the present invention relates to a method for altering the volatile fatty acid composition in a manure pit which comprises administering an effective amount of the producing strain(s) of the enzyme, one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) or mixtures thereof for animals whose manure is stored in the manure pit. In another embodiment, the enzyme-producing strains can be contacted directly with the manure well.
[0015] In yet another embodiment, the present invention relates to a method for altering gas emissions that accumulate in an animal housing room that comprises administering the producing strain(s) ) enzyme, combination of one or more of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed including one or more strain(s) or mixtures thereof for animals in an effective amount to reduce gas emissions.
[0016] In yet another embodiment, the present invention relates to methods for attenuating an inflammatory response comprising administering the enzyme-producing strain(s), one or more combination(s) of ( s) strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) or mixtures thereof for animals in an amount effective to attenuate the inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Exemplary embodiments of the present invention are illustrated in the accompanying drawings.
[0018] Figure 1 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS3BP5.
[0019] Figure 2 is a partial sequence of Bacillus subtilis AGTP BS3BP5 16S rDNA.
[0020] Figure 3 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS442.
[0021] Figure 4 is a partial sequence of Bacillus subtilis AGTP BS442 16S rDNA.
[0022] Figure 5 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS521.
[0023] Figure 6 is a partial sequence of Bacillus subtilis AGTP BS521 16S rDNA.
[0024] Figure 7 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS918.
[0025] Figure 8 is a partial sequence of Bacillus subtilis AGTP BS918 16S rDNA.
[0026] Figure 9 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS 1013.
[0027] Figure 10 is a partial sequence of Bacillus subtilis AGTP BS 1013 16S rDNA.
[0028] Figure 11 is a photograph of a gel showing a RAPD PCR profile of Bacillus pumilus AGTP BS 1068.
[0029] Figure 12 is a partial sequence of Bacillus pumilus AGTP BS 1068 16S rDNA.
[0030] Figure 13 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP BS1069.
[0031] Figure 14 is a partial sequence of Bacillus subtilis AGTP BS 1069 16S rDNA.
[0032] Figure 15 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP 944.
[0033] Figure 16 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP 944.
[0034] Figure 17 is a photograph of a gel showing a RAPD PCR profile of Bacillus subtilis AGTP 944.
[0035] Figure 18 is the partial sequence of the 16S rDNA gene of Bacillus subtilis AGTP 944.
[0036] Figure 19 is a photograph of a gel showing a RAPD PCR profile of Bacillus pumilus KX1 1-1.
[0037] Figure 20 is a photograph of a gel showing a RAPD PCR profile of Bacillus pumilus KX1 1-1.
[0038] Figure 21 is the partial sequence of the 16S rDNA gene of Bacillus pumilus KX1 1-1.
[0039] Figure 22 is a representative schematic of a cell culture plate design for screening Bacillus strains for anti-inflammatory effects. LPS has been used to induce the inflammatory response but any agent that induces the inflammatory response can be used.
[0040] Figure 23 is a bar graph depicting the anti-inflammatory effects of Bacillus strains as shown in a representative macrophage cell line (HD11 chicken). The agent used to induce the inflammatory response was LPS. Effects on IL-1β gene expression are shown by white bars (P < 0.01). Effects on IL-8 gene expression are shown by black bars (P < 0.01). Different letters (a, b, c) indicate the means that differed statistically (P < 0.01).
[0041] Figure 24 is a representative schematic of a cell culture plate design for the selection of a direct microbial feed candidate. LPS was used as the agent to induce the inflammatory response.
[0042] Figure 25 is a bar graph depicting the anti-inflammatory effects of Bacillus strains from a mammalian cell line (mouse intestinal epithelial cell line (IEC-6)). LPS was used to induce the inflammatory response. A tumor necrosis factor (TNF-a) gene expression was measured. Different letters (a or b) indicate that the medium differed statistically (P < 0.10).
[0043] Figure 26 is a line graph showing foam characteristics in a well above 3 samplings in the 170 day evaluation period.
[0044] Figure 27 is a representative schematic of a bioluminescence measurement in each pen in the zone marked with an "x".
[0045] Before explaining the embodiments of the present invention in detail, it is to be understood that the present invention is not limited in its application to the construction details and arrangement of components shown in the following description or illustrated in the drawings. The present invention is capable of other embodiments or of being practiced or performed in various ways. Also, it is to be understood that the phraseology and terminology employed in the present invention is for the purpose of description and should not be considered as limiting.
[0046] The organizational structure of this present invention should not limit any embodiments or elements of the present invention. It is intended that the elements and applications cited within one embodiment can be applied to other embodiments within the scope of the present invention. DETAILED DESCRIPTION
[0047] Numerical proportions in this present invention are approximate, and thus may include values outside the proportions, unless otherwise indicated. Numerical proportions include all values of and include the lower and higher values, in increments of one unit, provided that there is a separation of at least two units between any lower value and any higher value. As an example, if a composition, physical or other properties, such as, for example, molecular weight, viscosity, etc., is from 100 to 1000, it is intended that all individual values, such as 100, 101, 102, etc., and sub-ranges such as 100 to 144, 155 to 170, 197 to 200, etc., are expressly enumerated. For proportions that contain values that are less than one or contain fractional numbers greater than one (e.g., 1.1, 1.5, etc.), a unit is considered to be 0.0001, 0.001, 0.01, or 0.1, as appropriate. For proportions that contain numbers from one digit to less than ten (eg, 1 to 5), a unit is normally considered to be 0.1. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest and highest enumerated value are to be considered as expressly reported herein. Numerical ratios are provided within this present invention for, among other things, the relative amounts of components in a mixture, and at various ranges of temperature and other parameters recited in the methods.
[0048] By "administering" is meant the action of introducing at least one strain and/or the supernatant of a culture of at least one strain described in the present invention into the gastrointestinal tract of the animal. More particularly, this administration is an oral administration. This administration can in particular be carried out by supplementing the feed intended for the animal with at least one strain, and the supplemented feed being thus to be ingested by the animal. Administration can also be carried out using a stomach tube or in any other way so that at least one strain can be directly introduced into the animal's gastrointestinal tract.
[0049] By "at least one strain," is meant a single strain, but also mixtures of strains comprising at least two strains of bacteria. By "a mixture of at least two strains," is meant a mixture of two, three, four, five, six or even more strains. In some embodiments of a mixture of strains, the proportions can range from 1% to 99%. In certain embodiments, the proportion of a strain used in the blend is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Other embodiments of a mixture of strains are from 25% to 75%. Additional modalities of a mixture of strains are approximately 50% for each of the strains. When a mixture comprises more than two strains, the strains may be present in substantially equal proportions in the mixture, or in different proportions.
[0050] By "contacting" is meant the action of bringing together at least one strain and/or the supernatant of a culture of at least one strain described in the present invention in close proximity to a substrate, container or a substance, which includes, but is not limited to, a manure storage unit. In some embodiments, the dung storage unit is a dung pit. As the contact can be through a direct or indirect way. As used in the present invention, it includes contacting, spraying, inoculating, spreading, spreading, pouring, and other similar terms.
[0051] By "effective amount", we mean an amount of strain and/or supernatant sufficient to allow improvement in at least one of the following characteristics: animal production efficiency, carcass characteristics, an animal's growth performance, growth performance when feeding high levels of DDGS to an animal, nutrient digestibility, breakdown of complex feed components, poultry growth performance, swine growth performance, feed efficiency, gain of average daily weight, average daily food intake, body weight gain: food or food: gain, intake and morality.
[0052] In other embodiments, "effective amount" means an amount of strain and/or supernatant sufficient to allow improvement in at least one of the following: manure residue problems, the amount of foaming in a storage unit of manure, the microbial ecology of a manure storage unit, the amount of volatile fatty acids in a manure storage unit, the amount of gas production in an animal housing room or a manure storage unit, including , but not limited to methane and hydrogen sulfide.
[0053] In another embodiment, "effective amount" means an amount of strain and/or supernatant sufficient to allow improvement in at least one of the following characteristics: the expression of a gene involved in the inflammatory response, the expression of a protein involved in the inflammatory response process, and the activity of a protein involved in the inflammatory response.
[0054] As used in the present invention, "performance" refers to the growth of an animal, such as a swine or poultry, measured by one or more of the following parameters: average daily weight gain (GPD), weight, neonatal diarrhea, mortality, feed conversion, which includes both feed conversion and feed gain and feed consumption. "A performance improvement" or "performance improvement" as used in the present invention refers to an improvement of at least one of the parameters indicated within the scope of the performance definition.
[0055] As used in the present invention, a "variant" has at least 80% genetic sequence identity to the strains described using random amplification polymorphic DNA polymerase chain reaction (RAPD-PCR). The degree of identity of genetic sequences may vary. In some embodiments, the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% genetic sequence identity with the strains described using RAPD-PCR analysis. Six primers that can be used for RAPD-PCR analysis include the following: Primer 1 (5'-GGTGCGGGA A-3') (.SEQ ID NO 1); primer 2 (5'-GTTTCGCTCC-3') (SEQ ID NO. 2); primer 3 (5'-GTAGACCCGT-3') (SEQ ID NO 3). PRIMER 4 (5'-AAGAGCCCGT-3') (SEQ ID NO 4); primer 5 (5'-AACGCGCAAC-3') (SEQ ID NO: 5); and primer 6 (5'-CCCGTCAGCA-3'.) (SEQ ID NO 6). RAPD analysis can be performed using Ready-to-Go™ RAPD Analysis Beads (Amersham Biosciences, Sweden), which are designed as the pre-mixed, pre-dispensed reactions for performing RAPD analysis.
[0056] The inventors have discovered that certain strains of Bacillus have enzyme activity(s) that break down fiber(s), lipid(s), carbohydrate(s), and protein ( s). These strain(s) is (are) referred to in the present invention as "enzyme-producing strain(s)", "Bacillus strain(s)," or "strain(s)". In some embodiments, the enzyme activity(s) is (are) cellulase, α-amylase, xylanase, esterase, casein protease, corn starch amylase, β-mannanase, lipase, and/or protease , for example, zeinase and soy protease.
[0057] The inventors have discovered that certain microorganisms can be used to treat the challenging components of still dry grains with solubles (DDGS).
[0058] The inventors have also found that enzyme-producing strains can improve at least one of the following: (1) breakdown of complex food components, (2) manure residue problems, (3) animal production efficiency; (4) the carcass characteristics of the animals, (5) the growth performance of an animal, and (6) the effects of an inflammatory response. Enzyme producing strains
[0059] Enzyme-producing strains include Bacillus strains, including, but not limited to, B. subtilis, B. licheniformis, B. pumilus, B. coagulans, B. amyloliquefaciens, B. stearothermophilus, B. brevis, B. alkalophilus, B. clausii, B. halodurans, B. megaterium, B. circulans, B. lautus, B. thuringiensis and B. lentus, and strains having all the characteristics thereof, any derivative or variant, and mixtures the same.
[0060] In at least some embodiments, the B. subtilis strain(s) is (are) from Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS1013 and Bacillus subtilis AGTP BS1069, and Bacillus subtilis AGTP 944, and strains having all characteristics thereof, any derivative or variant thereof, and mixtures thereof. In some embodiments, the B. pumilus strain(s) is/are Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX1 1-1, and those strains having all of the characteristics thereof, any derivative or variant thereof, and their mixtures.
[0061] These strains were deposited by Danisco USA, Inc. of Waukesha, Wisconsin, in the 1815 North Street University Agricultural Culture Service (NRRL) research program, Peoria, III., 61604. Original deposit dates and numbers accession are as follows: Bacillus subtilis AGTP BS3BP5, May 13, 2011 (NRRL B-50510), Bacillus subtilis AGTP BS442, August 4, 2011 (NRRL B-50542), Bacillus subtilis AGTP BS521, August 4, 2011 (NRRL B-50545), Bacillus subtilis AGTP BS918, May 13, 2011 (NRRL B-50508), Bacillus subtilis AGTP BS1013, May 13, 2011 (NRRL B-50509), Bacillus subtilis AGTP BS 1069, August 4 2011 (NRRL B-50544), Bacillus subtilis AGTP 944, August 01, 2011 (NRRL B-50548), Bacillus pumilus AGTP BS 1068, August 4, 2011 (NRRL B-50543), and Bacillus pumilus KX11-1 , August 5, 2011 (NRRL B-50546). All deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure,
[0062] In some embodiments, the strains that produce the enzyme have enzyme activity(s), including but not limited to cellulase, α-amylase, xylanase, esterase, casein protease, corn starch amylase, β- mannanase, lipase and/or protease, for example zeinase and soy protease.
[0063] In at least some embodiments, more than one of the strain(s) described in the present invention is (are) combined.
[0064] Any derivative or variant of Bacillus is also included and is useful in the methods described and claimed in the present invention. In some embodiments, strains that have all the characteristics of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS 1013, Bacillus subtilis AGTP BS1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX11-1 are also included and are useful in the methods described and claimed in the present invention.
[0065] In certain embodiments, any derivative or variant of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS 1013, Bacillus subtilis AGTP BS 1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068, and Bacillus pumilus KX11-1 are also included and are useful in the methods described and claimed in the present invention.
[0066] In at least some embodiments, the enzyme-producing strain(s) is (are) used in combination. In one embodiment, the enzyme-producing strain may be used in combination with strains of bacteria from the genus Bacillus, and other strains of bacteria from a different genus.
[0067] In at least some embodiments, the enzyme-producing strain(s) and methods provided in the present invention improve one or more of the following characteristics: breakdown of complex dietary components, manure, production efficiency, carcass characteristics, and performance when feeding high levels of DDGS when compared to a control.
[0068] Manure residue problems include, but are not limited to, undesirable manure nutrients and microbial composition, and undesirable gas emissions from manure storage units such as manure pits. An improvement in manure residue problems includes, but is not limited to, at least one of (1) fewer nutrients accumulated in manure, (2) microbial communities of manure displacement to populations favorable for solids breakdown, and (3) a decrease in ammonia, methane, and sulfide gas emissions.
[0069] An improvement in carcass traits can be measured in at least an increase in lean percentage yield and carcass yield, and a decrease in fat iodine values. Performance can be measured by average daily gain, average daily feed intake and feed required per unit of gain, and other measures known in the art.
[0070] When ingested, the enzyme-producing strain(s) produces (in) enzymes. In some embodiments, the enzyme-producing strain(s) produces (in) enzymes in vivo. In other embodiments, the enzyme-producing strain(s) survives (m) in the dung of animals to which the strain is administered and produces (in) enzymes excreted in the dung. Strain culture methods
[0071] Bacillus strains are produced through the fermentation of bacterial strains. Fermentation can be initiated by scaling up a seed culture. This involves repeatedly and aseptically transferring the culture to a larger and larger volume to serve as the inoculum for fermentation, which is obtained in large stainless steel fermenters in a medium containing proteins, carbohydrates and mineral salts necessary for optimal growth. An exemplary non-limiting medium is TSB. After the inoculum has been added to the fermentation vessel, temperature and agitation are controlled to allow maximum growth. Once the culture has reached maximum population density, the culture is harvested by separating the cells from the fermentation medium. This is commonly done by centrifugation.
[0072] The crop count can then be determined. CFU or Colony Forming Unit is the viable cell count of a sample obtained from standard microbiological plating methods. The term is derived from the fact that a single cell, when plated on an appropriate medium, will grow to become a viable colony on the agar medium. Since several cells can give rise to a visible colony, the term colony-forming unit is a more useful unit of measurement than the number of cells.
[0073] In one embodiment, each strain of Bacillus is fermented between a CFU/mL level of 5 x 10 to about a level of 4 x 109 CFU/mL. In at least one embodiment, a level of 2 x 109 CFU/ml. Bacteria are collected by centrifugation, and the supernatant is removed. The supernatant can be used in the methods described in the present invention. In at least some embodiments, the bacteria are sedimented. In at least some embodiments, the bacteria are lyophilized. In at least some embodiments, the bacteria are mixed with a carrier. However, it is not necessary to freeze-dry Bacillus before using them. Strains can also be used with or without preservatives, and in concentrated, unconcentrated, or diluted form. DFMS and methods of preparing a DFM
[0074] A composition that includes one or more strain(s) described in the present invention is provided. The composition can be transmitted to an animal as a direct microbial feed (DFM). One or more carrier(s) or other ingredients may be added to the DFM. DFM can be presented in various physical forms, for example, as a prepared top, as a water-soluble concentrate for use as a liquid potion, or to be added to a milk replacer, gelatin capsules, or gels. In one embodiment of the prepared top form, the fermentation product of the freeze-dried bacteria is added to a vehicle, such as whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice husks, yeast, dry starch, and/or sodium silica aluminate. In one embodiment the water-soluble concentrate for a liquid potion or milk substitute supplement, the fermentation product of the freeze-dried bacteria is added to a water-soluble vehicle, such as whey, maltodextrin, sucrose, dextrose, dry starch, silica of sodium aluminate, and a liquid is added to form the potion, or the supplement is added to milk or a milk replacer. In one embodiment of the form of gelatin capsules, the fermentation product of the freeze-dried bacteria is added to a vehicle, such as whey, maltodextrin, sugar, limestone (calcium carbonate), rice husks, dry yeast, starch , and/or sodium silica aluminate. In one embodiment, the bacteria and vehicle are placed in a degradable gelatin capsule. In one embodiment of the form of gels, the fermentation product of the lyophilized bacteria is added to a vehicle, such as vegetable oil, sucrose, silicon dioxide, polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid, ethoxyquin, and/or coloring. artificial gel that forms.
[0075] The strain(s) may (m) optionally be mixed with a dry formulation of additives, including but not limited to growth substrates, enzymes, sugars, carbohydrates, extracts and micro-ingredient growth promoters. Sugars may include the following: lactose; maltose; dextrose; malto-dextrin, glucose, fructose, mannose, tagatose, sorbose, raffinose, and galactose. The sugars range from 50 to 95%, either individually or in combination. Extracts may include yeast or dry yeast fermentation solubles ranging from 5 to 50%. Growth substrates may include: trypticase, which ranges from 5 to 25%; sodium lactate, which ranges from 5 to 30%, and Tween 80, which ranges from 1 to 5%. Carbohydrates can include mannitol, sorbitol, adonitol and arabitol. Carbohydrates range from 5 to 50% individually or in combination. Micro-ingredients may include the following: calcium carbonate, ranging from 0.5 to 5.0%, calcium chloride, ranging from 0.5 to 5.0%; dipotassium phosphate, which varies from 0.5 to 5.0% or calcium phosphate, which varies from 0.5 to 5.0%; manganese proteinate, which ranges from 0.25 to 1.00%, and manganese, which ranges from 0.25 to 10.0%.
[0076] To prepare DFMS described in the present invention, the culture(s) and vehicle(s) (if used) can be added to a ribbon or paddle mixer. and mixed for about 15 minutes, although the period can be lengthened or shortened. The components are mixed in such a way that a uniform mixture of cultures and vehicles results. The final product is preferably a dry, flowable powder. The strain(s) may then be added to animal feed or a feed premix, added to an animal's water, or administered in other ways known in the art. An animal feed may be supplemented with one or more strain(s) described in the present invention or with a composition described in the present invention.
[0077] The DFM provided in the present invention can be administered, for example, as the solution containing the culture strain, the strain production supernatant, or the bacterial product of a culture solution.
[0078] Administration of a DFM provided in the present invention to an animal can enhance the performance of the animal. In one embodiment, administration of a DFM in the present invention given to an animal can increase mean daily feed intake (adfi), mean daily gain (adg), or feed efficiency (gain: feed; G: F or feed: gain; F:G) (collectively, the "performance indicators"). One or more than one of these metric performances can be improved.
[0079] DFM can be administered to the animal in one of many ways. For example, the strain(s) may be administered in a solid form as a veterinary pharmaceutical product, may be distributed in an excipient, preferably water, and introduced directly into the animal, may be physically mixed with the feed material in a dry form, or the strain(s) may be formed in a solution and then sprayed onto the feed material. The method of administering the strain(s) to the animal is considered to be within the general knowledge of a person skilled in the art. Methods of administration to an animal
[0080] In one embodiment, the strains can be administered in an amount effective to animals. In at least some embodiments, the present invention relates to a method comprising administering to an animal an effective amount of the enzyme-producing strain(s), one or more combination(s) of ( s) strain(s), one or more supernatant(s) from a culture of the strain(s), or from animal feed, including one or more strain(s) or mixtures thereof. In one embodiment, the animal is a swine. In another embodiment, the animal is a bird. In yet another embodiment, the animal is a ruminant.
[0081] The administration of one or more enzyme-producing strain(s) to animals is carried out by any convenient method, including the addition of strains to the animals' drinking water, to their food, or to bedding, or by direct oral insertion, such as by aerosol or injection.
[0082] In another embodiment, the administration of one or more enzyme-producing strains is by spraying the animal with the enzyme-producing strains. The animal can clean or smooth and ingest the enzyme-producing strains.
[0083] In one embodiment, the Bacillus strains are administered as spores.
[0084] As used in the present invention, the term "animal" includes, but is not limited to, humans, mammals, amphibians, birds, reptiles, swine, swine, cows, cattle, goats, horses, sheep, birds and others. animals kept or raised on farms or ranches, sheep, big horn sheep, buffalo, antelope, oxen, donkey, mule, deer, elk, reindeer, water buffalo, camel, llama, alpaca, rabbit, mouse, mouse, little pig of india, hamster, ferret, dog, cat and other pets, primate, monkey, and gorilla.
[0085] In some modalities, the animals are birds of different ages, such as starters, producers and finishers. In certain embodiments, the animals are exotic birds and fowl, including, but not limited to, chicks, turkeys, ducks, goose, corral keets, chickens, hens, cocks, cockerels and capons.
[0086] In some embodiments, the animals are pigs, including, but not limited to, newborn pigs, rearing piglets, lactating piglets, piglets, , golden piglets, wild boar, females, lactating piglets, and terminal pigs. The strain(s) may be fed to a gilt during the lactation period, although the strain(s) may be fed for different durations and at different times. In certain embodiments, the strain(s) is (are) administered to piglets by feeding the strain(s) of a gilt or piglet. It is believed that transfer to piglets from the golden piglet is achieved via the fecal-oral route and/or by other routes.
[0087] Enzyme producing strains can be administered to an animal to improve at least one of nutrient digestibility, swine growth performances, bird growth performance responses, feed efficiency (intake: feed or feed: intake ), body weight, feed intake, average daily gain, average daily feed intake, breakdown of complex feed components, poultry production efficiency, swine production efficiency, and mortality. These benefits can be particularly helpful when diets containing high levels of DDGS are provided. Initially, DDGS was 0% to 10% of the animal's diet. Currently, DGGS is from 30% to 60%
[0088] The amount of enhancement can be measured as described in the present invention, or by other methods known in the art. Such effective amounts can be administered to the animal by providing ad libitum access to food containing the DFM. DFM can also be administered in one or more doses.
[0089] In certain modalities, the improvement is at least 1 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35%, 35 to 40%, 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85%, 85 to 90%, 90 to 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to an untreated control.
[0090] In at least some embodiments, the improvement in these measurements in an animal to which the strain(s) is/are administered, is at least 1%, 2%, 3%, 4% , 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37% , 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99%, and more than 99% compared to a control animal.
[0091] In other embodiments, the improvement in these measurements in an animal to which the strain(s) is/are administered is 2 to 8% compared to a control animal. In certain other embodiments, the improvement in these measurements in an animal to which the strain(s) is/are administered is at least 8% compared to a control animal.
[0092] In some embodiments, a control animal is an animal that has not been administered the enzyme-producing strains.
[0093] This effective amount may be administered to the animal in one or more doses. In some embodiments, one or more Bacillus strain(s) is (are) added to an animal's food at a rate of at least 1 x 10 4 CFU/animal/day.
[0094] In one embodiment, administration improves at least one of nutrient digestibility, growth performance responses, e.g. feed efficiency, breakdown of complex food components, production efficiency, body weight gain , food consumption, and mortality.
[0095] In certain embodiments of the method, the strain(s) is (are) administered at about 1 x 105 CFU/animal/day at about 1 x 10" CFU/animal/day. In in some embodiments, the animal is a swine. In another embodiment, the animal is a bird.
[0096] In at least some embodiments, the method is used when the animal is fed high levels of soluble dry still grains (DDGS). High levels of DDGS can be a proportion of more than 10% of the animal's diet. High levels of DDGS can also be a proportion of more than 30% of the animal's diet.
[0097] In at least some embodiments, the effective amount of at least one strain of bacteria is administered to an animal by supplementing a food intended for the animal with the effective amount of at least one strain of bacteria. As used in the present invention, "supplementation" means the act of incorporating the effective amount of bacteria provided in the present invention directly into the feed intended for the animal. In this way, the animal, when being fed, ingests the bacteria provided in the present invention.
[0098] Enzyme-producing strains can be administered as a single strain, or as multiple strains. The supernatant from one or more enzyme-producing strains can be administered to an animal. When ingested, the enzyme-producing strains produce the enzymes.
[0099] In certain embodiments, one or more enzyme producing strain(s) is (are) fed to swine. The production of one or more enzyme producing strain(s) addresses (m) the challenging components in soluble dry distillers grains (DDGS).
[00100] In one embodiment, the enzyme producing strain(s) is (are) added to the animal feed at a ratio of 1 x 103.1 x 104.1 x 105.1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x1011, 1 x 1012, 1 x 1013 and greater than 1 x 1013 CFU per gram of animal feed.
[00101] In another embodiment, the enzyme-producing strain(s) is (are) added to the animal feed, at a ratio of 1 x 103, 1 x 104, 1 x 105, 1x106, 1x107, 1x108, 1x109, 1x1010, 1x1011, 1x1012, 1x1013 and greater than 1x1013 CFU per animal per day.
[00102] In one embodiment, one or more Bacillus strain(s) is (are) added to the swine feed, at a ratio of about 3.75 x 105 CFU per gram of feed. He(s) can also be fed at about 1 x 10 4 to about 1 x 10 10 CFU/animal/day. In some embodiments, one or more Bacillus strain(s) is (are) fed at about 1 x 10 8 CFU/animal/day.
[00103] For ruminants, one or more Bacillus strain(s) is (are) fed at about 5 x 109 CFU/hour/day.
[00104] For birds, one or more Bacillus strain(s) is (are) added to the feed at about 1 x 104 CFU/g of feed at about 1 x 1010 CFU/g of feed. In at least some embodiments, one or more Bacillus strain(s) is (are) fed at about 1 x 10 5 CFU/bird/day at about 1 x 10 8 CFU/bird/day. feeding material
[00105] In another embodiment, an animal feed comprises at least one strain of bacteria described in the present invention. In at least some embodiments, the feed is supplemented with an effective amount of at least one strain of bacteria. As used in the present invention, "supplementation" means the act of incorporating the effective amount of bacteria provided in the present invention directly into the feed intended for the animal. In this way, the animal, when it comes to food, ingests the bacteria provided in the present invention.
[00106] When used in combination with a feed material, for monogastric diets, the feed material may include corn, soybean meal, by-products such as still dry grain with solubles (DDGS), and vitamin/mineral supplement. The feed material for ruminants may be grain or hay or silage or grass, or combinations thereof. Included in these feed materials are corn, dry grain, alfalfa, food ingredients and industrial food or by-products, as well as the bio fuel industry of the same by-products and corn flour and mixtures thereof. Other feeding materials may also be used.
[00107] Administration time may vary as long as an improvement is shown in one or more of the following: (1) breakdown of complex feed components, (2) nutrient digestibility, (3) manure residue problems , (4) production efficiency (5), carcass characteristics (6), growth performance (7), growth performance when fed high levels of DDGS (8), growth performance responses of (9) swine growth performance responses, (10) poultry production efficiency, (1 1) swine production efficiency (12), body weight gain (13), feed intake (14) feed efficiency, and (15) mortality. Administration is possible at any time, with or without food. However, the bacterium is preferably administered with or immediately before feeding. Methods to improve an animal's growth performance
[00108] In one embodiment, the present invention relates to a method for improving the growth performance of an animal which comprises using the strains producing one or more enzymes or supernatants therefrom to improve the growth performance of the animal. animal relative to an animal that was not administered the enzyme-producing strains. In one embodiment, the animal is a swine. In another embodiment, the animal is a bird. In another embodiment, the animal is a ruminant.
[00109] In one embodiment, growth performance includes, but is not limited to, nutrient digestibility, bird growth performance responses, swine growth performance responses, feed efficiency, breakdown of complex feed components , mean daily gain, mean daily feed intake, weight gain, feed intake, carcass traits and mortality. In yet another embodiment, the methods described in the present invention are used to improve the growth performance of an animal fed an animal feed comprising DDGS.
[00110] In certain embodiments, the improvement in growth performance is at least 15%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35% , 35 to 40%, 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85% , 85 to 90%, 90 to 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to an untreated control.
[00111] In at least some embodiments, the improvement in growth performance of an animal to which the strain(s) is/are administered at least 1%, 2%, 3%, 4% , 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37% , 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99%, and over 99% compared to a control animal.
[00112] In one embodiment, the enzyme-producing strain to enhance the growth performance of an animal comprises a strain of Bacillus. In one embodiment, the Bacillus strain is Bacillus subtilis. In another embodiment, the Bacillus strain is Bacillus pumilus.
[00113] In another embodiment, the enzyme-producing strain to enhance growth performance includes, but is not limited to, Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS 1013, and Bacillus subtilis AGTP BS 1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KXL 1-1, and strains having all characteristics thereof, any derivative or variant thereof, and mixtures thereof.
[00114] The strain(s) producing enzymes to enhance an animal's growth performance may be administered as a single strain, or a combination of more of the strain(s). ) strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s), or mixtures thereof. A. Nutrient Digestibility
[00115] In yet another embodiment, the present invention relates to a method of increasing the digestibility of an animal feed comprising administering an enzyme-producing strain to an animal in an amount effective to increase the digestibility of an animal. feed compared to an animal not administered the enzyme-producing strain. In another embodiment, the method further comprises measuring the amount of nutrients accumulated in an animal manure pit administered with the enzyme-producing strain and comparing these amounts of nutrients to the amount of nutrients in a manure pit of an unspecified animal. -administered to enzyme-producing strain. In yet another embodiment, the animal feed comprises DDGS.
[00116] In yet another embodiment, the present invention relates to a method of increasing the digestibility of an animal feed, comprising administering to an animal a feed supplemented with an enzyme-producing strain in an amount effective to increase the digestibility of the animal feed compared to an animal not administered the enzyme-producing strain.
[00117] In one embodiment, methods for enhancing the growth performance of an animal, comprising administering an enzyme-producing strain to an animal, and reducing the amount of nutrients undigested by the animal relative to an animal that is not was administered with the enzyme-producing strain.
[00118] In another embodiment, methods for improving an animal's growth performance comprise reducing the amount of undigested nutrients by an animal by administering an enzyme-producing strain to the animal compared to an animal that was not administered with the enzyme-producing strain.
[00119] In another embodiment, methods for improving the growth performance of an animal comprising administering an enzyme-producing strain to an animal, measuring the amount of nutrients accumulated in a well of animal manure administered with the enzyme-producing strain, and comparing the amount of nutrients in the manure well of an animal administered the enzyme-producing strains to the amount of nutrients in a second manure well of an animal not administered the enzyme-producing strain.
[00120] In one embodiment, the digestibility of an animal feed can be measured by the amount of nutrients in a manure pit. Any nutrient can be measured from the manure pit, including but not limited to dry matter, ash, total nitrogen, ammonia nitrogen, phosphorus and calcium.
[00121] The enzyme producing strain(s) to improve nutrient digestibility may be administered as a single strain, or one or more combination(s) of the strain(s) (s), one or more supernatant(s) from a culture of the strain(s), the feed including one or more strain(s), or mixtures thereof. B. Bird Growth Performance
[00122] In one embodiment, the present invention relates to a method of enhancing the growth performance of birds which comprises administering an enzyme-producing strain of birds in an amount effective to increase the growth performance of birds, in compared to birds not administered the enzyme-producing strain. The methods described in the present invention can be used to improve growth performance irrespective of the feed or diet of the birds.
[00123] In one embodiment, the present invention relates to a method of increasing growth performance in birds fed a diet rich in fiber by-products comprising administering an enzyme-producing strain to birds, which are fed with a diet high in fibrous by-product, in an amount effective to increase the growth performance of birds, compared to birds not administered the enzyme-producing strain.
[00124] In another embodiment, the present invention relates to a method of increasing average daily weight gain in birds which comprises administering an enzyme-producing strain of birds in an amount effective to increase average daily weight gain of birds compared to birds not administered the enzyme-producing strain.
[00125] In another embodiment, the present invention relates to a method of increasing the average daily food intake in birds which comprises administering an enzyme-producing strain of birds in an amount effective to increase the average food intake. daily feed compared to birds not given the enzyme-producing strain.
[00126] In another embodiment, the present invention relates to a method of improving the feeding efficiency of an animal feed in poultry, comprising administering to the birds an animal feed supplemented with an enzyme-producing strain. in an amount effective to increase the feeding efficiency of birds compared to birds not administered the enzyme-producing strain.
[00127] In yet another embodiment, the present invention relates to a method of improving carcass characteristics, comprising administering an enzyme-producing strain of birds in an amount effective to improve carcass characteristics of birds, in compared to birds not administered the enzyme-producing strain. Carcass traits that can be improved include, but are not limited to, fat depth, organ weight, breast traits, prepared weight, carcass grade, and carcass value.
[00128] In one embodiment, the measured value of the housing characteristics can be increased or decreased.
[00129] In yet another embodiment, the measured value of one or more of the following carcass characteristics is increased: fat depth, organ weight, breast characteristics, prepared weight, carcass grade, and carcass value.
[00130] In yet another embodiment, the measured value of one or more of the following carcass characteristics is decreased: fat depth, organ weight, breast characteristics, prepared weight, carcass grade, and carcass value.
[00131] In yet another embodiment, the present invention relates to a method of reducing mortality in birds which comprises administering an enzyme-producing strain of birds in an amount effective to reduce said bird mortality as compared to with birds not administered the enzyme-producing strain.
[00132] In another embodiment, the present invention relates to a method of improving the digestibility of lignin which comprises administering an enzyme-producing strain of birds in an amount effective to improve the digestibility of lignin as compared to birds not administered the enzyme-producing strain.
[00133] In another embodiment, the present invention relates to a method for improving the digestibility of lignin in highly fibrous diets comprising administering an enzyme-producing strain of birds in an amount effective to improve the lignin digestibility of birds. highly fibrous diets compared to birds not fed the enzyme-producing strain. In another embodiment, highly fibrous diets comprise by-product diets. In yet another embodiment, the diet comprises DDGS.
[00134] In another embodiment, the present invention relates to a method of improving apparent ileal digestibility which comprises administering an enzyme-producing strain of birds in an amount effective to improve apparent ileal digestibility compared to birds not administered with the enzyme-producing strain.
[00135] In yet another embodiment, the present invention relates to a method of improving total digestibility comprising administering an enzyme-producing strain of birds in an amount effective to improve total digestibility as compared to non-powdered birds. administered with the enzyme-producing strain.
[00136] In yet another embodiment, the present invention relates to a method of lowering the pH of ileal digestion, comprising administering an enzyme-producing strain of birds in an amount effective to lower the pH of ileal digestion. , compared to birds not administered the enzyme-producing strain.
[00137] In yet another embodiment, the methods described above further comprise administering a food supplemented with an enzyme-producing strain.
[00138] The strain(s) producing enzyme(s) to enhance the growth performance of birds may(m) be administered as a single strain(s), or one or more combination(s) of the(s) ) strain(s), one or more supernatant(s) from a culture of the strain(s), animal feed, including one or more strain(s), or mixtures thereof. C. Pig Growth Performance
[00139] In one embodiment, the present invention relates to a method of increasing the growth performance of a swine which comprises administering an enzyme-producing strain to a swine in an amount effective to increase the growth performance of the swine. , compared to a swine that was not administered the enzyme-producing strain. The methods described in the present invention can be used to improve growth performance irrespective of the feed or diet of the swine.
[00140] In one embodiment, the present invention relates to a method of increasing growth performance in swine fed a diet rich in fibrous by-products comprising administering an enzyme-producing strain to a swine which is fed with a highly fibrous by-product diet, in an amount effective to increase the growth performance of the swine, compared to a swine that was not fed the enzyme-producing strain.
[00141] In another embodiment, the present invention relates to a method for increasing the average daily weight gain of a swine comprising administering an enzyme-producing strain to a swine in an amount effective to increase the average of daily weight gain of the swine compared to a swine that was not given the enzyme-producing strain.
[00142] In another embodiment, the present invention relates to a method of increasing the average daily feed intake in a swine comprising administering an enzyme-producing strain to a swine in an amount effective to increase the average of daily feed intake, compared to a swine that was not fed the enzyme-producing strain.
[00143] In another embodiment, the present invention relates to a method of increasing the feed efficiency of animal feed in a swine which comprises administering to a swine a feed supplemented with an enzyme-producing strain in an amount effective in increasing feed efficiency in swine compared to a swine not administered the enzyme-producing strain.
[00144] In yet another embodiment, the present invention relates to a method for improving the carcass characteristics of a swine which comprises administering an enzyme-producing strain to a swine in an amount effective to improve the carcass characteristics of swine compared to a swine that was not administered the enzyme-producing strain. Carcass characteristics that can be improved include, but are not limited to, fat depth, loin depth; lean meat percentage, hot carcass weight, organ weight, carcass grade, and carcass value.
[00145] In one embodiment, the measured value of the housing characteristics can be increased or decreased.
[00146] In yet another embodiment, the measured value of one or more of the following carcass characteristics is increased: fat depth, loin depth; lean meat percentage, hot carcass weight, organ weight, carcass grade, and carcass value.
[00147] In yet another embodiment, the measured value of one or more of the following carcass characteristics is decreased: fat depth, the depth of the loin; lean meat percentage, hot carcass weight, organ weight, carcass grade, and carcass value.
[00148] In yet another embodiment, the present invention relates to a method of reducing the mortality rate in swine, comprising administering an enzyme-producing strain of swine in an amount effective to reduce the mortality of said swine, compared to pigs that were not administered the enzyme-producing strain.
[00149] In another embodiment, the present invention relates to a method of improving the digestibility of lignin comprising administering an enzyme-producing strain of a swine in an amount effective to improve the digestibility of lignin as compared to a swine that was not administered with the enzyme-producing strain.
[00150] In another embodiment, the present invention relates to a method for improving the digestibility of lignin in highly fibrous diets comprising administering an enzyme-producing strain of a swine in an amount effective to improve the digestibility of lignin of the highly fibrous diets compared to a swine that was not fed the enzyme-producing strain. In another embodiment, highly fibrous diets comprise by-product diets. In yet another embodiment, the diet comprises DDGS.
[00151] In another embodiment, the present invention relates to a method of improving apparently ileal digestibility which comprises administering an enzyme-producing strain of a swine in an amount effective to improve apparently ileal digestibility in the swine, in compared to a swine that was not administered the enzyme-producing strain.
[00152] In yet another embodiment, the present invention relates to a method of improving total digestibility comprising administering an enzyme-producing strain of a swine in an amount effective to improve swine digestibility as compared to a swine that was not administered with the enzyme-producing strain.
[00153] In yet another embodiment, the present invention relates to a method of lowering the pH of ileal digestion, comprising administering an enzyme-producing strain of a swine in an amount effective to lower the pH of ileal digestion. , in the swine and from a swine that was not administered with the enzyme-producing strain.
[00154] In yet another embodiment, the methods described above further comprise administering a food supplemented with an enzyme-producing strain.
[00155] In another embodiment, the enzyme-producing strains in the methods described above related to swine growth performance is a composition comprising Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS 1013 (NRRL B- 50509) and AGTP BS3BP5 (NRRL B-50510).
[00156] The enzyme-producing strain(s) to improve swine growth performance may (m) be administered as a single strain, or one or more combination(s) of the ) strain(s), one or more supernatant(s) from a culture of the strain(s), animal feed, including one or more strain(s), or mixtures thereof. Methods to improve manure storage units
[00157] In one embodiment, the present invention relates to a method for improving manure storage units, comprising administering the strain(s) producing the enzyme(s), a or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) or the mixtures from an animal in an amount effective to improve the manure storage unit. In one embodiment, the animal is a swine. In certain embodiments, the dung storage unit is a dung pit.
[00158] In yet another embodiment, the present invention relates to a method for improving air quality in an animal housing room which comprises administering the strain(s) producing the ) enzyme(s), combination of one or more of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) s) or mixtures thereof to an animal in an effective amount to improve the air quality in the room. In one embodiment, improving air quality comprises reducing odors in the environment. In another embodiment, improving air quality comprises reducing production of one or more of the following: volatile fatty acids, ammonia, methane, or hydrogen sulfide.
[00159] In at least some embodiments, administration improves at least one of the following characteristics: lower incidence of foaming, lower solids accumulation, and less nitrogen, sulfur, phosphorus, fiber-bound nitrogen, total protein, fat, fiber and when compared to a control manure pit.
[00160] In one embodiment, the strains producing enzymes to improve manure storage units comprise a strain of Bacillus. In one embodiment, the Bacillus strain is Bacillus subtilis. In another embodiment, the Bacillus strain is Bacillus pumilus.
[00161] In another embodiment, the enzyme-producing strain for improving a manure storage unit includes, but is not limited to, Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, and Bacillus subtilis AGTP BS 1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX11-1, and strains having all characteristics thereof, any derivative or variant, and mixtures thereof.
[00162] In another embodiment, the present invention relates to a method for improving a manure storage unit comprising contacting the enzyme-producing strain(s), one or more combination(s) of the enzyme(s) ) strain(s), one or more supernatant(s) from a culture of the strain(s), compositions including one or more strain(s), or mixtures thereof directly to a manure storage unit , such as a manure pit. Improvements resulting from contacting the enzyme producing strain(s) directly to a manure storage unit include at least one of a lower incidence of foaming, less solids build-up, and less nitrogen. , sulfur, phosphorus, fiber-bound nitrogen, total protein, fat, and fiber content from control manure wells.
[00163] In another embodiment, the methods described above can be used to ameliorate manure residue problems, which include but are not limited to manure pit foaming, solids accumulation, increases in (the ) nitrogen, (b) sulfur, (c) phosphorus, (d) fiber-bound nitrogen, (e) total protein, (f) fat, and (g) fiber content. A. Methods for Controlling or Reducing Foam from a Manure Storage Unit
[00164] In another embodiment, the present invention relates to a method of controlling or reducing the foam of a manure storage unit which comprises administering an effective amount of the producing strain(s) of the enzyme(s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) or mixtures thereof to an animal in an amount effective to control or reduce the amount of foam in a manure storage unit as compared to a manure storage unit where animals have not been administered with the enzyme-producing strains. In yet another embodiment, the foam:liquid manure storage ratio is reduced.
[00165] In another embodiment, the present invention relates to a method for controlling or reducing foam in a storage well, comprising contacting the enzyme(s) producing strain(s) ( s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), compositions including one or more strain(s), or mixtures thereof directly into a manure storage well in an amount effective to control and reduce foam in a manure storage well as compared to a manure storage well without the enzyme producing strains. In another embodiment, the foam:liquid manure storage ratio is reduced.
[00166] The amount of foam in a manure storage unit is related to the amount of solids in the manure storage unit. Manure storage units with a higher percentage of solids generally have a higher foam:liquid ratio, and therefore more foam.
[00167] In another embodiment, the present invention relates to a method of controlling or reducing the foam of a manure storage unit which comprises administering an effective amount of the producing strain(s) of the enzyme(s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including a or more strain(s) or mixtures thereof to an animal in an amount effective to reduce the amount of solids, and thereby reduce the amount of foam, in a manure storage unit as compared to a manure storage unit. manure storage, where the animals were not administered the enzyme-producing strains. In yet another embodiment, the foam: the net proportion of manure storage is reduced.
[00168] In another embodiment, the present invention relates to a method for controlling or reducing the foam of a manure storage unit which comprises contacting the strain(s) producing the enzyme(s) (s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), the compositions including one or more strain(s) ), or mixtures thereof directly into a manure storage unit in an amount effective to reduce the amount of solids in the manure storage unit, compared to a manure storage unit without the enzyme-producing strains. In another embodiment, the foam:liquid manure storage ratio is reduced. B. Methods for altering the microbial ecology in a manure storage unit
[00169] In one embodiment, the present invention relates to a method of altering the microbial ecology in a manure storage unit, comprising administering the strain(s) producing the enzyme(s) ( s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), feed, including one or more strain(s) or admixing the same to an animal in an amount effective to modify the microbial ecology in the manure storage unit, as compared to a manure storage unit, where the animals were not administered the enzyme-producing strains.
[00170] In another embodiment, the present invention relates to a method for altering a microbial ecology in a manure storage unit which comprises contacting the strain(s) producing the enzyme(s) ( s), one or more combination(s) of the strain(s), one or more supernatant(s) from a culture of the strain(s), the compositions including one or more strain(s) , or mixtures thereof directly into the manure storage unit in an amount effective to alter the microbial ecology in the manure storage unit, compared to a manure storage unit, where enzyme-producing strains were not used.
[00171] In one embodiment, enzyme-producing strains can be altered, either directly or indirectly, with respect to microbial ecology in a manure storage unit and cause an increase in the population of certain species of bacteria and a decrease in the population of other bacterial species. Bacterial species that can be altered, either directly or indirectly, with enzyme-producing strains include, but are not limited to, methanogens, Bacteroides, Clostridium I cluster, Clostridium IV cluster, Clostridium XlVa cluster, and sulfate.
[00172] In one embodiment, the strains that produce the enzyme have the ability to shift nutrient usage through the microbial population and subsequently alter the microbial ecology such that incidents of aggregate foaming are alleviated, either through decreasing the production of gas available to be trapped in the foam matrix, altering the availability of the molecular compounds that make up the foam matrix, or directly inhibiting the growth of bacteria associated with foaming incidents. C. Methods of altering the composition of volatile fatty acids
[00173] In one embodiment, the present invention relates to a method for altering the volatile fatty acid composition in manure which comprises administering an enzyme-producing strain to an animal in an amount effective to alter the fatty acid composition in the manure from said animal, such as with respect to the manure of a second animal not administered the enzyme-producing strain. In one embodiment, changing the fatty acid composition can result in an increase in certain fatty acids and a decrease in other fatty acids. In another embodiment, the alteration of fatty acid compositions can occur either directly or indirectly.
[00174] In one embodiment, the present invention relates to a method for altering the volatile fatty acid composition in a manure storage unit comprising administering an enzyme-producing strain to an animal in an amount effective to altering the fatty acid composition of said animal that is stored in said manure storage unit as manure compared to a second animal not administered with the enzyme producing strain. In one embodiment, the animal is a swine. In another embodiment, the dung storage unit is a dung pit.
[00175] In yet another embodiment, the present invention relates to a method for altering the volatile fatty acid composition in a manure storage unit comprising administering an enzyme-producing strain of an animal, measuring the amount of volatile fatty acid in the manure of animals fed the enzyme-producing strain; and adjusting the concentration of enzyme-producing strain fed to the animal to achieve a desired concentration of volatile fatty acid in the manure stored in the manure pit.
[00176] In yet another embodiment, the present invention relates to a method for altering the volatile fatty acid composition in a manure storage unit which comprises contacting an enzyme-producing strain directly to the manure storage unit. manure in an amount effective to change the fatty acid composition in the manure storage unit compared to a manure storage unit without an enzyme-producing strain.
[00177] In another embodiment, volatile fatty acids that can be altered by the methods described in the present invention include, but are not limited to, acetate, propionate, butyrate, I-butyrate, 4-methyl-valerate. In another embodiment, the methods described in the present invention increase the fatty acid butyrate in manure. In yet another embodiment, the methods described in the present invention decrease 4-methyl-valerto fatty acids in manure.
[00178] In another embodiment, the total volatile fatty acids can be altered. In another embodiment, the methods described in the present invention reduce total volatile fatty acids in manure. Methods to change gas emissions
[00179] In one embodiment, the present invention relates to a method for altering gas emissions comprising administering an enzyme-producing strain to an animal in an amount effective to alter gas emissions as compared to a animal not administered with an enzyme-producing strain. In one embodiment, changing gas emissions may result in an increase in certain emissions of gases and a decrease in other gases. In another modality, changes in gas emissions can occur directly or indirectly.
[00180] In one embodiment, the strains producing enzymes to alter gas emissions comprise a strain of Bacillus. In one embodiment, the Bacillus strain is Bacillus subtilis. In another embodiment, the Bacillus strain is Bacillus pumilus.
[00181] In another embodiment, strains producing enzymes to alter gas emissions include, but are not limited to, Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP strains BS 1013, and Bacillus subtilis AGTP BS1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus X11-1, strains having all the characteristics thereof, any derivative or variant thereof, and mixtures thereof.
[00182] The strain(s) producing the enzyme(s) to alter gas emissions may be administered as a single strain, or one or more combination(s) of the strain(s) (s), one or more supernatant(s) from a culture of the strain(s), food including one or more strain(s), or mixtures thereof.
[00183] Gases that can be altered through enzyme-producing strains include, but are not limited to, ammonia, carbon dioxide, methane, and hydrogen sulfide.
[00184] In another embodiment, the present invention relates to a method for altering the gaseous emissions of an animal dwelling room which comprises administering an enzyme-producing strain to an animal in an effective amount. to alter gas emissions into the environment, compared to a room-dwelling animal that were not administered the enzyme-producing strains. In one embodiment, the animal is a swine. In another embodiment, the room is located in a barn. In one embodiment, methane and hydrogen sulfide gas emissions are reduced in the housing rooms of an animal that has been administered the enzyme-producing strains.
[00185] In another embodiment, the present invention relates to a method for altering gas emissions in an animal housing room which comprises administering an enzyme-producing strain to an animal in an amount effective to alter the gas emissions in the animal's dwelling environment, and the measurement of the amount of gas in the room.
[00186] In another embodiment, the present invention relates to a method of altering gas emissions in a manure storage unit which comprises administering an enzyme-producing strain to an animal in an amount effective to alter the gas emissions in the manure storage unit compared to a manure storage unit with manure from animals that were not administered the enzyme producing strains. In one embodiment, the animal is a swine. In another embodiment, the dung storage unit is a dung pit.
[00187] In another embodiment, the present invention relates to a method for altering gas emissions in a manure storage unit which comprises contacting an enzyme-producing strain directly to the manure storage unit in a effective amount to change gas emissions, compared to a manure storage unit without the enzyme-producing strain. In one embodiment, the animal is a swine. In another embodiment, the dung storage unit is a dung pit.
[00188] In one embodiment, methane and hydrogen sulfide gas emissions are reduced. Methods to alleviate an inflammatory response
[00189] In another embodiment, the present invention relates to a method of alleviating inflammatory effects in animals, comprising administering an enzyme-producing strain to the animal in an amount effective to alleviate or inhibit the inflammatory response. In one embodiment, the animal is a mammal. In another embodiment, the animal is a bird. In another embodiment, the animal is a chicken. In yet another embodiment, the animal is a swine.
[00190] Enzyme producing strains can alleviate or inhibit the inflammatory response by 2 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35% , 35 to 40%, 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85% , 85 to 90%, 90 to 95%, and greater than 95%, compared to a reference control (e.g., an agent with no anti-inflammatory properties, such as a buffered saline solution or a strain without anti-inflammatory properties ).
[00191] In one embodiment, the enzyme producing strains for alleviating inflammatory effects in an animal comprises a strain of Bacillus. In one embodiment, the Bacillus strain is Bacillus subtilis. In another embodiment, the Bacillus strain is Bacillus pumilus.
[00192] In another embodiment, the enzyme-producing strains for alleviating the inflammatory effects in an animal comprise the strains Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus subtilis AGTP BS918, Bacillus subtilis AGTP BS1013 , and Bacillus subtilis AGTP BS1069, Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX11-1, strains having all the characteristics thereof, any derivative or variant thereof, and mixtures thereof.
[00193] In another embodiment, the enzyme-producing strains to alleviate the inflammatory effects in an animal is a composition comprising Bacillus subtilis AGTP BS 1013, Bacillus subtilis AGTP BS3BP5, and Bacillus subtilis AGTP 944.
[00194] Enzyme-producing strains can alleviate or inhibit the inflammatory response by reducing the expression of genes involved in the inflammatory response. In one embodiment, enzyme-producing strains can decrease expression of a gene by 2 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35 %, 35 to 40%, 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85 %, 85 to 90%, 90 to 95%, and greater than 95%, compared to a reference control (e.g., an agent with no anti-inflammatory properties, such as a buffered saline solution or a strain with no anti-inflammatory properties). inflammatory).
[00195] In another embodiment, the enzyme-producing strains can alleviate or inhibit the inflammatory response by reducing the expression of a protein involved in the inflammatory response.
[00196] In yet another embodiment, the enzyme-producing strains can alleviate or inhibit the inflammatory response by reducing the activity of a protein involved in the inflammatory response.
[00197] In another embodiment, enzyme-producing strains can reduce the expression or activity of a protein by 2 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35%, 35 to 40%, 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85%, 85 to 90%, 90 to 95%, and greater than 95% compared to a reference control (e.g., an agent lacking anti-inflammatory properties, such as buffered saline or a strain without anti-inflammatory properties).
[00198] In another embodiment, production of enzyme strains can reduce the expression of a gene or reduce the activity of a protein involved in any pathway involved in the inflammatory response, including but not limited to adhesion between extravasation migration ; apoptosis signaling; calcium signaling; complement cascade; cytokines, and cytokine signaling; eicosanoid synthesis and signaling, glucocorticoid / PPAR signaling; G protein-coupled receptor signaling; detection of innate pathogens; leukocyte signaling; MAPK signaling; natural killer cell signaling; NK-kappa B signaling; antigen presentation; PI3K / AKT signaling; ROS/glutathione/cytotoxic granules, and TNF superfamily and signaling.
[00199] In one embodiment, the enzyme-producing strains can reduce the activity or expression of cytokines, including but not limited to interleukins, interferons, tumor necrosis factor, erythropoietin, Tpo, Fit-3L, SCF, M-CSF , and DME.
[00200] In one embodiment, the interleukins include but are not limited to interleukin (IL)-1, IL-1a, IL-1 such as, IL-β, IL-IRA, IL-2, IL-3, IL-4 , IL-5, IL-6, IL-6 as, IL-7, IL-8, IL-9, IL-10, IL-10 as IL-11, IL-12, IL-13, IL -14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, GM-CSF, and OSM.
[00201] In another embodiment, the interferons include, but are not limited to, the production of IFN-a, IFN-β and IFN-gamma.
[00202] In another embodiment, tumor necrosis factor includes, but is not limited to, CD154, LT-β, TNF-a, TNF-β, TGF-βl, TOP-β2, TOP-β3, 4-1BBL , APRIL, CD70, CD 153, CD 178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE.
[00203] In another embodiment, the enzyme-producing strain can be used to reduce the activity of or reduce the expression of chemokines, including but not limited to C chemokines, CC chemokines, CXC chemokines, and CXC3 chemokines.
[00204] In one embodiment, the C chemokines include, but are not limited to, XCL1, and XCL2.
[00205] In another embodiment, CC chemokines include, but are not limited to, CCL1, CCL 2, CCL 3, CCL 4, 5 CCL, CCL 6, 7 CCL, CCL 8, 9 CCL, CCL 10, CCL 1 1 and CCL 28.
[00206] In another embodiment, the CXC chemokines include, but are not limited to, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL1 1, CXCL12, CXCL13 and CXCL14.
[00207] The strain(s) producing the enzyme to alleviate the inflammatory response may be administered as a single strain, or one or more combination(s) of the strain(s) ( s), one or more supernatant(s) from a culture of the strain(s), animal feed, including one or more strain(s), or mixtures thereof.
[00208] The strains, methods and compositions described in the present invention can be further described in the numerical paragraphs below. 1. An isolated Bacillus strain with enzymatic activity. 2. The strain according to paragraph 1, in which the enzyme activity is selected from the group consisting of cellulase activity, α-amylase activity, xylanase activity, esterase, β-mannanase, β-mannanase activity, lipase, protease activity, and combinations thereof. 3. The strain according to any one of the preceding paragraphs, wherein the enzyme activity is selected from the group consisting of zeinase activity and soy protease activity and combinations thereof. 4. The strain according to any one of the preceding paragraphs, wherein, when the strain is administered to an animal, the strain exhibits an improvement in at least one of the breakdown of complex feed components, manure residue problems, efficiency of swine production, carcass characteristics, and performance of swine fed with high levels of DDGS, compared to a control animal. 5. The strain according to any one of the preceding paragraphs, wherein, when the strain is administered to an animal, the strain shows an improvement in at least one of the breakdown of complex food components, manure residue problems, efficiency of swine production, carcass characteristics, and performance of swine during feeding with high levels of DDGS by at least 2% relative to a control animal. 6. The strain according to any of the preceding paragraphs, wherein, when the strain is administered to an animal, the strain provides an improvement in at least one of the following: body weight, average daily gain, average daily feed intake , feed efficiency, carcass traits, nutrient digestibility and manure residue problems compared to a control animal. 7. The strain according to any of the preceding paragraphs, wherein, when the strain is administered to an animal, the strain provides an improvement in at least one of the following: body weight, average daily gain, average daily feed intake , feed efficiency, carcass traits, nutrient digestibility and manure residue problems by at least 2% compared to a control. 8. The strain according to any one of the preceding paragraphs, wherein the strain is selected from the group consisting of the species of B. subtilis and B. pumilus strains, strains possessing all the characteristics thereof, any derivative or variant of them and their mixtures. 9. The strain according to any of the preceding paragraphs, wherein the strain(s) is (are) selected from the group consisting of Bacillus subtilis AGTP BS3BP5 strains (NRRL B-50510), Bacillus subtilis AGTP BS442 (NRRL B-50542), Bacillus subtilis AGTP BS521 (NRRL B-50545), Bacillus subtilis AGTP BS918 (NRRL B-50508), Bacillus subtilis AGTP BS1013 (NRRL B-50509), Bacillus subtilis AGTP BS 1069 ( NRRL B-50544), Bacillus subtilis AGTP 944 (NRRL B-50548), Bacillus pumilus AGTP BS 1068 (NRRL B-50543) and Bacillus pumilus KX11-1 (NRRL B-50546) and the strains that have all the same characteristics and any variant or derivative thereof, and mixtures thereof. 10. The strain according to any of the preceding paragraphs, wherein the strain(s) is (are) selected from the group consisting of Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis AGTP BS442 (NRRL B-50542), Bacillus subtilis AGTP BS521 (NRRL B-50545), Bacillus subtilis AGTP BS918 (NRRL B-50508), Bacillus subtilis AGTP BS 1013 (NRRL B-50509), Bacillus subtilis AGTP BS 1069 ( NRRL B-50544), Bacillus subtilis AGTP 944 (NRRL B-50548), Bacillus pumilus AGTP BS 1068 (NRRL B-50543) and Bacillus pumilus KX1 1-1 (NRRL B-50546) any variant or derivative thereof, and their mixtures. 11. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP BS3BP5 (NRRL B-50510). 12. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP BS442 (NRRL B-50542). 13. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP BS521 (NRRL B-50545). 14. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP BS918 (NRRL B-50508). 15. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP BS 1013 (NRRL B-50509). 16. The strain of any of the preceding numbers, wherein the Bacillus strain is Bacillus pumilus AGTP BS 1068 (NRRL B-50543). 17. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis strain AGTP BS1069 (NRRL B-50544). 18. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus subtilis AGTP 944 (NRRL B-50548). 19. The strain according to any one of the preceding paragraphs, wherein the Bacillus strain is Bacillus pumilus KX1 1-1 (NRRL B-50546). 20. A composition comprising the supernatant one or more culture(s) of the strain(s) according to any one of paragraphs 1 to 19 and mixtures thereof. 21. A composition comprising one or more strain(s) according to any one of paragraphs 1-19 and mixtures thereof. 22. The composition of points 20 or 21, wherein the Bacillus subtilis strains are AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis AGTP BS918 (NRRL B-50508), and Bacillus subtilis AGTP BS1013 (NRRL B-50509). 23. The composition of points 20 or 21, wherein the Bacillus subtilis strains are AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis AGTP BS944 (NRRL B-50509) and Bacillus subtilis AGTP BS 1013 (NRRL B-50509) . 24. An animal feed, wherein the feed is supplemented with the isolated strain(s) according to any one of paragraphs 1 to 19 or the composition(s) according to any of paragraphs 20 to 23 or mixtures thereof. 25. A method comprising the step of administering to an animal an effective amount of the strain(s) according to any one of paragraphs 1 to 19 or the composition(s) according to any one of paragraphs 20 to 23, the food according to paragraph 24 or mixtures thereof, in which administration by the enzymatic route breaks down at least one of the fibres, proteins, carbohydrates and lipids in the diet for the animal when the food is at high levels of DDGS for the animal. 26. A method comprising the step of administering to an animal an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, wherein the administration improves at least one of the breakdown of complex feed components, manure residue problems, pig production efficiency, carcass characteristics and performance of pigs. 27. A method comprising the step of administering to an animal an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, wherein administration improves at least one of the following body weight, average daily gain, average daily feed intake, feed conversion , carcass traits, nutrient digestibility and manure residue problems compared to a control animal. 28. A method comprising the step of administering to birds an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, wherein administration improves at least one of the following body weight, average daily gain, average daily feed intake, feed conversion, carcass traits, nutrient digestibility and manure residue problems compared to a control animal. 29. A method comprising the step of administering to a swine an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, wherein administration improves at least one of the following body weight, average daily gain, average daily feed intake, feed conversion , carcass traits, nutrient digestibility and manure residue problems compared to a control animal. 30. The method of paragraphs 25 to 29, wherein the composition is the composition according to paragraph 22 or 23. 31. The method of any one of paragraphs 25 to 30, wherein the strain(s) ) is (are) administered at about 1 x 10 5 to about 1 x 10 11 CFU/animal/day. 32. The method according to any one of paragraphs 25 to 27, and 29 to 31, wherein the animal is a swine. 33. The method according to any one of paragraphs 25 to 32, wherein the animal is fed high levels of soluble dry still grains (DDGS). 34. The method according to any one of paragraphs 25 to 33, wherein the animal is fed soluble dry still grains (DDGS) at a rate of more than 10% of the animal's diet. 35. The method according to any one of paragraphs 25 to 34, wherein the animal is fed soluble dry still grains (DDGS) at a rate of more than 30% of the animal's diet. 36. A method comprising the step of administering an effective amount of the strain(s) according to any one of paragraphs 1 to 19 or the composition(s) according to any one of paragraphs 20 to 23 for a swine manure storage unit. 37. The method according to paragraph 36, wherein the swine waste storage unit is a manure pit. 38. The method in accordance with paragraph 36 or 37, which further comprises improving at least one of the following characteristics: lower incidence of foaming, lower solids build-up, and less nitrogen, sulfur, phosphorus, fiber-bound nitrogen , total protein, fat, and fiber content when compared to a control manure well. 39. A method of forming a composition, the method comprising: (a) growing in a liquid broth, a culture including one of the isolated strain(s) according to any one of paragraphs 1 to 19, and (b) separating the strain from the liquid broth. 40. The method of paragraph 39, which further comprises lyophilizing the isolated strain and adding the lyophilized strain to a carrier. 41. The method according to paragraph 39 or 40, which further comprises retaining the liquid broth after the strain has been separated therefrom to generate a supernatant. 42. A method of improving the growth performance of an animal comprising administering to an animal an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) ) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, compared to a control animal. 43. The method in accordance with paragraph 42 in which management improves at least one of the following body weight, average daily gain, average daily feed intake, feed conversion, carcass characteristics, nutrient digestibility and problems of manure residues. 44. A method of improving manure storage units, comprising administering to an animal an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof, in an amount effective to improve the manure storage unit, as compared to the manure of a control animal, which is stored in a second manure storage unit. 45. A method of improving manure storage units, comprising contacting an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any of paragraphs 20 to 23, the feed in accordance with paragraph 24, or mixtures thereof directly to the manure storage unit. 46. The method in accordance with paragraph 44 or 45, wherein the improvement comprises at least one of the following: a lower incidence of foaming, less solids build-up, and less nitrogen, sulfur, phosphorus, fiber-bound nitrogen, total protein, fat, fiber content and manure well control. 47. A method of controlling or reducing foam in a manure pit which comprises administering an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof for animals whose manure is stored in the manure pit. 48. A method of controlling or reducing foam in a manure pit comprising contacting an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof directly to the manure pit. 49. A method of altering the microbial ecology in a manure well comprising administering an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) of according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof for animals whose manure is stored in the manure pit. 50. A method of altering the microbial ecology in a manure well comprising contacting an effective amount of the strain(s) in accordance with any one of paragraphs 1 to 19, the composition(s) of according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof directly to the manure pit. 51. A method for altering the volatile fatty acid composition in a manure pit which comprises administering an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) ( s) in accordance with any one of paragraphs 20 to 23, the feed in accordance with paragraph 24, or mixtures thereof for animals whose manure is stored in the manure pit. 52. A method for altering the volatile fatty acid composition in a manure pit comprising contacting an effective amount of the strain(s) in accordance with any one of paragraphs 1 to 19, the composition(s) ( s) in accordance with any one of paragraphs 20 to 23, the feed in accordance with paragraph 24, or mixtures thereof directly into the manure pit. 53. A method of altering the gaseous emissions of an animal housing room which comprises administering an effective amount of the strain(s) in accordance with any one of paragraphs 1 to 19, the composition(s) ) in accordance with any one of paragraphs 20 to 23, feed in accordance with paragraph 24, or mixtures thereof for animals in an amount effective to reduce gas emissions. 54. A method for altering gas emissions in a manure storage unit comprising administering an effective amount of the strain(s) in accordance with any one of paragraphs 1 to 19, the composition(s) ( s) in accordance with any one of paragraphs 20 to 23, the feed in accordance with paragraph 24, or mixtures thereof for animals in an amount effective to reduce gas emissions. 55. A method for altering gas emissions in a manure storage unit comprising contacting an effective amount of the strain(s) in accordance with any one of paragraphs 1 to 19, the composition(s) ( s) in accordance with any one of paragraphs 20 to 23, the feed in accordance with paragraph 24, or mixtures thereof directly into the manure storage unit in an amount effective to reduce gas emissions. 56. A method of attenuating the inflammatory response comprising administering an effective amount of the strain(s) according to any one of paragraphs 1 to 19, the composition(s) according to any one of paragraphs 20 to 23, the feed according to paragraph 24, or mixtures thereof for animals in an amount effective to alleviate the inflammatory response. 57. An isolated strain according to paragraphs 1 to 19 or composition according to paragraphs 20 to 23 or a feed according to paragraph 24 for use as a medicament to ameliorate at least one of the breakdown of complex food components , manure residue problems, swine production efficiency, carcass characteristics and swine performance when fed high levels of DDGS. 58. Use of isolated strain according to paragraphs 1 to 19 or composition according to paragraphs 20 to 23 or a feed according to paragraph 24 in the preparation of a medicament to provide one or more enzyme(s). 60. An isolated Bacillus strain described in paragraphs 1 to 19 for use in improving the breakdown of complex feed components, manure residue problems, the efficiency of swine production, carcass characteristics and performance of swine when fed high levels of DDGS 61. Use of the isolated Bacillus strain described in paragraphs 1 to 19 in the preparation of a medicament to provide enzyme activity. 62. Use of the isolated Bacillus strain described in paragraphs 1 to 19 in the preparation of a medicament to improve at least one of the breakdown of complex feed components, manure residue problems, swine production efficiency, carcass characteristics and performance of pigs when fed high levels of DDGS. EXAMPLES
[00209] The following examples are provided for illustrative purposes only. The Examples are included in the present invention only to aid in a more complete understanding of the presently described present invention. The examples do not limit the scope of the present invention described and claimed in the present invention in any way. EXAMPLE 1
[00210] Isolation of environmental bacteria and identification of enzymatic activities
[00211] Agricultural and environmental waste samples were collected from various sources over a period of several years. Upon arrival, samples were diluted in a 1% peptone solution, spores treated for 35 minutes at 65 °C and serially diluted on tryptic soy agar plates (Difco BD, Franklin Lakes, NJ). After incubation at 32 °C for 48 hours, the growth of several unknown environmental colonies were cultured from the tryptic soy broth (TSB) plates, similarly reincubated and stored frozen at -85 °C for further analysis.
[00212] Approximately 4000 presumptive Bacillus isolates of environmental origin were collected and selected for their ability to degrade a variety of substrates of interest. Environmental cultures were picked from frozen library stocks and incubated in 0.5 ml TSB at 32 °C for 24 hours in an orbital shaker incubator, with the speed set to 130 (Gyromax 737). Highly thorough screening of these test strains was performed by replicating the local plating of 2 microliters of liquid culture to 15.0 ml of various types of substrate medium of interest on 100x100x15mm grid plates. Cellulase, a-amylase, zeinase, soy protease, esterase, lipase and xylanase activities were determined based on the use of the specific substrate by the individual strains. Media components used to assay substrate use properties from the enzymatic activity of the environment-derived strains are described in Table 1. Assay plates were allowed to dry for 30 minutes following application of culture, and then then incubated at 32 °C for 24 hours. Enzyme activities for each strain were determined by measuring the substrate degradation zone, in millimeters, as indicated by compensating the surrounding edge of colony growth. Mean values of identical plates were recorded.
[00213] Nine strains were selected from the approximately 4000 selected as candidates for direct feeding microbial strains demonstrating a proportion of substrate activities representing the 10 % and the upper 2 % of the enzyme activity of all strains analyzed (Table two). Based on their ability to utilize or degrade a variety of relevant substrates associated with the inclusion of DDGS in foods, nine isolates were chosen as candidates for one or more direct-feeding microbial(s) (DFM s). RAPD PCR profiles and partial 16S rDNA sequences from each strain were determined. The genus and determination of each presumptive species were made by amplifying the 16S rDNA gene using a set of 8F and 1541R primers. Purified PCR products were sequenced from both the forward and reverse sides, and a contiguous sequence generated using a CAP3 assembly program. The nine strains selected are: Bacillus subtilis AGTP BS3BP5 (Figures 1 & 2), Bacillus subtilis AGTP BS442 (Figures 3 and 4), Bacillus subtilis AGTP BS521 (Figures 5 and 6), Bacillus subtilis AGTP BS918 (Figures 7 and 8), Bacillus subtilis AGTP BS1013 (Figures 9 and 10), Bacillus pumilus AGTP BS 1068 (Figures 11 and 12), Bacillus subtilis AGTP BS 1069 (Figures 13 and 14), Bacillus subtilis AGTP 944 (Figures 15 to 18), and Bacillus pumilus KXL 1-1 (Figures 19 to 21). Table 1 - Components of the media used to analyze the enzymatic activities illustrated using the substrate use properties of an environmentally derived strain of Bacillus

Table 2. Summary of enzyme strain activity of the direct-feed microbial candidate a Isolated number b CMCase (cells e) Stearas and Amylas and corn starch Proteins and soy proteins Proteins and zein Xylanas e

a Values represent the substrate degradation zone, in millimeters (mm), as indicated by compensating the surrounding edge of colony growth for each strain. b For strain isolate designations, BS = Bacillus subtilis; BP = Bacillus pumilus 1 Values represent the top 2% of enzymatic activity in the specific class of all 4000 strains selected. 2 Values represent the first 10% of enzymatic activity in the specific class of all 4000 strains selected. EXAMPLE 2
[00214] Comparison of the enzymatic activity of the Novel Bacillus Strains and the Three Commercial Microbial Strains of Direct Feed Bacillus, Microsource ® S.
[00215] The three Microsource ® S strains of Bacillus (B. subtilis 27 (BS 27), B. licheniformis (previously thought to be B. amyloliquefaciens) 842 and B. licheniformis 21 (Bl 21)) were chosen from stocks individual frozen library samples and incubated in 0.5 ml TSB at 32 °C for 24 hours in an orbital shaker incubator, with the speed set to 130 (Gyro max 737R). Highly thorough screening of these strain products was performed by replicating local plating of 2 microliters of liquid culture to 15.0 ml of various types of substrate medium of interest in 100x100x15mm grid plates. Cellulase, soy protease, and esterase/lipase activities were determined based on specific substrate usage by the individual strains. Media components used to assay substrate use properties from the enzymatic activity of the environment-derived strains are described in Table 3. Assay plates were allowed to dry for 30 minutes following application of culture, and then , incubated at 32 °C for 24 hours. Enzyme activities for each strain were determined by measuring the substrate degradation zone, in millimeters, as indicated by compensating the surrounding edge of colony growth. Mean values from replicated plaques were recorded and compared with values obtained from the Bacillus strains identified in Example 1 (Table 4). Only one strain of three Bacillus MicroSource S ® strains demonstrated any substantial enzymatic activity when compared to Bacillus strains selected for their substrate degrading activity, which was exemplified by the soy protease activity of the Microsource ® S strain. Bacillus subtilis BS27. Table 3 - Medium components used to analyze the enzymatic activities illustrated through substrate use properties
Table 4. The enzyme activity of strains of Microsource ® S Bacillus products compared to selected Bacillus strains for improved substrate degradation.

a Values represent the substrate degradation zone, in millimeters (mm), as indicated by compensating the surrounding edge of colony growth for each strain. 1 Values represent the top 2% of enzymatic activity in the specific class of all 4000 strains selected. 2 Values represent the first 10% of enzymatic activity in the specific class of all 4000 strains selected. EXAMPLE 3 Animal Feed Trial Demonstrating Improved Growth Performance in Response to Bacillus subtilis 3BP5 Strain Added to Swine Diet.
[00216] A swine feeding trial was conducted to evaluate the effects of a direct feed feed additive based on microbial Bacillus (DFM) on body weight gain, feed intake and feed conversion of swine from growth to finish. . Approximately 180 pigs (Monsanto Genetic Choice GPK 35 sows mated to Ultra EB bulls) were locked into three weight blocks per initial body weight and written into groups of 5 pigs/pen upon completion of the care period. The pigs were transferred to a weaning facility and housed 5 pigs/pen in fully slatted pens (1.52 mx 3.05 m), equipped with a single hole feeder, and cup-finish drinkers. Minimum ambient initial ambient temperature was maintained at approximately 78 °C. During the finishing phase, the minimum temperature was reduced to 70 °C. Feed and water were freely available throughout the study.
[00217] One of two dietary treatments were assigned to each pen (18 pens/treatment) within each block, and administered during Phase 1 (50 to 90 pounds), Phase 2 (90 to 130 pounds), Phase 3 (130 pounds). to 180 pounds), Phase 4 (180 to 230 pounds) and Phase 5 (230 pounds to market at around 270 pounds). The two dietary treatments consisted of a basal control diet devoid of DFM 3BP5 and the basal diet with DFM 3BP5 in a five-phase growth-finish study of pigs. Diets were formulated to meet or exceed NRC (1988) requirements and consisted predominantly of corn, soybean meal, and DDGS in 47%, 18.6%, and 30% of the diet, respectively. Bacillus subtilis AGTP BS3BP5 strain was added to the diet at 7.3 x 10 CFU/lb feed and provided about 1 x 10 CFU/head/day based on average daily feed intake (adfi). Data collected were mean daily gain, mean daily feed intake, and feed required per unit of gain at each of the five growth and termination phases. The pigs were withdrawn from the study when the average pig weight of the entire barn reached about 270 lbs.
[00218] Performance data were analyzed as a randomized block design, with the pen as the experimental unit and blocks based on initial body weight. Analysis of variance was performed using SAS GLM procedures (SAS Institute, Inc., Cary, NC).
[00219] Diets containing swine fed the Bacillus subtilis AGTP BS3BP5 strain had higher (P < 0.01) average daily weight gain (adg) and gain:feed during Phase 1 of the growth period and tended (P < 0.08) to have higher ADG and gain: feeding in the combined periods of Phase 1 and Phase 2 compared to swine fed the control diet (Table 5). The increase in ADG during the first growing period resulted in pigs fed Bacillus subtilis AGTP BS3BP5 having higher (P < 0.01) body weight at the end of the phase 1 period compared to pigs fed the control diet. . Table 5. Growth performance responses of pigs fed Bacillus subtilis AGTP 3BP5 compared to pigs fed the control diets.

1 IF = standard error EXAMPLE 4
[00220] Animal Feeding and Manure Pit Mass Balance Test Demonstrating the Effects of a Combination of Bacillus subtilis Strain Added to the Swine Diet.
[00221] A swine feeding trial was carried out to evaluate the effects of a direct microbial feed based on Bacillus (DFM) administered in the diet of swine at grow-finish on growth performance responses (average daily gain (adg) , average daily feed intake (adfi) and gain: feed), carcass yield and quality measurements, manure nutrient composition, microbial composition of the manure pit, and gas emissions (ammonia, methane, and hydrogen) from the manure pit. A total of 720 pigs (Landrace x Yorkshire-Duroc genotype) were housed in 12 rooms with 12 pens/room and 5 pigs/pen. Each room contained two manure pits capable of storing manure for an entire weaning period at the end. Each manure pit is located below the 6 pens with a wall under the central walkway that divides the two pits in each room. Each of the twelve rooms was equipped to monitor gas emissions from each ventilated room independently. The pigs were weaned and placed in pens prior to the start of the study and started to receive experimental test feed when they had reached an average body weight of 29.5 kg. The pigs were fed through five feeding phases lasting three weeks each. , and ended when the pigs reached an average slaughter weight of 120 kg.
[00222] Two dietary treatments were administered to trial pigs, consisting of a control diet and a diet supplemented with a combination of Bacillus strains (Strains BS1013, BS918 and BS3BP5). Diets were formulated to meet or exceed NRC (1988) requirements and consisted predominantly of corn, soybean meal, and DDGS in 50%, 20%, and 30% of the diet, respectively. The three strains in the Bacillus DFM combination were equally represented in the experimental test material which contained 1.47 x 10 CFU of the DFM per gram of material. The Bacillus DFM combination was added to the diet at 7.3 x 10 CFU/lb of feed and provided about 1 x 10 CFU/head/day based on average daily feed intake.
[00223] Pig performance measures (average daily gain (adg), average daily feed intake (adfi), gain: feed) were determined at the end of each feeding phase. These data were represented by 72 replicates/treatment). Manure wells were vacuum sampled at week 0 (initially and before swine receiving treatment), week 9 and week 15 and centesimal analysis was performed on nutrients contained in swine manure residues (12 replicates/treatment). The subsample on each day was also obtained to determine volatile fatty acid content and microbial community analysis (12 replicates/treatment). In addition, on week 15 of sampling at the end of the trial, the wells were emptied into a mixing vessel to homogenize the entire manure well contents, determine the manure well volume and sample for nutrient analysis. Gas emissions were measured in each room to determine ammonia, methane and hydrogen sulfide gas production (6 replicates/treatment). At the end of the study, the animals were sent to a commercial slaughterhouse and carcass data such as percentage of lean meat yield, carcass yield and iodine index were collected (72 replicates/treatment).
[00224] Referring now to Table 6, preliminary performance data from the study indicates that pigs fed the Bacillus DFM combination had higher (P < 0.05) and gain: ADG feed during the last phase of the trial. In addition, pigs fed the DFM tended (P = 0.15) to weigh 4.4 lb more at the end of the trial than pigs fed the control diet. Analysis of data on carcass characteristics, manure nutrients and microbial composition, and gas emissions from manure storage units, including, but not limited to, manure pits has not yet been completed, but expectations are that DFM treatment will increase the percentage of lean meat yield and preparing the percentage, decreasing fat iodine values, resulting in less nutrients accumulating in the manure, shifting the manure microbial communities to populations favorable for solids breakdown and decreasing ammonia, methane and sulfide gas emissions. Table 6. Effect of a combination of three Bacillus DFM strains administered as a dietary supplement on the growth-finish performance responses of pigs compared to pigs fed a control diet. *

The data is the means of 24 pens/treatment. 1 MSE = means standard error EXAMPLE 5
[00225] Demonstration of the effectiveness of a Bacillus-based Pig Manure Additive Treatment for the Storage, Management and Handling of Manure Waste.
[00226] A study will be conducted to evaluate the effectiveness of a Bacillus-based swine manure pit additive on solids accumulation, nutrient composition, and manure foaming characteristics. Several production sites will be identified which contain at least three barns with separate manure treatment and storage units. The manure pits at each site will be treated with a Bacillus-based additive in two doses and one manure pit will be left untreated. The low dose well treatment will be added to a manure well at each production site at 500 g of test material per 100,000 liters of manure formulated to contain 4 x 1010 CFU per gram of test material. The high dose treatment will be added to a different manure pit than the low dose of each production unit at 500g of test material per 100,000 liters of manure formulated to contain 1 x 10" CFU per gram of test material. manure pit at each site will be left untreated as a control.
[00227] Samples will be obtained from each manure pit at each production site under test, initially before any treatment and periodically (approximately once a month) over a period of three to six months. Data from manure pits will be collected to assess the incidence of foaming and manure samples will be analyzed to assess solids accumulation and nutrient composition. Expectations are that treated swine wells will have less foaming, less solids buildup and less nitrogen, sulfur, phosphorus, fiber bound nitrogen, total protein, fat and fiber content from control manure wells. EXAMPLE 6 Demonstration of Poultry Feeding Test to Improve Growth Performance in Response to Bacillus Strain Combinations Added to Poultry Diets.
[00228] Poultry feeding trials will be carried out to evaluate the effects of a direct feed of Bacillus-based microbial additive (DFM) on weight gain, feed consumption, feed conversion and mortality of turkeys, broilers, and broilers. . In these studies, approximately 22 birds per repeat treatment will be randomly assigned to the dietary treatments. Dietary treatments may consist of various combinations of Bacillus strains administered as a DFM and experimental DFM treatments combined with enzymes, compared to a relative control group of birds. DFM Bacillus treatments will be added to the diet of 1.5 x 105 CFU / g feed and will provide about 1 x 107 5 x 107 CFU / head / day based on the average daily consumption of various production systems (turkeys, broilers , shoots). The diets will be composed of corn-soybean-DDGS-based diets. All other nutrient and energy levels will be formulated to meet or exceed test bird requirements. The diets will be fed for an approximate test period of 42 days and will be fed in three feeding phases: starting (dl-20) and growing (d 21-38) and finishing (d38-42). Diets will be pelleted (approximately 75°C), and the starter feed will be crumbled.
[00229] Data from the treated groups will be compared with those from your relevant control group using the appropriate statistical tests. Body weight, weight gain, food consumption, HRR, EHR and mortality will be analyzed using analysis of variance (ANOVA) and the least significant difference tests. When completed, the data are expected to support the effectiveness of the FMD treatment(s). Specifically, it is expected that the DFM treatment will increase lean meat yield and carcass yield, shift gastrointestinal microbial communities to populations favorable for nutrient use and improve poultry growth efficiency, and improve body weights. egg case. EXAMPLE 7 Effect of Direct Feed Microbial Bacillus on Pig Growth Performance, Carcass Measurements, Manure Pit Characteristics, and Environmental Gas Emissions.
[00230] A total of 444 pigs (200 barrows and 244 females) were used in a 15-week grow-to-finish study to investigate the use of a direct-feed microbial Bacillus supplement on growth performance, carcass measurements, traits from the manure pit and gas emissions. The pigs were housed in an environmentally controlled barn, which contained 12 identical rooms with 12 pens per room. Two manure pits were contained in each under each of the 12 rooms with 6 more pens from each manure pit. Before the start of the experiment, the manure pits were carefully cleaned. The manure pits were then loaded with a small amount of water (~600 gallons).
[00231] The pigs allocated to the test were weaned, blocked by weight and sex, and randomly assigned to dietary treatments (Control and Bacillus DFM) with 4 to 5 pigs (2 to 3 gilts and 2 to 3 sows per pen). Before the start of the experimental treatments, the pigs were fed an adjustment diet for two weeks in the manure wells. Pigs were fed a control diet or a control diet supplemented with Bacillus DFM. Microbial Bacillus DFM consisted of equal proportions of Bacillus subililis strains AGTP BS918 (NRRL B-50508), AGTP BS1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) adding up to a guaranteed 3.0 x 108 CFU/g of DFM product, and included at a rate of 1 lb/ton, in animal feed, resulting in a concentration of 1.5 x 105 CFU/g of feed.
[00232] Dietary treatments were maintained throughout the experiment, but diets were adjusted every three weeks to best meet the nutritional needs of the pigs, resulting in 5 feeding phases (3 growing phases, 2 finishing phases) formulated to meet or exceed the nutritional requirements of pigs at each stage of production in each of the five stages (NRC, 1998). Diet formulations were corn and soybean based with different grain levels of corn based dry distillers with solubles (DDGS) of more than five phases. Specifically, diets for growth phases 1, 2, and 3 were formulated to contain 25% DDGS, the Phase 4 finisher diet contained 20% DDGS, and the Phase 5 finisher diet contained 10% DDGS.
[00233] Pig body weight and feed intake in the pen were recorded every three weeks at the end of each phase. Manure wells were sampled at the beginning and end of each of the growth and final stages using a vacuum core sampler designed with a vacuum pump attached to two clear plastic tube vacuum flasks with a core end disk. of the plastic sampler. Core samples from the manure pit were obtained by sampling from four locations under each pen above each well under test. Manure pit sampling locations in relation to the pen include: (1) below the center of the pen, (2) under the pen water, (3) under the front of the pen feeder, and (4) under the corner from the corral opposite the feeder. Manure content was analyzed for total N, ammonium N, dry matter (DM), ash content, Ca and P (AOAC 2007). Throughout the experiment, gas concentrations in the well ventilation duct and in front of the exhaust fan were monitored in real time using continuous measurement equipment. These data were combined with ventilation ratios to determine the emission ratios per room per day for ammonia, methane and hydrogen sulfide. These data were expressed in grams of gas per pound of swine body weight gain. The concentrations of methane and hydrogen sulfate were also measured during 10 consecutive days (days 70 to 80 for CH4, days 80 to 90 for H2S) and an average of a base of a room for analysis of the total gas production (n = 12).
[00234] Well samples were analyzed for nutrients (AOAC 2007) and volatile fatty acid (VFA) composition. By high pressure liquid chromatography (HPLC) detection of volatile fatty acids, 10 mL of each sample was aliquoted into 15 mL Falcon tubes and stored at -20 °C until HPLC analysis. After thawing, samples were centrifuged at 16.1 rad for 15 minutes. One milliliter (1 mL) of the supernatant was diluted in 9 mL of 16.8 mM phosphoric acid in water/acetonitrile (98:2, v/v). The diluted supernatant was vortexed for 10 seconds and then centrifuged at 16.1 rad for 15 minutes. The supernatant was filtered (0.22 μπi) and analyzed for acetic acid, propionic acid, butyric acid, I-butyric acid, I-valeric, valeric acid, 4-methylvalerate using a Waters 2695 separation module (Waters Corporation, Milford , MA) equipped with a 300 x 7.8 mm Aminex HPX-87H column (Biorad Laboratories, Inc., Hercules, CA). An isocratic method was applied with a mobile phase solvent consisting of 16.8 mM phosphoric acid in water/acetonitrile (98:2, v/v), flow ratio of 0.85 mL/min and 65 °C of column temperature. All analytes were detected with a PDA 2996 detector (Waters) at 211 nm absorption.
[00235] Data were analyzed using the SAS General Linear Model procedure to test treatment and replicate differences. The corral was the experimental unit for growth performance and carcass data, the pit was the experimental unit for excretion and VFA data, and room was the experimental unit for gas emission data. Results
[00236] The pigs averaged 64.5 lbs at the start of the experiment and had an average weight of 257.1 lbs after 15 weeks of feeding. Pigs fed the diet containing the DFM supplement were 4 pounds heavier (P = 0.10) at the end of the experiment compared to control fed pigs (Table 7). This response resulted from faster growth when pigs were fed the DFM supplement compared to control pigs (2.01 vs 1.93 lbs/d, respectively, p <0.03; Table 8). Mean daily food consumption (adfi) was not affected by dietary treatment (Table 9). This lack of difference in response between treatments for feed intake, together with a greater average daily weight gain in the DFM treated pigs resulted in improved efficiency (P < 0.08) of the feed during the two phases of the finisher (Phase 4). and 5) and overall in a 15-week trial when pigs were fed the DFM supplemented diet compared to pigs fed the control diet. (Table 10). Table 7. Effects of direct feeding microbial Bacillus (DFM) diet supplementation on body weight of pigs.
Table 8. Effects of direct feeding microbial Bacillus diet (DFM) supplementation on average daily gain (adg).
Table 9. Effects of direct feeding microbial Bacillus diet supplementation (DFM) on average daily food intake (adfi).
Table 10. Effects of Direct Feed Microbial Bacillus (DFM) Diet Supplementation on Feed Efficiency (body weight gain in pound per pound of food consumed).

[00237] Hot carcass weights were 4.5 pounds heavier (P < 0.01) for pigs fed diets supplemented with DFM compared to control fed pigs (Table 1) 1. In addition, the rewards of carcass quality tended to be higher (P = 0.15) when pigs were supplemented with DFM. The observed increase in carcass weight from DFM supplementation resulted in $0.39 plus carcass value relative to carcass control. Table 11. Effects of direct feeding microbial Bacillus (DFM) diet supplementation on carcass traits.

[00238] The measured values of nutrient manure from samples obtained over the test period are shown in Table 12. Dry matter (P = 0.20), ash (P = 0.1 1), and ammonium nitrogen (P = 0.15) tended to be reduced in manure pits associated with pigs fed Bacillus DFM relative to control pigs. Bacillus DFM supplementation decreased dry matter by 7%, ash by 8%, and ammonium nitrogen by 5% in manure of treated pigs compared to controls. The observed reductions in dry matter and ash excretion can be attributed to improvements in feed efficiency. Table 12. Effects of direct feeding microbial Bacillus diet (DFM) supplementation on nutrient accumulation in the manure pit (g/lbs body weight gain) on the total trial.

[00239] The absence of difference in total nitrogen excretion in manure between treatments suggests that the observed reductions in ammonia nitrogen from the DFM treatment is a result of changes in microbial ecology and activity in manure pits associated with the DFM treatment versus to control. When expressed in grams per pound of pig body weight, methane gas emissions tended to be reduced (P = 0.16; 17% reduction) when pigs were fed DFM from supplemented diets (Table 13). Hydrogen sulphide emissions, expressed in grams per pound of pig body weight, were not significantly different from the control, but were reduced by 10% when pigs were fed DFM from supplemented diets. Ammonia emissions were numerically lower for DFM from fed pigs at all times. Total methane and hydrogen sulfide gas emissions (grams/day) were reduced (P = 0.08) by 14% and 19%, respectively, in rooms housing the DFM of supplemented pigs compared to control pigs. (Table 14). Table 13. Effects of direct feeding microbial Bacillus diet (DFM) supplementation on environmental gas emissions (g/lb of live weight gain). Diet
Table 14. Effects of DFM dietary supplementation on gas emissions in methane (CH4) and hydrogen sulfide (H2S) media.
1 Data in g/day SEM = standard error of the mean of the mean
[00240] Total volatile fatty acids (VFA) were reduced (P = 0.01) in manure from pigs fed the diets supplemented with Bacillus DFM relative to manure from control pigs (Table 15). Specifically, this decrease was the result of lower production of 1-butyrate (P = 0.04), 4-methylvalerate (P = 0.05), and ethyl propionate (P = 0.12) during anaerobic microbial fermentation. in the dung. On the other hand, DFM supplementation resulted in an increase (P = 0.06) in butyrate production. Table 15. Effects of dietary DFM supplementation on manure volatile fatty acid (VFA) composition. 1

1 Data on ppm dry matter weighted by body weight gain.
[00241] The data from this experiment indicate that pigs fed diets supplemented with this Bacillus DFM during the growing and finishing phases of production result in a better growth ratio, feed efficiency and final hot carcass weight. DFM supplementation can also reduce dry matter, ash, and ammonium N in the manure pit. In addition, reductions in methane and hydrogen sulfide emissions from stored swine waste were evident when Bacillus DFM was supplemented to swine feed. EXAMPLE 8 Effect of direct-feed microbial Bacillus on microbial ecology in stored swine waste.
[00242] Well manure samples were obtained from the 15 week growth-finish study described in Example 7. Manure samples for microbial analysis were collected at the end of the assay as described above in Example 7 at from each of the two individual wells per room and analyzed individually, resulting in a total of 24 observations. Archaeal methane production (Spence et al., 2008) and bacterial groups of interest were enumerated via quantitative polymerase chain reaction (qPCR) analysis (Metzler-Zebeli et al, 2010, Yu et al., 2005). Data were analyzed by ANOVA using the SAS PROC MIXED procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC) with a significance level of a = 0.10. Trends were stated for 0.20 > P > 0.10.
[00243] The addition of Bacillus DFM to swine diets resulted in changes in microbial populations in stored swine waste. The Clostridium proteolytic cluster of group I bacteria was reduced (P < 0.01) in the stored manure resulting from DFM-fed pigs relative to the manure of control pigs (Table 16). Administration of Bacillus DFM to swine resulted in an increase in the fibrolytic cluster of Clostridium XlVa (P = 0.09) associated with butyrate production. This increase in Clostridium XlVa cluster supports the observed increase in butyrate production associated with DFM treatment, as reported in Table 15 in Example 7. Bacteroides and Prevotella species, producing a wide variety of VFA, were significantly reduced (P = 0.08) in swine manure supplemented with DFM. Methanogens tended to be reduced (P = 0.13) in manure stored from pigs fed Bacillus DFM relative to manure from control pigs, and sulfate-reducing bacteria were numerically reduced. The observed reductions in these sulfate-reducing bacteria and archaea support the observed decreases in methane and hydrogen sulfide gas production with DFM supplementation documented in Table 13 and Table 14 in Example 7. Table 16. Effects of Bacillus microbial diet supplementation Direct Feeding (DFM) on microbial populations in stored swine waste.
1 Data in Δct in relation to total bacteria and adjusted for manure dry matter (DM) and weighted by body weight gain; SEM = standard error of the mean. EXAMPLE 9
[00244] The effect of supplementation of a direct feed microbial Bacillus (DFM) for pigs raised in a commercial facility from weaning to finish and fed diets formulated with a high level of by-products and limited energy levels.
[00245] To determine the growth performance of pigs fed commercial corn and soybean meal-based diets with increasing amounts of by-product, a weaning-to-finish study was carried out. A total of 1024 pigs were weaned at about 3 weeks of age, separated by sex and weight category, allocated to 32 trial pens and feeding phase for 105 days. The animals were weighed every two weeks during the three initial phases of breastfeeding. Early phase diets contained up to 20% corn distillers and soluble grains (cDDGS). The pigs were continued in two growth phases and a final phase of 21 days each. The two growing phases as well as the finishing phase diets contained 35% cDDGS and 15% wheat bran in place of corn in the diet (Table 17). Table 17. Feeding phases and diet composition. 1

1 SBM, soybean meal; CP, crude protein; cDDGS, corn dry still grains with solubles with ~10% oil content; treatment included corn costs.
[00246] The diets were formulated to simulate normal commercial diets with excess crude protein but limited energy. Except for the first 6 weeks of the trial, none of the growth-promoting antibiotics were fed. Treatment consisted of direct feeding microbial supplementation (DFM) in relation to diet control without DFM. Direct microbial feed consisted of equal proportions of Bacillus subtilis AGTP BS918 (NRRL B-50508), AGTP BS 1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) strains adding up to a guarantee of 3.0 x 108 CFU / g of DFM product, included at a rate of 1 lb / ton in the feed, resulting in a concentration of 1.5 x 105 CFU / g of diet. Growth performance and losses were analyzed using the SAS Proc Mixed procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC) with a significance level of a = 0.10. Trends were stated for 0.15 > P > 0.10. Data were locked by sex and weight category and balanced to baseline weight.
[00247] The average daily gain of pigs fed DFM was higher (P < 0.05) from d 0 to 14 and d 14 to 28 of the trial compared to control pigs (Table 18), which resulted in a increase (P < 0.05) in body weight of supplemented pigs with DFM at DL4 and d 28 of the study (Table 19). This increase in body weight gain exhibited by the DFM-supplemented pigs was a result of a higher (P < 0.10) mean daily feed intake during periods d 0 to 14 and d 14 to 28 (Table 20). Feed efficiency was also improved (P = 0.03) during the first two-week trial period (Table 20b). Table 18. Average daily gain (adg) over the duration of the study. DFM Control WITHOUT P-Value

1 SEM = standard error of the mean. Table 19. Pig body weight (lb) and percentage of health loss (mortality and slaughter) throughout the study period.
1 SEM = standard error of the mean. Table 20. Average daily food consumption (adfi) over the duration of the study.

1 SEM = standard error of the mean. Table 20b. Feed conversion (feed: gain, fg) over the duration of the study.
1 SEM = standard error of the mean.
[00248] Direct feeding microbial supplementation resulted in higher (P < 0.10) GPD and DCM during the early growth phase (d 42 to 63 of the trial). During the finishing phase of the trial, ADG was higher (P = 0.02) from days 84 to 105 when pigs were fed diets supplemented with DFM relative to control pigs, and tended to be higher (P = 0.14) for the overall time period d 0 to 105. The best response in ADG with DFM treatment from days 84 to 105 and lack of CMD response resulted in improvement (P < 0.01) in feed conversion during this period. time course. Improvements in ADG from DFM supplementation throughout the trial resulted in approximately a 3lb heavier pig at the end of the study (d 105) relative to control pigs (Table 19). In addition, health losses due to mortality and slaughter as a result of influenza, Streptococcus suis infection, etc. were reduced (P = 0.07; Table 19). EXAMPLE 10
[00249] The effect of supplementation of a direct feed microbial Bacillus (DFM) for pigs raised in a commercial facility from weaning to finish and fed diets formulated with a high level of by-products and limited energy levels on feed conversion efficiency .
[00250] The effect of a direct feed microbial Bacillus on feed use efficiency by animals, which are raised in a weaning facility at the end, was evaluated. A total of 2,160 animals were weaned at approximately 3 weeks of age, separated by sex, balanced for initial weight, and allocated to more than 68 pens in two rooms at the same trial site. The animals were fed for 105 days. The animals were weighed every two weeks, during the initial suckling phase until day 42, post-weaning. Early stage diets contained up to 20% corn distillers and soluble grains (cDDGS). The pigs were continued on trial through two growing phases and a finishing phase, every 21 days. The growing phase as well as the finishing phase diets contained 35% cDDGS and 15% wheat bran in place of corn in the diet (Table 21). Diets were formulated to simulate standard commercial diets with limited protein and crude energy. Table 21. Feeding phases and diet composition.

1 SBM, soybean meal; CP, crude protein; cDDGS, still dry corn grains with solubles with ~10% oil content; treatment included corn costs.
[00251] With the exception of the first 6 weeks of the trial, none of the growth promoting antibiotics were fed. Treatments consisted of direct feeding microbial supplementation (DFM) compared to a control diet without DFM. Direct microbial feed consisted of equal proportions of Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) adding to a guarantee of 3.0 x 108 CFU / g of DFM product, included at a rate of 1 lb/ton in the feed, resulting in a concentration of 1.5 x 105 CFU/g of diet. Feed conversion was analyzed using the SAS Proc Mixed Procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC) with a significance level of α = 0.10. Trends were stated for 0.15 > P > 0.10. Data were locked to room and gender for analysis.
[00252] Less feed (P=0.02) was required per pound of weight gain from d 0 to 14 of the trial when pigs were fed diets containing the Bacillus DFM supplement, and this response tends to show the feed efficiency (P = 0.13) which is evident throughout the entire breastfeeding phase from d 0 to 42 of the study. (Table 22). Direct feed microbial supplementation also improved (P = 0.08) feed efficiency during the finishing phase (d 84 to 105) Table 22. Feed conversion (feed: gain, fg) over the duration of the study.
1 SEM = standard error of the mean EXAMPLE 11
[00253] The effect of supplementation of a direct-feeding microbial Bacillus (DFM) in response to the feed efficiency of lactating pig diets formulated with high levels of fibrous by-products.
[00254] A total of 480 pigs (initial weight: approximately 6.0 lbs) were weaned at 21 days of age and separated into 10 pigs/pen in a controlled environment pig nursing facility. Animals were tested from 21 days of age to 63 days of age and fed a two-phase feeding program with formulated diets based on corn, soybean meal and 40% corn DDGS (Table 23) and to meet the nutritional requirements of pigs in each of the two stages of production (Table 24). Table 23. Composition of the basal diet of Phases 1 and 2 of the swine suckling diets.
Table 24. Calculated composition of basal diets, %. Phase 1 Phase 2


[00255] All diets contained phytase (500 FTU/lb of food). One of the three dietary treatments was randomly assigned to the pens such that each treatment was represented by eight identical pens. Treatments consisted of direct feed microbial supplementation (DFM) at two inclusion levels (0.5 and 1.0 lb/ton feed) compared to a control diet without the DFM supplement (Table 25).
[00256] Direct microbial feed consisted of equal proportions of Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) adding to a guarantee of 3.0 x 108 CFU / g of DFM product, included at a ratio of 0.5 or 1.0 lb / ton of feed resulting in a concentration of 7.5 x 104 CFU / g or 1.5 x 105 CFU / g of feed, respectively. Pig body weight gain and feed consumption were determined in the pen on d 21 and d 42 using the trial to calculate feed efficiency as gain:feed. Feed efficiency can also be calculated as feed:gain. Table 25. Dietary treatments and DFM inclusion proportions
1 Diets were processed as a blend, non-pelleted feed 2 All diets contained 500 FTU/kg phytase feed.
[00257] Pigs fed the Bacillus DFM treated diets had greater weight gain (P = 0.03) per unit of feed intake compared to pigs fed the control diet during the early suckling phase (d 0 to 21, after weaning, Table 26). Table 26. Body weight and feed efficiency of suckling pigs fed high fiber based diets supplemented with Bacillus DFM at two levels of dietary inclusion.
* Pigs per pen= 10 EXAMPLE 12
[00258] Effect of a direct-feed microbial Bacillus on energy and nutrient digestibility in diets in growing swine feed containing 40% corn DDGS.
[00259] A digestibility study was performed in growing swine to measure the effects of a direct-feed microbial Bacillus (DFM) on ileal and total digestibility of energy and nutrients in diets containing 40% dry still grain maize, including solubles (DDGS). Twenty-four pigs (initial BW: about 25 lb) from the matings of G-Performer boars to F-25 females (Genetiporc, Alexandria, MN) were surgically equipped with a T-cannula in the distal ileum. After the surgeries, the pigs were allowed on the 21st to recover. A meal based on the standard corn and soybean meal diet was provided on an ad libitum basis during this period. Three weeks after surgery, pigs were assigned to two dietary treatments consisting of a basal control diet and a DFM Bacillus. The animals were housed in individual pens (1.2 x 1.5 m) in a controlled environment room. Each corral was equipped with a feeder and a teat and had fully slatted concrete floors.
[00260] The experimental basal diet was formulated based on corn, soybean meal and 40% corn DDGS (Table 27). Dietary treatments were: (1) a basal diet without DFM, or (2) with the basal diet with 0.05% DFM added to cornstarch costs. Direct microbial feed consisted of equal proportions of Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) adding to a guarantee of 3.0 x 108 CFU / g of DFM product, included at a rate of 10.0 pounds / ton of feed resulting in a concentration of 1.5 x 105 CFU / g of diet. All diets were formulated to meet or exceed the nutrient requirements of growing pigs (NRC, 1998). Table 27. Composition of the experimental basal diet
Ingredient, %1 Direct microbial feed treatment was added to 0.05% of the diet, at the expense of corn starch. 3 The vitamin micromineral premix, provided the following amounts of vitamins and minerals per kilogram of complete diet: Vitamin A, 10,990 IU; vitamin D3, 1, 648 IU; vitamin E, 55 IU; vitamin K, 4.4 mg; thiamine, 3.3 mg; riboflavin, 9.9 mg; pyridoxine, 3.3 mg; vitamin B12, 0.044 mg; D-pantothenic acid, 33 mg; niacin, 55 mg, folic acid, 1.1 mg; biotin, 0.17 mg, Cu, 16 mg as copper sulfate; Fe, 165 mg as the iron sulfate, I, 0.36 mg as the potassium iodate; Mn, 44 mg as the manganese sulfate; SE, 0.3 mg sodium selenite; Zn, 165 mg as zinc oxide.
[00261] Titanium dioxide was used as a non-digestible marker in all diets. Diets were fed to 12 pigs, offering six pigs per diet for 17 days. Pigs were allowed ad libitum consumption of diets and water throughout the experiment. To minimize cross-contamination of control pens with DFM, pens fed diets without DFM were fed first, followed by pens treated with DFM. After each treatment was fed, the feed distribution carts were completely cleaned. Pigs fed diets without DFM were also weighed and collected before pigs fed diets containing DFM.
[00262] Stool samples were collected on day 12 via sampling and ileum samples were collected on days 13 and 14. Ileum samples were collected continuously for 9 h from 0800 on each collection day. The cannulas were opened and 225 ml of plastic bags were attached to the cannula barrel using clamps, which allowed digesta to flow from the cannula into the bag. The bags were changed whenever the digesta was filled or at least once every 30 min. Digesta pH was measured in the first bag collected after 0900, 1100, 1300 and 1500 on each collection day. After harvesting the final ileum, the pigs were fed their respective experimental diets for an additional 3 days. The morning meal (at 0700), which is fed the day after the last ileal collection, contained a green marker. During the following 36 h, ileal and faeces were marked every 30 minutes from all pigs, and for the first time, the marker appears at any of these sites which were recorded and used as a measure of the rate of passage to this diet. Special.
[00263] At the conclusion of the experiment, samples were thawed and homogenized within animal and diet and a sub-sample was collected for chemical analysis. All samples were lyophilized and grounded before analysis. All samples were also analyzed for dry matter (DM), acid detergent fiber (ADF), neutral detergent fiber (NDF) and lignin. The apparent ileal (AID) and total digestive tract (ATTD) values of nutrient digestibility were calculated as previously described (Stein et al., 2007). The homogeneity of variances was confirmed and outliers were tested using the UNIVARIATE procedure (SAS Institute Inc., Cary, NC). No outliers were detected. Data were analyzed using the MIXED procedure. The model included dietary treatment as a fixed effect while swine was a random effect. Least squares were calculated for each independent variable. The swine was the experimental unit for all calculations, and a level used to determine significance and trends between means was 0.05 and < 0.10, respectively.
[00264] Ileal pH was lower (P=0.03) in animals fed the diet containing the Bacillus DFM compared to the pH of the ileum of control pigs (Table 28). Passage ratio and fecal pH were not affected by dietary treatment. Although ADF and NDF were not affected, the addition of Bacillus DFM to the diet resulted in improved (P < 0.03) AID (Table 29) and ATTD (Table 30) of lignin compared to the control diet.
[00265] These data indicate that Bacillus DFM reduces ileal pH and improves lignin digestibility in diets based on highly fibrous by-products. Table 28. Effect of Bacillus DFM on pH and the rate of passage of ileal digesta and feces in soybean-corn meal diets in growing swine containing 40% DDGS1
Table 29. Effect of Bacillus DFM on apparently ileal digestibility (AID, %) of fibrous nutrients in soy-corn meal diets in growing swine containing 40 % DDGS1
1 Data are least squares means of 6 observations for all treatments. Table 30. Effect of Bacillus DFM on total apparent digestibility (ATTD, %) of fibrous nutrients in soybean-corn meal diets in growing swine containing 40% DDGS1
1 Data are least squares means of 6 observations for all treatments. EXAMPLE 13 Anti-inflammatory effects of Bacillus strains on a HD11 chicken macrophage cell line.
[00266] The HD1 1 chicken macrophage cell line was used to determine the inflammatory response to LPS and to determine the potential of direct-feeding microbial Bacillus strains to alleviate inflammation associated with a gram negative bacterial infection. Bacillus strains were screened in a cell culture assay to determine changes in gene expression of inflammatory cytokine responses to LPS and of each of the Bacillus strains (Bacillus subtilis AGTP BS1013 (NRRL B-50509), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), and Bacillus subtilis AGTP BS944 (NRRL B-50548).
[00267] HD11 cells were incubated with either: (1) alone (unstimulated), (2) with LPS, and (3) with each Bacillus strain, and (4) with LPS + Bacillus strain. The board template design is illustrated in Figure 22.
[00268] HD11 cells were grown to confluence and plated in 24-well tissue culture plates with Roswell Park Memorial Institute Antibiotic Free 1640 (RPMI) containing 10% fetal bovine serum media (FBS; Atlanta Biologicals). , Inc., Lawrenceville, GA). Once confluent, the medium was removed and treatments were administered in antibiotic-free medium and then incubated for 1 hour at 41°C. After incubation, cells were washed twice with PBS and incubated at 380°C. of TRIzol (Invitrogen, Life Technologies Corp, Carlsbad, CA) for 5 minutes. Samples were removed from the plates, placed in two microcentrifuge tubes, quickly frozen and stored at -80 °C until RNA was isolated. To separate the RNA from the organic phase, 2 ml heavy phase blocking gel tubes (Five Prime, Inc., Gaithersburg, MD) were used. RNA cleaning was performed using the RNeasy mini kit (Qiagen, Inc., Valencia, CA) and DNase digestion was performed using the DNase-free RNase kit (Qiagen). cDNA was synthesized using qScript SuperMix cDNA (VWR, Radnor, PA) immediately following RNA isolation.
[00269] Real Time PCR was used to determine gene expression from HD11 cells, using primer sets shown in Table 31. β-actin was used as a reference gene. One-way ANOVA was performed using the SAS Proc Mixed procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC). Means were separated using the Student-Newman-Keuls test, significance level a = 0.10. Table 31. Chicken-specific primer sets used in the screening assay


[00270] The challenge of lipopolysaccharide in the HDL 1 chicken macrophage cell line resulted in an increase (P < 0.01) in the expression of the inflammatory cytokine genes, interleukin (IL)-l β and IL-8, compared to unstimulated HDL11 cells (Figure 23). When AGTP strain BS1013 was added to HDL11 cells with an LPS in the spore state, this Bacillus strain decreased (P < 0.01) the expression of the inflammatory cytokine genes, IL-1 β and IL-8, resulting from the administration of LPS alone and was most similar to the unstimulated HDL22 cell profile of gene expression. Furthermore, the response of the chicken cell to LPS in the presence of the vegetative Bacillus strain BS 1013 was numerically inferior, as well as the Bacillus AGTP strain BS3BP5 in the spore state, and the spores and vegetative cells of the Bacillus AGTP strain BS944.
[00271] These data demonstrate the effectiveness of Bacillus DFM strains to alleviate inflammation associated with a bacterial infection, and their effectiveness in avian species. Bacillus DFM strains can be used to alleviate macrophage inflammation. In addition, Bacillus DFM strains can be used to alleviate gram-negative bacterial infections, and the effects of these bacterial infections. EXAMPLE 14
[00272] The anti-inflammatory effects of Bacillus strains from a mouse intestinal epithelial cell line (IEC-6)
[00273] The mouse gut epithelial cell line IEC-6 was used to determine the inflammatory response to LPS and to determine the potential of direct-feeding microbial Bacillus strains to alleviate inflammation associated with a gram negative bacterial infection. Bacillus strains were screened in a cell culture assay to determine changes in the expression of cytokine genes in inflammatory responses to LPS and of each of the Bacillus strains (Bacillus subtilis AGTP BS 1013 (NRRL B-50509), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), and Bacillus subtilis AGTP BS944 (NRRL B-50548), Bacillus subtilis AGTP BS1069 (NRRL B-50544), Bacillus subtilis AGTP BS 442 (NRRL B-50542), Bacillus subtilis AGTP BS521 (NRRL B-50545), and Bacillus subtilis AGTP BS918 (NRRL B-50508)). Additional strains of Bacillus may be used including, but not limited to, Bacillus pumilus AGTP BS 1068, (NRRL B-50543) and Bacillus pumilus KX1 1-1 (NRRL B-50546).
[00274] IEC-6 cells were incubated with either: (1) alone (unstimulated), (2) with LPS, (3) with each Bacillus DFM strain, and (4) with LPS + Bacillus strain. The board template design is illustrated in Figure 24.
[00275] IEC-6 cells were grown to confluence and seeded into 24-well tissue culture plates with Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Life Technologies Corp, Carlsbad, CA) containing 10% FBS (Atlanta Biologicals, Inc., Lawrenceville, GA) and 1% antibiotic-antimycotic (Atlanta Biologicals). Once the plates were confluent, the IEC-6 cells were washed three times with phosphate buffered saline (PBS). Treatments were administered in antibiotic-free medium and then incubated for 1 hour at 37 °C. After incubation, cells were washed twice with PBS and incubated in 380 μl of TRIzol (Invitrogen) for 5 minutes. .
[00276] The samples were removed from the plates, placed in 2 ml microcentrifuge tubes, quickly frozen and stored at -80°C until RNA was isolated. To separate the RNA from the organic phase, 2 ml heavy-phase blocking gel tubes (Five Prime, Inc., Gaithersburg, MD) were used. RNA cleaning was performed using the RNeasy mini kit (Qiagen, Inc., Valencia, CA) and DNase digestion was done using the DNase-free RNase kit (Qiagen). cDNA was synthesized using qScript SuperMix cDNA (VWR, Radnor, PA) immediately following RNA isolation.
[00277] Real-time PCR was used to determine gene expression from IEC-6 cells, using primer sets shown in Table 32. β-actin was used as a reference gene. One-way ANOVA was performed using the SAS Proc Mixed procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC). Means were separated using the Student-Newman-Keuls test, significance level a = 0.10. Table 32. Mouse-specific primer sets used in the screening assay.

[00278] Lipopolysaccharide challenge in the IEC-6 mouse intestinal epithelial cell line resulted in an increase (P < 0.01) in the expression of the inflammatory cytokine gene, TNF-a, compared to IEC-6 cells not stimulated (Figure 25). Bacillus strains BS1013 and BS1069 decreased (P < 0.10) TNF-a gene expression resulting from administration of LPS alone when in either spore or vegetative states. Bacillus strains BS3BP5, BS442, and BS521 also decreased (P < 0.10) TNF-a gene expression resulting from administration of LPS alone, but only when the spore form. Conversely, Bacillus strain BS918 decreased (P < 0.10) TNF-a gene expression resulting from administration of LPS alone, but only in its vegetative form.
[00279] These data demonstrate the effectiveness of Bacillus DFM strains to alleviate inflammation associated with a bacterial infection, and their effectiveness in a mammalian species. Bacillus DFM strains can be used to alleviate macrophage inflammation. In addition, Bacillus DFM strains can be used to alleviate gram-negative bacterial infections, and the effects of these bacterial infections. EXAMPLE 15
[00280] Efficacy of a Bacillus DFM to reduce foaming in commercial deep well swine manure storage systems
[00281] Deep swine manure pit systems are common in the Midwest US and have high foaming potential. This is believed to be the result of the progressive increase in the inclusion of fiber by-products in swine feed and the resulting shifts in microbial ecology and fermentation characteristics in the stored manure. The effectiveness of one of three Bacillus DFMs was evaluated to determine whether its application to swine manure wells could positively alter the microbial fermentation profile in the manure well and provide a tool for well foam control. Five production units each with three barns grow with identical finish (1400 head each) over the individual deep well systems that have been selected for evaluation. All sites have traditionally been at high risk for foam production based on high levels of inclusion of dry distillers grains that contain soluble products (DDGS) and other fibrous by-products in diets and past historical incidences of foaming.
[00282] For each of the three wells per site, a baseline sample was established before the trial began using a 1' PVC tube equipped with a ball valve to clamp the sample. For each sample, the depth of liquid and foam depth were measured and the proportion of foam calculated to accommodate different well volumes throughout the duration of the study as well volumes varied enormously after 21 days of sampling. The product tested consisted of equal proportions of AGTP BS918 (NRRL B-50508), AGTP BS1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) strains as for examples 9 and 10. Other Bacillus strains may be used including, but not limited to, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, and Bacillus subtilis AGTP BS1069 and Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX11-1.
[00283] Two inclusion ratios of Bacillus products were applied directly to the manure well and tested against the untreated wells. Bacillus well inoculant was applied at a ratio of 5.3 x 104 CFU / mL of manure to be equivalent to the inoculation ratio if fed to the animal at 1.5 x 105 CFU / g of feed and a dose of 2 .5-fold (2.5-fold) applied to the manure pit, at a ratio of 1.3 x 106 CFU/mL of manure. The Bacillus product was reapplied every 60 days for the full 170-day test period. Data were analyzed by ANOVA using the SAS PROC MIXED procedure (v. 9.1.3, SAS Institute, Inc., Cary, NC) with repeated measures for detection over time. The significance level a = 0.10, means were separated using least square difference (LSD) test.
[00284] There was no difference in foam depth, liquid depth or foam:net ratio between wells at the identified test locations (Table 33). However, three weeks after application of treatments, foam depth decreased (P = 0.03) in wells treated with Bacillus inoculant at each application rate compared to untreated wells. Liquid depth did not differ between the three treatments at three weeks after the Bacillus inoculant well was applied, resulting in a decrease (P = 0.01) of foam:liquid when the Bacillus inoculant well was applied at each application ratio. compared to untreated wells. Foam: The proportion of liquid was also reduced (P < 0.10) with the Bacillus inoculant in each application proportion relative to the control, when values were calculated over the three sampling points over the 170-day trial ( Table 34). The data indicate that the higher proportion of Bacillus Inoculant inclusion resulted in more consistent foam reduction over the course of the study (Figure 26).
[00285] These data indicate that the use of a three strain Bacillus inoculant applied directly to swine manure pit depth storage facilities controls foam accumulation. Table 33. Comparison of foam characteristics between baseline and 21 days of sampling averaging more than 5 si
a,'b means with different upper indices are significantly different (P < 0.10), means were separated using the standard error of the mean. Table 34. Comparison of foam characteristics on average with more than 3 samplings within a 170-day trial period.
a,b means with different upper indices are significantly different (P < 0.10), means were separated using the standard error of the mean. EXAMPLE 16
[00286] Direct application of Bacillus product to manure pits at commercial grow-end-phase sites alters manure characteristics.
[00287] To compare the efficacy of a three-well product Bacillus strains containing strains AGTP BS918 (NRRL B-50508), AGTP BS 1013 (RRL B-50509) and AGTP BS3BP5 (NRRL B-50510) in equal proportions to the residues of Commercial swine manure from the current treatment product (MicroSource S®; DSM), the three-strain Bacillus product was applied directly to the manure wells of three commercial production units in the Midwest that were currently using the feed. MicroSource S ®. Other strains of Bacillus may be used including, but not limited to, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, and Bacillus subtilis AGTP BS1069 and Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX1 1-1.
[00288] The Bacillus product was tested at three production sites for a period of 60 days to determine if it could improve waste management characteristics above the effect of administering MicroSource S ® in swine feed. Each site consisted of two identical rooms with individual manure pits and capacity for 2250 market pigs. Per site, one barn was used as an untreated control while the other barn was treated with the Bacillus well. For the treated wells, the Bacillus product inclusion ratio was based on waste volume, with an application ratio of 5.3 x 104 CFU/mL manure. The initial volume of the swine manure wells in the test was estimated to be 120,000 liters of manure, so a total of 2.4 x 10° CFU of Bacillus Product was applied directly to the well.
[00289] Control and treatment wells were sampled before and 60 days after application of the Bacillus Product. Sampling along the entire depth of the well was performed using a 1" PVC pipe equipped with a ball valve to intercept the sample. The improved manure characteristics test indicator was determined to be reduced relative to % of manure. solids after 60 days of treatment. A non-parametric Jonckheere-Terpstra test tail with exact statistics and significance level a = 0.10 was performed using SPSS statistical software (v. 17.0, IBM, Armonk, NY) to analyze difference between % solids before and after application of the treatment test the average differences.
[00290] There was no difference in mean manure solids between any of the test sites, where MicroSource S ® was included as standard operating procedure at all (Table 35). However, there was a 24.3 % (P = 0.10) reduction in dry matter across the 3 monitored sites when Bacillus inoculant from three strains was added to the manure pit. These data indicate that application of the three-strain Bacillus Inoculant improves manure management characteristics as indicated by a reduction in percent solids over and above the commercial product MicroSource S ®. Table 35. Reduction of solid after 60 days of application of the product from the Bacillus well to the treatment of manure wells in relation to the control of the manure well at the same production site.
1 SEM, standard error of the mean. EXAMPLE 17
[00291] Comparison of the effect of a three-strain direct-feed microbial Bacillus and MicroSource S ® on the performance of growing pigs.
[00292] A study was conducted to compare the effectiveness of a new three-strain Bacillus DFM and MicroSource S® (DSM) to improve the growth performance of growing pigs. A total of 144 pigs (initial weight: about 23 lb) were tested and placed in 36 pens with four pigs/pen in a controlled swine producer environment facility. One of three dietary treatments was assigned to each pen (12 replicates/treatment) and fed for the 6-week duration of the study. Treatments consisted of a basal control diet, a three-strain direct-feed microbial Bacillus (DFM) and MicroSource S ® (DSM), which is a Bacillus-based commercial swine waste DFM treatment.
[00293] The basal diet was formulated to contain 50% by-product (35% DDGS and 15% wheat bran; Table 36). Phytase (500 FTU/lb) was added to all diets. The new Bacillus DFM consisted of equal proportions of Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS 1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) to add up to a guaranteed 3.0 x 10 CFU / g of DFM product, included at a rate of 0.25 lb / ton of feed, resulting in a concentration of 3.75 x 104 CFU / g of diet. Other strains of Bacillus may be used including, but not limited to, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, and Bacillus subtilis AGTP BS 1069, and Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus X11-1. Table 36. Basal Diet Compositions


[00294] MicroSource S ® was included in the diet at 1 lb/ton of feed, resulting in 7.5 x 104 CFU/g of diet. Pig body weight gain and feed intake in the pen were determined on days 21 and 42 of the trial, and mean daily weight gain (adg), mean daily feed intake (adfi) and gain: feed/feed (G:F) ) were calculated.
[00295] Pigs fed diets supplemented with the novel Bacillus DFM had higher adg from d 0 to 21 than trial pigs fed control diets or diets supplemented with commercial DFM, Microsource S ® (Table 37). This increase in daily weight gain tends to result in higher (P < 0.10) body weight in animals fed the novel Bacillus DFM on day 21 of the study compared to the other two treatments. These data indicate that the new Bacillus DFM improves body weight gain of growing pigs compared to existing commercial Bacillus-based DFM (MicroSource S ®). Table 37. The growth performance of pigs fed a new Bacillus DFM compared to MicroSource S ®.
a,b Means without common superscripts are different, P < 0.05. c,d means without common superscripts are different, P < 0.10. EXAMPLE 18 Identification of the enzymatic activities of Bacillus strains.
[00296] In vitro assays were performed to test the enzymatic activity of the new strains of Bacillus against fibrous feed substrates commonly found in feed ingredients used to formulate swine and poultry feeds. Highly thorough screening of these test strains was performed by replicating the local plating of 2 microliters of liquid culture to 15.0 ml of various types of substrate medium of interest in 100x100x15mm grid plates. Cellulase, xylanase and β-mannanase activities were determined based on specific substrate usage by individual strains.
[00297] The components of the media used to assay the substrate use properties from the enzymatic activity of the strains derived from the environment are described in Table 38. The assay plates were allowed to dry for 30 minutes following the application of culture, and , then incubated at 32 °C for 24 hours. Enzyme activities for each strain were determined by measuring the substrate degradation zone, in millimeters, as indicated by compensating the surrounding edge of colony growth. The mean values of identical plates were recorded. Table 38. Components of media used to analyze enzyme activities illustrated by use properties of Bacillus substrate derived from the environment.


[00298] The activities of the fibrolytic degrading enzymes of various strains of Bacillus subtilis and Bacillus pumilus are reported in Table 39. All strains show degrading activity against at least two of the three fibrous substrates evaluated. These data indicate that these Bacillus strains have enzyme degradative capacity against cellulose, xylan, and β-mannose. Table 39. Cellulase, xylanase and β-mannanase activities of Bacillus strains.
EXAMPLE 19
[00299] The effect of supplementation of a direct-feed microbial Bacillus (DFM) on feeding residual bacterial load after washing in commercial grow-finish phase facilities.
[00300] To determine the growth performance of swine fed commercial corn and soybean meal-based diets with increasing amounts of by-product, a grow-finish phase study was conducted. A total of 1040 animals were weaned at approximately 3 weeks of age and weaned on a standard commercial starter diet. The animals were separated by sex, distributed to more than 40 pens on trial and fed. From day 42 onwards, a direct microbial feed consists of equal proportions of Bacillus subtilis strains AGTP BS918 (NRRL B-50508), AGTP BS 1013 (NRRL B-50509) and AGTP BS3BP5 (NRRL B-50510) plus a guaranteed 3.0 x 108 CFU/g of DFM product was included at a rate of 1 lb/ton in the feed, resulting in a concentration of 1.5 x 105 CFU/g in the diet (Table 40). Other strains of Bacillus may be used including, but not limited to, Bacillus subtilis AGTP BS442, Bacillus subtilis AGTP BS521, and Bacillus subtilis AGTP BS 1069, and Bacillus subtilis AGTP 944, Bacillus pumilus AGTP BS 1068 and Bacillus pumilus KX11-1. Table 40. Feeding phases and diet composition. 1

1 SBM, soybean meal; CP, crude protein; cDDGS, still dry corn grains with solubles with ~10% oil content; treatment included corn costs.
[00301] After the animals were carried out, washed and exposed to 24 hours of air drying in the facility, the residual bacterial load was determined as an indicator for pen cleanliness. Samples were collected in the area, which is at the back of each pen in the corner closest to the feeder, approximately 1 foot from a side and end panel (Figure 27).
[00302] The 16 in2 area of the installation floor was swabbed with a sterile pre-moistened cotton swab (PocketSwab Plus, Charme Sciences, Lawrence, MA). The sample area was passed 10 times on each strand and analyzed in triplicate. Within 15 seconds after the scraping procedure, the swab was placed in a LUMT bioluminescence reader (Ciências Charme, Lawrence, MA). Resulting relative light unit (RLU) values were recorded and corral average before statistical analysis.
[00303] Data were analyzed using the SAS NPAR1 WAY procedure (v. 9.1 0.3, SAS Institute, Inc., Cary, NC) with a significance level a = 0.05. Data indicated a significant (P < 0.05) reduction in bacterial load in pens fed diets containing DFM compared to load control diets after the pen was treated, washed and dried (Table 41). Table 41. Relative light unit (RLU) comparison, indicating residual bacterial load in commercial pens fed control diets or diets with direct microbial feed (DFM) included after barn washing and air drying
a, b means with different superscripts were significantly different (P <0.05); 1 SEM, standard error of the mean.
[00304] While specific embodiments have been illustrated and described in the present invention, it will be appreciated by those skilled in the art that any arrangement that is calculated to achieve the same objective may be substituted for the specific embodiments shown. This patent application is intended to cover any adaptations or variations that operate in accordance with the principles of the present invention as described. Therefore, the present invention is intended to be limited only by the claims and their equivalents. The patent descriptions, references and publications cited in the application are hereby incorporated by reference in their entirety herein. BIBLIOGRAPHY Association of Analytical Chemists (AOAC) (2007). Official methods of analysis, 18th ed. AOAC, Washington, D.C. Liu K. 201 1. Chemical composition of distillers grains, a review. J. Agric. Food CheM 59: 1508-1526. Metzler-Zebeli, B.U., Hooda, S., Pieper, R., Zijlstra, R.T., Van Kessel, A.G., Mosenhin, R. and G. Ganzle (2010). Polysaccharides Modulate Bacterial Microbiota, Pathways for Butyrate Production, and Abundance of Pathogenic Escherichia coli in the Pig Gastrointestinal Tract; J Appl Env Microbiol 76(11), 3692-3701. NRC. 1998. Nutrient Requirements of Swine. 10th rev. ed. natl. academy Press, Washington, DC. Stein, H.H. and G.C. Shurson. 2009. The use and application of distillers dried grains with solubles in swine diets. J. Anim. Sci. 87: 1292-1303. Stein, H. PL, B. Seve, M. F. Fuller, P. J. Moughan, and C. F. M. de Lange. 2007. Invited review: Amino acid bioavailability and digestibility in pig feed ingredients: Terminology and application. J. Anim. Sci. 85: 172-180. Spence, C, Whitehead, T.R. and M.A. Cotta (2008). Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate reducing bacteria in stored swine manure. J Appl Microbiol 105, 2143-2152. Yegani M., and D.R. Korver. 2008. Factors affecting intestinal health in poultry. Poult. Sci 87:2052-2063. Yu, Y., Lee, C, Kim, J. and S. Hwang (2005). Group-Specific Primer and Probe Sets to Detect Methanogenic Communities Using Quantitative Real-Time Polymerase Chain Reaction. Biotechnol Bioeng 89, 670-679. 1/1

权利要求:
Claims (4)
[0001]
1. Use of an isolated Bacillus strain, which has enzyme activity selected from the group consisting of: Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis AGTP BS442 (NRRL B-50542), Bacillus subtilis AGTP BS521 (NRRL B- 50545), Bacillus subtilis AGTP BS918 (NRRL B-50508), Bacillus subtilis AGTP BS 1013 (NRRL B-50509), Bacillus subtilis AGTP BS1069 (NRRL B-50544), Bacillus subtilis AGTP 944 (NRRL B-50548) and Bacillus pumilus AGTP BS 1068 (NRRL B-50543), characterized in that it is for administration to an animal, wherein, when the strain is administered to an animal, it provides a non-therapeutic improvement in at least one of the following characteristics: body weight, average daily gain, average daily feed intake, feed efficiency, carcass characteristics, nutrient digestibility and manure residue problems.
[0002]
2. Use of a strain according to claim 1, characterized in that it is for administration to an animal, wherein, when the strain is administered to an animal, it provides a non-therapeutic improvement in at least one of the following characteristics : body weight, average daily gain, average daily feed intake, feed efficiency, carcass characteristics, nutrient digestibility and manure residue problems by at least 2% compared to a control animal.
[0003]
3. Use of a strain, according to claim 1, characterized in that the animal is a bird or pig.
[0004]
4. Feed for an animal, characterized in that the feed is supplemented with Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis AGTP BS918 (NRRL B-50508) and Bacillus subtilis AGTP BS1013 (NRRL B-50509).

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同族专利:

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公开号 | 公开日

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EP2748300A1|2014-07-02|

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CN103930540B|2017-05-17|

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BR112014003950A2|2020-10-27|

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CA2845576A1|2013-02-28|

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ES2702230T3|2019-02-28|

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PL2748300T3|2019-05-31|

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US20150230498A1|2015-08-20|

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CA2845576C|2020-09-15|

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RU2014119583A|2015-11-20|

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CN107418908A|2017-12-01|

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DK2748300T3|2019-01-14|

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US20130064927A1|2013-03-14|

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US10058110B2|2018-08-28|

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CN107418908B|2021-01-08|

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CN103930540A|2014-07-16|

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WO2013029013A8|2017-09-28|

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US9089151B2|2015-07-28|

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EP2748300B1|2018-09-19|

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WO2013029013A1|2013-02-28|

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WO2021146582A1|2020-01-17|2021-07-22|AgBiome, Inc.|Compositions and methods for controlling undesirable microbes and improving animal health|

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WO2021163148A1|2020-02-11|2021-08-19|Locus Ip Company, Llc|Methods and compositions for reducing deleterious enteric atmospheric gases in livestock|

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WO2021173974A1|2020-02-28|2021-09-02|Dupont Nutrition Biosciences Aps|Feed compositions|

法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-06-01| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-01-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161526881P| true| 2011-08-24|2011-08-24|

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US61/526,881|2011-08-24|

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US201161527371P| true| 2011-08-25|2011-08-25|

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US61/527,371|2011-08-25|

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PCT/US2012/052360|WO2013029013A1|2011-08-24|2012-08-24|Enzyme producing bacillus strains|

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