Direct fed microorganisms using bacillus coagulans

Abstract

The present invention relates to direct fed microorganisms for improving animal performance and improving animal health, and combinations thereof. More specifically, the present invention relates to isolated Bacillus coagulans strains and strains having all identifying characteristics of these strains for use comprising the above-mentioned use.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 413,412, filed on October 5, 2022, under 35 U.S.C.§119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to direct - fed microbials for improving animal performance and improving animal health and combinations thereof. More particularly, the present invention relates to isolated strains of Bacillus coagulans and strains having all the identifying characteristics of these strains for uses including the above - mentioned uses.

[0004] Background Art and Summary of the Invention

[0005] The present invention relates to direct - fed microbial (DFM) compositions and methods for improving animal performance and improving animal health and combinations thereof. These improvements enhance the commercial value of animal populations.

[0006] The gastrointestinal tract of animals is constantly under attack from a large number of bacteria, viruses, and protozoa present in feed, bedding, and the environment. The gastrointestinal tract has a complex system composed of physical, chemical, and immunological defenses against these potential pathogens. Beneficial bacteria are an important part of this system. Pathogens, stress, metabolic disorders, the use of antimicrobials, and other factors can disrupt the balance of gut bacteria, which may impair digestion and make animals more susceptible to disease.

[0007] Direct - fed microbial products are products containing live (viable) microorganisms (e.g., bacteria). Over time, many direct - fed microbial products that were previously thought to be useful for improving animal performance (e.g., directly through improved feed conversion ratio) have lost their overall efficacy. Accordingly, there is a need for microbial strains that will improve animal performance, including average daily feed intake, average daily gain, feed conversion ratio, nutrient absorption, energy utilization, and overall growth performance.

[0008] The applicant has developed a direct-fed microbial composition comprising a strain of Bacillus coagulans that inhibits inflammation in animals, resulting in increased growth performance (e.g., increased nutrient absorption, increased average daily gain, increased average daily feed intake, and improved feed conversion ratio), improved metabolizable energy (i.e., improved energy utilization), and reduced disease concerns from animal pathogens. The direct-fed microbial compositions described herein provide a commercial benefit by providing all of these properties or combinations thereof in a single direct-fed microbial composition. In addition, the direct-fed microbial compositions described herein result in reduced antibiotic use and increased feed efficiency, which reduces the total cost of animal feed.

[0009] Methods and compositions are provided for inhibiting inflammation in animals, resulting in increased growth performance (e.g., increased nutrient absorption, increased average daily gain, increased average daily feed intake, and improved feed conversion ratio), improved metabolizable energy (i.e., improved energy utilization), and reduced disease concerns from animal pathogens, and combinations thereof. In various embodiments, the animal can be selected from poultry species, swine species, bovine species, ovine species, equine species, and companion animals. In an embodiment where the animal is a poultry species, the poultry species can be a broiler chicken. In an embodiment where the animal is a swine species, the swine species can be selected from grow finish pigs, nursery pigs, sows, and breeding stock pigs.

[0010] In one embodiment, a method of raising an animal is provided. The method includes the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof, wherein the Bacillus strain results in an effect selected from improved animal performance and improved animal health and combinations thereof.

[0011] In another embodiment, a method of raising an animal is provided. The method includes the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0012] In various embodiments, the compositions for use in the methods described herein can be a commercial package, a feed additive for an animal feed composition, an additive for animal drinking water, or an animal feed composition (e.g., a complete feed), each comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0013] The following clauses and combinations thereof provide various additional illustrative aspects of the invention described herein. Any of the various embodiments described in any other part of this patent application, including the part titled "Detailed Description" and the examples, can be applied to any of the following embodiments of the invention described in the numbered clauses below.

[0014] 1. A method of raising an animal, the method including the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof, wherein the Bacillus strain results in an effect selected from improving animal performance and improving animal health and combinations thereof.

[0015] 2. The method of clause 1, wherein the animal is selected from poultry species, swine species, bovine species, ovine species, equine species, and companion animals.

[0016] 3. The method of clause 2, wherein the poultry species is a broiler.

[0017] The method of any one of clauses 1 - 3, wherein the effect is to improve animal performance.

[0018] The method of clause 4, wherein the improvement in animal performance is selected from increased nutrient absorption, increased average daily gain, increased average daily feed intake, decreased feed conversion ratio, increased digestibility of the diet, increased ratio of metabolizable energy to total energy, improved performance consistency, and combinations thereof.

[0019] The method of clause 2, wherein the pig species is selected from growing pigs, nursery pigs, sows, and breeding pigs.

[0020] The method of any one of clauses 1 - 6, wherein the effect is to improve animal health.

[0021] The method of any one of clauses 1 - 7, wherein the Bacillus strain produces enzymes selected from α - galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0022] The method of any one of clauses 1 - 8, wherein at least one of the Bacillus strains has antimicrobial activity.

[0023] The method of clause 9, wherein the antimicrobial activity is against bacteria selected from Escherichia coli (E.coli), Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, and combinations thereof.

[0024] The method of clause 7, wherein the improvement in animal health is a reduction in the bioburden in the animal.

[0025] The method of clause 7, wherein the improvement in animal health is an increase in the immune response in the animal.

[0026] The method of any one of clauses 1 - 12, further comprising the step of administering to the animal another bacterial strain selected from another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0027] The method of any one of clauses 1 - 13, wherein the administered strain is Bacillus strain BC1 (NRRL No. B - 67744) or a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) or combinations thereof.

[0028] 15. The method of any one of clauses 1 - 13, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B - 67745) or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or a combination thereof.

[0029] 16. The method of any one of clauses 1 - 13, wherein the strain administered is Bacillus strain BC1 (NRRL No. B - 67744).

[0030] 17. The method of any one of clauses 1 - 13, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B - 67745).

[0031] 18. The method of any one of clauses 1 - 13, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or a combination thereof are administered in combination in a single composition.

[0032] 19. The method of any one of clauses 1 - 13, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or a combination thereof are administered in combination in separate compositions.

[0033] 20. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 5.0x10 12 CFU / gram of feed composition.

[0034] 21. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 1.0x10 7 CFU / gram of feed composition.

[0035] 22. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered at a dose greater than about 7.0x10 4The dose of CFU / gram of the feed composition is administered in the feed composition.

[0036] The method of any one of clauses 1 - 22, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

[0037] The method of any one of clauses 1 - 23, further comprising the step of administering an enzyme to the animal, the enzyme being selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase and combinations thereof.

[0038] The method of any one of clauses 1 - 24, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0039] The method of any one of clauses 1 - 24, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0040] The method of any one of clauses 1 - 24, wherein the feed composition is administered to the animal daily.

[0041] The method of any one of clauses 1 - 27, wherein the animal is selected from chickens, pigs, horses, ponies, cattle, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans and combinations thereof.

[0042] A commercial package comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) and combinations thereof.

[0043] 30. A feed additive for animal feed, which comprises an isolated Bacillus coagulans strain selected from the following: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0044] 31. An additive for animal drinking water, which comprises an isolated Bacillus coagulans strain selected from the following: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0045] 32. An animal feed composition, which comprises an isolated Bacillus coagulans strain selected from the following: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0046] 33. A commercial package, feed additive, feed composition or additive for animal drinking water according to any one of clauses 29 - 32, wherein the Bacillus strain results in an effect selected from improving animal performance, improving animal health, and combinations thereof.

[0047] 34. A commercial package, feed additive, feed composition or additive for animal drinking water according to any one of clauses 29 - 33, wherein the Bacillus strain inhibits pathogens selected from Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, and Clostridium.

[0048] 35. The feed additive or additive for animal drinking water according to clause 30 or 31, which is in the form of a concentrate.

[0049] 36. The feed additive or additive for animal drinking water according to clause 30 or 31, which is in the form of a superconcentrate.

[0050] 37. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30 - 34, in dry form.

[0051] 38. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30 - 34, in the form of a pellet.

[0052] 39. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30 - 34, in powder form.

[0053] 40. A commercial package, feed additive, feed composition, or additive for animal drinking water according to any one of clauses 29 - 34, wherein the strain is in a form selected from powder, liquid, gel, freeze - dried form, surface dressing, and pellet form.

[0054] 41. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 40, further comprising a carrier for the Bacillus strain.

[0055] 42. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 41, wherein the carrier is selected from bran, rice husk, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

[0056] 43. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 42, in a bag.

[0057] 44. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 43, wherein the bag is a plastic bag.

[0058] 45. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 44, further comprising instructions for use of one or more of the Bacillus strains.

[0059] 46. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 45, in a 20 - pound bag.

[0060] 47. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 46, in a 50 - pound bag.

[0061] 48. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29 - 47, in a container for commercial use.

[0062] 49. The commercial package, feed additive, additive for animal drinking water or feed composition of clause 48, wherein the container comprises plastic.

[0063] 50. The commercial package, feed additive, additive for animal drinking water or feed composition of clause 48, wherein the container comprises paper.

[0064] 51. The commercial package, feed additive, additive for animal drinking water or feed composition of any one of clauses 29 - 50, further comprising an adhesive.

[0065] 52. The commercial package, feed additive, additive for animal drinking water or feed composition of clause 51, wherein the adhesive is selected from clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

[0066] 53. The commercial package, feed additive, additive for animal drinking water or feed composition of any one of clauses 29 - 52, which is in the form of a dietary nutritional composition.

[0067] 54. The commercial package, feed additive, additive for animal drinking water or feed composition of any one of clauses 29 - 53, further comprising an exogenously added nutritional component selected from vitamins, antibiotics, enzymes, water-soluble or water-insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorus, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starch.

[0068] 55. The commercial package, feed additive, additive for animal drinking water or feed composition of clause 54, wherein the exogenously added component is an enzyme.

[0069] 56. The commercial package, feed additive, additive for animal drinking water or feed composition of clause 55, wherein the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0070] 57. A method of raising an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), and combinations thereof.

[0071] 58. The method of clause 57, wherein the animal is selected from poultry species, swine species, bovine species, ovine species, equine species, and companion animals.

[0072] 59. The method of clause 58, wherein the poultry species is a broiler.

[0073] 60. The method of clause 58, wherein the swine species is selected from growing pigs, nursery pigs, sows, and breeding pigs.

[0074] 61. The method of any one of clauses 57 - 60, wherein the Bacillus strain produces enzymes selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0075] 62. The method of any one of clauses 57 - 61, wherein at least one of the Bacillus strains has antimicrobial activity.

[0076] 63. The method of clause 62, wherein the antimicrobial activity is against bacteria selected from Escherichia coli, Salmonella spp., Staphylococcus spp., Enterococcus spp., Clostridium spp., Campylobacter spp., and combinations thereof.

[0077] 64. The method of any one of clauses 57 - 63, further comprising the step of administering to the animal another bacterial strain selected from another different Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0078] 65. The method of any one of clauses 57 - 64, wherein the administered strain is Bacillus strain BC1 (NRRL No. B - 67744) or a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) or a combination thereof.

[0079] 66. The method of any one of clauses 57 - 64, wherein the administered strain is Bacillus strain BCP2 (NRRL No. B - 67745) or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or a combination thereof.

[0080] 67. The method of any one of clauses 57 - 64, wherein the administered strain is Bacillus strain BC1 (NRRL No. B - 67744).

[0081] 68. The method of any one of clauses 57 - 64, wherein the administered strain is Bacillus strain BCP2 (NRRL No. B - 67745).

[0082] The method of any one of clauses 57 - 64, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or a combination thereof are administered in combination in a single composition.

[0083] The method of any one of clauses 57 - 64, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or a combination thereof are administered in combination in separate compositions.

[0084] The method of any one of clauses 57 - 70, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 5.0x10 12 CFU / gram of feed composition.

[0085] The method of any one of clauses 57 - 70, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 1.0x10 7 CFU / gram of feed composition.

[0086] The method of any one of clauses 57 - 72, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

[0087] The method of any one of clauses 57 - 73, further comprising the step of administering an enzyme to the animal, wherein the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0088] A method according to any one of clauses 57 - 74, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0089] A method according to any one of clauses 57 - 74, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0090] A method according to any one of clauses 57 - 74, wherein the feed composition is administered to the animal daily.

[0091] A method according to any one of clauses 57 - 74, wherein the animal is selected from the group consisting of chickens, pigs, horses, ponies, cattle, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1A -C shows the relative quantification of the expression of inflammatory chemokines and cytokines in the IEC - 6 cell line treated with Bacillus coagulans (BCP2) at 10 3 、10 4 and 10 5 CFU / ml and challenged with 10 ng LPS. Different letters indicate significant differences (p < 0.05).

[0093] Figure 2A -C shows the relative quantification of the expression of inflammatory cytokines and pathogen receptors in the IEC - 6 cell line treated with Bacillus coagulans (BC1) at 10 3 、10 4 and 10 5 CFU / ml and challenged with 10 ng LPS. Different letters indicate significant differences (p < 0.05).

[0094] Figure 3 Shows the expression of pathogen receptors and cytokines in pigs exposed to a high bioburden environment and treated with Bacillus DFM, or Bacillus DFM and Bacillus coagulans (BC1). Asterisks indicate values that are significantly different from the negative control (p < 0.05). From left to right in each group, the bars are the negative control, high bioburden control, high bioburden control + DFM, and high bioburden control + DFM + Bacillus strain BC1.

[0095] Figure 4 Shows the body weight, live weight gain, gain - to - feed ratio, and feed intake of chickens fed Bacillus coagulans. DETAILED DESCRIPTION

[0096] Methods and compositions are provided for suppressing inflammation in an animal, resulting in increased growth performance (e.g., increased nutrient absorption, increased average daily gain, increased average daily feed intake, and improved feed conversion rate), improved metabolizable energy (i.e., improved energy utilization), and reduced disease concerns from animal pathogens, and combinations thereof. In various embodiments, the animal may be selected from poultry species, swine species, bovine species, ovine species, equine species, and companion animals. In embodiments where the animal is a poultry species, the poultry species may be broiler chickens. In embodiments where the animal is a swine species, the swine species may be selected from growing pigs, nursery pigs, sows, and breeding pigs.

[0097] In one embodiment, a method of raising an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof, wherein the Bacillus strain results in an effect selected from improving animal performance and improving animal health, and combinations thereof.

[0098] In another embodiment, a method of raising an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0099] In various embodiments, the compositions for use in the methods described herein can be a commercial package, a feed additive for an animal feed composition, an additive for animal drinking water, or an animal feed composition (e.g., a complete feed), each comprising a Bacillus coagulans strain isolated from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0100] The following clauses and combinations thereof provide various additional illustrative aspects of the invention described herein. Each of the embodiments described in the section entitled "Detailed Description" may be applicable to any of the following embodiments of the invention described in the numbered clauses below.

[0101] 1. A method of raising an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising a Bacillus coagulans strain isolated from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRR No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof, wherein the Bacillus strain results in an effect selected from improving animal performance, improving animal health, and combinations thereof.

[0102] 2. The method of clause 1, wherein the animal is selected from the group consisting of poultry species, swine species, bovine species, ovine species, equine species, and companion animals.

[0103] 3. The method of clause 2, wherein the poultry species is a broiler.

[0104] 4. The method of any one of clauses 1-3, wherein the effect is improving animal performance.

[0105] 5. The method of clause 4, wherein the improvement in animal performance is selected from increased nutrient absorption, increased average daily gain, increased average daily feed intake, decreased feed conversion ratio, increased digestibility of the diet, increased ratio of metabolizable energy to total energy, improved performance consistency, and combinations thereof.

[0106] 6. The method of clause 2, wherein the swine species is selected from growing pigs, nursery pigs, sows, and breeding pigs.

[0107] 7. A method according to any one of clauses 1-6, wherein the effect is to improve animal health.

[0108] 8. A method according to any one of clauses 1-7, wherein the Bacillus strain produces an enzyme selected from α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0109] 9. A method according to any one of clauses 1-8, wherein at least one of the Bacillus strains has antimicrobial activity.

[0110] 10. The method of clause 9, wherein the antimicrobial activity is against bacteria selected from Escherichia coli (E. coli), Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, and combinations thereof.

[0111] 11. The method of clause 7, wherein the improvement in animal health is a reduction in the bioburden in the animal.

[0112] 12. The method of clause 7, wherein the improvement in animal health is an increase in the immune response in the animal.

[0113] 13. A method according to any one of clauses 1-12, further comprising the step of administering to the animal another bacterial strain selected from another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0114] 14. A method according to any one of clauses 1-13, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744) or a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) or combinations thereof.

[0115] 15. A method according to any one of clauses 1-13, wherein the administered strain is Bacillus strain BCP2 (NRRL No. B-67745) or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or combinations thereof.

[0116] 16. A method according to any one of clauses 1-13, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744).

[0117] 17. A method according to any one of clauses 1-13, wherein the administered strain is Bacillus strain BCP2 (NRRL No. B-67745).

[0118] 18. The method of any one of clauses 1 - 13, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or a combination thereof are combined and administered in a single composition.

[0119] 19. The method of any one of clauses 1 - 13, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or a combination thereof are combined and administered in separate compositions.

[0120] 20. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 5.0x10 12 CFU / gram of feed composition.

[0121] 21. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 1.0x10 7 CFU / gram of feed composition.

[0122] 22. The method of any one of clauses 1 - 19, wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0x10 4 CFU / gram of feed composition.

[0123] 23. The method of any one of clauses 1 - 22, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

[0124] 24. The method of any one of clauses 1-23 further comprises the step of administering an enzyme to the animal, the enzyme being selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0125] 25. The method of any one of clauses 1-24, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0126] 26. The method of any one of clauses 1-24, wherein the animal is a sow and the Bacillus strain is administered during pregnancy.

[0127] 27. The method of any one of clauses 1-24, wherein the feed composition is administered to the animal daily.

[0128] 28. The method of any one of clauses 1-27, wherein the animal is selected from chickens, pigs, horses, ponies, cattle, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

[0129] 29. A commercial package comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0130] 30. A feed additive for animal feed comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0131] 31. An additive for animal drinking water comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0132] 32. An animal feed composition comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0133] 33. A commercial package, feed additive, feed composition, or additive for animal drinking water according to any one of clauses 29-32, wherein the Bacillus strain results in an effect selected from improving animal performance, improving animal health, and combinations thereof.

[0134] 34. A commercial package, feed additive, feed composition, or additive for animal drinking water according to any one of clauses 29-33, wherein the Bacillus strain inhibits pathogens selected from Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, and Clostridium.

[0135] 35. A feed additive or additive for animal drinking water according to clause 30 or 31, in the form of a concentrate.

[0136] 36. A feed additive or additive for animal drinking water according to clause 30 or 31, in the form of a super-concentrate.

[0137] 37. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30-34, in a dry form.

[0138] 38. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30-34, in the form of a pellet.

[0139] 39. A feed additive, feed composition, or additive for animal drinking water according to any one of clauses 30-34, in the form of a powder.

[0140] 40. A commercial package, feed additive, feed composition, or additive for animal drinking water according to any one of clauses 29-34, wherein the strain is in a form selected from powder, liquid, gel, freeze-dried form, surface dressing, and pellet form.

[0141] 41. A commercial package, feed additive, additive for animal drinking water, or feed composition according to any one of clauses 29-40, further comprising a carrier for the Bacillus strain.

[0142] 42. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 41, wherein the carrier is selected from bran, rice husk, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

[0143] 43. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 42, which is in a bag.

[0144] 44. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 43, wherein the bag is a plastic bag.

[0145] 45. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 44, further comprising instructions for use of one or more of the Bacillus strains.

[0146] 46. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 45, which is in a 20 - pound bag.

[0147] 47. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 46, which is in a 50 - pound bag.

[0148] 48. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 47, which is in a container for commercial use.

[0149] 49. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 48, wherein the container comprises plastic.

[0150] 50. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 48, wherein the container comprises paper.

[0151] 51. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 50, further comprising an adhesive.

[0152] 52. The commercial package, feed additive, additive for animal drinking water, or feed composition of clause 51, wherein the adhesive is selected from clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

[0153] 53. The commercial package, feed additive, additive for animal drinking water, or feed composition of any one of clauses 29 - 52, which is in the form of a dietary nutritional composition.

[0154] 54. The commercial package, feed additive, additive for animal drinking water or feed composition of any one of Clauses 29 - 53 further comprises an exogenously added nutrient component selected from vitamins, antibiotics, enzymes, water-soluble or water-insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorus, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants and starch.

[0155] 55. The commercial package, feed additive, additive for animal drinking water or feed composition of Clause 54, wherein the exogenously added component is an enzyme.

[0156] 56. The commercial package, feed additive, additive for animal drinking water or feed composition of Clause 55, wherein the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase and combinations thereof.

[0157] 57. A method of raising animals, the method comprising the step of administering to the animals a feed composition or drinking water comprising an effective amount of an additive, the additive comprising isolated Bacillus coagulans strains selected from: Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) and combinations thereof.

[0158] 58. The method of Clause 57, wherein the animals are selected from poultry species, swine species, bovine species, ovine species, equine species and companion animals.

[0159] 59. The method of Clause 58, wherein the poultry species is broiler chickens.

[0160] 60. The method of Clause 58, wherein the swine species is selected from growing pigs, nursery pigs, sows and breeding pigs.

[0161] 61. The method of any one of Clauses 57 - 60, wherein the Bacillus strain produces enzymes selected from α - galactosidase, protease, lipase, amylase, xylanase, cellulase and combinations thereof.

[0162] 62. The method of any one of Clauses 57 - 61, wherein at least one of the Bacillus strains has antimicrobial activity.

[0163] 63. The method of clause 62, wherein the antimicrobial activity is against bacteria selected from Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Clostridium, Campylobacter, and combinations thereof.

[0164] 64. The method of any one of clauses 57 - 63, further comprising the step of administering to the animal another bacterial strain selected from another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0165] 65. The method of any one of clauses 57 - 64, wherein the strain administered is Bacillus strain BC1 (NRRL No. B - 67744) or a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) or combinations thereof.

[0166] 66. The method of any one of clauses 57 - 64, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B - 67745) or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or combinations thereof.

[0167] 67. The method of any one of clauses 57 - 64, wherein the strain administered is Bacillus strain BC1 (NRRL No. B - 67744).

[0168] 68. The method of any one of clauses 57 - 64, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B - 67745).

[0169] 69. The method of any one of clauses 57 - 64, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or combinations thereof are administered in combination in a single composition.

[0170] 70. The method of any one of clauses 57 - 64, wherein at least two of Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745), or combinations thereof are administered in combination in separate compositions.

[0171] 71. A method according to any one of clauses 57 - 70, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 5.0x10 12 CFU / gram of feed composition.

[0172] 72. A method according to any one of clauses 57 - 70, wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0x10 3 CFU / gram of feed composition - about 1.0x10 7 CFU / gram of feed composition.

[0173] 73. A method according to any one of clauses 57 - 72, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

[0174] 74. A method according to any one of clauses 57 - 73, further comprising the step of administering an enzyme to the animal, wherein the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase and combinations thereof.

[0175] 75. A method according to any one of clauses 57 - 74, wherein the animal is a sow, and the Bacillus strain is administered during lactation.

[0176] 76. A method according to any one of clauses 57 - 74, wherein the animal is a sow, and the Bacillus strain is administered during pregnancy.

[0177] 77. A method according to any one of clauses 57 - 74, wherein the feed composition is administered to the animal daily.

[0178] 78. A method according to any one of clauses 57 - 74, wherein the animal is selected from chickens, pigs, horses, ponies, cattle, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans and combinations thereof.

[0179] In various embodiments, the animals to which the feed additives, feed compositions, or drinking water described herein are administered can be selected from poultry species, swine species, bovine species, ovine species, equine species, companion animals, or humans. In embodiments where the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In embodiments where the animal is a swine species, the swine species can be selected from finishing pigs, nursery pigs, sows, and breeding stock pigs. In various exemplary embodiments, the animal can be selected from chickens (e.g., broilers or laying hens), pigs, horses, ponies, cattle, turkeys, goats, sheep, quail, pheasants, ostriches, ducks, fish (e.g., tilapia, catfish, flounder, or salmon), crustaceans (e.g., shrimp or crab), and combinations thereof. In various embodiments described herein, the feed additive, feed composition, or additive for animal drinking water can be a dietary nutritional composition (e.g., a probiotic composition). In one embodiment, the commercial package described herein can contain a dietary nutritional composition that includes a Bacillus coagulans strain described herein.

[0180] In one embodiment of the invention, an effective amount of a Bacillus strain can be administered to improve animal performance or improve animal health or a combination thereof. The term "effective amount" means an amount of a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all of the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof) that is capable of improving animal performance or improving animal health or a combination thereof by any mechanism, including those mechanisms described herein.

[0181] In the embodiments described herein of the present invention where the composition of the present invention comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof is administered to an animal, the composition is preferably administered orally to the animal in a feed composition or drinking water, but any other effective method of administration known to those skilled in the art may be utilized. In an illustrative embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof is provided in the form of an additive to be added to a feed composition (i.e., food) or the drinking water of the animal.

[0182] In another illustrative embodiment, the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all of the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all of the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof, is provided in the form of a feed additive for addition to a feed composition. The feed composition may contain the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all of the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all of the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof, in a mixture with an animal feed blend (including any recognized animal feed blend or any animal feed blend described herein). As used herein, "feed composition" or "animal feed composition" means a feed composition containing the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all of the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all of the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof, in a mixture with an animal feed blend and any other optional components that may be used in the feed composition, said other components including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0183] Any animal feed blend can be used in accordance with the methods and compositions described in this patent application, including those known in the art and those described herein, such as rapeseed meal, cottonseed meal, soybean meal, corn meal, barley, wheat, silage, and ensiled haylage. In various embodiments, the animal feed blend can be supplemented with the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all of the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all of the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof, but other ingredients can optionally be added to the animal feed blend, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0184] In various illustrative embodiments, optional components of the animal feed blend include sugars and complex carbohydrates such as water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional components include cereal distillers' solubles, fats (e.g., crude fat), phosphorus, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, oil derived from fish meal, raw seeds (e.g., flaxseed), antioxidants, and starch. In another embodiment, minerals can be added in the form of a mineral premix.

[0185] Optional amino acid components that can be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HCl, and the like, and their salts. Vitamins that can be optionally added are thiamine HCl, riboflavin, pyridoxine HCl, niacin, nicotinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, etc. In another embodiment, vitamins can be added in the form of a vitamin premix. In yet another embodiment, protein components can be added to the animal feed blend and include proteins obtained from meat meal, bone meal, or fish meal, liquid or powdered eggs, fish solubles, crude protein, and the like.

[0186] In another illustrative aspect, any pharmaceutical component known in the art can be added to the animal feed blend or an additive for animal drinking water, such as antibiotics. In various embodiments, the antibiotics are selected from ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, Denagard TM (i.e., tylosin), BMD TM (i.e., bacitracin methylene disalicylate), Carbadox TM (i.e., carbadox), Stafac TM (i.e., virginiamycin), erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM (i.e., tylosin), Pulmotil TM (i.e., tilmicosin), vancomycin, avilamycin (Kavault TM ), gentamicin, neomycin, and combinations thereof. In another embodiment, the animal feed blend, feed composition, feed additive, or additive for animal drinking water can be free of antibiotics.

[0187] In another illustrative embodiment, one or more enzymes may be added to an animal feed blend. In various embodiments, enzymes that may be added include galactosidase, phytase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, combinations thereof, and any other enzymes that enhance the effectiveness of a feed composition for improving animal performance or health. In yet another embodiment, yeast, fungi (e.g., Aspergillus or Trichoderma), or micronutrients may be added to animal feed. Any of the foregoing ingredients suitable for addition to an additive for animal drinking water may be added as a component of the additive for animal drinking water as described herein.

[0188] In various illustrative embodiments, a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or combinations thereof) or any other bacterial strain added in addition to Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or combinations thereof may be administered in the feed composition at a dose of from about 1.0 x 10 3 CFU / gram of feed composition to about 5.0 x 10 12 CFU / gram of feed composition or at a dose of from about 1.0 x 10 3 CFU / gram of feed composition to about 1.0 x 10 7 CFU / gram of feed composition. In other embodiments, a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B - 67744), Bacillus strain BCP2 (NRRL No. B - 67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B - 67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B - 67745) or combinations thereof) is administered at a dose greater than about 1.0 x 10 3 CFU / gram of feed composition, greater than about 1.1 x 10 3 CFU / gram of feed composition, greater than about 1.25 x 10 3The dose of CFU / gram of the feed composition is greater than about 1.5x10 3 The dose of CFU / gram of the feed composition is greater than about 1.75x10 3 The dose of CFU / gram of the feed composition is greater than about 1.0x10 4 The dose of CFU / gram of the feed composition is greater than about 2.0x10 4 The dose of CFU / gram of the feed composition is greater than about 3.0x10 4 The dose of CFU / gram of the feed composition is greater than about 4.0x10 4 The dose of CFU / gram of the feed composition is greater than about 5.0x10 4 The dose of CFU / gram of the feed composition is greater than about 6.0x10 4 The dose of CFU / gram of the feed composition is greater than about 7.0x10 4 The dose of CFU / gram of the feed composition is greater than about 8.0x10 4 The dose of CFU / gram of the feed composition is greater than about 1.0x10 5 The dose of CFU / gram of the feed composition is greater than about 1.0x10 6 The dose of CFU / gram of the feed composition is greater than about 1.0x10 7 The dose of CFU / gram of the feed composition is greater than about 1.0x10 8 The dose of CFU / gram of the feed composition is greater than about 1.0x10 9 The dose of CFU / gram of the feed composition is greater than about 1.0x10 10 The dose of CFU / gram of the feed composition is greater than about 1.0x10 11 The dose of CFU / gram of the feed composition or greater than about 1.0x10 12 The dose of CFU / gram of the feed composition is administered in the feed composition. In yet another embodiment, a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof) is at about 7x10 4 The dose of CFU / gram of the feed composition is administered in the feed composition.

[0189] In various embodiments, the Bacillus coagulans strains (e.g., Bacillus strains BC1 and / or BCP2) used in accordance with the methods and compositions described herein are optionally selected from Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), strains having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or strains having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or combinations thereof. These strains are Bacillus coagulans strains. Bacillus strain MDGBC1 (strain BC1) and Bacillus strain MDGBCP2 (strain BCP2) were deposited on February 6, 2019, with the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, Illinois 61604-3999, and were given accession numbers B-67744 and B-67745, respectively. The deposits were made under the provisions of the internationally recognized requirements for the deposit of microorganisms for the purposes of patent procedures in accordance with the Budapest Treaty. The NRRL strains are named MDGBC1 and MDGBCP2, which are equivalent to the Bacillus coagulans strains BC1 and BCP2 as referred to in the present application, respectively.

[0190] Any of these strains can be administered alone or in combination in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or in drinking water for an animal. In one embodiment, multiple strains are administered in combination in a single composition. In another embodiment, multiple strains are administered in combination in separate compositions.

[0191] In another embodiment, one or more of the Bacillus strains described in the foregoing paragraphs (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof) can be administered to an animal together with a different bacterial strain selected from another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. In yet another embodiment, one or more of the Bacillus strains described in the foregoing paragraphs (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof) can be administered to an animal together with any other different bacterial strain that effectively improves animal performance or health.

[0192] As used herein, a "strain having" all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) or Bacillus strain BCP2 (NRRL No. B-67745) can be a mutant strain having all the identifying characteristics of Bacillus strain BC1 or Bacillus strain BCP2 (e.g., a DNA fingerprint corresponding to the DNA fingerprint of Bacillus strain BC1 or Bacillus strain BCP2 based on DNA analysis; enzyme activity corresponding to Bacillus strain BC1 or Bacillus strain BCP2; antimicrobial activity corresponding to Bacillus strain BC1 or Bacillus strain BCP2; antibiotic sensitivity and tolerance patterns corresponding to Bacillus strain BC1 or Bacillus strain BCP2, or a combination thereof). In an alternative embodiment, the mutation can be a natural mutation or a genetically engineered mutation. In another embodiment, a "strain having" all the identifying characteristics of Bacillus strain BC1 or Bacillus strain BCP2 can be, for example, a strain produced by isolating one or more plasmids from Bacillus strain BC1 or Bacillus strain BCP2 and introducing the one or more plasmids into another bacterium, such as another Bacillus strain, provided that the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain BC1 or Bacillus strain BCP2 (e.g., a DNA fingerprint corresponding to the DNA fingerprint of Bacillus strain BC1 or Bacillus strain BCP2 based on DNA analysis).

[0193] A feed composition or drinking water comprising the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof can be administered to an animal for any period of time that is effective to continuously improve animal performance or improve animal health or a combination thereof. For example, in one embodiment, the feed composition or drinking water can be provided to the animal daily. In an alternative embodiment, the feed composition or drinking water can be administered to the animal during lactation and / or during pregnancy. The periods of administration of the above feed composition or drinking water are non-limiting examples, and it should be understood that any period of time or schedule of administration that is determined to be effective to improve animal performance or improve animal health or a combination thereof can be employed.

[0194] As described herein, one method embodiment is a method of raising an animal by administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus strain selected from the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof, wherein the Bacillus strain results in an effect selected from improving animal performance and / or improving animal health and a combination thereof.

[0195] In embodiments where the effect is to improve animal performance, the improvement in animal performance can be selected from reducing feed conversion rate (e.g., reducing the feed to gain ratio (F / G)), increasing average daily feed intake (ADFI), increasing average daily gain (ADG), improving the consistency of performance, improving or increasing the digestibility of the diet, improving or increasing the ratio of metabolizable energy to total energy, increasing nutrient absorption, and combinations thereof. The improvement can be relative to animals not fed the bacterial strain. In one embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof can increase the digestibility of the diet by producing enzymes that increase the digestibility of the nutrients consumed, wherein the enzymes are selected from α-galactosidase, protease, phytase, lipase, amylase, xylanase, cellulase, and combinations thereof. The enzyme can also be any other enzyme that degrades long-chain fatty acids, such as an enzyme that degrades stearic acid, palmitic acid, and / or oleic acid (but not limited to these fatty acids). This increase in the digestibility of the diet results in an improvement in animal performance, which is selected from reducing feed conversion rate (e.g., reducing the feed to gain ratio (F / G)), increasing average daily feed intake (ADFI), increasing average daily gain (ADG), improving the consistency of animal performance (e.g., reducing performance variation, such as reducing variation in F / G, ADFI, and ADG and increasing the consistency of F / G, ADFI, and ADG), improving the ratio of metabolizable energy to total energy, and combinations thereof.

[0196] In embodiments where the effect is to improve animal health, the improvement can be caused by a mechanism that includes, but is not limited to, the antimicrobial activity of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof. In various embodiments, the antimicrobial activity is against bacteria selected from Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Clostridium, Campylobacter, and combinations thereof. Thus, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof can improve the intestinal health of animals and reduce pathogens in the animals and the animals' environment. In yet another embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof can reduce the bioburden in animals or increase the immune response in animals to improve animal health. The improvements described herein can be relative to animals that have not been fed the bacterial strains.

[0197] In embodiments of the method, the method can improve animal health by improving the animal's environment through an effect selected from reducing the respiratory problems of the animal, improving the intestinal health of the animal, improving the consistency of animal performance, reducing diseases in the animal related to environmental toxicity, and reducing pathogens in the animal. In embodiments where the animal is a poultry species, the method can improve animal health through an effect selected from reducing the respiratory problems of the poultry species, reducing chest blisters in the poultry species, improving the consistency of poultry species performance, and reducing damage to the feet of the poultry species. These animal health improvement mechanisms are non-limiting examples.

[0198] In a further embodiment of the present invention, there is provided a composition comprising the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof. In one embodiment, there is provided a commercial package comprising an isolated Bacillus strain selected from the group consisting of the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0199] In another embodiment, there is provided a feed additive for animal feed, the feed additive comprising an isolated Bacillus strain selected from the group consisting of the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0200] In yet another embodiment, there is provided an additive for animal drinking water, the additive comprising an isolated Bacillus strain selected from the group consisting of the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0201] In yet another illustrative aspect of the present invention, there is provided an animal feed composition comprising an isolated Bacillus strain selected from the group consisting of the Bacillus strain BC1 (NRRL No. B-67744), the Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of the Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of the Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

[0202] In one embodiment, a feed additive for addition to an animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, using an automated micronutrient delivery system or, for example, by hand weighing and adding, to achieve any dosage of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof, for administration to an animal in the form of a complete feed composition. Mixing can also be carried out by any other suitable method known in the art for combining a direct-fed microbe with an animal feed blend to obtain a homogeneous mixture. In various embodiments, mixing can be carried out for any suitable period of time (e.g., about 1 - about 4 minutes).

[0203] In embodiments in which Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof are in the form of an additive for animal drinking water, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof can be in the form of, for example, a powder, liquid, gel, freeze-dried form, top dressing, or pellet and can be mixed with the drinking water using any suitable method known in the art to achieve any dosage of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof for administration to the animal in the animal's drinking water.

[0204] Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof may also be fed directly to the animal orally (i.e., by oral insertion) in the form of a powder, liquid, gel, freeze-dried form, surface dressing, or bolus.

[0205] In any of the composition embodiments described herein, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof may result in an effect selected from improving animal performance or improving animal health and combinations thereof. The commercial packages, feed additives, feed compositions, or additives for animal drinking water described herein may also inhibit pathogens selected from Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Clostridium, Campylobacter, and combinations thereof. These effects are non-limiting examples of the types of effects that Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof may result in.

[0206] In an illustrative aspect, the feed additive, the additive for animal drinking water, or the feed composition may be in a commercial package, such as in the form of a dietary nutritional composition (e.g., a probiotic composition). In another illustrative embodiment, the feed additive or the additive for animal drinking water or Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745) or a combination thereof in a commercial package may be in a concentrate (e.g., about 1x10 8 - about 5x10 9 CFU / g) or a super-concentrate (e.g., about 1x10 10 - about 5x1012 in the form of CFU / g). In another embodiment, the Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof, in a feed additive, a feed composition, an additive for animal drinking water, or a commercially packaged composition, may be in a dry form (e.g., powder), pellet form, liquid form, freeze-dried form, top-dressing form, or gel form, or any other suitable form.

[0207] In another illustrative embodiment, the commercially packaged product, feed additive, additive for animal drinking water, or feed composition may further comprise a carrier for the Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or a combination thereof. The carrier may be selected from bran, rice husk, salt, mineral oil, dextrin (e.g., maltodextrin), whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof. In another embodiment, the carrier may be any suitable carrier known in the art for microorganisms for direct feeding. The carrier is added exogenously to the bacterial strain (i.e., not naturally occurring, or not present in nature together with the bacterial strain). In another embodiment, the commercially packaged product, feed additive, additive for animal drinking water, or feed composition may further comprise an adhesive, such as clay, yeast cell wall components, aluminum silicate, dextran, or other known adhesives. The adhesive is added exogenously to the bacterial strain (i.e., not naturally occurring, or not present in nature together with the bacterial strain).

[0208] In still other embodiments, commercial packages, feed additives, additives for animal drinking water, or feed compositions comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), strains having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or strains having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or combinations thereof, are present in containers for commercial use. In various embodiments, the container can be, for example, a bag (such as a 20-pound bag, 50-pound bag, 2-ounce bag, 1-pound bag, or 1-kilogram bag), sachet, drum, bottle, or box. In an illustrative aspect, containers for commercial packages, feed additives, additives for animal drinking water, or feed compositions comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), strains having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) and / or strains having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), or combinations thereof, can comprise plastic, metal, foil, paper, fiber, or cardboard (such as a plastic drum, paper bag, foil bag, fiber drum, etc.). The commercial package, feed additive, additive for animal drinking water, or feed composition can further comprise instructions for use of one or more of the Bacillus strains.

[0209] In one aspect, the commercial packages, feed additives, additives for animal drinking water, or feed compositions described herein can further comprise exogenously added nutritional components (i.e., nutritional components that do not occur in nature with the bacterial strains), which are selected from vitamins, antibiotics, enzymes, water-soluble or water-insoluble monosaccharides, disaccharides, or polysaccharides, fats, phosphorus, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starch.

[0210] In one embodiment, the exogenously added nutritional component is an enzyme, and the enzyme is selected from galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0211] The following examples are for illustrative purposes only. The examples are non-limiting and are not intended to limit the invention in any way.

[0212] Example 1

[0213] Immunomodulation by Bacillus coagulans in vitro

[0214] IEC-6 rat intestinal epithelial cell line was challenged with 10 ng of LPS and treated with 10 3 、10 4 or 10 5 CFU / ml of Bacillus coagulans strain BCP2 or Bacillus coagulans strain BC1. The cell line was incubated at 37 °C in 5% CO2 for 2 hours. RNA was extracted from IEC-6 cells and qPCR was used to measure the expression of immune markers, including inflammatory cytokines, chemokines, and pathogen recognition receptors.

[0215] Figure 1A -C showed that compared with the LPS-challenged control, Bacillus coagulans strain BCP2 significantly reduced the expression of inflammatory immune markers CXCL2, IL-6, and TNF. Figure 2A -C showed that compared with the LPS-challenged control, Bacillus coagulans strain BC1 significantly reduced the expression of inflammatory markers IL-6, TLR2, and TNF. The anti-inflammatory effects of both strains were similar in the dose range from 10 3 to 10 5 CFU / ml.

[0216] Example 2

[0217] In vivo animal experiment

[0218] Pigs were exposed to an environment with a microbial bioburden higher than that of the negative control group, which was achieved by conforming to commercial-set management practices and diet. High-bioburden animals were fed Bacillus DFM without Bacillus coagulans strain BC1 described herein or Bacillus DFM containing Bacillus coagulans strain BC1 described herein for 24 weeks. RNA was extracted from whole blood samples and qPCR was used to measure the expression of immune markers.

[0219] Figure 3 It was shown that the high-bioburden control group had significantly higher expression of immune markers NOD2, SOCS3, TLR4, and TLR2 than the negative control group, indicating that microbial bioburden induced an immune response. The high-bioburden group fed with Bacillus DFM also had significantly higher expression of markers NOD2, SOCS3, and TLR4 than the negative control. The high-bioburden group fed with Bacillus DFM containing Bacillus coagulans strain BC1 was not different from the negative control, indicating that Bacillus coagulans BC1 alleviated the inflammation caused by high microbial bioburden.

[0220] Example 3

[0221] Evaluation of Bacillus coagulans in broiler chickens up to 6 weeks post-hatch

[0222] Broiler chickens were fed nutritionally adequate and nutritionally restricted diets until 6 weeks post-hatch to determine the efficacy of Bacillus coagulans in improving growth performance and feed efficiency. Experimental treatment groups included Treatment A: positive control nutritionally adequate and high-fat diet, without DFM; Treatment B: negative control nutritionally restricted diet, without DFM (CP, AA, and ME reduced by 5%); Treatment C: as B + 3.68×10 1 CFU / g Bacillus coagulans (0.5X TrtD); Treatment D: as B + 7.35×10 1 CFU / g Bacillus coagulans (as 1X, replicate / confirmation of 16-P092); Treatment E: as B + 1.10×10 2 CFU / g Bacillus coagulans (1.5X TrtD); and Treatment F: as B + 1.47×10 2 CFU / g Bacillus coagulans (2.0X TrtD).

[0223] Prior to the start of the experimental trial, the chickens received coccidia and vaccine 2. Using the six (6) pens of similar weight, the chickens were assigned to the experiment according to the SOP allocation procedure. At the start of the study, the pens were randomly assigned to the dietary treatments within the replicates. The pens were maintained in the treatment until the end of the experiment. When raising, providing water, and monitoring the health decline in the chickens, the daily management was carried out according to the standard operating procedure. The facility temperature was set at 95°F and decreased by 1°F daily starting from Day 0 until the temperature reached 85°F, where it was maintained. The water troughs were checked daily and cleaned and refilled if necessary. Measurements included total pen weight (at assignment (Day 0) and weekly thereafter); feed disappearance (at assignment (Day 0) and weekly thereafter); live weight gain, feed intake, and feed efficiency; morbidity and mortality; and high and low temperatures (with HOBO devices).

[0224] Using the Ross Nutrient Specification Manual (2014), the Treatment A diet was formulated to be adequate in EAA, aP, Ca, Na, Mn, and Cu. The Treatment B - F diets consisted of a basal restricted diet and a component premix. The diets were budgeted daily (Table 2a). The final diets were manufactured with corn, SBM, DDGS, protein sources, VTM, and crystalline AA in the DEO. The final experimental diets were sampled from the manufactured feed in each feeder. Each diet sample was stored for proximate and AA analysis.

[0225] Initial data analysis was performed for all metrics to determine distribution normality and outliers (from the total mean ± >3 standard deviations). The statistical (SAS) program utilized "pen" as the experimental unit, "treatment" as the fixed effect, and "replicate" as the random effect. Body weight, growth rate, feed intake, and feed efficiency were analyzed using the Mixed procedure in SAS. Mean separation was performed using pdiff for single-degree-of-freedom comparisons. Continuous (repeated) body weight, growth rate, feed intake, and feed efficiency were analyzed using the Mixed procedure in SAS. For each matrix, the repeated effect (period)–covariance matrix modeling (TOEP, CS, AR(1), UN) was run; the matrix with the closest value to zero (0) for the AICC was used for the final analysis. Mean separation of treatments (orthogonal single-degree-of-freedom comparisons) – main effects: the same as for cumulative data; interaction between treatment and period (P<0.05). Mortality was analyzed using NPar1Way in SAS as nonparametric data analysis.

[0226] Compared with chickens fed a full diet, chickens fed a restricted diet had a 5.0% reduction in live weight gain from day 1 to day 43 (P<0.05). Only chickens fed 7.35×10 1 CFU / g Bacillus coagulans were not different from chickens fed a full diet (P>0.1) and recovered 100% of the lost live weight gain; there was an additional numerical increase in live weight gain of more than 4.3% over the full diet. There were no other differences between chickens fed Bacillus coagulans and a restricted diet. Overall, there were no differences in feed efficiency; however, chickens fed a restricted diet had a numerically reduced gain / feed (5.1%; P>0.1). Chickens fed 3.68×10 1 or 7.35×10 1 CFU / g Bacillus coagulans recovered 71.0% of the lost feed efficiency. Supplementation with 7.35×10 1 of Bacillus coagulans improved the growth and feed efficiency of chickens fed a nutritionally restricted diet.

[0227] Table 1: Required power: 6.2, 6 treatments, 9 replicates, 43d duration

[0228]

[0229] Table 2a: Formulation of the basal negative control diet

[0230]

[0231]

[0232] Table 2b: Experimental ingredients to be added at the expense of lost corn

[0233]

[0234] Table 3: Effect of Bacillus coagulans fed to broiler chickens on a nutritionally restricted diet 1,2,3

[0235]

[0236]

[0237] abcd Within columns, means without a common superscript are different over the time period, P < 0.05.

[0238] 1 Data are means of nine replicates of eight birds / pen (male) fed the experimental diets continuously for 43 d starting at hatch.

[0239] 2 Treatments included: ad libitum nutrition [Trt.A, positive control]; restricted nutrition [Trt.B, negative control]; or restricted and containing Bacillus coagulans [Trts.C–F].[[]]

[0240] 3 Significance levels (P-values) were according to the main effect of diet treatment.

[0241] 4 Live weight gain = (pen weight at end - pen weight at start) / number of birds at end.

[0242] 5 Gain / feed = g live weight gain / kg feed intake.

[0243] 6 Feed intake = total feed disappearance per pen / number of birds at end.

[0244] 7 Mortality = exact count of instances.

[0245] Table 4: Replicate effect of Bacillus coagulans fed to broiler chickens on a nutritionally restricted diet 1,2,3

[0246]

[0247] Example 4

[0248] Evaluation of the efficacy of Bacillus coagulans DFM on the growth performance of broiler chickens

[0249] Experimental treatment groups included Treatment A: positive control ad libitum diet, no DFM; Treatment B: as A + 7.35×10 4CFU / g Bacillus subtilis (1 lb / ton Novela; 73,500 CFU / g); Treatment C: Negative control nutritionally restricted diet, no DFM (5% reduction in CP+EAA); Treatment D: As C + 7.35×10 1 CFU / g Bacillus coagulans (4 lb / ton MDG mixture 2; 73.5 CFU / g); Treatment E: As C + 7.35×10 2 CFU / g Bacillus coagulans (4 lb / ton MDG mixture 3; 735 CFU / g); and Treatment F: As C + 7.35×10 3 CFU / g Bacillus coagulans (4 lb / ton MDG mixture 4; 7,350 CFU / g).

[0250] Chickens were allocated to pens according to chicken weight. Pens were then randomly assigned to dietary treatments and the study was started immediately. Pens were maintained on the dietary treatments until the end of the experiment. Daily management followed standard operating procedures while the chickens were reared, given water, and monitored for health decline. Facility lighting was turned on for the duration of the experiment. Facility temperature was set at 95°F and decreased 1°F daily starting on Day 0 (when they arrived at the chicken house). Once the temperature was as low as 85°, the room was maintained at 85°. Farm employees checked the water troughs daily and, if needed, they cleaned and refreshed them by emptying the troughs and refilling with fresh water. Pens were lined with 60 lb of corrugated kraft paper for the entire experiment to induce a health challenge. Litter from a previous experiment (16-P082, control diet) was used for all tiered cages to attempt to induce a health response. Approximately 35 g of used litter was placed in each cage above the corrugated kraft paper. Paper and litter were changed on Day 14 at the time of weight and feed recording.

[0251] Measurements included total pen weight (Day 0, Day 14, and Day 20); feed disappearance (Day 14 and Day 20); feed / gain ratio (lb / lb and kcal / lb; Days 1-14, Days 14-20, and Days 1-20; feed / gain can be adjusted for mortality by the formula: total feed consumed / (pen live weight gain + mortality weight gain)); morbidity and mortality; and high and low temperatures (with HOBO devices).

[0252] Experimental test materials used in diet formulation included Novela (MDG) and Bacillus coagulans (MDG). Diets remained the same during the trial. Final diets were manufactured at DEO from a basal diet with the addition of the experimental mixtures. Four different concentrations of the product were manufactured to incorporate into the final diets. The premix weighed 0.25 lb, which could be included in its entirety in a 125 lb treatment batch. Before final manufacture for addition to the experimental diets, the MDG products were pre-mixed with an additional 5 lb of corn to ensure thorough mixing.

[0253] Litter from previous experiments was used in the tiered cages to attempt to induce an immune response. Approximately 30 g of used litter was placed in the cages above the corrugated kraft paper. For the additional Novela work, as a preliminary study, procedures for collecting excreta and measuring NH3 were conducted according to Hossain et al (2015) and Zhang et al (2013) (Trt. A & B). On day 12, the paper was removed from the excreta trays and replaced with clean corrugated kraft paper. On day 15, for treatments A and B, three samples of 300 g of excreta were collected from 12 replicates and placed in plastic GLAD containers with 2, 4, and 6 ounces of water added. The samples were then sealed with Parafilm and allowed to ferment for 24 hours. After 24 hours (day 16), the samples were manually shaken for 30 seconds before inserting the Gastec air pump tube to break any possible crust formation and to homogenize the samples. The Gastec air pump tube was then inserted into the container through a hole previously sealed with tape. The air pump tube was inserted into the container until the tip of the tube was approximately 2 cm above the material, where the sample was obtained. At 96 hours or day 19 of the trial, the staff conducted the NH3 reading according to the aforementioned procedure.

[0254] Initial data analysis was performed for all metrics to determine distribution normality and outliers (from the total mean ± > 3 standard deviations). The statistical (SAS) procedure used "pen" as the experimental unit, "treatment" as the fixed effect, and "replicate" as the random effect. For period and cumulative weight gain, feed intake, and feed efficiency: PROC MIXED. For period and cumulative mortality: PROC NPAR1WAY.

[0255] The chickens in the trial were fed a control diet with or without Novela, or a nutritionally restricted diet with one of three concentrations of Bacillus coagulans with or without. The chickens were healthy, with a relatively low mortality rate (only 6.6%). There were no problems with the diet or chicken handling during the trial.

[0256] From day 1 to day 14, there were no differences between the chickens fed the control diet with or without Novela. The chickens fed the restricted diet had a reduced BW and live weight gain of 13.9 and 15.1% (P < 0.01), respectively, and a reduced feed efficiency and MAFCR of 7.3 and 7.5% (P < 0.01), respectively. Inclusion of 73.50, 735.0, and 7350 CFU / g of Bacillus coagulans allowed for the recovery of 60.2, 20.0, and 36.7% of the lost BW, respectively, and 59.6, 19.3, and 36.8% of the lost live weight gain, respectively. Inclusion of 73.50, 735.0, and 7350 CFU / g of Bacillus coagulans allowed for the recovery of 47.5, 34.4, and 54.1% of the lost feed efficiency, respectively, and 55.6, 33.3, and 55.6% of the lost MAFCR, respectively.

[0257] From days 15 to 20, there were no differences between chickens fed a control diet with or without Novela. Chickens fed a restricted diet had reduced BW, live weight gain, total weight gain, and feed intake by 11.5, 7.8, 6.6, and 6.1% (P<0.01), respectively. Inclusion of 73.50 CFU / g Bacillus coagulans allowed recovery of 82.1, 140.9, 133.3, and 137.5% of the losses in BW, live weight gain, total weight gain, and feed intake, respectively. Compared to chickens fed a restricted nutrient diet, inclusion of 735.0 CFU / g Bacillus coagulans reduced live weight gain, total weight gain, and feed intake by 10.8, 11.8, and 6.2% (P<0.05), respectively. There were no differences between chickens fed a restricted diet and those receiving 7350 CFU / g Bacillus coagulans.

[0258] From days 1 to 20, there were no differences between chickens fed a control diet with or without Novela. Compared to chickens fed a control diet, chickens fed a restricted diet had a reduced (P<0.01) live weight gain of 12.0% (79 g). Inclusion of 73.50 CFU / g Bacillus coagulans recovered (P<0.05) 82.3% of the loss in live weight gain in chickens fed a restricted diet. Inclusion of 7350 CFU / g Bacillus coagulans numerically recovered (P>0.05) 22.8% of the loss in live weight gain in chickens fed a restricted diet. Compared to chickens fed a restricted diet, inclusion of 735.0 CFU / g Bacillus coagulans numerically reduced (P>0.05) live weight gain by 4.5%.

[0259] Inclusion of Novela in the control diet made no difference; however, feed efficiency numerically increased by 3% compared to the control diet, similar to previous studies. Inclusion of only 73.50 CFU / g Bacillus coagulans in a nutritionally restricted diet allowed recovery of the lost BW, weight gain, and feed efficiency to levels not different from the control diet. Compared to chickens fed a nutritionally restricted diet (without DFM), chickens fed 73.5 CFU / g Bacillus coagulans had increased weight gain. Bacillus coagulans microorganisms added to broiler diets seem to produce enzymes to increase protein and fat digestibility.

[0260] Table 5: Formulation of the basal negative control diet

[0261]

[0262]

[0263] Table 6: Experimental ingredients to be added at the expense of lost corn

[0264]

[0265] Table 7: Effects of DFM supplementation on the growth performance of broiler chickens 1

[0266]

[0267]

[0268] 1 Data are the mean of 8 replicates of 6 chickens / pen fed the experimental diet from d0 to d20. The experimental period started immediately after arrival at the DEO Poultry Unit. Data were analyzed using the Mixed procedure of SAS as a randomized complete design.

[0269] 2 Live weight gain was calculated as the pen weight at the end minus the pen weight at the start, divided by the number of chickens in the pen at the end of the experiment.

[0270] 3 Total weight gain was calculated as the pen weight at the end plus the weight of dead chickens, minus the pen weight at the start, divided by the number of chickens in the pen at the start of the experiment.

[0271] 4 Feed intake represented the total feed disappearance per pen divided by the number of chickens in the pen at the end of the experiment.

[0272] 5 Gain / feed was calculated as gain divided by kg feed intake.

[0273] Table 8: Effects of DFM supplementation on the body weight and mortality of broiler chickens 1

[0274]

[0275]

[0276] 1 Data are the mean of 8 replicates of 6 chickens / pen fed the experimental diet from d0 to d20. The experimental period started immediately after arrival at the DEO Poultry Unit. Data were analyzed using the Mixed procedure of SAS as a randomized complete design.

[0277] 2 End wt was calculated as the total pen weight divided by the number of chickens in the pen at the end.

[0278] 3 Mortality-adjusted feed conversion ratio (FCR) was calculated as feed intake during the experiment divided by total weight gain.

[0279] Table 9: Effects of Novela on ammonia volatilization

[0280]

[0281] *The sample was collected on 5 / 24 / 16, with a room temperature of 85.5°F and an atmospheric pressure of 1021 hPa.

Claims

1. A method of raising an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof, wherein the Bacillus strain results in an effect selected from the group consisting of improved animal performance and improved animal health and combinations thereof.

2. The method according to claim 1, wherein the improvement in animal health is a reduction in the bioburden in the animal.

3. The method according to claim 1, wherein the improvement in animal health is an increase in the immune response in the animal.

4. The method according to claim 1, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744).

5. The method according to claim 1, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B-67745).

6. The method according to any one of claims 1-5, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

7. A commercial package comprising an isolated Bacillus coagulans strain selected from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

8. A feed additive for animal feed, comprising an isolated Bacillus coagulans strain selected from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

9. An additive for animal drinking water, comprising an isolated Bacillus coagulans strain selected from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

10. An animal feed composition comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

11. The commercial package, feed additive, feed composition or additive for animal drinking water according to any one of claims 7-10, wherein the Bacillus strain results in an effect selected from improving animal performance, improving animal health, and combinations thereof.

12. The commercial package, feed additive, additive for animal drinking water or feed composition according to any one of claims 7-10, further comprising a carrier for the Bacillus strain.

13. The commercial package, feed additive, additive for animal drinking water or feed composition according to claim 12, wherein the carrier is selected from bran, rice husk, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

14. The commercial package, feed additive, additive for animal drinking water or feed composition according to any one of claims 7-10, further comprising a binder.

15. The commercial package, feed additive, additive for animal drinking water or feed composition according to claim 14, wherein the binder is selected from clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

16. The commercial package, feed additive, additive for animal drinking water or feed composition according to any one of claims 7-10, which is in the form of a dietary nutritional composition.

17. A method of raising animals, the method comprising the step of administering to the animals a feed composition or drinking water comprising an effective amount of an additive, the additive comprising an isolated Bacillus coagulans strain selected from: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain BCP2 (NRRL No. B-67745), a strain having all the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all the identifying characteristics of Bacillus strain BCP2 (NRRL No. B-67745), and combinations thereof.

18. The method according to claim 17, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744).

19. The method according to claim 17, wherein the strain administered is Bacillus strain BCP2 (NRRL No. B-67745).

20. The method according to any one of claims 17-19, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan TM , Pulmotil TM and vancomycin.

21. The method according to any one of claims 17 - 19, wherein the animal is selected from chickens, pigs, horses, ponies, cattle, turkeys, goats, sheep, quails, pheasants, ostriches, ducks, fish, crustaceans, and combinations thereof.

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