Microbial strain composition

Abstract

The present invention relates to microorganisms for improving human and animal health. More particularly, the present invention relates to isolated Bacillus strains, as well as strains having all identification characteristics of these strains, for use including the above-mentioned use, in particular for improving gastrointestinal health in animals or humans.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application Serial No. 63 / 413,419, filed on October 5, 2022, and U.S. Provisional Application Serial No. 63 / 472,076, filed on June 9, 2023, the entire disclosures of which are incorporated herein by reference.

[0003] public domain

[0004] The present invention relates to microorganisms for improving human and animal health. More particularly, the present invention relates to isolated Bacillus strains and strains having all the identifying characteristics of these strains for use in the uses described above, particularly for improving gastrointestinal health in animals or humans.

[0005] Background and Overview of the Invention

[0006] The present invention relates to probiotic compositions and methods for improving the health of animals or humans, particularly the gastrointestinal health. The present invention also relates to such probiotic strains and methods for evaluating their contribution to the beneficial functions of the animal or human ecosystem; such as digestive enzyme production, antimicrobial activity (E. coli, Salmonella, Campylobacter, Clostridium perfringens, Candida albicans, Candida auris, Enterococcus cecorum, Fusarium graminearium and Aspergillus flavus), immunomodulation, antioxidant capacity and quorum quenching capacity. Although a "healthy gut microbiome" has not yet been precisely defined, it has been well established over the years that the diversity and balance of microorganisms are key components. Beneficial bacteria, including the strains described in this application, are an important part of the gastrointestinal environment because they provide animals and humans with bacteria that help establish (or reconstitute) a normal bacterial profile, they boost the immune system, they help target disease (e.g., those caused by the aforementioned Gram-negative bacteria, Gram-positive bacteria, yeasts, and molds) through their antimicrobial metabolites and quorum sensing quenching capabilities, and they help reduce inflammation through the production of antioxidant enzymes.

[0007] The gastrointestinal tract of animals or humans is constantly challenged by a large number of bacteria and viruses that exist in the environment. The gastrointestinal tract has a complex system consisting of physical, chemical, and immunological defense lines to fight back against these potential pathogens. Beneficial bacteria are an important part of this system. Pathogens, stress, metabolic disorders, use of antimicrobials, and other causes can disturb the balance of the intestinal bacteria, which can impair digestion and make the animal or human more susceptible to disease.

[0008] Probiotics (i.e., also referred to as direct fed microbial in this application) are products that contain live (viable) microorganisms (e.g., bacteria). Over time, many probiotic products that were previously thought to be useful in improving health have lost overall efficacy. Therefore, there is a need for additional microbial strains that will improve animal and human health. The applicants have developed probiotic compositions comprising Bacillus subtilis and Bacillus coagulans strains to improve animal health and human health.

[0009] Methods and compositions for improving animal and human health are provided. In various embodiments, the animal can be selected from the group consisting of 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 can be broiler chickens. In embodiments where the animal is a swine species, the swine species can be selected from the group consisting of growing-finishing swine, nursery swine, sows, and breeding swine. In embodiments where the animal is a companion animal, the companion animal can be a dog or a cat or any other companion animal. In another embodiment, the methods and compositions described herein are used to treat humans.

[0010] In one embodiment, a method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof, wherein the Bacillus strain improves the health of the animal.

[0011] In another embodiment, a method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0012] In still another embodiment, a method of improving human health is provided. The method includes the step of administering to the human a probiotic composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0013] 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 or human food, an additive for animal or human drinking water, or an animal feed composition (e.g., a complete feed) or human food composition each comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0014] The following clauses, and combinations thereof, provide various additional illustrative aspects of the application described herein. The various embodiments described in any other section of this patent application, including the section entitled "Detailed Description of Illustrative Embodiments" and the Examples section, can be applicable to any of the following embodiments of the application described in the following numbered clauses.

[0015] 1. A method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof, wherein the Bacillus strain improves the health of the animal.

[0016] 2. The method of clause 1, wherein the animal is selected from the group consisting of a poultry species, a swine species, a bovine species, an ovine species, an equine species, and a companion animal.

[0017] 3. The method of clause 2, wherein the poultry species is broiler chickens.

[0018] 4. The method of any one of clauses 1-2, wherein the animal is a companion animal, and the companion animal is a dog or a cat.

[0019] 5. The method of clause 2, wherein the swine species is selected from the group consisting of grow-finish pigs, nursery pigs, sows, and breeding stock pigs.

[0020] 6. The method of any one of clauses 1-5, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0021] 7. The method of any one of clauses 1-6, wherein at least one of the Bacillus strains has antimicrobial activity.

[0022] 8. The method of clause 7, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0023] 9. The method of any one of clauses 1-8, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0024] 10. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0025] 11. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0026] 12. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0027] 13. The method of any of clauses 1-10, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744).

[0028] 14. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68053).

[0029] 15. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68054).

[0030] 16. The method of any of clauses 1-15, wherein at least two of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are administered in a single composition.

[0031] 17. The method of any one of clauses 1-15, wherein at least two of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are administered in separate compositions.

[0032] 18. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 5.0 x 10 12 CFU / gram of feed composition.

[0033] 19. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 1.0 x 10 7 CFU / gram of feed composition.

[0034] 20. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of greater than about 7.0 x 10 4 CFU / gram of feed composition.

[0035] 21. The method of any one of clauses 1-20, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from the group consisting of Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM , Pulmotil TM , chloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0036] 22. The method of any one of clauses 1-21, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0037] 23. The method of any one of clauses 1-22, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0038] 24. The method of any one of clauses 1-22, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0039] 25. The method of any one of clauses 1-22, wherein the feed composition is administered to the animal daily.

[0040] 26. The method of any one of clauses 1-3 or 5-25, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0041] 27. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0042] 28. A feed additive for animal feed or human food comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0043] 29. An additive for animal or human drinking water comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0044] 30. An animal feed composition or a human food composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0045] 31. The commercial package, feed additive for animal feed or human food, animal feed composition or human food composition, or additive for animal or human drinking water according to any one of Clauses 27-30, wherein the Bacillus strain improves the health of an animal or human.

[0046] 32. The commercial package, feed additive for animal feed or human food, animal feed composition or human food composition, or additive for animal or human drinking water according to any one of Clauses 27-31, wherein the Bacillus strain inhibits a pathogen selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, Clostridium, Candida, Mucor, Penicillium, and Aspergillus.

[0047] 33. The feed additive for animal feed or human food or additive for animal or human drinking water in the form of a concentrate according to Clause 28 or 29.

[0048] 34. The feed additive for animal feed or human food or additive for animal or human drinking water in the form of a super concentrate according to Clause 28 or 29.

[0049] 35. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in dry form according to any one of Clauses 28-34.

[0050] 36. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in pellet form according to any one of Clauses 28-35.

[0051] 37. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in powder form according to any one of Clauses 28-35.

[0052] 38. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to any one of Clauses 27-34, wherein the strain is in a form selected from the group consisting of a powder, a liquid, a gel, a lyophilized form, a poultice, and a pellet.

[0053] 39. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to any one of Clauses 27-38, further comprising a carrier for the Bacillus strain.

[0054] 40. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to Clause 39, wherein the carrier is selected from the group consisting of bran, rice hulls, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

[0055] 41. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in a bag according to any one of Clauses 27-40.

[0056] 42. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to Clause 41, wherein the bag is a plastic bag.

[0057] 43. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-42 further comprising instructions for using one or more of the Bacillus strain.

[0058] 44. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a 20 pound bag of any one of clauses 27-43.

[0059] 45. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a 50 pound bag of any one of clauses 27-43.

[0060] 46. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a container for commercial use of any one of clauses 27-45.

[0061] 47. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 46, wherein the container comprises plastic.

[0062] 48. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 46, wherein the container comprises paper.

[0063] 49. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-48 further comprising a binder.

[0064] 50. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 49, wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

[0065] 51. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-50 in the form of a dietary nutritional composition.

[0066] 52. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-51 further comprising an exogenously added nutritional ingredient selected from the group consisting of vitamins, antibiotics, enzymes, water soluble or water insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorous, bicarbonate of soda, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starch.

[0067] 53. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 52, wherein the exogenously added ingredient is an enzyme.

[0068] 54. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 53, wherein the enzyme is selected from the group consisting of galactosidases, proteases, lipases, amylases, hemicellulases, arabinoxylanases, xylanases, cellulases, NSP enzymes, phytases, methionine reductases, methionine synthases, uricases, prolyl endopeptidases, and combinations thereof.

[0069] 55. A method of feeding 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 strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0070] 56. The method of clause 55, wherein the animal is selected from the group consisting of poultry species, swine species, bovine species, ovine species, equine species, and companion animals.

[0071] 57. The method of clause 56, wherein the poultry species is broiler chickens.

[0072] 58. The method of clause 56, wherein the swine species is selected from the group consisting of grow-finish pigs, nursery pigs, sows, and breeding stock.

[0073] 59. The method of clause 56, wherein the animal is a companion animal, and the companion animal is a dog or a cat.

[0074] 60. The method of any of clauses 55-59, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0075] 61. The method of any of clauses 55-60, wherein at least one of the Bacillus strains has antimicrobial activity.

[0076] 62. The method of clause 61, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0077] 63. The method of any of clauses 55-62, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0078] 64. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0079] 65. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0080] 66. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0081] 67. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744).

[0082] 68. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68053).

[0083] 69. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68054).

[0084] 70. The method of any of clauses 55-63, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in a single composition.

[0085] 71. The method of any of clauses 55-63, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in separate compositions.

[0086] 72. The method of any of clauses 55-71, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 5.0 x 10 12 CFU / gram of feed composition.

[0087] 73. The method of any of clauses 55-71, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 1.0 x 10 7 CFU / gram of feed composition.

[0088] 74. The method of any of clauses 55-73, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from the group consisting of Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM , Pulmotil TMchloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0089] 75. The method of any one of clauses 55-74, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0090] 76. The method of any one of clauses 55-58 or 60-75, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0091] 77. The method of any one of clauses 55-58 or 60-75, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0092] 78. The method of any one of clauses 55-75, wherein the feed composition is administered to the animal daily.

[0093] 79. The method of any one of clauses 55-58 or 60-75, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0094] 80. A method of improving human health, the method comprising the step of administering to the human a probiotic composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0095] 81. The method of clause 80, wherein the improvement in health is an improvement in gastrointestinal health.

[0096] 82. The method of any one of clauses 80-81, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0097] 83. The method of any one of clauses 80-82, wherein at least one of the Bacillus strains has antimicrobial activity.

[0098] 84. The method of clause 83, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0099] 85. The method of any one of clauses 80-84, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0100] 86. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0101] 87. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0102] 88. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0103] 89. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744).

[0104] 90. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68053).

[0105] 91. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68054).

[0106] 92. The method of any one of clauses 80-85, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in a single composition.

[0107] 93. The method of any one of clauses 80-85, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in separate compositions.

[0108] 94. The method of any one of clauses 80-93, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of probiotic composition - about 5.0 x 10 12 CFU / gram of probiotic composition.

[0109] 95. The method of any one of clauses 80-93, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of probiotic composition - about 1.0 x 10 7 CFU / gram of probiotic composition.

[0110] 96. The method of any one of clauses 80-95, further comprising the step of administering an antibiotic to the human, wherein the antibiotic is selected from the group consisting of Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM , Pulmotil TMchloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0111] 97. The method of any one of clauses 80-96, further comprising the step of administering to the human an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0112] 98. The method of any one of clauses 1-26 or 55-97, wherein the Bacillus strain does not cause hemolysis.

[0113] 99. The method of any one of clauses 1-26 or 55-98, wherein the Bacillus strain does not cause cytotoxicity.

[0114] 100. The method of any one of clauses 1-26 or 55-99, wherein the Bacillus strain does not produce a toxin selected from the group consisting of hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), emetic toxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK).

[0115] 101. The method of any one of clauses 1-26 or 55-100, wherein the Bacillus strain produces an anti-inflammatory biomarker selected from the group consisting of SOCS1, TOLLIP, IL-10, and CXCL12.

[0116] 102. The method of any one of clauses 1-26 or 55-101, wherein the Bacillus strain has antioxidant activity.

[0117] 103. The method of any one of clauses 1-26 or 55-102, wherein the Bacillus strain has quorum quenching activity.

[0118] 104. The method of any one of clauses 1-26 or 55-103, wherein the composition or additive is a dietary nutritional composition. SUMMARY

[0120] Figure 1Gel electrophoresis results from PCR assays with Bacillus strain BC1 (NRRL No. B-67744). Lanes are the same for each image, lane 1 is NTC, lane 2 is Bc control, lane 3 is Bt control, and lane 4 is NRRL 67744. A) Pre-mix 1 containing primer sequences for hblA, hblC, hblD, and emetic strain specific sequences. B) Pre-mix 2 containing primer sequences for nheA, nheB, and nheC. C) Pre-mix 3 containing primer sequences for entFM, bceT, and cytK.

[0121] Figure 2 Photograph showing gels displaying RAPD PCR profiles (primers 1-6) for Bacillus strain BC1 (NRRL No. B-67744). The far left and far right lanes include markers, and each set of two consecutive lanes between the markers, from left to right, correspond to primers 1-6.

[0122] Figure 3 shows relative quantification of inflammatory cytokine and pathogen receptor expression in IEC-6 cell lines challenged with 10 ng LPS and treated with B. coagulans NRRL 67744 at 10 3 , 10 4 , and 10 5 CFU / ml. Different letters indicate significant differences (p < 0.05).

[0123] Figure 4 Gel electrophoresis results from PCR assays. Lanes are the same for each image, lane 1 is NTC, lane 2 is Bc control, lane 3 is Bt control, and lane 4 is NRRL 68053. A) Pre-mix 1 containing primer sequences for hblA, hblC, hblD, and emetic strain specific sequences. B) Pre-mix 2 containing primer sequences for nheA, nheB, and nheC. C) Pre-mix 3 containing primer sequences for entFM, bceT, and cytK.

[0124] Figure 5 RAPD-PCR gel electrophoresis results for NRRL No. B-68053. Lanes 1 and 8 are a molecular weight ladder with increments of 200 kb. Strain DNA was run in duplicate for each of the 6 primers in the PCR reaction. Lanes 2 and 3 are primer 1. Lanes 4 and 5 are primer 2. Lanes 6 and 7 are primer 3. Lanes 9 and 10 are primer 4. Lanes 11 and 12 are primer 5. Lanes 13 and 14 are primer 6.

[0125] Figure 6RAPD-PCR gel electrophoresis results showing NRRL number B-68054. Lanes 1 and 8 are a molecular weight ladder with 200 kb increments. Strain DNA was run in duplicate for each of the six primers in the PCR reaction. Lanes 2 and 3 are primer 1. Lanes 4 and 5 are primer 2. Lanes 6 and 7 are primer 3. Lanes 9 and 10 are primer 4. Lanes 11 and 12 are primer 5. Lanes 13 and 14 are primer 6.

[0126] Figure 7 Gel electrophoresis results from PCR assays are shown. The lanes are the same for each image, lane 1 is NTC, lane 2 is Bc control, lane 3 is Bt control, and lane 4 is NRRL 68054. A) Primer mix 1 containing hblA, hblC, hblD, and emetic strain specific sequences. B) Primer mix 2 containing nheA, nheB, and nheC. C) Primer mix 3 containing entFM, bceT, and cytK.

[0127] Detailed description of illustrative embodiments

[0128] Methods and compositions for improving the health of animals and humans are provided. In various embodiments, the animal can be selected from the group consisting of a poultry species, a swine species, a bovine species, an ovine species, an equine species, and a companion animal. In embodiments where the animal is a poultry species, the poultry species can be a broiler chicken. In embodiments where the animal is a swine species, the swine species can be selected from the group consisting of a grow-finish pig, a nursery pig, a sow, and a breeding stock pig. In embodiments where the animal is a companion animal, the companion animal can be a dog or a cat or any other companion animal. In another embodiment, the methods and compositions described herein are used to treat a human.

[0129] In one embodiment, a method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL number B-67744), Bacillus strain (NRRL number B-68053), Bacillus strain (NRRL number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL number B-68054), and combinations thereof, wherein the Bacillus strain improves the health of the animal.

[0130] In another embodiment, a method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0131] In still another embodiment, a method of improving human health is provided. The method includes the step of administering to the human a probiotic composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0132] 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 or human food, an additive for animal or human drinking water, or an animal feed composition (e.g., a complete feed) or human food composition each comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0133] The following clauses, and combinations thereof, provide various additional illustrative aspects of the application described herein. The various embodiments described in the section entitled "Detailed Description of Illustrative Embodiments" can be applicable to any of the following embodiments of the application described in the following numbered clauses.

[0134] 1. A method of feeding 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 comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof, wherein the Bacillus strain improves the health of the animal.

[0135] 2. The method of clause 1, wherein the animal is selected from the group consisting of a poultry species, a swine species, a bovine species, an ovine species, an equine species, and a companion animal.

[0136] 3. The method of clause 2, wherein the poultry species is broiler chickens.

[0137] 4. The method of any one of clauses 1-2, wherein the animal is a companion animal, and the companion animal is a dog or a cat.

[0138] 5. The method of clause 2, wherein the swine species is selected from the group consisting of growing-finishing pigs, nursery pigs, sows, and breeding stock pigs.

[0139] 6. The method of any one of clauses 1-5, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0140] 7. The method of any one of clauses 1-6, wherein at least one of the Bacillus strains has antimicrobial activity.

[0141] 8. The method of clause 7, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0142] 9. The method of any one of clauses 1-8, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0143] 10. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0144] 11. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0145] 12. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0146] 13. The method of any of clauses 1-10, wherein the strain administered is Bacillus strain BC1 (NRRL No. B-67744).

[0147] 14. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68053).

[0148] 15. The method of any of clauses 1-9, wherein the strain administered is Bacillus strain (NRRL No. B-68054).

[0149] 16. The method of any of clauses 1-15, wherein at least two of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are administered in a single composition.

[0150] 17. The method of any one of clauses 1-15, wherein at least two of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are administered in separate compositions.

[0151] 18. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 5.0 x 10 12 CFU / gram of feed composition.

[0152] 19. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of about 1.0 x 10 3 CFU / gram of feed composition - about 1.0 x 10 7 CFU / gram of feed composition.

[0153] 20. The method of any one of clauses 1-17, wherein the Bacillus strain is administered in a dose of greater than about 7.0 x 10 4 CFU / gram of feed composition.

[0154] 21. The method of any one of clauses 1-20, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from the group consisting of Denagard® TM , BMD® TM , Carbadox® TM , Stafac® TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan® TM , Pulmotil® TM , chloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0155] 22. The method of any one of clauses 1-21, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0156] 23. The method of any one of clauses 1-22, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0157] 24. The method of any one of clauses 1-22, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0158] 25. The method of any one of clauses 1-22, wherein the feed composition is administered to the animal daily.

[0159] 26. The method of any one of clauses 1-3 or 5-25, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0160] 27. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0161] 28. A feed additive for animal feed or human food comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0162] 29. An additive for animal or human drinking water comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0163] 30. An animal feed composition or a human food composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0164] 31. The commercial package, feed additive for animal feed or human food, animal feed composition or human food composition, or additive for animal or human drinking water according to any one of Clauses 27-30, wherein the Bacillus strain improves the health of an animal or human.

[0165] 32. The commercial package, feed additive for animal feed or human food, animal feed composition or human food composition, or additive for animal or human drinking water according to any one of Clauses 27-31, wherein the Bacillus strain inhibits a pathogen selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, Clostridium, Candida, Mucor, Penicillium, and Aspergillus.

[0166] 33. The feed additive for animal feed or human food or additive for animal or human drinking water in the form of a concentrate according to Clause 28 or 29.

[0167] 34. The feed additive for animal feed or human food or additive for animal or human drinking water in the form of a super concentrate according to Clause 28 or 29.

[0168] 35. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in dry form according to any one of Clauses 28-34.

[0169] 36. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in pellet form according to any one of Clauses 28-35.

[0170] 37. The feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in powder form according to any one of Clauses 28-35.

[0171] 38. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to any one of Clauses 27-34, wherein the strain is in a form selected from the group consisting of a powder, a liquid, a gel, a lyophilized form, a poultice, and a pellet form.

[0172] 39. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to any one of Clauses 27-38, further comprising a carrier for the Bacillus strain.

[0173] 40. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to Clause 39, wherein the carrier is selected from the group consisting of bran, rice hulls, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

[0174] 41. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition in a bag according to any one of Clauses 27-40.

[0175] 42. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to Clause 41, wherein the bag is a plastic bag.

[0176] 43. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-42 further comprising instructions for using one or more of the Bacillus strains.

[0177] 44. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a 20 pound bag of any one of clauses 27-43.

[0178] 45. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a 50 pound bag of any one of clauses 27-43.

[0179] 46. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition in a container for commercial use of any one of clauses 27-45.

[0180] 47. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 46, wherein the container comprises plastic.

[0181] 48. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 46, wherein the container comprises paper.

[0182] 49. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-48 further comprising a binder.

[0183] 50. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 49, wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

[0184] 51. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-50 in the form of a dietary nutritional composition.

[0185] 52. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of any one of clauses 27-51 further comprising an exogenously added nutritional ingredient selected from the group consisting of vitamins, antibiotics, enzymes, water soluble or water insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorous, bicarbonate of soda, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starch.

[0186] 53. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 52, wherein the exogenously added ingredient is an enzyme.

[0187] 54. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of clause 53, wherein the enzyme is selected from the group consisting of galactosidases, proteases, lipases, amylases, hemicellulases, arabinoxylanases, xylanases, cellulases, NSP enzymes, phytases, methionine reductases, methionine synthases, uricases, prolyl endopeptidases, and combinations thereof.

[0188] 55. A method of feeding 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 strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

[0189] 56. The method of clause 55, wherein the animal is selected from the group consisting of poultry species, swine species, bovine species, ovine species, equine species, and companion animals.

[0190] 57. The method of clause 56, wherein the poultry species is broiler chickens.

[0191] 58. The method of clause 56, wherein the swine species is selected from the group consisting of grow-finish pigs, nursery pigs, sows, and breeding stock.

[0192] 59. The method of clause 56, wherein the animal is a companion animal, and the companion animal is a dog or a cat.

[0193] 60. The method of any of clauses 55-59, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0194] 61. The method of any of clauses 55-60, wherein at least one of the Bacillus strains has antimicrobial activity.

[0195] 62. The method of clause 61, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0196] 63. The method of any of clauses 55-62, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0197] 64. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0198] 65. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0199] 66. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0200] 67. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744).

[0201] 68. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68053).

[0202] 69. The method of any of clauses 55-63, wherein the administered strain is Bacillus strain (NRRL No. B-68054).

[0203] 70. The method of any of clauses 55-63, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in a single composition.

[0204] 71. The method of any of clauses 55-63, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in separate compositions.

[0205] 72. The method of any of clauses 55-71, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of feed composition to about 5.0 x 10 12 CFU / gram of feed composition.

[0206] 73. The method of any of clauses 55-71, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of feed composition to about 1.0 x 10 7 CFU / gram of feed composition.

[0207] 74. The method of any of clauses 55-73, further comprising the step of administering an antibiotic to the animal, wherein the antibiotic is selected from the group consisting of Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM , Pulmotil TMchloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0208] 75. The method of any one of clauses 55-74, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0209] 76. The method of any one of clauses 55-58 or 60-75, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0210] 77. The method of any one of clauses 55-58 or 60-75, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0211] 78. The method of any one of clauses 55-75, wherein the feed composition is administered to the animal daily.

[0212] 79. The method of any one of clauses 55-58 or 60-75, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0213] 80. A method of improving human health, the method comprising the step of administering to the human a probiotic composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), and combinations thereof.

[0214] 81. The method of clause 80, wherein the improvement in health is an improvement in gastrointestinal health.

[0215] 82. The method of any one of clauses 80-81, wherein the Bacillus strain produces an enzyme selected from the group consisting of alpha-galactosidase, protease, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0216] 83. The method of any one of clauses 80-82, wherein at least one of the Bacillus strains has antimicrobial activity.

[0217] 84. The method of clause 83, wherein the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

[0218] 85. The method of any one of clauses 80-84, further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another different Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0219] 86. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744), or a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), or combinations thereof.

[0220] 87. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68053), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), or combinations thereof.

[0221] 88. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68054), or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0222] 89. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain BC1 (NRRL No. B-67744).

[0223] 90. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68053).

[0224] 91. The method of any one of clauses 80-85, wherein the administered strain is Bacillus strain (NRRL No. B-68054).

[0225] 92. The method of any one of clauses 80-85, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in a single composition.

[0226] 93. The method of any one of clauses 80-85, wherein at least two of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof are administered in combination in separate compositions.

[0227] 94. The method of any one of clauses 80-93, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of probiotic composition - about 5.0 x 10 12 CFU / gram of probiotic composition.

[0228] 95. The method of any one of clauses 80-93, wherein the Bacillus strain is administered at a dosage of about 1.0 x 10 3 CFU / gram of probiotic composition - about 1.0 x 10 7 CFU / gram of probiotic composition.

[0229] 96. The method of any one of clauses 80-95, further comprising the step of administering an antibiotic to the human, wherein the antibiotic is selected from the group consisting of Denagard TM , BMD TM , Carbadox TM , Stafac TM , erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM , Pulmotil TMchloramphenicol, clindamycin, ciprofloxacin, gentamicin, kanamycin, linezolid, streptomycin, tetracycline, tigecycline, and vancomycin.

[0230] 97. The method of any one of clauses 80-96, further comprising the step of administering to the human an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof.

[0231] 98. The method of any one of clauses 1-26 or 55-97, wherein the Bacillus strain does not cause hemolysis.

[0232] 99. The method of any one of clauses 1-26 or 55-98, wherein the Bacillus strain does not cause cytotoxicity.

[0233] 100. The method of any one of clauses 1-26 or 55-99, wherein the Bacillus strain does not produce a toxin selected from the group consisting of hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), emetic toxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK).

[0234] 101. The method of any one of clauses 1-26 or 55-100, wherein the Bacillus strain produces an anti-inflammatory biomarker selected from the group consisting of SOCS1, TOLLIP, IL-10, and CXCL12.

[0235] 102. The method of any one of clauses 1-26 or 55-101, wherein the Bacillus strain has antioxidant activity.

[0236] 103. The method of any one of clauses 1-26 or 55-102, wherein the Bacillus strain has quorum quenching activity.

[0237] 104. The method of any one of clauses 1-26 or 55-103, wherein the composition or additive is a dietary nutritional composition.

[0238] In various embodiments, the animal to which the feed additive, feed composition, or potable water as described herein is administered can be selected from the group consisting of poultry species, swine species, bovine species, ovine species, equine species, and companion animals. In embodiments in which the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In embodiments in which the animal is a swine species, the swine species can be selected from the group consisting of growing-finishing swine, nursery swine, sows, and breeding stock swine. In various exemplary embodiments, the animal can be selected from the group consisting of chickens (e.g., broilers or layers), pigs, horses, ponies, cows, turkeys, goats, sheep, quail, pheasants, ostriches, ducks, fish (e.g., tilapia, catfish, flounder, or salmon), crustaceans (e.g., shrimp or crabs), and combinations thereof.

[0239] In another embodiment, the additive for food, food composition, consumable liquid, or potable water comprising the probiotic composition described herein is administered to a human. As used herein, "administered," "administering," "administer," "administration," and the like mean administered by another or self-consumption by a human of the probiotic composition described herein. As used herein with respect to consumption by a human, "consumable liquid" means any consumable liquid, including water, sports or other nutritional beverages, soft drinks, fruit juices, tea, coffee, milk, lactic acid bacteria beverages, and the like. In various embodiments described herein, the probiotic composition can also be referred to as a "direct fed microbial composition," and both types of compositions can be dietary nutritional compositions. In one embodiment, the commercial package described herein can contain a probiotic composition, a direct fed microbial composition, or a dietary nutritional composition comprising a Bacillus strain described herein.

[0240] In one embodiment of the present application, an effective amount of a Bacillus strain can be administered to improve the health of an animal or a human. By "effective amount" is meant the amount of a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof) that is capable of improving the health of an animal or a human by any mechanism, including those described herein.

[0241] In embodiments described herein in which the composition of the application comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, is administered to an animal or human, the composition is preferably administered orally to the animal in a feed composition or in drinking water, or to a human in food or a consumable liquid or drinking water, but can be administered using any other effective method known to those of skill in the art. In an illustrative embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, is provided in the form of an additive for addition to a feed composition for an animal or to drinking water for an animal or to food, to a consumable liquid, or to drinking water for a human.

[0242] In one illustrative embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, are provided in the form of a feed additive for addition to a feed composition for an animal. The feed composition can contain Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, in admixture with an animal feed admixture, including any art-recognized animal feed admixture or any animal feed admixture described herein. As used herein, "feed composition" or "animal feed composition" means a feed composition comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, in admixture with an animal feed admixture, and optionally any other ingredients that can be used in a feed composition, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0243] Any animal feed admixture, including those known in the art and those described herein, such as rapeseed meal, cottonseed meal, soybean meal, corn meal, barley, wheat, silage, and semi-dry silage, can be used according to the methods and compositions described in the present patent application. In various embodiments, the animal feed admixture can be supplemented with Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, although other ingredients can optionally be added to the animal feed admixture.

[0244] In another illustrative aspect, any pharmaceutical ingredient known in the art, such as an antibiotic, can be added to the animal feed admixture or to an additive for animal drinking water. In various embodiments, the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, Denagard TM (i.e., tylosin), BMD TM (i.e., methylene salicylate bacitracin), Carbadox TM (i.e., carbadox), Stafac TM (i.e., virginiamycin), erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampin, Tylan TM (i.e., tylosin), Pulmotil TM (i.e., tilmicosin), vancomycin, avilamycin (Kavault TM ), gentamicin, neomycin, ciprofloxacin, kanamycin, linezolid, streptomycin, tigecycline, and combinations thereof. In another embodiment, the animal feed admixture, feed composition, feed additive, or additive for animal drinking water can be free of antibiotics.

[0245] In embodiments in which the Bacillus strain is used as an additive for food or for consumable liquids or for drinking water or by direct consumption, the Bacillus strain can be added to food and consumable liquids such as yogurt, beverages (such as water, sports or other nutritional beverages), beverage powders, soft drinks, fruit juices, tea, coffee, milk, lactic acid bacteria beverages, cheese, ice cream, desserts and any dessert products (such as fruit or cream fillings, frosting or cheesecake fillings), bread products (such as sandwich bread), cookies, cake mixes, rolls, muffins, and any other food or consumable liquid for human consumption suitable for the addition of the Bacillus strain described herein in the form of a probiotic composition. In another embodiment, the Bacillus strain described herein can be directly consumed by humans, for example, in the form of tablets, capsules, gels, gelatin capsules, powders, granules, liquids, sprays, or any other suitable form for direct consumption by humans. Any other ingredients useful in food compositions or consumable liquids can be optionally added, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0246] In various illustrative embodiments, optional ingredients of the animal feed admixture or food composition include sugars and complex carbohydrates, such as water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional ingredients include dried distillers solubles, fat (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.

[0247] Optional amino acid ingredients that can be added to the animal feed admixture or food composition are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl ester hydrochloride, alanine, aspartic acid, glutamic acid sodium, glycine, proline, serine, cysteine ethyl ester hydrochloride, and analogs and salts thereof. Vitamins that can be optionally added are thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, niacin, nicotinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. In another embodiment, vitamins can be added in the form of a vitamin premix. In still another embodiment, protein ingredients can be added to the animal feed admixture or food composition and include protein obtained from meat meal, bone meal or fish meal, liquid or powdered egg, fish solubles, crude protein, and the like.

[0248] In another illustrative embodiment, one or more enzymes can be added to an animal feed admixture or a human food or consumable liquid. In various embodiments, the enzymes that can be added include galactosidases, phytases, proteases, lipases, amylases, hemicellulases, arabinoxylanases, xylanases, cellulases, NSP enzymes, methionine reductases, methionine synthases, uricases, prolyl endopeptidases, combinations thereof, and any other enzyme that improves the effectiveness of a feed composition, food, or consumable liquid for improving the performance or health of an animal or human. In still another embodiment, a yeast, fungus (e.g., Aspergillus or Trichoderma) or micronutrient can be added to an animal feed or human food. Any of the above described ingredients suitable for addition to an additive for animal or human drinking water or to a consumable liquid can be added as an ingredient of an additive for animal or human drinking water or a consumable liquid as described herein.

[0249] In various illustrative embodiments, a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof), or any other bacterial strain added in addition to Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can be added to an animal feed or human food at a dose of about 1.0 x 10 3 CFU / gram of feed composition or human food composition - about 5.0 x 10 12 CFU / gram of feed composition or food composition, or at a dose of about 1.0 x 10 3 CFU / gram of feed composition or food composition - about 1.0 x 10 7The dosage of CFU / gram feed composition or food composition is administered in an animal feed composition or a human food composition. In other embodiments, a Bacillus strain (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or a combination thereof) is administered in a feed composition or food composition at a dosage of greater than about 1.0 x 10 3 CFU / gram feed composition or food composition, at a dosage of greater than about 1.1 x 10 3 CFU / gram feed composition or food composition, at a dosage of greater than about 1.25 x 10 3 CFU / gram feed composition or food composition, at a dosage of greater than about 1.5 x 10 3 CFU / gram feed composition or food composition, at a dosage of greater than about 1.75 x 10 3 CFU / gram feed composition or food composition, at a dosage of greater than about 1.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 2.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 3.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 4.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 5.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 6.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 7.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 8.0 x 10 4 CFU / gram feed composition or food composition, at a dosage of greater than about 1.0 x 10 5 CFU / gram feed composition or food composition, at a dosage of greater than about 1.0 x 10 6 CFU / gram feed composition or food composition, at a dosage of greater than about 1.0 x 10 7 CFU / gram feed composition or food composition, at a dosage of greater than about 1.0 x 10 8CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10 9 CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10 10 CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10 11 CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10 12 CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10 4 CFU / gram of feed composition or food composition, at a dose of greater than about 1.0 x 10

[0250] In another embodiment, the Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054) can be dosed at about 10 5 CFU / day, about 10 6 CFU / day, about 10 7 CFU / day, about 10 8 CFU / day, about 10 9 CFU / day, about 10 10 CFU / day, about 10 11 CFU / day, about 10 12 CFU / day, or any suitable strain amount expressed in CFU / day.

[0251] In various embodiments, Bacillus strains for use in accordance with the methods and compositions described herein can be selected from Bacillus strain BC1 (NRRL Accession No. B-67744), Bacillus strain (NRRL Accession No. B-68053), Bacillus strain (NRRL Accession No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Accession No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Accession No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Accession No. B-68054), or combinations thereof. Bacillus strain MDG BC1 (strain BC1) was deposited 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 on February 6, 2019, and given Accession No. B-67744. Bacillus strains MDG-HP20 and MDG-HP29 were deposited 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 on July 6, 2021, and given Accession Nos. B-68053 and B-68054, respectively. The deposits were made in compliance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. NRRL strain designations MDG BC1, MDG-HP20, and MDG-HP29 are equivalent to Bacillus coagulans strain BC1 and Bacillus subtilis strains NRRL Accession No. B-68053 and NRRL Accession No. B-68054, respectively, as referred to in this application.

[0252] 61604-3999, and given Accession No. B-67744. Bacillus strains MDG-HP20 and MDG-HP29 were deposited 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 on July 6, 2021, and given Accession Nos. B-68053 and B-68054, respectively. The deposits were made in compliance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. NRRL strain designations MDG BC1, MDG-HP20, and MDG-HP29 are equivalent to Bacillus coagulans strain BC1 and Bacillus subtilis strains NRRL Accession No. B-68053 and NRRL Accession No. B-68054, respectively, as referred to in this application.

[0253] Any of these strains can be administered alone or in combination in a feed composition (e.g., a complete feed comprising an animal feed admixture), a food composition for a human, or a drinking water or consumable liquid for an animal or human, or the strains can be administered directly without the addition of any feed or food ingredient. In one embodiment, multiple strains are administered in a single composition. In another embodiment, multiple strains are administered in separate compositions.

[0254] In another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof) can be administered to an animal or human with another different bacterial strain selected from another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. In still another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof) can be administered to an animal or human with any other different bacterial strain that is effective to improve the performance or health of the animal or human.

[0255] As used herein, a "strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744) or Bacillus strain (NRRL No. B-68053) or Bacillus strain (NRRL No. B-68054)" can be a mutant strain having all of the identifying characteristics of these Bacillus strains (e.g., DNA analysis-based DNA fingerprints corresponding to the DNA fingerprints of these strains, enzyme activities corresponding to these strains, antimicrobial activities corresponding to these strains, antibiotic sensitivity and resistance profiles corresponding to these strains, or combinations thereof). In alternative embodiments, the mutations can be natural mutations or genetically engineered mutations. In another embodiment, a "strain having all of the identifying characteristics of Bacillus strain BC1 or Bacillus strain (NRRL B-68053) or Bacillus strain (NRRL B-68054)" can be a strain produced, for example, by isolating one or more plasmids from Bacillus strain BC1 or Bacillus strain (NRRL B-68053) or Bacillus strain (NRRL B-68054) and introducing the one or more plasmids into another bacterium, such as another Bacillus strain, so long as the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain BC1 or Bacillus strain (NRRL B-68053) or Bacillus strain (NRRL B-68054) (e.g., DNA analysis-based DNA fingerprints corresponding to the DNA fingerprints of Bacillus strain BC1 or Bacillus strain (NRRL B-68053) or Bacillus strain (NRRL B-68054)).

[0256] The feed composition or drinking water for animals or food composition or drinking water or consumable liquid for humans described above can be administered to the animal or human for any period of time effective to improve the health of the animal or human. For example, in one embodiment, the feed or food composition or drinking water or consumable liquid can be provided to the animal or human on a daily basis. In alternative embodiments, the feed composition or drinking water can be administered to the animal during lactation and / or during pregnancy. The above-described periods of time for administering the feed or food composition or drinking water or consumable liquid are non-limiting examples, and it should be appreciated that any period of time or schedule of administration determined to be effective to improve the health of the animal or human can be used. In another embodiment, the period of time for administration can be determined by the human.

[0257] As described herein, one of the method embodiments is a method of feeding an animal or human by administering to the animal or human a feed or food composition or drinking water or consumable liquid comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof, wherein the Bacillus strain improves the health of the animal, particularly the gastrointestinal health of the animal.

[0258] The health improvement can be relative to an animal or human that is not fed the bacterial strain. In one embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can increase the digestibility of a diet by producing enzymes that increase the digestibility of consumed nutrients, wherein the enzymes are selected from the group consisting of alpha galactosidase, protease, phytase, lipase, amylase, xylanase, cellulase, methionine reductase, methionine synthase, uricase, prolyl endopeptidase, and combinations thereof. The enzymes can also be any other enzyme that degrades long chain fatty acids, such as enzymes that degrade stearic acid, palmitic acid, and / or oleic acid, but are not limited to these fatty acids. Such an increase in the digestibility of the diet results in an improvement in the health of the animal and human.

[0259] In embodiments where the effect is to improve the health of an animal or human, 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 (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof. In various embodiments, the antimicrobial activity is against a microorganism selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof. Thus, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can improve the intestinal health of an animal or human, and reduce pathogens in an animal or human, and in the animal's environment. In still another embodiment, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can reduce the bioburden of an animal or human, or increase the immune response of an animal or human, to improve the health of the animal or human. The improvements described herein can be relative to an animal or human that is not fed a bacterial strain.

[0260] In this method embodiment, the method can improve the health of the animal by improving the animal's environment through effects selected from the group consisting of reducing respiratory problems in the animal, improving gut health in the animal, improving consistency of performance in the animal, reducing disease associated with environmental toxicity in the animal, and reducing pathogen in the animal. In embodiments where the animal is a poultry species, the method can improve the health of the animal by effects selected from the group consisting of reducing respiratory problems in the poultry species, reducing keel cysts in the poultry species, improving consistency of performance in the poultry species, and reducing damage to the poultry species' feet. These mechanisms of improving the health of the animal are non-limiting examples.

[0261] In further embodiments of the application, compositions comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are provided. In one embodiment, commercial packages comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof are provided.

[0262] In another embodiment, feed additives for animal feed or human food are provided, the additives comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof.

[0263] In still another embodiment, an additive for animal or human drinking water or for a consumable liquid is provided, the additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof.

[0264] In still another illustrative aspect of the present application, an animal feed composition or a human food composition is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof.

[0265] In one embodiment, a feed additive for addition to an animal feed mix to produce a complete feed composition or for drinking water or an additive for addition to human food, drinking water or a consumable liquid can be mixed with the animal feed mix or drinking water, or with the human food, drinking water or consumable liquid, to achieve any dosage of Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof described herein, for administration to an animal or human in the form of a complete feed composition or human food composition or drinking water or consumable liquid, for example, with an automated micro-nutrient delivery system, or for example, by manual weighing and addition. Mixing can also be carried out by any other suitable method known in the art to combine the direct fed microbial or probiotic with the animal feed mix or with the human food or with the drinking water or consumable liquid to obtain a uniform mixture. In various embodiments, mixing can be carried out for any suitable period of time, for example, from about 1 to about 4 minutes.

[0266] In embodiments wherein Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof, are in the form of a drinkable water for animals or humans or an additive for a consumable liquid, the Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof, can be in the form of, for example, a powder, a liquid, a gel, a lyophilized form, a poultice, or a pellet, and can be mixed with the drinkable water or consumable liquid using any suitable method known in the art to achieve any dose of the Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof, described herein for administration to an animal or human in the drinkable water or consumable liquid for the animal or human.

[0267] The Bacillus strain BC1 (NRRL Number B-67744), Bacillus strain (NRRL Number B-68053), Bacillus strain (NRRL Number B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL Number B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL Number B-68054), or combinations thereof, can also be directly fed to an animal or human orally (i.e., by oral insertion) in the form of a powder, a liquid, a gel, a lyophilized form, a poultice, a capsule, a tablet, a granule, a spray, a paste, a liquid bolus, or a pellet.

[0268] In any of the composition embodiments described herein, Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can cause improvements in animal or human health, particularly gastrointestinal health. The commercial package, feed or food additive, feed or food composition, or drinking water for animals or humans, or additive for consumable liquids described herein can also inhibit a pathogen selected from the group consisting of: E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof. These effects are non-limiting examples of the types of effects that Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can cause.

[0269] In an illustrative aspect, the feed or food additive, additive for drinking water for animals or humans, or consumable liquids, or feed or food composition can be in the form of a commercial package, such as a dietary nutritional composition (e.g., a probiotic composition or a direct fed microbial composition). In another illustrative embodiment, the feed or food additive, or additive for drinking water for animals or humans, or consumable liquids, or Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, in a commercial package can be in the form of a concentrate (e.g., about 1 x 10 8 - about 5 x 10 9 CFU / g) or a superconcentrate (e.g., 1 x 10 10 - about 5 x 10 12In another illustrative embodiment, the commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition can further comprise a carrier for Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof. The carrier can be selected from the group consisting of bran, rice hulls, salt, mineral oil, dextrin (e.g., malt dextrin), whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, chicory root, and combinations thereof. In another embodiment, the carrier can be any suitable carrier known in the art for direct fed microbial or probiotic compositions. The carrier is exogenously added to the bacterial strain (i.e., not naturally occurring or absent in nature with the bacterial strain). In another embodiment, the commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, dextran, bentonite, zeolite, hydrous calcium sodium aluminosilicate, charcoal, chlorella, and sodium metabisulfite, or other known binders. The binder is exogenously added to the bacterial strain (i.e., not naturally occurring or absent in nature with the bacterial strain).

[0270] In another illustrative embodiment, the commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition can further comprise a carrier for Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof. The carrier can be selected from the group consisting of bran, rice hulls, salt, mineral oil, dextrin (e.g., malt dextrin), whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, chicory root, and combinations thereof. In another embodiment, the carrier can be any suitable carrier known in the art for direct fed microbial or probiotic compositions. The carrier is exogenously added to the bacterial strain (i.e., not naturally occurring or absent in nature with the bacterial strain). In another embodiment, the commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, dextran, bentonite, zeolite, hydrous calcium sodium aluminosilicate, charcoal, chlorella, and sodium metabisulfite, or other known binders. The binder is exogenously added to the bacterial strain (i.e., not naturally occurring or absent in nature with the bacterial strain).

[0271] In any of the method or composition embodiments described herein, a prebiotic can also be added to the final feed composition, to the feed additive or to the drinking water, or can be added separately. Exemplary prebiotics include, but are not limited to, indigestible carbohydrates, fructooligosaccharides (FOS), galactooligosaccharides (GOS), transgalactooligosaccharides, short- and long-chain fructopolysaccharides (e.g., FOS and inulin), and lactulose, oligosaccharide carbohydrates (OSC), oligofructose or oligofruftose, galactooligosaccharides (e.g., GOS with excess galactose at C3, C4, or C6 and GOS manufactured from lactose by enzymatic transglycosylation to provide a mixture of, e.g., tri- to pentasaccharides with galactose in beta (1→6), beta (1→3, and beta (1→4) linkages, i.e., transgalactooligosaccharides or TOS), resistant starches, glucose-derived oligosaccharides (e.g., polydextrose), pecticoligosaccharides (POS), and non-carbohydrate oligosaccharides (e.g., cocoa-derived flavanols).

[0272] In still other embodiments, a commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, is in a container for commercial use. In various embodiments, the container can be, for example, a bag (e.g., a 20 pound bag, a 50 pound bag, a 2 ounce bag, a 1 ounce bag, or a 1 kilogram bag), a pouch, a tub, a bottle, or a box. In illustrative aspects, the container for a commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition comprising Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all of the identifying characteristics of Bacillus strain BC1 (NRRL No. B-67744), a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68053), and / or a strain having all of the identifying characteristics of Bacillus strain (NRRL No. B-68054), or combinations thereof, can comprise plastic, metal, foil, paper, fiber, or paperboard (e.g., a plastic tub, a paper bag, a foil pouch, a fiber tub, etc.). The commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition can further comprise instructions for using one or more of the Bacillus strains.

[0273] In one aspect, the commercial package, feed or food additive, additive for animal or human drinking water or consumable liquid, or feed or food composition described herein can further comprise an exogenously added nutritional component (i.e., a nutritional component that does not naturally occur with the bacterial strain) selected from the group consisting of vitamins, antibiotics, enzymes, water soluble or water insoluble monosaccharides, disaccharides or polysaccharides, fats, phosphorous, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oil, raw seeds, antioxidants, and starches.

[0274] In one embodiment, the exogenously added nutritional component is an enzyme, and the enzyme is selected from the group consisting of galactosidases, proteases, lipases, amylases, hemicellulases, arabinoxylanases, xylanases, cellulases, NSP enzymes, phytases, methionine reductases, methionine synthases, uricases, prolyl endopeptidases, and combinations thereof.

[0275] In any of the embodiments described herein, the Bacillus strain can not cause hemolysis, can not cause cytotoxicity, can have antioxidant activity, and / or can have quorum quenching activity. In any of the embodiments described herein, the Bacillus strain does not produce a toxin selected from the group consisting of hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), emetic toxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK). In any of the embodiments described herein, the Bacillus strain can produce an anti-inflammatory biomarker selected from the group consisting of SOCS1, TOLLIP, IL-10, and CXCL12.

[0276] The following examples are for illustrative purposes only. The examples are non-limiting and are not intended to limit the application in any way.

[0277] Bacillus strain BC1 (NRRL No. B-67744)

[0278] Example 1

[0279] Antibiotic resistance

[0280] Resistance of microbial pathogens to therapeutic antibiotics is currently considered one of the biggest challenges in the field of medicine and public health, as if some infectious diseases become unresponsive to current therapies, they can become almost untreatable. Antibiotic resistance can be divided into two types; intrinsic / natural or acquired / adventitious. Intrinsic / natural is when resistance is inherent to the bacterial species, and is a characteristic usually shared by all members of that species. Acquired / adventitious is when a strain of a generally susceptible species is resistant to a given antimicrobial drug. Acquired / adventitious resistance can come from acquisition of foreign DNA or mutation of intrinsic genes. While intrinsic resistance can present a very low risk of spread, acquired resistance, especially when the relevant genes are associated with mobile genetic elements such as plasmids and transposons, can be transferred to pathogenic or other commensal bacteria. It is generally recommended to evaluate resistance to antibiotics in all probiotic strains prior to marketing. Phenotypic assessment of antibiotic resistance involves testing the ability of a microorganism to survive in media containing different concentrations of antibiotics. Given that most microorganisms can survive under low concentrations of many antibiotics, resistance is defined as the ability to grow at antibiotic concentrations similar to those reached in the human body during therapeutic intervention.

[0281] The inventors have phenotypically evaluated resistance. EFSA defines a bacterial strain as susceptible when its growth is inhibited at a specific antibiotic concentration equal to or lower than the established cut-off value for that particular species. A bacterial strain is defined as resistant when it is able to grow at a specific antibiotic concentration higher than the established cut-off value. Both EFSA and CLSI guidelines for determining susceptibility and resistance are interpreted by their defined cut-off values. See Table 1.

[0282] Table 1

[0283]

[0284]

[0285] Example 2

[0286] Hemolytic activity

[0287] The hemolytic activity of Bacillus strain BC1 (NRRL No. B-67744) was tested by using blood agar plates to determine zero, partial or complete lysis of red blood cells. The hemolytic activity of this strain was zero. Beta-hemolysis is defined as complete or true lysis of red blood cells. A clear zone of color and transparency close to the base medium surrounds the colony. Alpha-hemolysis (or partial hemolysis) is the reduction of red blood cell hemoglobin to methemoglobin (surrounding the medium of the colony). Discoloration of the surrounding medium to a brown or green color of the cells is a common phenomenon of alpha hemolysis. Unlike beta hemolysis, alpha hemolysis maintains the structure of the cell membrane. The strain was cultured and tested for antibiotic susceptibility by the following method. First, the strain was cultured from a frozen stock by using a sterile inoculating loop to iso-streak cells on MRS medium and incubated anaerobically at 45°C for 24 hours. When colonies were visible the next day, they were checked for purity morphologically and a single colony was collected using a sterile inoculating loop. The single colony was cultured to a higher density by inoculating into BHI Broth + Yeast Extract + Cysteine and incubated anaerobically at 45°C for 24 hours. The next day, the growth culture was standardized to an OD of 0.5 using 0.1% peptone as a diluent. Blood agar plates were purchased pre-made and contained trypticase soy agar and 5% sheep blood. Using a sterile inoculating loop with a diameter that held about 10 μl, the culture was streaked horizontally across the surface of the plate in triplicate. The plates were then incubated aerobically at 45°C for 24 hours. The next day, the plates were removed from the incubator and the results were recorded. For no discoloration of the medium, the result was determined to be "zero hemolysis", partial hemolysis and green-brown discoloration of the medium was determined to be "alpha hemolysis", and for complete lysis of the medium and showing a clear zone around the cells, it was determined to be "beta hemolysis". The strain produced zero hemolysis around the cells streaked on blood agar plates for all triplicate streaks. 600nm 0.5 using 0.1% peptone as a diluent. Blood agar plates were purchased pre-made and contained trypticase soy agar and 5% sheep blood. Using a sterile inoculating loop with a diameter that held about 10 μl, the culture was streaked horizontally across the surface of the plate in triplicate. The plates were then incubated aerobically at 45°C for 24 hours. The next day, the plates were removed from the incubator and the results were recorded. For no discoloration of the medium, the result was determined to be "zero hemolysis", partial hemolysis and green-brown discoloration of the medium was determined to be "alpha hemolysis", and for complete lysis of the medium and showing a clear zone around the cells, it was determined to be "beta hemolysis". The strain produced zero hemolysis around the cells streaked on blood agar plates for all triplicate streaks.

[0288] Example 3

[0289] Enterotoxin

[0290] Using a multiplex PCR method adapted from Yang et al., Bacillus strain BC1 (NRRL No. B-67744) was tested for the negative presence of all known toxin genes commonly associated with Bacillus cereus and Bacillus thuringiensis. Whole genome sequence analysis was also used to query these known Bacillus toxin gene sequences and no significant similarities were found in the database. The strain (NRRL No. B-67744) was cultured, DNA was extracted, and the DNA was tested for toxins by the following method. First, the strain was cultured from a frozen stock by performing a separate streak of cells on MRS media using a sterile inoculating loop and incubating them aerobically at 45°C for 24 hours. When colonies were visible the next day, they were checked for purity morphologically and a single colony was collected using a sterile inoculating loop for DNA extraction. The single colony was cultured to a higher density by inoculating it into BHI + Yeast Extract + Cysteine Broth and incubating it anaerobically at 45°C for 24 hours. The next day, 1 ml of culture was transferred to a 1.5 ml microfuge tube and the cells were pelleted in a centrifuge at 10,000 rpm for 10 minutes. DNA extraction was performed on these cells using Qiagen’s DNeasy Blood and Tissue Single Column Kit. The supernatant was discarded from the pelleted cells and the pellet was resuspended in 180 μl of lysis buffer containing 20 mg / ml lysozyme. NRRL No. B-67744 + lysis solution was incubated at 37°C for 45 minutes to disintegrate the cell wall and lyse the cellular components. Then, 20 μl of proteinase K and 200 μl of Buffer AL were added and the sample was incubated at 56°C for another 30 minutes to degrade the protein components. After the incubation was complete, 200 μl of EtOH was added and vortexed to a homogenous solution to aggregate the insoluble DNA. A spin column was used to isolate and purify the DNA from the solution according to the manufacturer’s recommended protocol. The DNA was eluted in 100 μl of Low-TE Buffer purchased from ThermoFisher.

[0291] Primer sequences and the multiplex PCR method were adapted from Yang et al. Primers for the toxins hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), vomitoxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK) were used to test for the presence of toxin-producing Bacillus species. These primer sequences were purchased from Eurofins and diluted to working concentrations of 40–350 nM (Table 2). Three separate master mixes were prepared for the multiplex toxin assay, as shown in Table 1. For each master mix, a total volume of 49 μl was used, consisting of 200 μM DNTP, 1X PCR buffer, 2 U Fast Start Taq polymerase, and the remaining volume in ddH2O. The total reaction volume was 55 μl, including 49 μl of the corresponding master mix and 6 μl of template DNA. Bacillus cereus and Bacillus thuringiensis genomic DNA were used as positive controls, and ddH2O was used for a no-template control.

[0292] PCR reactions were performed using an Applied Biosystems 2720 thermal cycler using the endpoint PCR technique. All three master mixes were tested against NRRL 67744, a Bc positive control, a Bt positive control, and a NT negative control. Reaction conditions included a denaturation step at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds, and concluded with an extension step at 72°C for 7 minutes. Amplification products were analyzed on 2% agarose gels, electrophoresed at 100 volts for 80 minutes, and visualized using a gel imaging system with a UV transilluminator and GeneSnap software tools.

[0293] like Figure 1 As shown, NRRL No. B-67744 genomic DNA was negative for the presence of any toxin in all three multiplex assays after 30 cycles of amplification. Genomic DNA controls for Bc and Bt produced amplification products of the expected band size for each multiplex assay, and no-template controls were negative for all three master mixes.

[0294] The results of the whole genome sequence analysis did not produce significant similarity of NRRL Number B-67744 to these known B. cereus and B. thuringiensis toxin gene sequences (Table 3). The multiplex PCR assay did not produce amplification of the B. cereus-like toxin genes hemolysin BL, non-hemolytic enterotoxin, enterotoxin FM, enterotoxin T, or cytotoxin K for NRRL Number B-67744 DNA. Amplified PCR products were seen at the expected band sizes, and additionally, there were no discrete bands of unexpected sizes for the genomic DNA positive controls, and no amplification was seen in the no template controls. Furthermore, the multiplex PCR assay has a sensitivity of approximately 100 pg as described in Yang et al., and the primer pairs are specific for their target. All of these data indicate that these primer sequences, in the multiplex reaction, are an appropriate method of analysis.

[0295] These results were confirmed by aligning the whole genome sequence against toxin gene sequences via the BLASTn database, shown in Table 3.

[0296] Table 2. Premix set and primer concentrations for each primer set

[0297]

[0298] Table 3. Results of whole genome sequencing analysis from Bc and Bt toxin genes

[0299] Toxin genes GenBank Accession No. Query results for NRRL 67744 hbl (subunits C / D) U63928.1 No significant similarity found nhe (subunits A / B / C) DQ885236.1 No significant similarity found cytK AJ277962.1 No significant similarity found entFM AY789084.1 No significant similarity found bceT D17312.1 No significant similarity found

[0300] Example 4

[0301] Cytotoxicity assay

[0302] EFSA requires Bacillus spp. to be non-toxigenic, so Bacillus coagulans NRRL Number B-67744 was tested for in vitro cytotoxicity using Vero (epithelial) cells. Cytotoxicity was measured with a lactate dehydrogenase (LDH) assay and followed the EFSA "Guideline on the characterisation of microorganisms used as feed additives or production organisms." An absorbance value higher than 20% of the absorbance obtained from the maximum LDH release control (Promega 10x lysis) indicates cytotoxicity. As shown in Tables 4 and 5 below, supernatant from Bacillus coagulans NRRL Number B-67744 had no cytotoxic effect on Vero cells at 10% concentration.

[0303] Table 4. Bacillus coagulans NRRL Number B-67744 LDH percentage released by Vero cells after 60 minute exposure

[0304]

[0305] As a control for endogenous LDH signal in the bacterial culture medium used to generate the test article, the test article was also tested in EC buffer (in the absence of Vero cells). The results of this assay are in the table above, as shown in the equation below the table.

[0306] Table 5. Bacillus coagulans NRRL No. B-67744 LDH signal percentage in EC buffer and supernatant control

[0307]

[0308] Example 5

[0309] RAPD-PCR DNA profile

[0310] Genetic variability of strains was identified using the random amplified polymorphic DNA PCR method (RAPD-PCR). Preparation of DNA to be used in RAPD-PCR reactions was performed using Qiagen’s Blood and Tissue Single Column Kit. To obtain DNA, overnight cultures were prepared, purity was checked, pelleted, and DNA was extracted following the manufacturer’s protocol. Preparation of RAPD-PCR reactions was performed by using Cytiva RAPD Bead Kit, which requires the use of one bead per reaction, sterile water, and DNA template, along with one of six primers previously designed as random amplified polymorphic DNA. All six primers were used in separate reactions for each strain. Each 25 μΐ reaction contained one Cytiva RAPD bead, 16 μΐ sterile water, 5 μΐ of the corresponding primer, and 4 μΐ of DNA template. Before running the reactions, each sample was sealed, vortexed, and centrifuged briefly. RAPD-PCR reactions were run in an AB2720 thermal cycler with the following run conditions; 95°C for 5 min, followed by 45 cycles (95°C for 1 min, 36°C for 1 min, 72°C for 2 min), then 72°C for 7 min, and ending with a 4°C hold indefinitely to preserve the product. RAPD-PCR products were analyzed by gel electrophoresis using 1% (wt / vol) agarose in lx TBE buffer and a UV imager. The agarose gel contained 1 ul / 10 ml of SyberSafe gel stain. The gel was run at 120 volts for 3 hours. At the end of the run, a digital camera connected to the UV imager uploaded the gel image to the GeneSnap software, which then inverted the saturation values and adjusted the contrast for viewing purposes. See Figure 2 .

[0311] Example 6

[0312] Digestive enzyme production

[0313] Phenotypic enzyme assays using NRRL 67744 resulted in positive enzyme activity for lipase, amylase, protease, and cellulase, and genomic analysis further yielded genomic potential for alpha and beta galactosidases, as well as methionine reductase and synthetase. These enzymes are extraordinary biocatalysts that increase the rate of biochemical reactions, particularly during digestion. Enzymes and reactions are responsible for hydrolyzing complex carbohydrates, lipids, and proteins into more bioavailable constituents, thereby increasing the rate of absorption and nutrient intake.

[0314] Phenotypic enzyme activity plates were performed to determine the enzymatic potential of digestive enzymes well known to be produced by resident microbiota. Trypticase soy agar (TSA) is the base agar medium used with selective reagents specific to each enzyme. For lipase activity, 50 mL Tween 80 and 2.5 mL polysorbate 80 were added to 1 L of TSA. For amylase activity, 10 g of corn starch was added to 1 L of TSA. For protease activity, 10 g of casein was added to 1 L of TSA. For cellulase activity, 5 g of CMC was added to 1 L of TSA. All media were autoclaved at 122°C for 30 min and then poured into agar plates and dried. For each set of plates, 10 ul of NRRL 67744 was spotted in triplicate and incubated anaerobically at 37°C for 24 hours. The next day, positive enzyme activity was determined by clearing zones around the culture spots. If there was no clearing zone, it indicated a negative reaction.

[0315] Example 7

[0316] Immunomodulation

[0317] Bacillus species are known to modulate immunity through interactions with the host gastrointestinal tract. This immune modulation can help the human body maintain a delicate balance between eliminating invading pathogens while still maintaining a modulated level of inflammatory response that is able to restore homeostasis. Research has found that 70% of the human immune system is located within the epithelial tissue of the gastrointestinal tract. Bacillus, and specifically NRRL No. B-67744, is able to directly and indirectly communicate with these immune cells to provide the necessary response to the surrounding environment, provide essential health benefits to the host, and modulate immune homeostasis.

[0318] The rat IEC-6 cell line is derived from rodent intestinal epithelial cells that form many microvilli and the characteristics of typical crypt cells with tight junctions connecting adjacent cells. Rat IEC-6 cells represent a well-characterized model to study the response of the intestinal epithelium to bacterial infection. This cell line expresses the characteristics of intestinal cells and mediates responses from many different immune cell types. To test the immunomodulatory ability of NRRL No. B-67744, this strain was exposed to IEC-6 cells in the presence and absence of LPS cell wall components. IEC-6 cells were cultured using DMEM cell culture medium supplemented with 10% FBS and 1% antibiotic / antimycotic and passaged twice to achieve viability consistency and were incubated at 37°C and 5% C02. For the immunization assay, cells were cultured in 24-well plates with a 500 μΐ volume and seeded at a density of 100 k / well. The confluent monolayer was maintained for 2 days prior to exposure to allow for full maturation of the immune cells within the mucosal epithelial monolayer. Once matured, the antibiotic / antimycotic was removed from the culture medium and a 4-hour equilibration period was given.

[0319] To prepare the NRRL No. B-67744 test article, overnight cultures were grown in 5 ml BHI + yeast extract + cysteine and incubated at 42°C. Cultures were quantified using optical density measurements (OD at 600 nm) using an OD of 0.5 as a reference for 10 7 cells of Bacillus sp. Samples were diluted using 1 ml of sterile PBS to yield 10 6 , 10 5 , 10 4 , and 10 3 concentrations. Precise cell counts were also taken of the cultures. To prepare the LPS compound, an aliquot of the 100 ng / ul solution was thawed and diluted using PBS down to 10 ng / ul. For the immunization assay, 5 ul of 10 ng / ul LPS was used as a stimulant and 10 ul of culture was used per dose of NRRL No. B-67744 strain as shown in the following figure. Each test condition and control had 6 replicates. Plates were incubated at 37°C, 5% C02 for 2 hours.

[0320] Cells were harvested and RNA extracted for expression analysis after the 2-hour incubation period. Briefly, one well per treatment was removed to quickly perform and not allow for expression changes, the culture medium was removed and 200 ul of Tri reagent was added. The Tri reagent was allowed to work for 5 minutes at room temperature, then once the cells were released from the binding with the plate well, they were removed from the plate and placed in a 96-well 2 ml round bottom block. The block was covered with a gas permeable sterile membrane on the first layer and adhesive foil on the second layer. The block was then snap frozen in liquid nitrogen and stored at -80°C until ready for RNA extraction and expression work.

[0321] RNA extraction was completed using Qiagen's RNeasy 96 well kit using DTT added to the RLT buffer. RLT+DTT buffer was prepared by adding 40ul / mL of 1M DTT to the RLT buffer. Following the manufacturer's protocol, and eluting two rounds of 45uL. RNA was stabilized into cDNA immediately after extraction using QuantaBio reverse transcriptase. For the reaction, 16ul of RNA and 4ul rt enzyme were added to a 96 well PCR plate. The reaction was run on a Bio-Rad machine using the standard reverse transcription protocol provided by QuantaBio. For the immunomodulatory assay, immune biomarker expression was quantified and expressed as mean relative quantity (RQ) values. GAPDH was used as a reference housekeeping gene. The results in the following Figure 3 illustrate the mean RQ value differences when compared to the LPS control.

[0322] Immune response results from IEC-6 cells show significant differences in biomarker expression of pro-inflammatory cytokines and pathogen receptors when compared to LPS stimulant (IL-6, TLR2, and TNF alpha). This data indicates that strain NRRL No. B-67744 has an anti-inflammatory effect after the stimulatory event has occurred.

[0323] Example 8

[0324] Antioxidant activity

[0325] Genomic analysis revealed the potential of many natural enzymatic antioxidants, including both primary (superoxide dismutase gene for manganese, catalase activity, and glutathione peroxidase) and secondary (glucose-6-phosphate dehydrogenase) and inhibition of protein oxidation by thioredoxin and glutaredoxin activity. While oxygen is an essential element for many functions of an organism, oxygen concentrations above the normal range cause oxidative stress by producing reactive oxygen species (ROS). ROS are naturally produced by both endogenous and exogenous sources that occur on a daily basis. Endogenous sources include byproducts of metabolic processes, NADPH oxidases, mitochondrial electron transport chain leakage, and cytokine and growth factor receptors; while exogenous sources come from UV light, radiation, drugs, pollutants, and / or pathogens. The resulting oxidative stress can cause damaging injury to DNA / RNA, proteins, and lipids, and also leads to cellular responses such as inflammation and carcinogenesis. If excess ROS is not controlled, these damaging changes often lead to chronic diseases, including atherosclerosis, arthritis, diabetes, Alzheimer's disease, neurodegenerative diseases, and cardiovascular disease.

[0326] Antioxidants are ROS scavengers that can mask, remove, and repair oxidative damage, thereby protecting target components or molecules from oxidative damage. Organisms have enzymatic and nonenzymatic antioxidant mechanisms to inactivate ROS, and microorganisms have been identified as sources of both. Enzymes, including catalase, glutathione peroxidase, and superoxide dismutase, are endogenous antioxidants that control ROS damage, while carotenoids, flavonoids, coenzyme Q, vitamins, minerals, and phenolic acids are sources of exogenous antioxidants.

[0327] Bacillus strain (NRRL No. B-68053)

[0328] Example 9

[0329] Antibiotic resistance

[0330] The inventors have phenotypically evaluated resistance. EFSA defines a bacterial strain as susceptible when its growth is inhibited at a specific antibiotic concentration equal to or lower than the established cut-off value for that particular species. A bacterial strain is defined as resistant when it is able to grow at a specific antibiotic concentration higher than the established cut-off value. We used the cut-off values for their definition to determine both EFSA and CLSI guidelines for susceptibility and resistance. See Table 6.

[0331] Table 6. Results for the minimum inhibitory concentration (MIC) against medical antibiotics

[0332]

[0333] Example 10

[0334] Hemolytic activity

[0335] The method of determining zero, partial or complete lysis of red blood cells by blood agar plates was used to test the hemolytic activity of Bacillus sp. strain (NRRL No. B-68053). The hemolytic activity of NRRL 68053 was negative. Beta-hemolysis is defined as complete or true lysis of red blood cells. A clear zone of color and transparency close to the base media surrounds the colony. Alpha-hemolysis is the reduction of red blood cell hemoglobin to methemoglobin (media surrounding the colony). Discoloration of the media surrounding the cells to a brown or green color is a common occurrence of alpha hemolysis. Unlike beta hemolysis, alpha hemolysis maintains the structure of the cell membrane. NRRL 68053 was cultured and tested for antibiotic susceptibility by the following method. First, the strain was cultured from a frozen stock by isolating streaks of cells on tryptic soy agar media using a sterile inoculating loop and incubated overnight at 37°C. When colonies were visible the next day, they were checked for purity morphologically and a single colony was collected using a sterile inoculating loop. The single colony was cultured to a higher density by inoculating into tryptic soy broth and incubated overnight at 37°C, 230 rpm. The next day, the growth culture was standardized to an OD of 0.8 using 0.1% proteose peptone as a diluent. Blood agar plates were purchased pre-made and contained tryptic soy agar and 5% sheep blood. Using a sterile inoculating loop with a diameter that held approximately 10 μl, the culture was streaked horizontally across the surface of the plate in triplicate. The plates were then incubated overnight at 37°C. The next day, the plates were removed from the incubator and the results were recorded. For no discoloration of the media, the results were determined to be "no hemolysis", partial hemolysis and green-brown discoloration of the media were determined to be "alpha hemolysis", and for complete lysis of the media and showing a clear zone around the cells, "beta hemolysis" was determined. NRRL No. B-68053 produced no hemolysis around the cells streaked in triplicate on blood agar plates. 600nm 0.8 using 0.1% proteose peptone as a diluent. Blood agar plates were purchased pre-made and contained tryptic soy agar and 5% sheep blood. Using a sterile inoculating loop with a diameter that held approximately 10 μl, the culture was streaked horizontally across the surface of the plate in triplicate. The plates were then incubated overnight at 37°C. The next day, the plates were removed from the incubator and the results were recorded. For no discoloration of the media, the results were determined to be "no hemolysis", partial hemolysis and green-brown discoloration of the media were determined to be "alpha hemolysis", and for complete lysis of the media and showing a clear zone around the cells, "beta hemolysis" was determined. NRRL No. B-68053 produced no hemolysis around the cells streaked in triplicate on blood agar plates.

[0336] Example 11

[0337] Enterotoxin

[0338] Bacillus strain NRRL No. B-68053 was tested for the negative presence of all known toxin genes commonly associated with B. cereus and B. thuringiensis using a multiplex PCR method adapted from Yang et al. Whole genome sequence analysis was also used to query these known Bacillus toxin gene sequences and no significant similarities were found in the database. NRRL No. B-68053 was cultured, DNA was extracted, and toxins were tested by the following method. First, the strain was cultured from a frozen stock by performing a single streak of cells on tryptic soy agar medium using a sterile inoculation loop and incubated overnight at 37°C. When colonies were visible the next day, they were checked for purity morphologically and a single colony was collected using a sterile inoculation loop to extract DNA from the strain. It was cultured to a higher density by inoculating a single colony into tryptic soy broth and incubated overnight at 37°C. The next day, 1 ml of culture was transferred to a 1.5 ml microcentrifuge tube and the cells were pelleted in a centrifuge at 10,000 rpm for 10 minutes. DNA extraction was performed on these cells using Qiagen’s DNeasy Blood and Tissue Single Column Kit. The supernatant was discarded from the pelleted cells and the pellet was resuspended in 180 μΐ of lysis buffer containing 20 mg / ml lysozyme. NRRL No. B-68053 + lysis solution was incubated at 37°C for 45 minutes to disrupt the cell wall and lyse the cellular components. Then, 20 μΐ of proteinase K and 200 μΐ of Buffer AL were added and the sample was incubated at 56°C for another 30 minutes to degrade the protein components. After the incubation was complete, 200 μΐ of EtOH was added and vortexed to a homogenous solution to aggregate the insoluble DNA. A spin column was used to isolate and purify the DNA from the solution according to the manufacturer’s recommended protocol. The DNA was eluted in 100 μΐ of Low-TE Buffer purchased from ThermoFisher.

[0339] The primer sequences and multiplex PCR method were adapted from Yang et al. Primers for toxin hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), emetic toxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK) were used to test for the presence of toxin-producing Bacillus species. These primer sequences were purchased from Eurofins and diluted to a working concentration of 40-350 nM (Table 7). Three separate master mixes were prepared for the multiplex toxin assays as shown in Table 7. For each master mix, the total volume was 49 μΐ, including 200 μΜ DNTPs, 1X PCR buffer, 2 U of Fast-Start Taq polymerase, and the remaining volume of ddH2O. The total reaction volume was 55 μΐ, including 49 μΐ of the respective master mix and 6 μΐ of template DNA. B. cereus and B. thuringiensis genomic DNA were used as positive controls, and ddH2O was used for no-template controls.

[0340] PCR reactions were performed using endpoint PCR technology on an Applied Biosystems 2720 thermal cycler. All three master mixes were tested against NRRL B-68053, Bc positive control, Bt positive control, and NT negative control. Reaction conditions included a denaturation step at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 45 seconds, and ending with an extension step at 72 °C for 7 minutes. Amplification products were analyzed on 2% agarose gels, electrophoresed at 100 volts for 80 minutes, and visualized using a gel imaging system with UV transilluminator and GeneSnap software tools.

[0341] Results are shown in Figure 4 NRRL number B-68053 genomic DNA was negative for the presence of any toxin in the three multiplex assays by 30 amplification cycles. Bc and Bt genomic DNA controls produced amplification products of the expected band sizes for each multiplex assay, and the no-template controls for all three master mixes were negative. Whole genome sequence analysis did not yield significant similarity of NRRL number B-68053 to the gene sequences of these known B. cereus and B. thuringiensis toxins (Table 8).

[0342] For NRRL No. B-68053 DNA, the multiplex PCR assay did not produce amplification of Bacillus cereus-like toxin genes hemolysin BL, non-hemolytic enterotoxin, enterotoxin FM, enterotoxin T, or cytotoxin K. Amplified PCR products were seen at the expected band sizes, and additionally, there were no discrete bands of unexpected sizes for the genomic DNA positive control, and no amplification in the no template control. Furthermore, as described in Yang et al., the sensitivity of the multiplex PCR assay was approximately 100 pg, and the primer pairs were specific for their target. All of these data indicate that these primer sequences, paired in the multiplex reaction, are a suitable method of analysis. These results were confirmed by aligning the whole genome sequence against toxin gene sequences via BLASTn database, shown in Table 8.

[0343] Table 7. Premix set and primer concentrations for each primer set

[0344]

[0345] Table 8. Results from whole genome sequencing analysis of Bc and Bt toxin genes

[0346] Toxin genes GenBank Accession No. Query results for NRRL 68053 hbl (subunits C / D) U63928.1 No significant similarity found nhe (subunits A / B / C) DQ885236.1 No significant similarity found cytK AJ277962.1 No significant similarity found entFM AY789084.1 No significant similarity found bceT D17312.1 No significant similarity found

[0347] Example 12

[0348] Cytotoxicity assay

[0349] EFSA requires non-toxigenic activity of Bacillus spp. In vitro cytotoxicity testing was performed on Bacillus subtilis NRRL No. B-68053 using Vero (epithelial) cells. Cytotoxicity was measured with a lactate dehydrogenase (LDH) assay and followed the EFSA “Guideline on the characterisation of the microbial status of a production organism used as a feed additive or for production”. An absorbance value higher than 20% of the absorbance obtained from the maximum LDH release control (Promega 10x lysis solution) indicates cytotoxicity. As shown in the table below, supernatant from Bacillus subtilis NRRL No. B-68053 had no cytotoxic effect on Vero cells at a 10% concentration.

[0350] Table 9. Percentage of Bacillus subtilis NRRL No. B-68053 LDH released by Vero cells after 60 minute exposure

[0351]

[0352] As a control for endogenous LDH signal in the bacterial culture medium used to produce the test article, the test article was also tested in EC buffer (in the absence of Vero cells). The results of this assay are shown in the table above, as shown in the equation below the table.

[0353] Table 10. Percent Bacillus subtilis NRRL No. B-68053 LDH signal in EC buffer and supernatant controls

[0354]

[0355] Example 13

[0356] RAPD-PCR DNA profile

[0357] Genetic variability of strains was identified using the random amplified polymorphic DNA PCR method (RAPD-PCR). Preparation of DNA to be used in RAPD-PCR reactions was performed using Qiagen’s Blood and Tissue Single Column Kit. To obtain DNA, overnight cultures were prepared, purity was checked, pelleted, and DNA was extracted following the manufacturer’s protocol. Preparation of RAPD-PCR reactions was performed by using Cytiva RAPD Bead Kit, which requires the use of one bead per reaction, sterile water, and DNA template, and one of six primers pre-designed to random amplified polymorphic DNA. All six primers were used in separate reactions for each strain. Each 25 μΐ reaction contained one Cytiva RAPD bead, 16 μΐ sterile water, 5 μΐ of the respective primer, and 4 μΐ of DNA template. Before running the reactions, each sample was sealed, vortexed, and centrifuged briefly. RAPD-PCR reactions were run in an AB2720 thermocycler with the following run conditions; 95°C for 5 min, followed by 45 cycles of 95°C for 1 min, 36°C for 1 min, 72°C for 2 min, followed by 72°C for 7 min, and ending with a 4°C hold indefinitely to preserve the product. RAPD-PCR products were analyzed by gel electrophoresis using 1% (wt / vol) agarose in lx TBE buffer and a UV imager. Agarose gels contained 1 ul / 10 ml of SyberSafe gel stain. Gels were run at 120 volts for 3 hours. At the end of the run, a digital camera connected to a UV imager uploaded the gel image to GeneSnap software, which subsequently inverted the saturation values and adjusted the contrast for viewing purposes. See Figure 5 .

[0358] Example 14

[0359] Digestive enzyme production

[0360] Phenotypic enzyme assays using NRRL No. B-68053 resulted in positive enzyme activities for lipase, amylase, protease, xylanase, and cellulase, and genomic analysis further yielded genomic potential for α- and β-galactosidase, methionine reductase and synthase, uricase for uric acid, and prolyl endopeptidase for gluten degradation. These enzymes are extraordinary biocatalysts that increase the rate of biochemical reactions, particularly during digestion. The enzymes and reactions are responsible for hydrolyzing complex carbohydrates, lipids, and proteases into more bioavailable components, thereby increasing the rate of absorption and nutrient uptake.

[0361] Phenotypic enzyme activity plate assays were performed to determine the enzymatic potential of digestive enzymes known to be produced by resident microbiota. Tryptic soy agar (TSA) is a basic agar medium used with selective agents specific for each enzyme. For lipase activity, 50 mL of Tween 80 and 2.5 mL of polysorbate 80 were added to 1 L of TSA. For amylase activity, 10 g of corn starch were added to 1 L of TSA. For protease activity, 10 g of casein were added to 1 L of TSA. For xylanase activity, 10 g of xylan were added to 1 L of TSA. For cellulase activity, 5 g of CMC were added to 1 L of TSA. All culture media were autoclaved at 122° C. for 30 min and then poured into agar plates and dried. For each set of plates, 10 ul of NRRL No. B-68053 was sampled in triplicate and incubated overnight at 37° C. The next day, positive enzyme activity was determined by the clearing zone around the culture spot. If there is no clear zone, a negative reaction is indicated. Specific for xylanase activity, the plate is flooded with Gram's iodine and positive enzyme activity is determined by an unstained clear zone, while a negative reaction is indicated by the entire plate becoming stained with iodine.

[0362] Example 15

[0363] Antimicrobial activity

[0364] NRRL No. B-68053 has antimicrobial activity against gram-negative pathogens, gram-positive pathogens, yeasts, and molds. The antimicrobial secondary metabolites are naturally produced and provide a survival function for the organism that produces them. For decades, it has been known that Bacillus strains produce a wide variety of antimicrobial secondary metabolites and that the spectrum produced is considered to be broader than those produced by lactic acid producing bacteria. Antimicrobial susceptibility testing (AST) was performed using the cross-streak method. Bacillus strain NRRL No. B-68053 was inoculated from a frozen glycerol stock in a single 1 cm wide linear streak down the center of a TSA (E. coli, Salmonella, yeast and mold) or BHI + cysteine (Clostridia and Campylobacter) agar plate. The Bacillus streaked plates were incubated aerobically at 37°C for 24 hrs until the Bacillus growth streaks were clearly visible. In a biological safety cabinet, the organisms to be tested for susceptibility were streaked perpendicular to the Bacillus streak (up to 1 mm) and each organism was incubated by the following conditions. The cross-streaks of the organisms E. coli and Salmonella were incubated aerobically at 37°C, the yeast and mold strains were incubated aerobically at room temperature, the Campylobacter strains were incubated microaerobically at 42°C, and the Clostridia and Enterococcus strains were incubated anaerobically at 37°C; all for 24 hrs. No more than 5 staggered cross-streaks were applied per plate. After the incubation period, the plates were examined for zones of inhibition around the original Bacillus streaks and the width of each zone of inhibition was quantified in millimeters.

[0365] Bacillus subtilis strain NRRL No. B-68053 produced strong inhibition against Clostridium perfringens, Clostridium difficile, Candida albicans, and Candida auris strains. Moderate inhibition was produced against E. coli, Salmonella, Enterococcus, Penicillium, and Aspergillus strains. Low inhibition was produced against Fusarium and Mucor strains.

[0366] Example 16

[0367] Immunomodulation

[0368] It is known that Bacillus species modulate immunity through interactions with the host gastrointestinal tract. This immune modulation can help the human body maintain a delicate balance between eliminating invading pathogens while still maintaining a modulated level of inflammation that enables the body to return to homeostasis. Research has found that 70% of the human immune system is located within the epithelial tissue of the gastrointestinal tract. Bacillus, and specifically NRRL No. B-68053, is able to communicate directly and indirectly with these immune cells to provide the necessary response to the surrounding environment, provide essential health benefits to the host, and modulate immune homeostasis.

[0369] The HT29 cell line is developed from human colorectal adenocarcinoma cells and, in contrast to its Caco-2 epithelial cell counterpart, these cells secrete mucin, which is important because the mucus layer plays a role in modulating adhesion of living organisms to epithelial surfaces and bacterial cell components. HT29 cells represent a well-characterized model to study the response of the intestinal epithelium to bacterial infection. This cell line expresses characteristics of intestinal epithelial cells and mediates responses from many different immune cell types.

[0370] To test the immunomodulatory ability of NRRL Number B-68053, the strain was exposed to HT29 cells in the presence and absence of LPS cell wall components. HT29 cells were cultured using DMEM cell culture medium supplemented with 10% FBS and 1% antibiotic / antimycotic and passaged twice for viability consistency and incubated at 37°C and 5% C02. For the immunization assay, cells were cultured in 24 well plates with a 500ul volume and seeded at a density of 100k / well. Confluent monolayers were maintained for 21 days prior to exposure to allow for full maturation of immune cells within the mucosal epithelial monolayer. Once mature, antibiotic / antimycotic was removed from the media and a 4 hour equilibration period was given.

[0371] To prepare the NRRL Number B-68053 test article, overnight cultures were grown in 5ml TSB and incubated at 37°C. Cultures were quantified using optical density measurements (OD at 600nm) using an OD of 0.5 as a reference for 10 7 cells of Bacillus sp. Samples were diluted using 1ml of sterile PBS to yield concentrations of 10 6 , 10 5 , 10 4 , and 10 3 cells / ml. Cultures were also enumerated for precise cell counts. To prepare the LPS compound, an aliquot of 100ng / ul solution was thawed and diluted using PBS down to 10ng / ul. For the immunization assay, 5ul of 10ng / ul LPS was used as a stimulant and 10ul of culture was used per dose of NRRL Number B-68053 strain. Each test condition and control had 6 replicates. Plates were incubated at 37°C, 5% C02 for 2hrs (Table 11).

[0372] Table 11

[0373]

[0374] Cells were harvested after a 2 hour incubation period and RNA was extracted for expression analysis. Briefly, one well at a time was removed to quickly perform and not allow for expression changes, the media was removed and 200ul of Tri reagent was added. The Tri reagent was allowed to work for 5 minutes at room temperature, then once the cells were released from the bond with the well of the plate, they were removed from the plate and placed in a 96 well 2ml round bottom block. The block was covered with a gas permeable sterile membrane on the first layer and adhesive foil on the second layer. The block was then snap frozen in liquid nitrogen and stored at -80°C until ready for RNA extraction and expression work.

[0375] RNA extraction was completed using Qiagen's RNeasy 96 well kit using DTT added to the RLT buffer. The RLT+DTT buffer was prepared by adding 40ul / mL of 1M DTT to the RLT buffer. The protocol was followed by the manufacturer and 45uL was eluted in two rounds. RNA was immediately stabilized into cDNA using QuantaBio reverse transcriptase. For the reaction, 16ul of RNA and 4ul rt enzyme were added to a 96 well PCR plate. The reaction was run on a Bio-Rad machine using the standard reverse transcription protocol provided by QuantaBio. For the immune modulation assay, immune biomarker expression was quantified and expressed as mean relative quantity (RQ) values. GAPDH was used as a reference housekeeping gene. The table below results illustrate the mean RQ value differences when compared to LPS control.

[0376] Table 12. Gene expression data results for immune biomarkers

[0377] Immunological biomarkers Biomarker responses Average RQ values e5 dose TNFa Proinflammatory -33 TLR4 Proinflammatory +0.5 IL-6 Proinflammatory +2.12 CXCL2 Proinflammatory -4 SOCS1 Anti-inflammatory +.1 TOLLIP Anti-inflammatory +1.51 IL-10 Anti-inflammatory +1.79 CXCL12 Anti-inflammatory +0.5

[0378] Example 17

[0379] Antioxidant activity

[0380] The phenotypic antioxidant assay using NRRL number B-68053 resulted in positive chelation and DPPH scavenging activity, indicating positive antioxidant capacity. Genome analysis further revealed the genomic potential for many natural enzymatic antioxidants, including both primary (superoxide dismutase genes for copper, iron, zinc, and manganese, catalase activity, and glutathione peroxidase) and secondary (glucose-6-phosphate dehydrogenase) and flavonoid activity (quercetin).

[0381] While oxygen is an essential element for many functions of living organisms, oxygen concentrations above the normal range cause oxidative stress by generating reactive oxygen species (ROS). ROS are naturally produced through both endogenous and exogenous daily occurrences. Endogenous sources include byproducts of metabolic processes, NADPH oxidase, mitochondrial electron transport chain leakage, and cytokine and growth factor receptors; while exogenous sources come from UV light, radiation, drugs, pollutants, and / or pathogens. The resulting oxidative stress can cause damaging injury to DNA / RNA, proteins, and lipids, and also leads to cellular responses such as inflammation and carcinogenesis. If excess ROS is not controlled, these damaging changes often lead to chronic diseases, including atherosclerosis, arthritis, diabetes, Alzheimer's disease, neurodegenerative diseases, and cardiovascular disease.

[0382] Antioxidants are ROS scavengers that can mask, remove, and repair oxidative damage, thereby protecting target components or molecules from oxidative damage. Organisms have enzymatic and non-enzymatic antioxidant mechanisms to inactivate ROS, and microorganisms have been identified as sources of both. Enzymes, including catalase, glutathione peroxidase, and superoxide dismutase, are endogenous antioxidants that control ROS damage, while carotenoids, flavonoids, coenzyme Q, vitamins, minerals, and phenolic acids are sources of exogenous antioxidants.

[0383] To test the antioxidant capacity of NRRL No. B-68053 phenotypically, we used assays that quantified DPPH scavenging activity and chelation activity. DPPH free radicals are a method widely used to assess the free radical scavenging capacity of natural compounds. The assay is based on the measurement of the ability of antioxidant substances to scavenge stable free radicals. Ferrous ion chelation is another widely used assay to determine scavenging potential.

[0384] For DPPH assay, supernatant was prepared from NRRL No. B-68053 for these assays. This was done by inoculating from 10 ul of an overnight culture grown in TSB and then incubated in a 250 mL flask for 24 hrs, incubation conditions were at a temperature of 32 °C and shaking at 180 rpm. The flask was confirmed for purity and centrifuged at 6000 rpm for 20 minutes at 4 °C to separate the biomass from the supernatant. The supernatant was then filtered sterilized through a 0.2 uM filter and aliquoted into 1.5 ml microfuge tubes. The aliquots were snap frozen in liquid nitrogen and stored at -80 °C until use. Butylated hydroxytoluene (BHT) was used as a reference standard and prepared as a 1 mM solution in methanol. From this stock, a standard curve was prepared at different increments at a concentration of 0-1000 uM. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was prepared as a 0.1 mM working solution in methanol. For the assay, a 96 well microtiter plate was used and 100 ul of DPPH + 100 ul of sample / standard was added to the respective wells. The plate was wrapped in tin foil to avoid light and incubated at room temperature for 60 minutes. The results were read using a plate reader at a wavelength of 517 nm. DPPH produces a purple color in methanol solution and fades to a yellowish tone in the presence of antioxidants. The percent DPPH radical scavenging activity was calculated by the following equation where A0 is the absorbance of the control and Ai is the absorbance of the extract / standard.

[0385] % DPPH radical scavenging activity = {(A0-A1) / A0}*100

[0386] Chelate Iron Ion Assay was performed using the same supernatant extract preparation as above. EDTA was used as a reference standard, which was prepared as a lx working solution from a 10x stock by dissolving 0.05 g EDTA into 50 ml water, pH 8.0. Additional reagents prepared were 10x furazolidone stock (5 mM stock) and 10x FeCl2stock (2 mM stock). EDTA standard curve was prepared from the stock in 10 mg / L increments for concentrations of 0-50 mg / L. For experimental setup, a 96 well titer was used and contained controls, control blanks, samples, and sample blanks. The control contained 100 ul water + 50 ul lx FeCl2+ 100 ul lx furazolidone. The control blank contained 200 ul water + 50 ul lx FeCl2. The sample blank contained 100 ul standard / sample + 50 ul lx FeCl2+ 100 ul water. Finally, the sample contained 100 ul standard / sample + 50 ul lx FeCl2+ 100 ul lx furazolidone. All samples were run in triplicate. The plates were then incubated at room temperature for 5 minutes and read on a plate reader at 562 nm. The respective blanks were subtracted from the controls and samples, and the percent chelate activity was calculated by the following equation, where A0is the absorbance of the control, and Ai is the absorbance of the extract / standard. See Table 13.

[0387] % Chelate Activity = {(A0- Ai) / A0} * 100

[0388] Table 13. Results of antioxidant capacity test

[0389]

[0390] Example 18

[0391] Quorum quenching ability

[0392] Quorum sensing is a method of communication for cells and is a cell density-dependent bacterial response mediated by autoinducer compounds. This communication network controls phenotypic variations, including biofilm formation, virulence factor expression, and motility. Quorum sensing quenching is the ability of an organism to inhibit or interfere with these communications using chemical or enzymatic means to counteract behaviors regulated by quorum sensing.

[0393] Both Gram-negative and Gram-positive bacterial organisms utilize this type of communication signaling, though through different peptide molecules. Gram-negative bacteria primarily utilize acyl-hemoserine (AHL) molecules, such as AI-1, LuxI, or LuxR; while Gram-positive bacteria primarily utilize autoinducing peptides (AIP).

[0394] Chromobacterium violaceum is a well-studied quorum sensing reporter strain that contains a LuxIR-type system to detect and respond to changes in cell population density. A hallmark feature of C. violaceum is its production of the purple pigment, violacein, which is synthesized during active quorum sensing activity. However, when quorum sensing is inhibited, this reporter strain becomes colorless, thus making it a good screening tool for quorum sensing quenching activity.

[0395] In vitro screening of NRRL Number B-68053 for inhibition of C. violaceum quorum sensing communication was tested. Supernatant was prepared from NRRL Number B-68053 for these assays. This was accomplished by inoculating 10 ul of an overnight culture grown in TSB and then incubating in a 250 mL flask for 24 hrs, incubation conditions were at a temperature of 32°C and shaking at 180 rpm. The flasks were confirmed for purity and centrifuged at 4°C, 6000 rpm for 20 minutes to separate the biomass from the supernatant. The supernatant was then filter sterilized through a 0.2 uM filter and aliquoted into 1.5 ml microfuge tubes. The aliquots were snap frozen in liquid nitrogen and stored at -80°C until use.

[0396] C. violaceum strain # 12472 was purchased from ATCC and used as the reporter strain for this screening assay. Frozen stock cultures were allowed to thaw at room temperature in a biological safety cabinet (BSC). In addition, frozen NRRL Number B-68053 supernatant was allowed to thaw at room temperature in a BSC. Using a 96 well microtiter plate, test wells were run as 180 ul TSB, 20 ul NRRL Number B-68053 supernatant and 2 ul ATCC 12472 C. violaceum strain. The positive control was 200 ul TSB and 2 ul C. violaceum. The negative control was 200 ul TSB. All samples were run in triplicate. The plates were incubated overnight at 32°C, approximately 16 hrs. The next day, color differences were quantified by reading the wells on a plate reader at absorbance of 562 nm. The percent quorum sensing quenching activity was calculated by the following equation, where A0 is the absorbance of the control and A1 is the absorbance of the extract / standard. See Table 14.

[0397] % Quorum Sensing Quenching Activity = {(A0-A1) / A0}*100

[0398] Table 14. Results of Quorum Sensing Quenching Activity

[0399]

[0400] Example 19

[0401] Human clinical tolerability study

[0402] A pilot, open-label study evaluated the safety and tolerability of Bacillus subtilis NRRL #68053 in healthy adult volunteers. See Tables 15 and 16. Ten participants enrolled, and all ten participants completed the study. The mean age of the participants was 35.1 ± 11.6 years, weight was 75.6 ± 7.1 kg, and body mass index was 23.8 ± 1.5. The basic inclusion criteria specified that participants must be in good health as determined by medical history and routine blood chemistry, and maintain their regular diet and exercise patterns during the study. The study also included more specific inclusion / exclusion criteria surrounding medical history, medications, and over-the-counter supplements. Participant compliance and completion rates were 100%.

[0403] Participants were given 1 billion total CFU capsules daily for 42 days. The study product used maltodextrin as a carrier and size 0 vegetarian capsules. Participants were instructed to take the capsules in a 24-hour rhythm, preferably with meals. The primary endpoints were adverse event monitoring and weekly visual analog scale (VAS) questionnaires to evaluate GI health and comfort, mood, and stress scores. The GI health questionnaire recorded scores for bloating, abdominal distension, abdominal discomfort, stool consistency, stool regularity, stool frequency, and constipation. The mood and stress questionnaire recorded scores for enthusiasm, happiness, vigor, fatigue, depression, anxiety, uneasiness, tension, worry, and feeling of bad luck. Additional secondary and tertiary endpoints measured various blood biomarkers to evaluate trends.

[0404] Adverse events were recorded weekly throughout the study and evaluated with a score of mild to severe (1-5), as well as CBC blood and lipid panels and vital signs for overall health. No serious adverse events were reported during the study. One case of mild constipation was reported as possibly related to the test product. Healthy adult volunteers consumed 1 x 10 9 CFU of Bacillus subtilis NRRL #68053 for 42 days was well-tolerated.

[0405] The study results were evaluated by statistical analysis using JASP version 0.17.2.1. Statistical significance was determined by one-way ANOVA, Tukey comparisons, and 95% confidence intervals. For the GI health questionnaire, Bacillus subtilis NRRL #68053 significantly improved scores for bloating and abdominal distension, and further produced a trend improvement in abdominal discomfort and a directional improvement in stool regulation. There was no difference in recorded constipation levels. For the mood and stress questionnaire, Bacillus subtilis NRRL #68053 significantly improved levels of enthusiasm, and significantly reduced feelings of fatigue and anxiety. Additionally, there was a trend improvement in reduced worry and irritability, and a directional improvement in increased happiness and vigor recorded over the six-week span; as well as reduced feelings of depression, tension, and difficulty relaxing. There was no difference in levels of uneasiness.

[0406] Additional sera were collected and analyzed for immune modulating biomarkers, and as expected, there was a large biological variability from person to person, especially for the cytokine markers IL-2, IL-8, and IL-10. However, the inflammatory cytokine TNFa was significantly reduced between the initial and final test after the 6-week study duration.

[0407] Table 15. Summary of result mean, standard deviation, and statistical significance.

[0408]

[0409] Table 16. Result mean interpretation; when greater is better, when less is better.

[0410]

[0411] Bacillus strain (NRRL No. B-68054)

[0412] Example 20

[0413] Antibiotic resistance

[0414] The inventors have phenotypically evaluated resistance. EFSA defines a bacterial strain as susceptible when its growth is inhibited at a specific antibiotic concentration equal to or lower than the established cut-off value for that particular species. A bacterial strain is defined as resistant when it is able to grow at a specific antibiotic concentration higher than the established cut-off value. The inventors have used both EFSA and CLSI guidelines to determine susceptibility and resistance, taking into account the interpretation of the cut-off values by which they are defined.

[0415] Table 17. Results for the minimum inhibitory concentration (MIC) against medical antibiotics

[0416]

[0417]

[0418] Example 21

[0419] Hemolytic activity

[0420] Bacillus strain (NRRL No. B-68054) was tested for hemolytic activity by blood agar plates to determine zero, partial, or complete lysis of erythrocytes. NRRL 68054 tested negative for hemolytic activity. β-hemolysis is defined as complete or true lysis of erythrocytes. A clear zone, approaching the color and clarity of the basal medium, surrounds the colonies. α-hemolysis is the reduction of erythrocyte hemoglobin to methemoglobin (in the medium surrounding the colonies). α-hemolysis is commonly observed as brown or green discoloration of the cells surrounding the medium. Unlike β-hemolysis, α-hemolysis maintains the structure of the cell membrane.

[0421] NRRL No. 68054 was cultured and tested for antibiotic susceptibility by the following method. First, the strain was cultured from a frozen stock by streaking the cells on tryptic soy agar medium using a sterile inoculating loop and incubated overnight at 37°C. When colonies were visible the next day, they were examined morphologically for purity and single colonies were collected using a sterile inoculating loop. A single colony was grown to a higher density by inoculating it into tryptic soy broth and incubating it overnight at 37°C at 230 rpm. The next day, the grown cultures were normalized to an OD of 0.1 using 0.1% peptone as a diluent. 600nm The cells were plated at a cell density of 0.8. Blood agar plates were purchased pre-made and contained tryptic soy agar and 5% sheep blood. Using a sterile inoculating loop with a diameter to accommodate approximately 10 μl, the culture was streaked horizontally across the surface of the plate in triplicate. The plates were then incubated overnight at 37°C. The next day, the plates were removed from the incubator and the results recorded. Results were determined as "no hemolysis" for no discoloration of the culture medium, "alpha hemolysis" for partial hemolysis and a green-brown discoloration of the culture medium, and "beta hemolysis" for complete lysis of the culture medium with a clearing zone around the cells. NRRL No. B-68054 produced no hemolysis for all triplicate streaks around the cells streaked on the blood agar plates.

[0422] Example 22

[0423] Enterotoxin

[0424] Using a multiplex PCR method adapted from Yang et al., Bacillus strain (NRRL No. B-68054) resulted in the negative presence of all known toxin genes typically associated with B. cereus and B. thuringiensis. Whole genome sequence analysis was also used to query these known Bacillus toxin gene sequences and the database did not find significant similarity. NRRL No. B-68054 was cultured, DNA was extracted, and toxins were tested by the following method. First, the strain was cultured from a frozen stock by using a sterile inoculation loop to make isolated streaks of cells on tryptic soy agar medium and incubated overnight at 37°C. When colonies were visible the next day, they were checked for purity morphologically and a single colony was collected using a sterile inoculation loop to extract DNA from the strain. It was cultured to a higher density by inoculating a single colony into tryptic soy broth and incubated overnight at 37°C. The next day, 1 ml of culture was transferred to a 1.5 ml microcentrifuge tube and the cells were pelleted in a centrifuge at 10,000 rpm for 10 minutes. DNA extraction was performed on these cells using Qiagen’s DNeasy Blood and Tissue Single Column Kit. The supernatant was discarded from the pelleted cells and the pellet was resuspended in 180 μΐ of lysis buffer containing 20 mg / ml lysozyme. NRRL No. B-68054 + lysis solution was incubated at 37°C for 45 minutes to disrupt cell walls and lyse cellular components. Then, 20 μΐ of proteinase K and 200 μΐ of Buffer AL were added and the sample was incubated at 56°C for another 30 minutes to degrade protein components. After incubation was complete, 200 μΐ of EtOH was added and vortexed to a homogenous solution to aggregate insoluble DNA. A spin column was used to isolate and purify DNA from the solution according to the manufacturer’s recommended protocol. DNA was eluted in 100 μΐ of Low-TE Buffer purchased from ThermoFisher.

[0425] The primer sequences and multiplex PCR method were adapted from Yang et al. Primers for toxin hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), emetic toxin, enterotoxin FM (entFM), enterotoxin T (bceT), and cytotoxin K (cytK) were used to test for the presence of toxin-producing Bacillus species. These primer sequences were purchased from Eurofins and diluted to a working concentration of 40-350 nM (Table 18). Three separate master mixes were prepared for the multiplex toxin assays as shown in Table 18. For each master mix, the total volume was 49 μΐ, including 200 μΜ DNTPs, 1X PCR buffer, 2 U of Fast-Start Taq polymerase, and the remaining volume of ddH2O. The total reaction volume was 55 μΐ, including 49 μΐ of the respective master mix and 6 μΐ of template DNA. B. cereus and B. thuringiensis genomic DNA were used as positive controls, and ddH2O was used for no-template controls.

[0426] PCR reactions were performed using end-point PCR technology on an Applied Biosystems 2720 thermal cycler. All three master mixes were tested against NRRL B-68054, Bc positive control, Bt positive control, and NT negative control. Reaction conditions included a denaturation step at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 45 seconds, and ending with an elongation step at 72 °C for 7 minutes. Amplification products were analyzed on 2% agarose gels, electrophoresed at 100 volts for 80 minutes, and visualized using a gel imaging system with UV transilluminator and GeneSnap software tools.

[0427] Table 19 shows the results of the multiplex PCR assays for NRRL B-68054, Bc and Bt positive controls, and NT negative controls. Figure 7 NRRL No. B-68054 genomic DNA was negative for the presence of any toxin in the three multiplex assays by 30 amplification cycles. The Bc and Bt genomic DNA controls produced the expected band sizes for amplification products for each multiplex assay, and the no-template controls for all three master mixes were negative. Whole genome sequence analysis did not yield significant similarity of NRRL No. B-68054 to these known B. cereus and B. thuringiensis toxin gene sequences (Table 19).

[0428] For NRRL No. B-68054 DNA, the multiplex PCR assay did not produce amplification of Bacillus cereus-like toxin genes hemolysin BL, non-hemolytic enterotoxin, enterotoxin FM, enterotoxin T, or cytotoxin K. Amplified PCR products were seen at the expected band sizes, and additionally, for the genomic DNA positive control, there were no discrete bands of unexpected sizes, and no amplification was seen in the no template control. Furthermore, as described in Yang et al., the sensitivity of the multiplex PCR assay was approximately 100 pg, and the primer pairs were specific for their target. All of these data indicate that these primer sequences, paired in the multiplex reaction, are a suitable method of analysis. These results were confirmed by aligning the whole genome sequence against toxin gene sequences via BLASTn databases, shown in Table 88.

[0429] Table 18. Premix set and primer concentrations for each primer set

[0430]

[0431] Table 19. Results from whole genome sequencing analysis of Bc and Bt toxin genes

[0432]

[0433]

[0434] Example 23

[0435] Cytotoxicity assay

[0436] EFSA requires non-toxigenic activity for Bacillus species. Bacillus subtilis NRRL No. B-68054 was tested for in vitro cytotoxicity using Vero (epithelial) cells. Cytotoxicity was measured with a lactate dehydrogenase (LDH) assay and followed the EFSA “Guideline on the characterisation of the microbial species used as feed additives or production organisms”. An absorbance value higher than 20% of the absorbance obtained from the maximum LDH release control (Promega 10x lysis solution) indicates cytotoxicity. As shown in the table below, supernatant from Bacillus subtilis NRRL No. B-68054 had no cytotoxic effect on Vero cells at a 10% concentration.

[0437] Table 20. Bacillus subtilis NRRL No. B-68054 LDH percentage released by Vero cells after 60 minute exposure

[0438]

[0439] As a control for the endogenous LDH signal in the bacterial culture medium used to generate the test article, the test article was also tested in EC buffer (in the absence of Vero cells). The results of this assay are shown in the table above, as shown in the equation below the table.

[0440] Table 21. Percent Bacillus subtilis NRRL No. B-68054 LDH signal in EC buffer and supernatant control

[0441]

[0442]

[0443] Example 24

[0444] RAPD-PCR DNA profile

[0445] Genetic variability of strain NRRL No. B-68054 was identified using the random amplified polymorphic DNA PCR method (RAPD-PCR). Preparation of DNA to be used in the RAPD-PCR reaction was performed using the Qiagen Blood and Tissue Single Column Kit. To obtain DNA, overnight cultures were prepared, purity was checked, pelleted, and DNA was extracted following the manufacturer’s protocol. Preparation of the RAPD-PCR reaction was performed by using the Cytiva RAPD Bead Kit, which requires the use of one bead per reaction, sterile water, and DNA template, along with one of six primers pre-designed as random amplified polymorphic DNA. All six primers were used in separate reactions for each strain. Each 25 μΐ reaction contained one Cytiva RAPD bead, 16 μΐ sterile water, 5 μΐ of the respective primer, and 4 μΐ of DNA template. Prior to running the reactions, each sample was sealed, vortexed, and centrifuged briefly. RAPD-PCR reactions were run in an AB2720 thermal cycler with the following run conditions; 95 °C for 5 min, followed by 45 cycles of 95 °C for 1 min, 36 °C for 1 min, 72 °C for 2 min, followed by 72 °C for 7 min, and ending with a 4 °C hold indefinitely to preserve the product. RAPD-PCR products were analyzed by gel electrophoresis using 1% (wt / vol) agarose in lx TBE buffer and a UV imager. The agarose gel contained 1 ul / 10 ml of SyberSafe gel stain. The gel was run at 120 volts for 3 hours. At the end of the run, a digital camera connected to the UV imager uploaded the gel image to the GeneSnap software, which then inverted the saturation values and adjusted the contrast for viewing purposes. See Figure 6 .

[0446] Example 25

[0447] Digestive enzyme production

[0448] Phenotypic enzyme assays using NRRL Number B-68054 resulted in positive enzyme activity for lipase, amylase, protease, xylanase, and cellulase, and genomic analysis further yielded genomic potential for alpha and beta galactosidases, methionine reductase and synthase, uricase for uric acid, and prolyl endopeptidase for gluten degradation. These enzymes are extraordinary biocatalysts that increase the rate of biochemical reactions, particularly during digestion. Enzymes and reactions are responsible for hydrolyzing complex carbohydrates, lipids, and proteins into more bioavailable constituents, thereby increasing the rate of absorption and nutrient intake.

[0449] Phenotypic enzyme activity plate assays were performed to determine the enzymatic potential of digestive enzymes well known to be produced by resident microbiota. Tryptic Soy Agar (TSA) is the base agar medium used with selective reagents specific to each enzyme. For lipase activity, 50 mL Tween 80 and 2.5 mL polysorbate 80 were added to 1 L of TSA. For amylase activity, 10 g of corn starch was added to 1 L of TSA. For protease activity, 10 g of casein was added to 1 L of TSA. For xylanase activity, 10 g of xylan was added to 1 L of TSA. For cellulase activity, 5 g of CMC was added to 1 L of TSA. All media were autoclaved at 122°C for 30 min and then poured into agar plates and dried. For each set of plates, 10 ul of NRRL Number B-68054 was spotted in triplicate and incubated overnight at 37°C. The next day, positive enzyme activity was determined by a clearing zone around the culture spot. If there was no clearing zone, then a negative reaction was indicated. Specific to xylanase activity, culture plates were flooded with Gram iodine and positive enzyme activity was determined by an unstained clearing zone, while a negative reaction was indicated by the entire plate turning iodine stained

[0450] Example 26

[0451] Immunomodulation

[0452] Bacillus species are known to modulate immunity through interactions with the host gastrointestinal tract. This immune modulation can help the human body maintain a delicate balance between eliminating invading pathogens while still maintaining a modulated level of inflammatory response that is able to restore homeostasis. Research has found that 70% of the human immune system is located within the epithelial tissue of the gastrointestinal tract. Bacillus, and specifically NRRL Number B-68054, is able to directly and indirectly communicate with these immune cells to provide the necessary response to the surrounding environment, provide essential health benefits to the host, and modulate immune homeostasis.

[0453] The HT29 cell line is developed from human colorectal adenocarcinoma cells and, in contrast to its Caco-2 epithelial cell counterpart, these cells secrete mucin, which is important because the mucus layer plays a role in modulating adhesion of living organisms to epithelial surfaces as well as bacterial cell components. HT29 cells represent a well-characterized model to study the response of the intestinal epithelium to bacterial infection. This cell line expresses characteristics of intestinal epithelial cells and mediates responses from many different immune cell types.

[0454] To test the immunomodulatory ability of NRRL Number B-68054, the strain was exposed to HT29 cells in the presence and absence of LPS cell wall components. HT29 cells were cultured using DMEM cell culture medium supplemented with 10% FBS and 1% antibiotic / antimycotic and passaged twice for viability consistency and were incubated at 37°C and 5% C02. For the immunization assay, cells were cultured in 24 well plates with a 500ul volume and seeded at a density of 100k / well. Confluent monolayers were maintained for 21 days prior to exposure to allow for full maturation of immune cells within the mucosal epithelial monolayer. Once mature, antibiotic / antimycotic was removed from the media and a 4 hour equilibration period was given.

[0455] To prepare the NRRL Number B-68054 test article, overnight cultures were grown in 5ml TSB and incubated at 37°C. Cultures were quantified using optical density measurements (OD at 600nm) using an OD of 0.5 as a reference for 10 7 cells of Bacillus sp. Samples were diluted using 1 mL of sterile PBS to yield 10 6 , 10 5 , 10 4 and 10 3 concentrations. Cultures were also enumerated for precise cell counts. To prepare the LPS compound, an aliquot of 100 ng / ul solution was thawed and diluted using PBS down to 10 ng / ul. For the immunization assay, 5ul of 10 ng / ul LPS was used as a stimulant and 10ul of culture was used per dose of NRRL Number B-68054 strain. Each test condition and control had 6 replicates. Plates were incubated at 37°C, 5% C02 for 2 hrs.

[0456]

[0457] Cells were harvested after a 2 hour incubation period and RNA was extracted for expression analysis. Briefly, one well at a time was removed to quickly perform and not allow for expression changes, the media was removed and 200ul of Tri reagent was added. The Tri reagent was allowed to work for 5 minutes at room temperature, then once the cells were released from the binding with the well of the plate, they were removed from the plate and placed in a 96 well 2ml round bottom block. The block was covered with a gas permeable sterile membrane on the first layer and adhesive foil on the second layer. The block was then snap frozen in liquid nitrogen and stored at -80°C until ready for RNA extraction and expression work.

[0458] RNA extraction was completed using Qiagen's RNeasy 96 well kit using DTT added to the RLT buffer. The RLT+DTT buffer was prepared by adding 40ul / mL of 1M DTT to the RLT buffer. The manufacturer's protocol was followed and 45uL was eluted in two rounds. RNA was immediately stabilized into cDNA using QuantaBio reverse transcriptase. For the reaction, 16ul of RNA and 4ul of rt enzyme were added to a 96 well pcr plate. The reaction was run on a Bio-Rad machine using the standard reverse transcription protocol provided by QuantaBio. For the immunomodulatory assay, the immune biomarker expression was quantified and expressed as a mean relative quantity (RQ) value. GAPDH was used as a reference housekeeping gene. The table results (Table 22) illustrate the mean RQ value differences when compared to the LPS control.

[0459] Table 22. Gene expression data results for immune biomarkers

[0460] Immunological biomarkers Biomarker responses Average RQ values e5 dose TNFa Proinflammatory -38 TLR4 Proinflammatory -0.3 IL-6 Proinflammatory +3.06 CXCL2 Proinflammatory -5 SOCS1 Anti-inflammatory +.41 TOLLIP Anti-inflammatory +2.2 IL-10 Anti-inflammatory +1.3 CXCL12 Anti-inflammatory +1.2

[0461] Example 27

[0462] Antioxidant activity

[0463] The phenotypic antioxidant assay using NRRL number B-68054 resulted in positive chelation and DPPH scavenging activity, indicating positive antioxidant capacity. Genome analysis further revealed the genomic potential for many natural enzymatic antioxidants, including both primary (superoxide dismutase genes for copper, iron, zinc, and manganese, catalase activity, and glutathione peroxidase) and secondary (glucose-6-phosphate dehydrogenase) as well as flavonoid activity (quercetin).

[0464] While oxygen is an essential element for many functions of living organisms, oxygen concentrations above the normal range cause oxidative stress by generating reactive oxygen species (ROS). ROS are naturally produced through both endogenous and exogenous daily occurrences. Endogenous sources include byproducts of metabolic processes, NADPH oxidase, mitochondrial electron transport chain leakage, and cytokine and growth factor receptors; while exogenous sources come from UV light, radiation, drugs, pollutants, and / or pathogens. The resulting oxidative stress can cause damaging injury to DNA / RNA, proteins, and lipids, and also leads to cellular responses such as inflammation and carcinogenesis. If excess ROS is not controlled, these damaging changes often lead to chronic diseases, including atherosclerosis, arthritis, diabetes, Alzheimer's disease, neurodegenerative diseases, and cardiovascular disease.

[0465] Antioxidants are ROS scavengers that can mask, remove, and repair oxidative damage, thereby protecting target components or molecules from oxidative damage. Organisms have enzymatic and non-enzymatic antioxidant mechanisms to inactivate ROS, and microorganisms have been identified as sources of both. Enzymes, including catalase, glutathione peroxidase, and superoxide dismutase, are endogenous antioxidants that control ROS damage, while carotenoids, flavonoids, coenzyme Q, vitamins, minerals, and phenolic acids are sources of exogenous antioxidants.

[0466] To test the antioxidant capacity of NRRL No. B-68054 phenotypically, we used assays that quantified DPPH scavenging activity and chelation activity. DPPH free radicals are a method widely used to assess the free radical scavenging capacity of natural compounds. The assay is based on the measurement of the ability of antioxidant substances to scavenge stable free radicals. Ferrous ion chelation is another widely used assay to determine scavenging potential.

[0467] For DPPH assay, supernatant was prepared from NRRL No. B-68054 for these assays. This was done by inoculating from 10 ul of an overnight culture grown in TSB and then incubated in 25 ml flasks for 24 hrs, incubation conditions were at a temperature of 32 °C and shaking at 180 rpm. The flasks were confirmed for purity and centrifuged at 6000 rpm for 20 minutes at 4 °C to separate the biomass from the supernatant. The supernatant was then filtered sterilized through a 0.2 uM filter and aliquoted into 1.5 ml microfuge tubes. The aliquots were snap frozen in liquid nitrogen and stored at -80 °C until use. Butylated hydroxytoluene (BHT) was used as a reference standard and prepared as a 1 mM solution in methanol. From this stock, a standard curve was prepared at different increments at concentrations of 0-1000 uM. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was prepared as a 0.1 mM working solution in methanol. For the assay, a 96 well microtiter plate was used and 100 ul of DPPH + 100 ul of sample / standard was added to the respective wells. The plate was wrapped in tin foil to avoid light and incubated at room temperature for 60 minutes. The results were read using a plate reader at a wavelength of 517 nm. DPPH produces a purple color in methanol solution and fades to a yellowish tone in the presence of antioxidants. The percent DPPH radical scavenging activity was calculated by the following equation where A0 is the absorbance of the control and Ai is the absorbance of the extract / standard.

[0468] % DPPH radical scavenging activity = {(A0-A1) / A0}*100

[0469] Chelated iron ion assay was performed using the same supernatant extract preparation as above. EDTA was used as a reference standard, which was prepared as a lx working solution from a 10x stock by dissolving 0.05 g EDTA into 50 ml water at pH 8.0. Additional reagents prepared were 10x furazolidone stock (5 mM stock) and 10x FeCl2stock (2 mM stock). EDTA standard curve was prepared from the stock at concentrations of 0-50 mg / L in 10 mg / L increments. For experimental setup, 96 well titer was used and it contained controls, control blanks, samples, and sample blanks. Controls contained 100 ul water + 50 ul lx FeCl2+ 100 ul lx furazolidone. Control blanks contained 200 ul water + 50 ul lx FeCl2. Sample blanks contained 100 ul standard / sample + 50 ul lx FeCl2+ 100 ul water. Finally, samples contained 100 ul standard / sample + 50 ul lx FeCl2+ 100 ul lx furazolidone. All samples were run in triplicate. Plates were then incubated at room temperature for 5 minutes and read at 562 nm on a plate reader. The respective blanks were subtracted from controls and samples, and the percent chelation activity was calculated by the following equation, where A0is the absorbance of the control and Ai is the absorbance of the extract / standard.

[0470] % Chelation Activity = {(A0- Ai) / A0}* 100

[0471] Table 23. Results of antioxidant capacity test

[0472]

[0473]

[0474] Example 28

[0475] Quorum quenching ability

[0476] Quorum sensing is a method of communication for cells and is a cell density-dependent bacterial response mediated by autoinducer compounds. This communication network controls phenotypic variations, including biofilm formation, virulence factor expression, and motility. Quorum sensing quenching is the ability of an organism to inhibit or interfere with these communications using chemical or enzymatic means to counteract behaviors regulated by quorum sensing.

[0477] Both gram-negative and gram-positive bacterial organisms utilize this type of communication signaling, although through different peptide molecules. Gram-negative bacteria primarily utilize acyl homoserine lactone (AHL) molecules, such as AI-1, LuxI, or LuxR; while gram-positive bacteria primarily utilize autoinducing peptides (AIP).

[0478] Chromobacterium violaceum is a well-studied quorum sensing reporter strain that contains a LuxIR-type system to detect and respond to changes in cell population density. A hallmark feature of C. violaceum is its production of the purple pigment, violacein, which is synthesized during active quorum sensing activity. However, when quorum sensing is inhibited, this reporter strain becomes colorless, thus making it a good screening tool for quorum sensing quenching activity.

[0479] In vitro screening of NRRL Number B-68054 for inhibition of C. violaceum quorum sensing communication was tested. Supernatant was prepared from NRRL Number B-68054 for these assays. This was done by inoculating 10 ul of an overnight culture grown in TSB and then incubating in a 250 mL flask for 24 hrs, incubation conditions were at a temperature of 32°C and shaking at 180 rpm. The flask was confirmed for purity and centrifuged at 4°C, 6000 rpm for 20 minutes to separate the biomass from the supernatant. The supernatant was then filter sterilized through a 0.2 uM filter and aliquoted into 1.5 ml microfuge tubes. The aliquots were snap frozen in liquid nitrogen and stored at -80°C until use.

[0480] C. violaceum strain #12472 was purchased from ATCC and used as the reporter strain for this screening assay. The frozen stock culture was allowed to thaw at room temperature in a biological safety cabinet (BSC). Additionally, the frozen NRRL Number B-68054 supernatant was allowed to thaw at room temperature in the BSC. Using a 96 well microtiter plate, test wells were run as 180 ul TSB, 20 ul NRRL Number B-68054 supernatant and 2 ul ATCC 12472 C. violaceum strain. The positive control was 200 ul TSB and 2 ul C. violaceum. The negative control was 200 ul TSB. All samples were run in triplicate. The plate was incubated overnight at 32°C for approximately 16 hrs. The next day, color difference was quantified by reading the wells on a plate reader at absorbance of 562 nm. The percent quorum sensing quenching activity was calculated by the following equation, where A0 is the absorbance of the control and A1 is the absorbance of the extract / standard.

[0481] % Quorum Sensing Quenching Activity = {(A0-A1) / A0}*100

[0482] Table 24. Results for quorum sensing quenching activity

[0483]

Claims

1. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

2. A feed additive for use in animal feed or human food, comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

3. An additive for drinking water for animals or humans comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

4. An animal feed composition or a human food composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

5. The commercial package, feed additive for animal feed or human food, animal feed composition or human food composition, or additive for drinking water for animals or humans according to any one of claims 1 to 4, wherein the Bacillus strain inhibits pathogens selected from the group consisting of Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, Clostridium, Candida, Mucor, Penicillium, Aspergillus.

6. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition according to claim 5, further comprising a carrier for the Bacillus strain.

7. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition of claim 6, wherein the carrier is selected from the group consisting of bran, rice hulls, salt, mineral oil, dextrin, whey, sugar, limestone, dry starch, sodium aluminosilicate, vegetable oil, and combinations thereof.

8. The commercial package, the feed additive for animal feed or human food, the additive for animal or human drinking water, or the animal feed composition or human food composition according to claim 5, further comprising a binder.

9. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition according to claim 8, wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, dextran, and combinations thereof.

10. The commercial package, feed additive for animal feed or human food, additive for drinking water for animals or humans or animal feed composition or human food composition according to any one of claims 1 to 4, in the form of a dietary nutritional composition.

11. The commercial package, feed additive for animal feed or human food, additive for animal or human drinking water, or animal feed composition or human food composition according to any one of claims 1 to 4, further comprising exogenously added nutrients 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.

12. A method of feeding an animal, the method comprising administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of: Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

13. The method of claim 12, wherein at least one of the Bacillus strains has antimicrobial activity.

14. The method of claim 13, wherein the antimicrobial activity is against a microorganism selected from the group consisting of Escherichia coli, Salmonella, Staphylococcus, Enterococcus, Clostridium, Campylobacter, Candida, Mucor, Penicillium, Aspergillus, and combinations thereof.

15. The method of claim 12, wherein the administered strain is a Bacillus strain (NRRL No. B-68053).

16. The method of claim 12, wherein the administered strain is a Bacillus strain (NRRL No. B-68054).

17. A method of improving the health of a human, the method comprising administering to the human a probiotic composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of: Bacillus strain BC1 (NRRL No. B-67744), Bacillus strain (NRRL No. B-68053), Bacillus strain (NRRL No. B-68054), 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 (NRRL No. B-68053), a strain having all the identifying characteristics of Bacillus strain (NRRL No. B-68054), and combinations thereof.

18. The method of claim 17, wherein the improvement in health is an improvement in gastrointestinal health.

19. The method according to any one of claims 17-18, wherein i) the Bacillus strain does not cause hemolysis; ii) the Bacillus strain does not cause cytotoxicity; iii) the Bacillus strain does not produce a toxin selected from the group consisting of hemolysin BL subunit A (hblA), hemolysin BL subunit C (hblC), hemolysin BL subunit D (hblD), non-hemolytic enterotoxin subunit A (nheA), non-hemolytic enterotoxin subunit B (nheB), non-hemolytic enterotoxin subunit C (nheC), vomitoxin, enterotoxin FM (entFM), enterotoxin T (bceT) and cytotoxin K (cytK); iv) the Bacillus strain produces an anti-inflammatory biomarker selected from SOCS1, TOLLIP, IL-10, and CXCL12; v) the Bacillus strain has antioxidant activity; or vi) the Bacillus strain has quorum sensing quenching activity.

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